异烟肼和利福平合用致肝细胞毒性及药物保护机制探讨
|
摘要
结核病是由结核分支杆菌感染引起的疾病,最早以肺结核病多见,近年来肺外结核菌感染及不典型结核病也较常见。结核病在世界范围内仍是发病率和死亡率最高的疾病之一。世界卫生组织(WHO)预计,如果得不到有效的治疗,今后10年内全球至少将有3000万人死于结核病。在我国,仅2008年1-9月就新发结核病77万人,因此目前抗结核治疗形势仍然十分严峻。
在结核病的治疗方案中,异烟肼(isoniazid,INH)和利福平(rifampicin,RFP)仍是世界卫生组织推荐的、不可替代的一线用抗结核药。异烟肼通过抑制结核杆菌细胞所特有的分枝菌酸(mycolic acid)的合成,使细菌丧失耐酸性、疏水性和增殖力而死亡;利福平则可以抑制细菌RNA多聚酶,阻碍mRNA合成,从而达到抑菌和杀菌作用。二者对于繁殖期和静止期状态的细菌均有较强的杀菌、抑菌作用,联合应用对杀死细胞内外结核杆菌有协同效果并可减少耐药性,但两药合用时肝脏毒性发生的几率却明显增加,有时甚至发生急性肝脏衰竭危及生命。虽然临床抗结核药物肝脏损伤现状仍十分严重,抗结核药物致肝损伤的机制却尚未阐明,因此对抗结核药物所致肝脏损害的机制进行更深入的研究就显得尤为重要。
研究认为异烟肼和利福平合用时,二者在体内的代谢转化过程改变,其特定代谢物水平升高是导致肝毒性的重要因素。肝脏细胞色素P450酶(cytochrome P450,CYPs)是催化异烟肼和利福平在体内代谢的主要酶类。目前已知的CYPs的种类超过1000多种,人肝脏中CYP 3A4的含量最丰富,约占其CYPs总量的40%,并参与50%以上常用药物种类的代谢。CYP 2E1在肝脏中的含量只占其中CYPs总量的7%左右,也只代谢2%左右种类的常用药物。但这两种酶均在异烟肼和利福平合用后其代谢产物乙酰肼和/或肼生成增加、肝毒性产生过程中发挥关键作用。
本课题组在前期工作中利用小鼠在体和原代培养的小鼠肝细胞体外实验研究了异烟肼和利福平单/合用及其代谢物对肝细胞的毒性作用,探讨了利福平与异烟肼合用肝毒性增加的发生机制,并对阿魏酸钠、黄苓苷、水飞蓟宾等具有保肝作用的中药成分对抗抗结核药物致肝损害的保护作用进行了研究。
本课题首先观察结核病患者血浆异烟肼及其代谢物水平的变化,并以人肝细胞株QSG-7701为对象,研究异烟肼及其代谢物及异烟肼和利福平合用对肝细胞的损伤和毒性作用,探讨细胞色素酶P450同工酶CYP 3A4、CYP 2E1在异烟肼、利福平所致肝毒性中的作用;筛选抗结核药物异烟肼、利福平所致肝毒害的特异性保护药物并探讨其作用机制。旨在阐明异烟肼和利福平合用导致肝毒性增加的机制,探索有效抑制其毒性的药物,为制定抗结核用药的新策略奠定基础。
方法
1受试者入选与分组给药
遵照《人体生物学医学研究国际道德指南》规定的原则选择受试者,并填写《患者知情同意书》。将24名入选的肺结核患者,随机分为异烟肼组、利福平组及异烟肼和利福平合用组。异烟肼组晨起空腹口服0.3 g异烟肼一次,早晚空腹口服0.2 g左氧氟沙星各一次,早中晚空腹口服0.1 g葡萄糖醛酸内酯各一次;利福平组晨起空腹口服0.45 g利福平一次,早晚空腹口服0.2 g左氧氟沙星各一次,早中晚空腹口服0.1 g葡萄糖醛酸内酯各一次;异烟肼和利福平合用组除服用与异烟肼组相同的药物外,晨起空腹口服0.45 g利福平一次,连续服药两周。
2血样采集与处理方法
患者连续服药两周后,即第15天晨起服药前及服药后2 h、4 h、12 h采用抗凝管收集肘静脉血5 mL,3500 rpm离心10 min,取血浆分装两份,于-70℃低温冰箱保存,备异烟肼及其代谢物浓度检测;分别于开始抗结核前和用药两周后晨起空腹抽取肘静脉血5 mL,检测谷丙转氨酶、谷草转氨酶和总胆红素水平。
3 QSG-7701肝细胞培养方法
利用QSG-7701肝细胞株研究异烟肼和利福平对离体肝细胞的毒性。将冻存于液氮中的QSG-7701肝细胞于37℃水浴中迅速解冻,离心弃上清后,沉淀加入含10%胎牛血清的高糖DMEM培养基,用吸管吹打,使成单细胞悬液,用台盼蓝染色后,显微镜下用计数板计数,并接种于70 mm培养皿中培养,接种密度为1×105 cells/mL,体积5 mL,于CO2培养箱中培养24 h后换全液,以后隔天换液,待细胞长满后吸出培养液,加入1 mmol/L的EDTANa2清洗,吸弃EDTANa2溶液后,用0.25%的胰酶消化液消化,加入培养液并用吸管吹打使成单细胞悬液,进行传代。用于加药处理的细胞,接种于培养板。
4 MTT法检测细胞存活率
向培养板培养的细胞培养液中加入MTT溶液使其终浓度为0.05%,继续培养4 h后吸弃培养液,每孔加入DMSO 150μL,振荡使甲瓒结晶溶解,以空白孔调零,用读板机或分光光度计读取570 nm波长处的吸光度值,以空白对照组吸光度的均值为100%,用各样本吸光度值/空白对照组吸光度的均值表示该样本细胞的存活率。
5细胞培养液及细胞裂解液乳酸脱氢酶活性的测定
药物处理完毕后,分别收集各组细胞培养上清液,-80℃冻存备测。将细胞用生理盐水冲洗一遍后,每孔加入生理盐水1 mL,用细胞刮刀将细胞刮下,将细胞悬液2000 rpm离心5 min,倾弃上清后,沉淀加入0.5 mL细胞裂解液,4℃下裂解30 min,12000 rpm离心20 min,收集上清-80℃冻存备测。用乳酸脱氢酶测试盒,比色法测定440 nm样品的吸光度值,并计算乳酸脱氢酶的活力。用细胞培养液中酶活力与细胞裂解液中酶活力的比值代表药物作用对细胞损伤及乳酸脱氢酶活力的影响。
6 HPLC-MS法测定血浆中异烟肼及其代谢物乙酰肼和肼的浓度
取血浆样品0.1 mL,依次加入内标溶液20μL,甲醇0.2 mL,涡旋混合3 min,4000 r·min-1离心10 min,取上清液0.2 mL,加入衍生化溶液10μL,室温下暗处反应24 h,取20μL用于HPLC-MS法测定。采用高效液相色谱质谱联用法测定人血浆中异烟肼、乙酰肼和肼的浓度。所采用的色谱方法为:Agilent C18分析柱(5μm,2.1mm×150 mm ID),Agilent SB C18保护柱(5μm),柱温40℃,流动相为甲醇-水(含0.1%冰醋酸),流速为150μL/min。质谱检测采用电喷雾(ESI)离子化源,毛细管电压4.0 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;正离子方式检测,碎片电压为分别为190V、130V、150V;选择离子监测(SIM)模式,用于定量分析的离子分别为m/z 256.1(异烟肼衍生物)、m/z 193.2(乙酰肼衍生物)、m/z 151.1(肼衍生物)。
7 HPLC-MS法测定细胞孵育液中咪达唑仑和对硝基儿茶酚的浓度
色谱条件:色谱柱:Gemini C18分析柱(2.0×50 mm,5μm),Phenomenex公司;C18保护柱(5μm,3.0×4.0 mm),Phenomenex公司;流动相:乙腈-水(含0.2%冰醋酸)=60:40;流速为0.2 mL·min-1;柱温为30℃;进样量:10μL。
质谱条件:电喷雾(ESI)离子化源,选择离子监测(SIM)模式。
对硝基儿茶酚检测条件:毛细管电压2 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;负离子方式检测,碎片电压为150 V,m/z [154.0]-(对硝基儿茶酚)。
咪达唑仑:毛细管电压3 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;正离子方式检测,碎片电压为50 V,m/z [326.0]+(咪达唑仑)。
8分组及加药处理
8.1异烟肼及其代谢物和利福平对细胞存活率的影响
细胞接种于96孔板,接种密度为每孔1×104 cells,将细胞分为空白对照组、利福平组、异烟肼组、乙酰肼组、肼组和利福平+异烟肼组,每组6孔。于接种后培养24 h时分别换含有空白溶剂、利福平(100μg/mL,200μg/mL,300μg/mL)、异烟肼(250μg/mL,500μg/mL,2500μg/mL)、乙酰肼(125μg/mL,250μg/mL,1250μg/mL)、肼(0.25μg/mL,2.5μg/mL,25μg/mL)、利福平+异烟肼(100+50μg/mL,200+100μg/mL,300+150μg/mL)的含药培养液,培养箱中培养48 h后采用MTT法测定细胞存活率。
8.2异烟肼及其代谢物和利福平对细胞乳酸脱氢酶释放的影响
将细胞接种于6孔培养板,接种后24 h换含药培养液培养48 h,分组及加药方法同“8.1”项下操作,收集培养液和细胞用于乳酸脱氢酶活性的测定。
8.3异烟肼和利福平对细胞CYP 2E1和3A4活性的的影响
将细胞分为8组,每组6孔,分别加入含有溶剂、异烟肼100μg/mL、利福平200μg/mL、异烟肼100μg/mL和利福平100μg/mL的培养液,各两组。继续培养48 h后,吸弃含药培养液,分别加入含有CYP 2E1作用底物对硝基酚(15μg/mL)或CYP 3A4作用底物咪达唑仑(1μg/mL)的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h,吸出培养液,用HPLC-MS法测定对硝基酚代谢产物对硝基儿茶酚和咪达唑仑的水平,用以代表各组细胞CYP 2E1和3A4的活性。
8.4 CYP 2E1和3A4抑制剂对异烟肼和利福平合用增加细胞乳酸脱氢酶释放的影响
将培养板中的细胞分为14组,分别为空白对照组、异烟肼和利福平合用组、柑桔素组、红霉素组、阿魏酸钠组和三七皂苷组,每组6孔。各组分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL)的培养液继续培养48 h。柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加入CYP 3A4抑制剂柑桔素(1μg/mL、5μg/mL、25μg/mL)和红霉素(1μg/mL、5μg/mL、25μg/mL);阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时分别加入CYP 2E1抑制剂阿魏酸钠(10μg/mL、50μg/mL、250μg/mL)和三七皂苷(4μg/mL、20μg/mL、100μg/mL)共同培养。收集细胞培养上清液和细胞,检测乳酸脱氢酶的活性
8.5 CYP 2E1和3A4抑制剂对异烟肼和利福平合用降低细胞存活率的影响
将接种于24孔培养板的细胞分为8组,每组6孔,于接种后24 h吸弃培养液,分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL)的培养液继续培养48 h。柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加入CYP 3A4抑制剂柑桔素(5μg/mL)和红霉素(5μg/mL);阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时分别加入CYP 2E1抑制剂阿魏酸钠(50μg/mL)和三七皂苷(20μg/mL)共同培养。采用MTT法测定培养后细胞的存活率的变化。
8.6酶抑制剂对细胞内CYP450 2E1和3A4酶活性的影响
将接种于24孔培养板的细胞分为8组,每组6孔,各组分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL);柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加含CYP 3A4抑制剂柑桔素(5μg/mL)和红霉素(5μg/mL)的培养液继续培养48 h,吸弃含药培养液,加入含有CYP 3A4作用底物咪达唑仑1μg/mL的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h;阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时加入含CYP 2E1抑制剂阿魏酸钠(50μg/mL)和三七皂苷(20μg/mL)的培养液继续培养48 h,吸弃含药培养液,加入含有CYP 2E1作用底物对硝基酚15μg/mL的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h。孵育后吸出培养液,用HPLC-MS法测定咪达唑仑和对硝基儿茶酚的浓度。
9数据处理与统计分析
统计分析采用SPSS 11.5 for windows软件,数据以均数±标准差(mean±SD)表示,组间差异采用成组设计的t检验;多组间比较采用方差分析(ANOVA)继于Dunnett t test;量效关系采用二元变量相关分析,计算Pearson相关系数,统计结果以P<0.05为具有显著性差异。
结果
1肺结核病患者血浆异烟肼及其代谢物乙酰肼和肼浓度的变化
经过对肺结核病患者服药后血浆中药物浓度的测定发现,与单用异烟肼组比较,异烟肼和利福平合用组肺结核病患者血浆中肼的浓度在服药后2 h即显著升高(P<0.05),在服药后4 h和12 h时仍显著高于单用异烟肼组(均为P<0.01)。与单用异烟肼组比较,异烟肼和利福平合用组患者血浆中异烟肼和乙酰肼的浓度未见显著性变化(P>0.05)。各组患者服药前后谷丙转氨酶、谷草转氨酶和总胆红素水平均未见异常(P>0.05)。
2异烟肼、利福平及异烟肼代谢物乙酰肼和肼对细胞存活率的影响
进一步对异烟肼、利福平及异烟肼代谢物乙酰肼和肼对离体肝细胞的毒性作用进行研究发现,与对照组比较,500μg/mL和2500μg/mL的异烟肼处理48 h,肝细胞的存活率均显著降低,其存活率分别为64.3% (P<0.01)和9.9% (P<0.01),异烟肼浓度与细胞存活率的Pearson相关系数为r = -0.959 (P<0.01);300μg/mL的利福平处理肝组细胞的存活率显著降低(56.2%, P<0.01);中、高剂量利福平和异烟肼合用组肝细胞存活率依次显著降低(分别为73.7%, P<0.05和33.3%, P<0.01),异烟肼和利福平合用浓度与细胞存活率的Pearson相关系数为r = -0.892 (P<0.01);2.5μg/mL和25μg/mL肼处理组肝细胞的存活率显著降低(分别为53.5%, P<0.05和46.0%, P<0.01)。结果表明,异烟肼和利福平合用的肝细胞毒性作用大于其单用,异烟肼代谢物肼对肝细胞的毒性作用最强。
3异烟肼、利福平及其异烟肼代谢物对细胞乳酸脱氢酶活性的影响
检测药物处理后肝细胞外/内乳酸脱氢酶活性的比值,反映细胞的损伤程度。与对照组比较,三种浓度的异烟肼处理后,细胞外/细胞内LDH的比值分别升高83.8%(P<0.01)、112.0%(P<0.01)和124.9%(P<0.01);300μg/mL的利福平处理使细胞外/细胞内LDH的比值显著升高50.3% (P<0.05);中、高浓度的利福平和异烟肼合用剂量依赖地使细胞外/细胞内LDH比值显著升高,升高幅度分别为76.2%(P<0.05)、210.8%(P<0.01)和1010.3% (P<0.01);异烟肼和利福平合用浓度与LDH比值的Pearson相关系数为r = 0.907 (P<0.01);三种浓度的肼处理后,细胞外/细胞内LDH比值分别升高73.3%(P<0.05)、102.1%(P<0.01)和115.5%(P<0.01);250μg/mL和1250μg/mL的乙酰肼处理后,细胞外/细胞内LDH比值分别升高121.1%(P<0.01)和153.1%(P<0.01);乙酰肼浓度与LDH比值的Pearson相关系数为r = 0.674 (P<0.01)。结果表明,异烟肼和利福平合用的对肝细胞的损伤作用大于其单用,异烟肼代谢物乙酰肼和肼均有损伤作用,其中肼对肝细胞的损伤作用最强。
4异烟肼和利福平对肝细胞CYP 2E1和CYP 3A4活性的影响
本实验关于异烟肼和利福平对肝细胞CYPs活性影响的结果显示,与对照组比较,单用异烟肼和异烟肼与利福平合用处理48 h,肝细胞CYP 2E1酶活性均显著升高(均为P<0.05);而单用利福平处理48 h,肝细胞CYP 2E1酶活性则未见明显变化(P>0.05);单用异烟肼和单用利福平处理48 h,分别使肝细胞CYP 3A4酶活性显著升高(P<0.05和P<0.01);异烟肼和利福平合用处理48 h,肝细胞CYP 3A4酶活性进一步升高(P<0.01)。表明异烟肼和/或利福平单用和合用均可上调细胞CYP 2E1和CYP 3A4的活性,二者合用更可进一步上调CYP 3A4的活性。
5 CYP450 3A4抑制剂对异烟肼和利福平致肝细胞毒性作用的影响
采用柑桔素或红霉素与异烟肼和利福平共同处理肝细胞发现,与异烟肼和利福平合用组比较,给予异烟肼和利福平处理的同时,加入柑桔素或红霉素共同培养,肝细胞中CYP 3A4的酶活性均显著低于异烟肼和利福平合用组(均为P<0.01);肝细胞的存活率显著升高(分别为95.2%和107.8%,均为P<0.01);中、高剂量的柑桔素和低、中、高剂量的红霉素均显著降低了细胞外和细胞内乳酸脱氢酶的比值(均为P<0.01),其中柑桔素浓度与乳酸脱氢酶比值的Pearson相关系数为r = -0.712 (P<0.01)。表明柑桔素和红霉素可以通过抑制异烟肼和利福平对CYP 3A4的诱导作用,减轻其对肝细胞的损伤。
6 CYP450 2E1抑制剂对异烟肼和利福平致肝细胞毒性作用的影响
采用阿魏酸钠和三七皂苷与异烟肼和利福平共同处理肝细胞发现,与异烟肼和利福平合用组比较,给予异烟肼和利福平处理的同时,加入阿魏酸钠或三七皂苷共同培养,肝细胞中CYP 2E1的酶活性均显著降低(均为P<0.01);阿魏酸钠组肝细胞的存活率显著升高(77.1%,P<0.05);中、高剂量的三七皂苷和低、中、高剂量的阿魏酸钠均显著降低了细胞外/内乳酸脱氢酶的比值(均为P<0.01),其中三七皂苷浓度与乳酸脱氢酶比值的Pearson相关系数为r = -0.659 (P<0.01)。表明阿魏酸钠和三七皂苷可以通过抑制异烟肼和利福平对CYP 2E1的诱导作用,减轻其对肝细胞的损伤。
结论
1.肺结核病患者使用异烟肼和利福平联合治疗时,血浆中异烟肼代谢物肼的浓度较单用异烟肼时显著升高,这可能是两药合用导致肝毒性增加的机制之一。
2.采用体外肝细胞培养的方法进一步证明,不同剂量的异烟肼、利福平、肼及异烟肼和利福平合用能明显造成肝细胞损伤、显著降低肝细胞的存活率,其中异烟肼和利福平合用的肝细胞毒性强于其单用,异烟肼代谢物肼对肝细胞的毒性作用远大于其母体异烟肼,是异烟肼产生肝毒性的重要代谢产物。
3.异烟肼和利福平单独或合用可不同程度地使肝细胞CYP 3A4和CYP 2E1的活性增加,推测此为异烟肼和其与利福平合用时异烟肼代谢产物水平升高,肝细胞存活率降低、损伤增加,乳酸脱氢酶向细胞外释放增加的机制之一。
4.柑桔素和红霉素可以显著降低肝细胞内CYP 3A4的活性,阿魏酸钠和三七皂苷可以显著降低肝细胞内CYP 2E1的活性。不同浓度的柑桔素、红霉素、阿魏酸钠和三七皂苷均可抑制异烟肼和利福平合用致肝细胞存活率降低和/或乳酸脱氢酶释放增加的作用,具有对其肝细胞毒性的保护作用,对相应酶活性的调节可能是这些药物发挥作用的途径之一。
Tuberculosis (TB) is one of the major causes of death from infectious disease. Recommended standard treatment by the World Health Organization (WHO) for adult respiratory TB is a regimen of isoniazid, rifampicin, and pyrazinamide for 2 months, followed by 4 months of isoniazid and rifampicin. The most frequent and most serious adverse effect of anti-tuberculosis treatment is hepatotoxicity. Evidence accumulated during the last years documented that cytochrome enzymes that mediate the metabolism of isoniazid play an important role in the development of hepatotoxicity. Moreover, the cytochrome enzymes CYP 3A4 and 2E1 are thought to mediate the hepatotoxicity. However, mechanisms that mediate anti-tuberculosis treatment induced hepatotoxicity are still not clear.
Studies by others and our previous studies in mice suggested that hydrazine, the metabolite of isoniazed, was the main hepatotoxic compound. And drugs administered, such as sodium ferulate, baicalin and silibinin are protective in isoniazid and rifampicin exacerbated heptotoxicity.
The present study was carried out to research the levels of isoniazid and its metabolites in the plasma of lung tuberculosis patients administered with isoniazid with or without rifampicin for two weeks. And the hepatocyte toxicity of isoniazid, its metabolites and rifampicin was investigated in cultured hepatocytes by using cell viability assays and lactate dehydrogenase release, a marker of cell death. Furthermore, the role of CYP 3A4, 2E1 and their inhibitors in the occurrence and preservation of isoniazid-rifampicin induced hepatoxicity was demonstrated.
Methods
1. Patients and drugs administration
Twenty-four lung tuberculosis patients were designated to three groups randomly. In the isoniazid group, patients were administered with isoniazid, levofloxacin and glucurolactone. In the rifampicin group, patients were administered with rifampicin, levofloxacin and glucurolactone. In the isoniazid and rifampicin group, patients were administered with isoniazid, rifampicin and glucurolactone.
2. Plasma sample collection and disposal
Plasma samples were collected before and 2 hours, 4 hours and 12 hours after the last administration of isoniazid and/or rifampicin following a two week duration of therapy and the samples were then stored at -70℃until HPLC-MS analysis. Alanine aminotransferase, aspartate aminotransferase and total bilirubin levels in serum were determined before and after therapy.
3. Culture of QSG-7701 hepatocellulars
The QSG-7701 cell strain was cultured in DMEM culture media that contained high glucose at a cell density of 1×105 cells/mL. Pancreatin (0.25%) was used for trypsinization and sub-culturing.
4. Cell viability assay
The MTT assay was used to evaluate the viability of the hepatocytes cultured in 96-well plates. Briefly, MTT was added to the culture media with a final concentration of 0.05%. After 4 hours of culture, the culture media was removed and 150μL DMSO was added to dissolve the crystals. The absorbance of the solution was read at 570nm on a spectrophotometer and the cell viability was calculated.
5. Measurement of lactate dehydrogenase activity in cells and culture media
The cells in the culture plate were flushed with sodium solution and scraped off. After centrifugation, the cells were lyzed with lysis buffer that was incubated with the cells for 30 minutes. The resultant supernatant was used for the assay. Lactate dehydrogenase activity in cell lysates and culture media were determined using a lactate dehydrogenase activity kit and absorbance spectrophotometery. The ratio of extracellular and intracellular activity of lactate dehydrogenase was calculated.
6. Determination of plasma isoniazid, acetylhydrazine and hydrazine concentrations using HPLC-MS
Internal standard and methanol were added to plasma samples that were then vortexed for 3 minutes. After centrifugation of the samples, the supernatants were incubated with derivative agent for 24 hours and then analyzed with HPLC-MS using a 10μL on each HPLC run. An Agilent C18 analyze column (5μm, 2.1mm×150 mm ID) was used with an Agilent SB C18 guard column (5μm). The mobile phase consisted of methanol: water (containing 0.1% glacial acetic acid) and was pumped into the system at a rate of 0.15 mL·min-1. ESI ionization was used in the mass detection in selected ion monitoring. The ions selected for isoniazid, acetylhydrazine and hydrazine- were m/z [256.1]+, m/z [193.2]+ and m/z [151.1]+ , respectively.
7. Determination of midazolam and 4-nitrocatichol concentrations in culture media using HPLC-MS
A Gemini C18 analyze column (2.0×50 mm, 5μm) was used with a C18 guard column (5μm,3.0×4.0 mm). The mobile phase consists of acetonitrile: water (60:40, v/v, containing 0.2% glacial acetic acid) and was pumped in 0.2 mL·min-1. Ten milliliters of sample was analyzed on the column. ESI ionization was used in mass detection in selected ion monitoring. The ions selected for 4-nitrocatechol and midazolam- were m/z [154.0]- and m/z [326.0]+, respectively.
8. Grouping and drug treatment
8.1 Effect of isoniazid, its metabolites and rifampicin on cellular viability
The cells were plated in 96-well plates at a cell density of 1×104 cells per well. The cells were designated to sixteen groups with six wells in each group. The six treatment groups consisted of the blank vehicle, isoniazid (250μg/mL, 500μg/mL, 2500μg/mL), rifampicin (100μg/mL, 200μg/mL, 300μg/mL), acetylhydrazine (125μg/mL, 250μg/mL, 1250μg/mL), hydrazine (0.25μg/mL, 2.5μg/mL, 25μg/mL) and isoniazid+rifampicin (100+50μg/mL, 200+100μg/mL, 300+150μg/mL). All treatments were added to the culture media and after 48 hours of culture, cell viability was determined using the MTT method.
8.2 Effect of isoniazid, its metabolites and rifampicin on lactate dehydrogenase activity
Cells were plated in to 6-well plates. The groups and treatments were the same as item“8.1”. Cells and culture media were collected for assay of lactate dehydrogenase.
8.3 Effect of isoniazid and rifampicin on the activity of CYP 2E1 and 3A4
Cells were designated to eight treatment groups with six wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL), rifampicin (200μg/mL), isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. After a 48 hour incubation period the culture media was removed. 4-nitrophenol (15μg/mL) and midazolam (1μg/mL), substrates of CYP 2E1 and 3A4 respectively, were added to the wells and cultured for an additional 2 hours. The concentration of 4-nitrocatechol, a metabolite of 4-nitrophenol, and midazolam were determined using HPLC-MS. The activity of CYP 2E1 and 3A4 were calculated by the increasing level of 4-nitrocatechol and the decreasing level of midazolam.
8.4 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin on lactate dehydrogenase release
Cells were plated onto 24-well plates, and were designated to fourteen groups with six-wells in each group. Three of the fourteen groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) treatments that were added to the culture media. The other groups consisted of the naringenin and erythromycin groups where naringenin (1μg/mL, 5μg/mL, 25μg/mL) or erythromycin (1μg/mL, 5μg/mL, 25μg/mL) were added in addition. In the sodium ferulate and sanchinoside groups, sodium ferulate (10μg/mL, 50μg/mL, 250μg/mL) and sanchinoside (4μg/mL, 20μg/mL, 100μg/mL) were added in addition. After 48 hours of culture, lactate dehydrogenase activity in the cells and culture media were determined.
8.5 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin decreased cellular viability
Cells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were add to the culture media. In naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. In sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the cell viability was determined using the MTT method.
8.6 Effect of enzyme inhibitors on the activity of CYP 2E1 and 3A4
Cells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. In the naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 3A4 determined. In the sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 2E1 determined.
9 Data and statistics
Statistic was performed with SPSS 11.5 for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA with the Dunnett t test and binary variable correlation analysis. Significance was considered when P<0.05.
Results
1. Concentrations of isoniazid, acetylhydrazine and hydrazine in the plasma of lung tuberculosis patients
The concentration of hydrazine increased significantly 2 h, 4 h and 12 h (P<0.05 or P<0.01) in plasma after isoniazid and rifampicin were co-administered to patients with lung tuberculosis compared with treatment with isoniazid alone. There were no significant differences between the concentrations of isoniazid and acetylhydrazine between the groups (P>0.05). There was no significant abnormality in the levels of GPT, GOT and total bilirubin before and after two weeks of drug therapy (P>0.05).
2. Viability of isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytes
After 48 hours of treatment with isoniazid (500μg/mL or 2500μg/mL), the viability of cultured QSG-7701 cells decreased significantly compared with the control group with the value of 64.3% (P<0.01) and 9.9% (P<0.01). The Pearson correlation coefficient was -0.959 (P<0.01) between the concentration of isoniazid and cell viability. The viability of cells decreased to 56.2% (P<0.01), 53.5% (P<0.05) and 46.0% (P<0.01) in the rifampicin (300μg/mL) and hydrazine (2.5μg/mL and 25μg/mL) treated groups. Co-administration of isoniazid and rifampicin dose dependently decreased cell viability and the Pearson correlation coefficient was -0.892 (P<0.01).
3. Activity of lactate dehydrogenase released from isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytes
Isoniazid, rifampicin, acetylhydrazine, hydrazine, isoniazid and rifampicin co-treatment increased the release of lactate dehydrogenase from cells after 48 hours of culture (P<0.01 or P<0.01). The effect of acetylhydrazine and isoniazid-rifampicin co-treatment was dose dependent and the Pearson correlation coefficient was 0.674 (P<0.01) and 0.907 (P<0.01), respectively.
4. Effect of isoniazid and rifampicin on activity of CYP 3A4 and 2E1 in hepatocytes
The activity of CYP 2E1 increased significantly in QSG-7701 cells cultured for 48 hours with isoniazid or isoniazid and rifampicin (P<0.05) compared with the control group. There was no significant change in CYP 2E1 activity in the rifampicin alone treated group (P>0.05). Treatment with isoniazid, rifampicin alone or isoniazid and rifampicin all increased the activity of CYP 3A4 in QSG-7701 cells compared with the control group(P<0.05 or P<0.01).
5.Effect of CYP 3A4 inhibitors on the hepatotoxicity of isoniazid and rifampicin
Naringenin and erythromycin inhibited the activity of CYP 3A4 enhanced by isoniazid and rifampicin, and attenuated the decrease in cell viability and depressed lactate dehydrogenase release (P<0.01) from cultured QSG-7701 cells. The effect of naringenin on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.712 (P<0.01).
6. Effect of CYP 2E1 inhibitors on the hepatotoxicity of isoniazid and rifampicin
Sodium ferulate and sanchinoside inhibited the activity of CYP 2E1 enhanced by isoniazid and rifampicin, attenuated the decrease in cell viability and depressed the increase in lactate dehydrogenase release (P<0.05 or P<0.01) from cultured QSG-7701 cells. The effect of sanchinoside on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.659 (P<0.01).
Conclusions
1 The concentration of hydrazine increased significantly in the plasma of lung tuberculosis patients that were co-administered isoniazid and rifampicin compared with patients that were treated with isoniazid alone, and the co-treatment may be the mechanism by which isoniazid and rifampicin induce hepatotoxicity.
2 Different co-administered doses of isoniazid, hydraine, rifampicin, isoniazid and rifampicin result in increasing lactate dehydrogenase release and decrease the viability of hepatocytes. The effect of co-administration is more intensive than isoniazid or rifampicin treatment alone. And hydrazine, the metabolite of isoniazid, induced more severe hepatocytes toxicity than its precursor, which suggests a pivotal and considerable role of hydrazine in anti-tuberculosis induced hepatotoxicity.
3 The activity of CYP 3A4 and 2E1 could be enhanced by isoniazid and rifampicin alone and their co-treatment, which may be responsible for the excessive production of hydrazine and the hepatocyte toxicity following isoniazid and rifampicin co-therapy.
4 The activity of CYP 3A4 could be depressed by naringenin and erythromycin and the activity of CYP 2E1 could be depressed by sodium ferulate and sanchinoside. Moreover, naringenin, erythromycin, sodium ferulate and sanchinoside attenuate isoniazid and rifampicin co-treatment induced hepatocyte viability and decrease lactate dehydrogenase release through their effect on CYPs.
在结核病的治疗方案中,异烟肼(isoniazid,INH)和利福平(rifampicin,RFP)仍是世界卫生组织推荐的、不可替代的一线用抗结核药。异烟肼通过抑制结核杆菌细胞所特有的分枝菌酸(mycolic acid)的合成,使细菌丧失耐酸性、疏水性和增殖力而死亡;利福平则可以抑制细菌RNA多聚酶,阻碍mRNA合成,从而达到抑菌和杀菌作用。二者对于繁殖期和静止期状态的细菌均有较强的杀菌、抑菌作用,联合应用对杀死细胞内外结核杆菌有协同效果并可减少耐药性,但两药合用时肝脏毒性发生的几率却明显增加,有时甚至发生急性肝脏衰竭危及生命。虽然临床抗结核药物肝脏损伤现状仍十分严重,抗结核药物致肝损伤的机制却尚未阐明,因此对抗结核药物所致肝脏损害的机制进行更深入的研究就显得尤为重要。
研究认为异烟肼和利福平合用时,二者在体内的代谢转化过程改变,其特定代谢物水平升高是导致肝毒性的重要因素。肝脏细胞色素P450酶(cytochrome P450,CYPs)是催化异烟肼和利福平在体内代谢的主要酶类。目前已知的CYPs的种类超过1000多种,人肝脏中CYP 3A4的含量最丰富,约占其CYPs总量的40%,并参与50%以上常用药物种类的代谢。CYP 2E1在肝脏中的含量只占其中CYPs总量的7%左右,也只代谢2%左右种类的常用药物。但这两种酶均在异烟肼和利福平合用后其代谢产物乙酰肼和/或肼生成增加、肝毒性产生过程中发挥关键作用。
本课题组在前期工作中利用小鼠在体和原代培养的小鼠肝细胞体外实验研究了异烟肼和利福平单/合用及其代谢物对肝细胞的毒性作用,探讨了利福平与异烟肼合用肝毒性增加的发生机制,并对阿魏酸钠、黄苓苷、水飞蓟宾等具有保肝作用的中药成分对抗抗结核药物致肝损害的保护作用进行了研究。
本课题首先观察结核病患者血浆异烟肼及其代谢物水平的变化,并以人肝细胞株QSG-7701为对象,研究异烟肼及其代谢物及异烟肼和利福平合用对肝细胞的损伤和毒性作用,探讨细胞色素酶P450同工酶CYP 3A4、CYP 2E1在异烟肼、利福平所致肝毒性中的作用;筛选抗结核药物异烟肼、利福平所致肝毒害的特异性保护药物并探讨其作用机制。旨在阐明异烟肼和利福平合用导致肝毒性增加的机制,探索有效抑制其毒性的药物,为制定抗结核用药的新策略奠定基础。
方法
1受试者入选与分组给药
遵照《人体生物学医学研究国际道德指南》规定的原则选择受试者,并填写《患者知情同意书》。将24名入选的肺结核患者,随机分为异烟肼组、利福平组及异烟肼和利福平合用组。异烟肼组晨起空腹口服0.3 g异烟肼一次,早晚空腹口服0.2 g左氧氟沙星各一次,早中晚空腹口服0.1 g葡萄糖醛酸内酯各一次;利福平组晨起空腹口服0.45 g利福平一次,早晚空腹口服0.2 g左氧氟沙星各一次,早中晚空腹口服0.1 g葡萄糖醛酸内酯各一次;异烟肼和利福平合用组除服用与异烟肼组相同的药物外,晨起空腹口服0.45 g利福平一次,连续服药两周。
2血样采集与处理方法
患者连续服药两周后,即第15天晨起服药前及服药后2 h、4 h、12 h采用抗凝管收集肘静脉血5 mL,3500 rpm离心10 min,取血浆分装两份,于-70℃低温冰箱保存,备异烟肼及其代谢物浓度检测;分别于开始抗结核前和用药两周后晨起空腹抽取肘静脉血5 mL,检测谷丙转氨酶、谷草转氨酶和总胆红素水平。
3 QSG-7701肝细胞培养方法
利用QSG-7701肝细胞株研究异烟肼和利福平对离体肝细胞的毒性。将冻存于液氮中的QSG-7701肝细胞于37℃水浴中迅速解冻,离心弃上清后,沉淀加入含10%胎牛血清的高糖DMEM培养基,用吸管吹打,使成单细胞悬液,用台盼蓝染色后,显微镜下用计数板计数,并接种于70 mm培养皿中培养,接种密度为1×105 cells/mL,体积5 mL,于CO2培养箱中培养24 h后换全液,以后隔天换液,待细胞长满后吸出培养液,加入1 mmol/L的EDTANa2清洗,吸弃EDTANa2溶液后,用0.25%的胰酶消化液消化,加入培养液并用吸管吹打使成单细胞悬液,进行传代。用于加药处理的细胞,接种于培养板。
4 MTT法检测细胞存活率
向培养板培养的细胞培养液中加入MTT溶液使其终浓度为0.05%,继续培养4 h后吸弃培养液,每孔加入DMSO 150μL,振荡使甲瓒结晶溶解,以空白孔调零,用读板机或分光光度计读取570 nm波长处的吸光度值,以空白对照组吸光度的均值为100%,用各样本吸光度值/空白对照组吸光度的均值表示该样本细胞的存活率。
5细胞培养液及细胞裂解液乳酸脱氢酶活性的测定
药物处理完毕后,分别收集各组细胞培养上清液,-80℃冻存备测。将细胞用生理盐水冲洗一遍后,每孔加入生理盐水1 mL,用细胞刮刀将细胞刮下,将细胞悬液2000 rpm离心5 min,倾弃上清后,沉淀加入0.5 mL细胞裂解液,4℃下裂解30 min,12000 rpm离心20 min,收集上清-80℃冻存备测。用乳酸脱氢酶测试盒,比色法测定440 nm样品的吸光度值,并计算乳酸脱氢酶的活力。用细胞培养液中酶活力与细胞裂解液中酶活力的比值代表药物作用对细胞损伤及乳酸脱氢酶活力的影响。
6 HPLC-MS法测定血浆中异烟肼及其代谢物乙酰肼和肼的浓度
取血浆样品0.1 mL,依次加入内标溶液20μL,甲醇0.2 mL,涡旋混合3 min,4000 r·min-1离心10 min,取上清液0.2 mL,加入衍生化溶液10μL,室温下暗处反应24 h,取20μL用于HPLC-MS法测定。采用高效液相色谱质谱联用法测定人血浆中异烟肼、乙酰肼和肼的浓度。所采用的色谱方法为:Agilent C18分析柱(5μm,2.1mm×150 mm ID),Agilent SB C18保护柱(5μm),柱温40℃,流动相为甲醇-水(含0.1%冰醋酸),流速为150μL/min。质谱检测采用电喷雾(ESI)离子化源,毛细管电压4.0 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;正离子方式检测,碎片电压为分别为190V、130V、150V;选择离子监测(SIM)模式,用于定量分析的离子分别为m/z 256.1(异烟肼衍生物)、m/z 193.2(乙酰肼衍生物)、m/z 151.1(肼衍生物)。
7 HPLC-MS法测定细胞孵育液中咪达唑仑和对硝基儿茶酚的浓度
色谱条件:色谱柱:Gemini C18分析柱(2.0×50 mm,5μm),Phenomenex公司;C18保护柱(5μm,3.0×4.0 mm),Phenomenex公司;流动相:乙腈-水(含0.2%冰醋酸)=60:40;流速为0.2 mL·min-1;柱温为30℃;进样量:10μL。
质谱条件:电喷雾(ESI)离子化源,选择离子监测(SIM)模式。
对硝基儿茶酚检测条件:毛细管电压2 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;负离子方式检测,碎片电压为150 V,m/z [154.0]-(对硝基儿茶酚)。
咪达唑仑:毛细管电压3 kV;氮气(N2)流速10 L·min-1,雾化气压力为210 kPa,干燥气温度为350℃;正离子方式检测,碎片电压为50 V,m/z [326.0]+(咪达唑仑)。
8分组及加药处理
8.1异烟肼及其代谢物和利福平对细胞存活率的影响
细胞接种于96孔板,接种密度为每孔1×104 cells,将细胞分为空白对照组、利福平组、异烟肼组、乙酰肼组、肼组和利福平+异烟肼组,每组6孔。于接种后培养24 h时分别换含有空白溶剂、利福平(100μg/mL,200μg/mL,300μg/mL)、异烟肼(250μg/mL,500μg/mL,2500μg/mL)、乙酰肼(125μg/mL,250μg/mL,1250μg/mL)、肼(0.25μg/mL,2.5μg/mL,25μg/mL)、利福平+异烟肼(100+50μg/mL,200+100μg/mL,300+150μg/mL)的含药培养液,培养箱中培养48 h后采用MTT法测定细胞存活率。
8.2异烟肼及其代谢物和利福平对细胞乳酸脱氢酶释放的影响
将细胞接种于6孔培养板,接种后24 h换含药培养液培养48 h,分组及加药方法同“8.1”项下操作,收集培养液和细胞用于乳酸脱氢酶活性的测定。
8.3异烟肼和利福平对细胞CYP 2E1和3A4活性的的影响
将细胞分为8组,每组6孔,分别加入含有溶剂、异烟肼100μg/mL、利福平200μg/mL、异烟肼100μg/mL和利福平100μg/mL的培养液,各两组。继续培养48 h后,吸弃含药培养液,分别加入含有CYP 2E1作用底物对硝基酚(15μg/mL)或CYP 3A4作用底物咪达唑仑(1μg/mL)的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h,吸出培养液,用HPLC-MS法测定对硝基酚代谢产物对硝基儿茶酚和咪达唑仑的水平,用以代表各组细胞CYP 2E1和3A4的活性。
8.4 CYP 2E1和3A4抑制剂对异烟肼和利福平合用增加细胞乳酸脱氢酶释放的影响
将培养板中的细胞分为14组,分别为空白对照组、异烟肼和利福平合用组、柑桔素组、红霉素组、阿魏酸钠组和三七皂苷组,每组6孔。各组分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL)的培养液继续培养48 h。柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加入CYP 3A4抑制剂柑桔素(1μg/mL、5μg/mL、25μg/mL)和红霉素(1μg/mL、5μg/mL、25μg/mL);阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时分别加入CYP 2E1抑制剂阿魏酸钠(10μg/mL、50μg/mL、250μg/mL)和三七皂苷(4μg/mL、20μg/mL、100μg/mL)共同培养。收集细胞培养上清液和细胞,检测乳酸脱氢酶的活性
8.5 CYP 2E1和3A4抑制剂对异烟肼和利福平合用降低细胞存活率的影响
将接种于24孔培养板的细胞分为8组,每组6孔,于接种后24 h吸弃培养液,分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL)的培养液继续培养48 h。柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加入CYP 3A4抑制剂柑桔素(5μg/mL)和红霉素(5μg/mL);阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时分别加入CYP 2E1抑制剂阿魏酸钠(50μg/mL)和三七皂苷(20μg/mL)共同培养。采用MTT法测定培养后细胞的存活率的变化。
8.6酶抑制剂对细胞内CYP450 2E1和3A4酶活性的影响
将接种于24孔培养板的细胞分为8组,每组6孔,各组分别加入含有溶剂、异烟肼(100μg/mL)和利福平(200μg/mL);柑桔素组和红霉素组在加入异烟肼和利福平的同时分别加含CYP 3A4抑制剂柑桔素(5μg/mL)和红霉素(5μg/mL)的培养液继续培养48 h,吸弃含药培养液,加入含有CYP 3A4作用底物咪达唑仑1μg/mL的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h;阿魏酸钠组和三七皂苷组在加入异烟肼和利福平的同时加入含CYP 2E1抑制剂阿魏酸钠(50μg/mL)和三七皂苷(20μg/mL)的培养液继续培养48 h,吸弃含药培养液,加入含有CYP 2E1作用底物对硝基酚15μg/mL的无血清DMEM培养液0.5 mL,在培养箱中孵育2 h。孵育后吸出培养液,用HPLC-MS法测定咪达唑仑和对硝基儿茶酚的浓度。
9数据处理与统计分析
统计分析采用SPSS 11.5 for windows软件,数据以均数±标准差(mean±SD)表示,组间差异采用成组设计的t检验;多组间比较采用方差分析(ANOVA)继于Dunnett t test;量效关系采用二元变量相关分析,计算Pearson相关系数,统计结果以P<0.05为具有显著性差异。
结果
1肺结核病患者血浆异烟肼及其代谢物乙酰肼和肼浓度的变化
经过对肺结核病患者服药后血浆中药物浓度的测定发现,与单用异烟肼组比较,异烟肼和利福平合用组肺结核病患者血浆中肼的浓度在服药后2 h即显著升高(P<0.05),在服药后4 h和12 h时仍显著高于单用异烟肼组(均为P<0.01)。与单用异烟肼组比较,异烟肼和利福平合用组患者血浆中异烟肼和乙酰肼的浓度未见显著性变化(P>0.05)。各组患者服药前后谷丙转氨酶、谷草转氨酶和总胆红素水平均未见异常(P>0.05)。
2异烟肼、利福平及异烟肼代谢物乙酰肼和肼对细胞存活率的影响
进一步对异烟肼、利福平及异烟肼代谢物乙酰肼和肼对离体肝细胞的毒性作用进行研究发现,与对照组比较,500μg/mL和2500μg/mL的异烟肼处理48 h,肝细胞的存活率均显著降低,其存活率分别为64.3% (P<0.01)和9.9% (P<0.01),异烟肼浓度与细胞存活率的Pearson相关系数为r = -0.959 (P<0.01);300μg/mL的利福平处理肝组细胞的存活率显著降低(56.2%, P<0.01);中、高剂量利福平和异烟肼合用组肝细胞存活率依次显著降低(分别为73.7%, P<0.05和33.3%, P<0.01),异烟肼和利福平合用浓度与细胞存活率的Pearson相关系数为r = -0.892 (P<0.01);2.5μg/mL和25μg/mL肼处理组肝细胞的存活率显著降低(分别为53.5%, P<0.05和46.0%, P<0.01)。结果表明,异烟肼和利福平合用的肝细胞毒性作用大于其单用,异烟肼代谢物肼对肝细胞的毒性作用最强。
3异烟肼、利福平及其异烟肼代谢物对细胞乳酸脱氢酶活性的影响
检测药物处理后肝细胞外/内乳酸脱氢酶活性的比值,反映细胞的损伤程度。与对照组比较,三种浓度的异烟肼处理后,细胞外/细胞内LDH的比值分别升高83.8%(P<0.01)、112.0%(P<0.01)和124.9%(P<0.01);300μg/mL的利福平处理使细胞外/细胞内LDH的比值显著升高50.3% (P<0.05);中、高浓度的利福平和异烟肼合用剂量依赖地使细胞外/细胞内LDH比值显著升高,升高幅度分别为76.2%(P<0.05)、210.8%(P<0.01)和1010.3% (P<0.01);异烟肼和利福平合用浓度与LDH比值的Pearson相关系数为r = 0.907 (P<0.01);三种浓度的肼处理后,细胞外/细胞内LDH比值分别升高73.3%(P<0.05)、102.1%(P<0.01)和115.5%(P<0.01);250μg/mL和1250μg/mL的乙酰肼处理后,细胞外/细胞内LDH比值分别升高121.1%(P<0.01)和153.1%(P<0.01);乙酰肼浓度与LDH比值的Pearson相关系数为r = 0.674 (P<0.01)。结果表明,异烟肼和利福平合用的对肝细胞的损伤作用大于其单用,异烟肼代谢物乙酰肼和肼均有损伤作用,其中肼对肝细胞的损伤作用最强。
4异烟肼和利福平对肝细胞CYP 2E1和CYP 3A4活性的影响
本实验关于异烟肼和利福平对肝细胞CYPs活性影响的结果显示,与对照组比较,单用异烟肼和异烟肼与利福平合用处理48 h,肝细胞CYP 2E1酶活性均显著升高(均为P<0.05);而单用利福平处理48 h,肝细胞CYP 2E1酶活性则未见明显变化(P>0.05);单用异烟肼和单用利福平处理48 h,分别使肝细胞CYP 3A4酶活性显著升高(P<0.05和P<0.01);异烟肼和利福平合用处理48 h,肝细胞CYP 3A4酶活性进一步升高(P<0.01)。表明异烟肼和/或利福平单用和合用均可上调细胞CYP 2E1和CYP 3A4的活性,二者合用更可进一步上调CYP 3A4的活性。
5 CYP450 3A4抑制剂对异烟肼和利福平致肝细胞毒性作用的影响
采用柑桔素或红霉素与异烟肼和利福平共同处理肝细胞发现,与异烟肼和利福平合用组比较,给予异烟肼和利福平处理的同时,加入柑桔素或红霉素共同培养,肝细胞中CYP 3A4的酶活性均显著低于异烟肼和利福平合用组(均为P<0.01);肝细胞的存活率显著升高(分别为95.2%和107.8%,均为P<0.01);中、高剂量的柑桔素和低、中、高剂量的红霉素均显著降低了细胞外和细胞内乳酸脱氢酶的比值(均为P<0.01),其中柑桔素浓度与乳酸脱氢酶比值的Pearson相关系数为r = -0.712 (P<0.01)。表明柑桔素和红霉素可以通过抑制异烟肼和利福平对CYP 3A4的诱导作用,减轻其对肝细胞的损伤。
6 CYP450 2E1抑制剂对异烟肼和利福平致肝细胞毒性作用的影响
采用阿魏酸钠和三七皂苷与异烟肼和利福平共同处理肝细胞发现,与异烟肼和利福平合用组比较,给予异烟肼和利福平处理的同时,加入阿魏酸钠或三七皂苷共同培养,肝细胞中CYP 2E1的酶活性均显著降低(均为P<0.01);阿魏酸钠组肝细胞的存活率显著升高(77.1%,P<0.05);中、高剂量的三七皂苷和低、中、高剂量的阿魏酸钠均显著降低了细胞外/内乳酸脱氢酶的比值(均为P<0.01),其中三七皂苷浓度与乳酸脱氢酶比值的Pearson相关系数为r = -0.659 (P<0.01)。表明阿魏酸钠和三七皂苷可以通过抑制异烟肼和利福平对CYP 2E1的诱导作用,减轻其对肝细胞的损伤。
结论
1.肺结核病患者使用异烟肼和利福平联合治疗时,血浆中异烟肼代谢物肼的浓度较单用异烟肼时显著升高,这可能是两药合用导致肝毒性增加的机制之一。
2.采用体外肝细胞培养的方法进一步证明,不同剂量的异烟肼、利福平、肼及异烟肼和利福平合用能明显造成肝细胞损伤、显著降低肝细胞的存活率,其中异烟肼和利福平合用的肝细胞毒性强于其单用,异烟肼代谢物肼对肝细胞的毒性作用远大于其母体异烟肼,是异烟肼产生肝毒性的重要代谢产物。
3.异烟肼和利福平单独或合用可不同程度地使肝细胞CYP 3A4和CYP 2E1的活性增加,推测此为异烟肼和其与利福平合用时异烟肼代谢产物水平升高,肝细胞存活率降低、损伤增加,乳酸脱氢酶向细胞外释放增加的机制之一。
4.柑桔素和红霉素可以显著降低肝细胞内CYP 3A4的活性,阿魏酸钠和三七皂苷可以显著降低肝细胞内CYP 2E1的活性。不同浓度的柑桔素、红霉素、阿魏酸钠和三七皂苷均可抑制异烟肼和利福平合用致肝细胞存活率降低和/或乳酸脱氢酶释放增加的作用,具有对其肝细胞毒性的保护作用,对相应酶活性的调节可能是这些药物发挥作用的途径之一。
Tuberculosis (TB) is one of the major causes of death from infectious disease. Recommended standard treatment by the World Health Organization (WHO) for adult respiratory TB is a regimen of isoniazid, rifampicin, and pyrazinamide for 2 months, followed by 4 months of isoniazid and rifampicin. The most frequent and most serious adverse effect of anti-tuberculosis treatment is hepatotoxicity. Evidence accumulated during the last years documented that cytochrome enzymes that mediate the metabolism of isoniazid play an important role in the development of hepatotoxicity. Moreover, the cytochrome enzymes CYP 3A4 and 2E1 are thought to mediate the hepatotoxicity. However, mechanisms that mediate anti-tuberculosis treatment induced hepatotoxicity are still not clear.
Studies by others and our previous studies in mice suggested that hydrazine, the metabolite of isoniazed, was the main hepatotoxic compound. And drugs administered, such as sodium ferulate, baicalin and silibinin are protective in isoniazid and rifampicin exacerbated heptotoxicity.
The present study was carried out to research the levels of isoniazid and its metabolites in the plasma of lung tuberculosis patients administered with isoniazid with or without rifampicin for two weeks. And the hepatocyte toxicity of isoniazid, its metabolites and rifampicin was investigated in cultured hepatocytes by using cell viability assays and lactate dehydrogenase release, a marker of cell death. Furthermore, the role of CYP 3A4, 2E1 and their inhibitors in the occurrence and preservation of isoniazid-rifampicin induced hepatoxicity was demonstrated.
Methods
1. Patients and drugs administration
Twenty-four lung tuberculosis patients were designated to three groups randomly. In the isoniazid group, patients were administered with isoniazid, levofloxacin and glucurolactone. In the rifampicin group, patients were administered with rifampicin, levofloxacin and glucurolactone. In the isoniazid and rifampicin group, patients were administered with isoniazid, rifampicin and glucurolactone.
2. Plasma sample collection and disposal
Plasma samples were collected before and 2 hours, 4 hours and 12 hours after the last administration of isoniazid and/or rifampicin following a two week duration of therapy and the samples were then stored at -70℃until HPLC-MS analysis. Alanine aminotransferase, aspartate aminotransferase and total bilirubin levels in serum were determined before and after therapy.
3. Culture of QSG-7701 hepatocellulars
The QSG-7701 cell strain was cultured in DMEM culture media that contained high glucose at a cell density of 1×105 cells/mL. Pancreatin (0.25%) was used for trypsinization and sub-culturing.
4. Cell viability assay
The MTT assay was used to evaluate the viability of the hepatocytes cultured in 96-well plates. Briefly, MTT was added to the culture media with a final concentration of 0.05%. After 4 hours of culture, the culture media was removed and 150μL DMSO was added to dissolve the crystals. The absorbance of the solution was read at 570nm on a spectrophotometer and the cell viability was calculated.
5. Measurement of lactate dehydrogenase activity in cells and culture media
The cells in the culture plate were flushed with sodium solution and scraped off. After centrifugation, the cells were lyzed with lysis buffer that was incubated with the cells for 30 minutes. The resultant supernatant was used for the assay. Lactate dehydrogenase activity in cell lysates and culture media were determined using a lactate dehydrogenase activity kit and absorbance spectrophotometery. The ratio of extracellular and intracellular activity of lactate dehydrogenase was calculated.
6. Determination of plasma isoniazid, acetylhydrazine and hydrazine concentrations using HPLC-MS
Internal standard and methanol were added to plasma samples that were then vortexed for 3 minutes. After centrifugation of the samples, the supernatants were incubated with derivative agent for 24 hours and then analyzed with HPLC-MS using a 10μL on each HPLC run. An Agilent C18 analyze column (5μm, 2.1mm×150 mm ID) was used with an Agilent SB C18 guard column (5μm). The mobile phase consisted of methanol: water (containing 0.1% glacial acetic acid) and was pumped into the system at a rate of 0.15 mL·min-1. ESI ionization was used in the mass detection in selected ion monitoring. The ions selected for isoniazid, acetylhydrazine and hydrazine- were m/z [256.1]+, m/z [193.2]+ and m/z [151.1]+ , respectively.
7. Determination of midazolam and 4-nitrocatichol concentrations in culture media using HPLC-MS
A Gemini C18 analyze column (2.0×50 mm, 5μm) was used with a C18 guard column (5μm,3.0×4.0 mm). The mobile phase consists of acetonitrile: water (60:40, v/v, containing 0.2% glacial acetic acid) and was pumped in 0.2 mL·min-1. Ten milliliters of sample was analyzed on the column. ESI ionization was used in mass detection in selected ion monitoring. The ions selected for 4-nitrocatechol and midazolam- were m/z [154.0]- and m/z [326.0]+, respectively.
8. Grouping and drug treatment
8.1 Effect of isoniazid, its metabolites and rifampicin on cellular viability
The cells were plated in 96-well plates at a cell density of 1×104 cells per well. The cells were designated to sixteen groups with six wells in each group. The six treatment groups consisted of the blank vehicle, isoniazid (250μg/mL, 500μg/mL, 2500μg/mL), rifampicin (100μg/mL, 200μg/mL, 300μg/mL), acetylhydrazine (125μg/mL, 250μg/mL, 1250μg/mL), hydrazine (0.25μg/mL, 2.5μg/mL, 25μg/mL) and isoniazid+rifampicin (100+50μg/mL, 200+100μg/mL, 300+150μg/mL). All treatments were added to the culture media and after 48 hours of culture, cell viability was determined using the MTT method.
8.2 Effect of isoniazid, its metabolites and rifampicin on lactate dehydrogenase activity
Cells were plated in to 6-well plates. The groups and treatments were the same as item“8.1”. Cells and culture media were collected for assay of lactate dehydrogenase.
8.3 Effect of isoniazid and rifampicin on the activity of CYP 2E1 and 3A4
Cells were designated to eight treatment groups with six wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL), rifampicin (200μg/mL), isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. After a 48 hour incubation period the culture media was removed. 4-nitrophenol (15μg/mL) and midazolam (1μg/mL), substrates of CYP 2E1 and 3A4 respectively, were added to the wells and cultured for an additional 2 hours. The concentration of 4-nitrocatechol, a metabolite of 4-nitrophenol, and midazolam were determined using HPLC-MS. The activity of CYP 2E1 and 3A4 were calculated by the increasing level of 4-nitrocatechol and the decreasing level of midazolam.
8.4 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin on lactate dehydrogenase release
Cells were plated onto 24-well plates, and were designated to fourteen groups with six-wells in each group. Three of the fourteen groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) treatments that were added to the culture media. The other groups consisted of the naringenin and erythromycin groups where naringenin (1μg/mL, 5μg/mL, 25μg/mL) or erythromycin (1μg/mL, 5μg/mL, 25μg/mL) were added in addition. In the sodium ferulate and sanchinoside groups, sodium ferulate (10μg/mL, 50μg/mL, 250μg/mL) and sanchinoside (4μg/mL, 20μg/mL, 100μg/mL) were added in addition. After 48 hours of culture, lactate dehydrogenase activity in the cells and culture media were determined.
8.5 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin decreased cellular viability
Cells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were add to the culture media. In naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. In sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the cell viability was determined using the MTT method.
8.6 Effect of enzyme inhibitors on the activity of CYP 2E1 and 3A4
Cells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. In the naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 3A4 determined. In the sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 2E1 determined.
9 Data and statistics
Statistic was performed with SPSS 11.5 for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA with the Dunnett t test and binary variable correlation analysis. Significance was considered when P<0.05.
Results
1. Concentrations of isoniazid, acetylhydrazine and hydrazine in the plasma of lung tuberculosis patients
The concentration of hydrazine increased significantly 2 h, 4 h and 12 h (P<0.05 or P<0.01) in plasma after isoniazid and rifampicin were co-administered to patients with lung tuberculosis compared with treatment with isoniazid alone. There were no significant differences between the concentrations of isoniazid and acetylhydrazine between the groups (P>0.05). There was no significant abnormality in the levels of GPT, GOT and total bilirubin before and after two weeks of drug therapy (P>0.05).
2. Viability of isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytes
After 48 hours of treatment with isoniazid (500μg/mL or 2500μg/mL), the viability of cultured QSG-7701 cells decreased significantly compared with the control group with the value of 64.3% (P<0.01) and 9.9% (P<0.01). The Pearson correlation coefficient was -0.959 (P<0.01) between the concentration of isoniazid and cell viability. The viability of cells decreased to 56.2% (P<0.01), 53.5% (P<0.05) and 46.0% (P<0.01) in the rifampicin (300μg/mL) and hydrazine (2.5μg/mL and 25μg/mL) treated groups. Co-administration of isoniazid and rifampicin dose dependently decreased cell viability and the Pearson correlation coefficient was -0.892 (P<0.01).
3. Activity of lactate dehydrogenase released from isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytes
Isoniazid, rifampicin, acetylhydrazine, hydrazine, isoniazid and rifampicin co-treatment increased the release of lactate dehydrogenase from cells after 48 hours of culture (P<0.01 or P<0.01). The effect of acetylhydrazine and isoniazid-rifampicin co-treatment was dose dependent and the Pearson correlation coefficient was 0.674 (P<0.01) and 0.907 (P<0.01), respectively.
4. Effect of isoniazid and rifampicin on activity of CYP 3A4 and 2E1 in hepatocytes
The activity of CYP 2E1 increased significantly in QSG-7701 cells cultured for 48 hours with isoniazid or isoniazid and rifampicin (P<0.05) compared with the control group. There was no significant change in CYP 2E1 activity in the rifampicin alone treated group (P>0.05). Treatment with isoniazid, rifampicin alone or isoniazid and rifampicin all increased the activity of CYP 3A4 in QSG-7701 cells compared with the control group(P<0.05 or P<0.01).
5.Effect of CYP 3A4 inhibitors on the hepatotoxicity of isoniazid and rifampicin
Naringenin and erythromycin inhibited the activity of CYP 3A4 enhanced by isoniazid and rifampicin, and attenuated the decrease in cell viability and depressed lactate dehydrogenase release (P<0.01) from cultured QSG-7701 cells. The effect of naringenin on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.712 (P<0.01).
6. Effect of CYP 2E1 inhibitors on the hepatotoxicity of isoniazid and rifampicin
Sodium ferulate and sanchinoside inhibited the activity of CYP 2E1 enhanced by isoniazid and rifampicin, attenuated the decrease in cell viability and depressed the increase in lactate dehydrogenase release (P<0.05 or P<0.01) from cultured QSG-7701 cells. The effect of sanchinoside on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.659 (P<0.01).
Conclusions
1 The concentration of hydrazine increased significantly in the plasma of lung tuberculosis patients that were co-administered isoniazid and rifampicin compared with patients that were treated with isoniazid alone, and the co-treatment may be the mechanism by which isoniazid and rifampicin induce hepatotoxicity.
2 Different co-administered doses of isoniazid, hydraine, rifampicin, isoniazid and rifampicin result in increasing lactate dehydrogenase release and decrease the viability of hepatocytes. The effect of co-administration is more intensive than isoniazid or rifampicin treatment alone. And hydrazine, the metabolite of isoniazid, induced more severe hepatocytes toxicity than its precursor, which suggests a pivotal and considerable role of hydrazine in anti-tuberculosis induced hepatotoxicity.
3 The activity of CYP 3A4 and 2E1 could be enhanced by isoniazid and rifampicin alone and their co-treatment, which may be responsible for the excessive production of hydrazine and the hepatocyte toxicity following isoniazid and rifampicin co-therapy.
4 The activity of CYP 3A4 could be depressed by naringenin and erythromycin and the activity of CYP 2E1 could be depressed by sodium ferulate and sanchinoside. Moreover, naringenin, erythromycin, sodium ferulate and sanchinoside attenuate isoniazid and rifampicin co-treatment induced hepatocyte viability and decrease lactate dehydrogenase release through their effect on CYPs.
引文
1叶任高,陆再英主编.内科学,第六版.人民卫生出版社.
2 Noda A, Hsu KY, Noda H, et al. Is isoniazid-hepatotoxicity induced by the metabolite, hydrazine? J UOEH, 1983; 5: 183~90
3 Gent WL, Seifart HI, Parkin DP, et al. Factors in hydrazine formation from isoniazid by paediatric and adult tuberculosis patients. Eur J Clin Pharmacol, 1992; 43: 131~6
4 Menzies D, Dion MJ, Rabinovitch B, et al. Treatment completion and costs of a randomized trial of rifampin for 4 months versus isoniazid for 9 months. Am J Respir Crit Care Med, 2004; 170: 445–449
5 Hong Kong Chest Service, Tuberculosis Research Centre, Madras, British Medical Research Council.Adouble-blind placebo-controlled clinical trial of three anti-tuberculosis chemoprophylaxis regimens in patients with silicosis in Hong Kong. Am Rev Respir Dis, 1992; 145: 36~41
6乐江,彭仁修,刘娟. RP–HPLC法测定血浆中异烟肼代谢物的含量.中国药科大学学报, 2004, 35(1): 47
7殷琦,车宁,赫广威, et al. HPLC法测定人体血浆中利福平和异烟肼的浓度.药物分析杂志, 2002, 22(5): 358
8 Bertino J Jr, Fish D. The safety profile of the fluoroquinolones. Clin Ther, 2000; 22: 798~817
9 Wolfson JS, Hooper DC. Overview of fluoroquinolone safety. Am J Med, 1991; 91: 153S~161S
10 Coleman CI, Spencer JV, Chung JO, Reddy P. Possible gatifloxacin induced fulminant hepatic failure. Ann Pharmacother, 2002; 36: 1162~1167
11 Kahn JB. Latest industry information on the safety profile of levofloxacin in the US. Chemotherapy, 2001; 47: 32~37
12 Steele MA, Burk RF, Desprez RM. Toxic Hepatitis with Isoniazid and Rifampin. A meta2analysis. Chest, 1991, 99: 465
13 Sarma GR, Immanuel C, Kailasam S, et al. Rifampin induced release of hydrazine from isoniazid: A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifampin. Am Rev Respir Dis, 1986, 133(6): 1072
14 Zhang RL, Wang ZY, Li D, et al. Effect of rifampicin on pharmacoki- netics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacologica Sinica, 1992, 13(6): 494
15 Rae JM, Johnson MD, Lippman ME, et al. Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrayds. J Pharmacol Exp Ther, 2001, 299(3): 849~857
1 World Health Organization. Tuberculosis. WHO Fact Sheet No. 104. Revised. March 2006
2 Kaona FA, Tuba M, Siziya S, Sikaona L. An assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment. BMC Public Health 2004; 4: 68. 5
3 Knowles SR, Uetrecht J, Shear NH. Idiosyncratic drug reactions: the reactive metabolite syndromes. Lancet 2000; 356: 1587–91
4 Tostmann A, Boeree MJ, Aarnoutse RE, et al. Antituberculosis drug-induced hepatotoxicity: concise up-to-date review. Journal of Gastroenterology and Hepatology, 2008, 23: 192~202
5 Sarich TC, Youssefi M, Zhou T, et al. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. Arch Toxicol, 1996, 70: 835~840
6 Zhang RL, Wang ZY, Li D, et al. Effect of rifampicin on pharmacokinetics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacol Sin, 1992
7 Rae JM, Johnson MD, Lippman ME, et al. Rifampin is a selective pleiotropic inducer of drug metabolism genes in human hepatocytes: sutdies with cDNA and oligonucleotide expression arrayds. J PharmacolExp Ther, 2001, 299(3): 849~857
8 Panchagnula R, Sood A, Sharda N, et al. Determination of rifampicin and its main metabolite in plasma and urine in presence of pyrazinamide and isoniazid by HPLC method. Journal of Pharmaceutical and Biomedical Analysis18 (1999) 1013–1020
9 Zhang RL , Wang ZY, L i D, et al. Effect of rifampicin on pharmacokinetics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacologica Sinica, 1992, 13 (6): 494
10 Noda A, H su KY, Noda H, et al. Is isoniazid hepatotoxicity.induced by the metabolite, hydrazine? Sangyo Ika DaigakuZasshi, 1983, 5 (2) : 183
11 Noda A , Sendo T, Ohno K, et al. Effect of rifamp icin andPhenobarbital on the fate of isoniazid and hydrazine in vivo in rats. Toxicol Lett, 1985, 25 (3): 313
12 Sendo T, Noda A , Noda H, et al. Metabolic hydrolysis of isoniazid by subcellular fractions of rat liver. J UOEH, 1984, 6(3): 249
13 Nilesh Mehta, MD. Emmanuel Gbadehan, MD, Lisa Ozick, MD, Drug-Induced Hepatotoxicity Updated: Mar 28, 2008
14 Lee WM. Drug-induced hepatotoxicity. N Eng J Med, 2003; 349(5): 474~85
15 Lee WM. Impact of Drug Induced Liver Toxicity on Hepatology and Practice of Medicine. US Food and Drug Administration, Center for Drug Evaluation and Research; 2001
16 Chalasani N. Statins and hepatotoxicity: focus on patients with fatty liver. Hepatology, 2005; 41(4): 690~5
1 Nelson DR, Koymans L, Kamataki T, et al. P450 superfamily: update on new sequences, gene mapping, accessionnumbers and nomenclature . Pharmacogenetics, 1996, 6(1): 1~42
2 Ingelman SM. Pharmacogenetics of cytochrome P450 and its aplications in drug therapy: the past, present and future . Trends Pharmacol Sci, 2004, 25(4): 193~200
3 Watkins PB. Role of cytochromes p450 in drug metabolism and hepatotoxicity. Semin Liver Dis. 1990 10(4): 235~250
4 Dickson RC. Glycyne cytoprotection durying lethal hepatocellular injury from adenosinetfiphate depletion. Gastrerol 2001, 6(1): 428~431
5 Kaplowit N. Biochem ical and cellular mechanisms of toxic liver injury. Sem in Liver Dis 2002, 22(2): 137~144
6 Wen X, Wang JS, Neuvonen PJ, Backman JT. Eur J Clin Pharmacol, 2002, 57(11): 799~804
7 Zeruesenay D, Siegmund W, Zschiesche M, Franke G, et al. J Pharm Pharmacol, 1992, 44(11): 893~897
8 Catania JR, McGarrigle BP, Rittenhouse-Olson K, Olson JR. Toxicol In Vitro, 2007, 21(1): 109~115
9 Chung HJ, Choi YH, Kim SH, Lee MG. J Pharm Pharmacol, 2006, 58(4): 449~457
10 Ozawa S, Ohta K, Miyajima N, Matsurnoto Y, Fukuoka M, Ohno Y. Xenobiotica, 2000, 30(10): 1005~1017
11 Ramaiah SK, Apte U, Mehendale HM. Drug Metab Dispos, 2001, 29(8): 1088~1095
12 Madan A, Graham RA, Carroll KM, Mudra DR, Burton LA, et al. Drug Metab Dispos, 2003, 31(4): 421~431
13 Graham RA, Downey A, Mudra D, Krueger L, et al. Drug Metab Dispos, 2002, 30(11): 1206~1213
14 Lee DY, Lee SJ, Lee MG. Int J Pharm, 2005, 298(1): 38~46
15 Wang T, Shankar K, Bucci TJ, Warbritton A, Mehendale HM. Toxicol Appl Pharmacol, 2001, 173(1): 27~37
16 Sarich TC, Adams SP, Petricca G, Wright JM. J Pharmacol Exp Ther, 1999, 289(2): 695~702
17 Kinzig-Schippers M, Tomalik-Scharte D, Jetter A, Scheidel B, Jakob V, Rodamer M, Cascorbi I, Doroshyenko O, Sorgel F, Fuhr U. Antimicrob Agents Chemother, 2005, 49(5): 1733~1738
18 Anundi I, Lahteenmaki T, Rundgren M, Lindros KO. Biochem Pharmacol, 1993, 45(6): 1251~1259
19 Wen X,Wang JS, Neuvonen PJ,Backman JT. Isoniazid is a mechanism-based inhibitor of cytochrome P450 1A2,2A6,2C19 and 3A4 isoforms in human liver microsomes.Eur.J.Clin. Pharmacol. 2002; 57: 799~804
20 Zand R, Nelson SD, Slattery JT, Thummel KE, Kalhorn TF, et al. ClinPharmacol Ther, 1993, 54(2): 142~149
21 Self TH, Chrisman CR, Baciewicz AM, Bronze MS. Am J Med Sci, 1999, 317(5): 304~311
22 Yue J, Peng RX, Yang J, Kong R, Liu J. Acta Sin, 2004, 25(5): 699~704
23 Huang YS, Chern HD, Su WJ et al. Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis. Hepatology 2003; 37: 924~930
24 Vuilleumier N, Rossier MF, Chiappe A et al. CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis.Eur.J.Clin.Pharmacol. 2006; 62: 423~9
1 Guengerich, FP. Human Cytochrome P450 Enzymes. Life Sci, 1992, 50, 1471~1478
2 Rendic S, di Carlo F. Human Cytochrome P450 Enzymes: a Status Report Summarizing Their Reactions, Substrates, Inducers and Inhibitors. Drug Metab Rev, 1997, 29, 413~580
3 From the Centers for Disease Control and Prevention. Fatal and severe hepatitis associated with rifampin and pyrazinamide for the treatment of latent tuberculosis infection -New York and Georgia, 2000. JAMA, 2001; 285: 2572~3
4薛洪源,侯艳宁,刘会臣, et al.水飞蓟宾胶囊对异烟肼和利福平肝损害小鼠的保护作用.中成药, 2003, 25(4)
5侯艳宁,朱秀媛,程桂芳.黄芩甙对小鼠肝细胞色素P450及其亚家族的诱导.解放军药学学报, 2000, 16(2)
6汪晖,彭仁琇.阿魏酸钠减轻对乙酰氨基酚致小鼠肝损伤.中国药理学报, 1994, 15(1): 81~83
7薛洪源,侯艳宁,刘会臣, et al.阿魏酸钠对异烟肼和利福平肝损害小鼠的保护作用.中国医院药学杂志, 2003, 23(10): 584~586
8张娴文,李凤贤,李辉.三七皂苷抗肝纤维化作用的现状.中国新药杂志, 2007, 16(4): 260~263
9 WEBER A, JAGER R, BORNER A, et al. Can Grapefruit Juice Influence Ethinylestradiol Bioavailability. Contraception, 1996, 53(1): 41~47.
10 Bailey DG. Food-durg interactions: enphasis on grapefruit juice effects. Can J Clin Pharmacol. 1995, 2: 10~14
11 Guengerich FP. Role of cytochrome P450 enzymes in drug-durg interactions. Adv Pharmacol, 1997, 43: 7~35
12 PARI L, GNANASOUNDARI M. Influence of naringenin on oxytetracyline mediated oxidative damage in rat liver. Basic Clin Pharmacol Toxicol, 2006, 98(5): 456~461
13 LEE MH, YOON S, MOON JO. The flavonoid naringenin inhibits dinethylnitrosamine-induced liver damage in rats. Bio Pharma Bull, 2004, 27(1): 72~76
1 World Health Organization. Tuberculosis. WHO Fact Sheet No.104. Revised. March 2006.
2卫生部,2008年我国卫生改革与发展情况,中国新闻网,2009.2.17
3 Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C.Tuberculosis. Lancet, 2003; 362: 887~99
4 Kaona FA, Tuba M, Siziya S, Sikaona L. An assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment. BMC Public Health, 2004; 4: 68
5 Wares DF, Singh S, Acharya AK, Dangi R. Non-adherence to tuberculosis treatment in the eastern Tarai of Nepal. Int J Tuberc Lung Dis, 2003; 7: 327~35
6 World Health Organization/IUATLD Global project on anti-tuberculous drug Resistance Surveillance. Anti-tuberculous Drug Resistance in the World. Third global report.WHO/HTM/TB/2004.343. Geneva: World Health Organization, 2004
7 Andres E. Drug-induced hepatotoxicity.N Engl J Med, 2003, 13; 349(20): 1974~6
8曾民德,萧树东.肝脏与内分泌.北京:人民卫生出版社, 1995: 132~145
9 Lee J, Boyer JL. Molecular alterations in hepatocyte transport. Semin Liver Dis, 2000; 20: 373~384
10 Hilsden RJ, Shaffer E. Liver structure and function. In: Thomson A, Shaffer E, editors. First principles of gastroenterology: the basis of disease and an approach to management, 4th ed. Edmonton, AB,Canada: Astra; 2000: 462~564
11 Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: its clinical and toxicological role. J Clin Pharm Ther, 2000; 25: 165~175
12 Lieber CS. Susceptibility to alcohol-related liver injury. Alcohol Alcohol Suppl, 1994; 2: 315~326
13 Kanathur N, Mathai MG, Byrd RP, Fields CL, Roy TM. Simvastatindiltiazem drug interaction resulting in rhabdomyolysis and hepatitis. Tenn Med, 2001; 94: 339~341
14 Lee WM. Drug-induced hepatotoxicity. N Engl J Med, 2003; 349: 474~85
15 Fountain FF, Tolley E, Chrisman CR, Self TH. Isoniazid hepatotoxicity associated with treatment of latent tuberculosisinfection: a 7-year evaluation from a public health tuberculosisclinic. Chest, 2005; 128: 116~23
16 Stuart RL, Grayson ML. A review of isoniazid-related hepatotoxicity during chemoprophylaxis. Aust N Z J Med, 1999; 29(3): 362~7
17 Walubo A, Smith P, Folb PI. The role of oxygen free radicals in isoniazid-induced hepatotoxicity. Methods Find Exp Clin Pharmacol, 1998; 20(8): 649~55
18李键,王修齐,杨桂仙,等.药源性消化系统疾病.北京:科学出版社, 2001: 265~67
19 SteeleMA,Burk RF,DesPrez RM. Toxic hepatitiswith isoniazid and rifamp in: a meta2analysis. Chest, 1991, 99: 465~71
20 Andrade RJ, Lucena MI, Melgarejo F.Hepatotoxicity caused by isoniazid or by paracetamol Gastroenterol Hepatol, 1998; 21(6): 314~5
21赵凤芹.抗结核药物诱导肝损伤的评价.国外医学呼吸系统分册, 2003, 23(6): 322~4
22 Girling DJ. The hepatic toxicity of antituberculosis regimens containing isoniazid, rifampicin and pyrazinamide. Tubercle, 1978; 59: 13~32
23 Villarino ME, Ridzon R, Weismuller PC et al. Rifampin preventive therapy for tuberculosis infection: experience with 157 adolescents. Am J Respir Crit Care Med, 1997; 155: 1735~8
24 Slivka IuI. Comparative characterization of the hepatotoxicity of isoniazid, rifampicin and pyrazinamide. Farmakol Toksikol, 1989; 52(4):82~5
25 Yew WW, Leung CC. Antituberculosis drugs and hepatotoxicity. Respirology, 2006, 11: 699~707
26 Jussi J, Saukkonen,David L, et al. An official ATS statement:hepatotoxicity of antituberculosis therapy. Am J Respir Crit Care Med, 2006, 174: 935~52
27 Yee D, Valiquette C, Pelletier M, Parisien I, Rocher I, Menzies D.Incidence of serious side effects from first-line antituberculosis drugs among patients treated for active tuberculosis. Am J Respir Crit Care Med, 2003; 167: 1472~7
28 Schaberg T, Rebhan K, Lode H. Risk factors for side-effects of isoniazid, rifampin and pyrazinamide in patients hospitalized for pulmonary tuberculosis. Eur Respir J, 1996; 9: 2026~30
29 Danan G, Pessayre D, Larrey D. Pyrazinamide fulminant hepatitis: an old hepatotoxin strikes again.Lancet. 1981, 7; 2(4): 1056~7
30 Corbella X, Vadillo M, Cabellos C. Hypersensitivity hepatitis due to pyrazinamide.Scand J Infect Dis, 1995; 27(1):93~4
31 Sarich TC, Youssefi M, Zhou T, Adams SP, Wall RA, Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. Arch. Toxicol. 1996; 70: 835~40
32 Steele MA ,Burk RF ,Desprez RM. Toxic Hepatitis with Isoniazid and Rifampin. A meta analysis. Chest, 1991, 99: 465
33 Sarma GR ,Immanuel C , Kailasam S , et al . Rifampin induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifampin. Am Rev Respir Dis, 1986, 133(6): 1072
34 Zhang RL ,Wang ZY,Li D , et al . Effect of rifampicin on pharmacoki netics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacologica Sinica, 1992, 13(6): 494
35 Jenner AM, Timbrell JA. In vitro microsomal metabolism of hydrazine. Xenobiotica, 1995; 25: 599~609
36 Jenner AM, Timbrell JA. Influence of inducers and inhibitors of cytochrome P450 on the hepatotoxicity of hydrazine in vivo. Arch Toxicol, 1994; 68: 349~57
37 Jenner AM, Timbrell JA. Effect of acute and repeated exposure to lowdoses of hydrazine on hepatic microsomal enzymes and biochemical parameters in vivo. Arch Toxicol 1994; 68: 240~5
38 Sodhi CP, Rana SV, Mehta SK, Vaiphei K, Attari S, Mehta S. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats. Drug Chem Toxicol, 1997; 20: 255~69
39 Sodhi CP, Rana SV, Mehta SK et al. Study of oxidative stress in isoniazid-induced hepatic injury in young rats with and without protein-energy malnutrition. J Biochem Toxicol, 1996; 11: 139~46
40 Chowdhury A, Santra A, Kundu S et al. Induction of oxidative stress in antitubercular drug-induced hepatotoxicity. Indian J Gastroenterol, 2001; 20: 97~100
41孙友乐译.临床药理学,第1版.科学技术文献出版社重庆分社,重庆: 1982: 240~247
42 Wen X, Wang JS, Neuvonen PJ, Backman JT. Isoniazid is a mechanism-based inhibitor of cytochrome P450 1A2, 2A6, 2C19 and 3A4 isoforms in human liver microsomes. Eur J Clin Pharmacol, 2002; 57: 799~804
43 Rae JM, Johnson MD, Lippman ME, et al. Rifamp in is a selective, pleiotropic inducer of drugmetabolism genes in human hepatocytes: studies with cDNA and oligonucleotide exp ression arrays. J Pharmacol Exp Ther, 2001, 299: 849~57
44 Kolars JC, Schmiedlin-Ren P, Schuetz JD, Fang C, Watkins PB. Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes. J Clin Invest, 1992; 90: 1871~8
45 Combalbert J, Fabre I, Fabre G et al. Metabolism of cyclosporin A. IV. Purification and identification of the rifampicin-inducible human liver cytochrome P-450 (cyclosporin A oxidase) as a product of P450IIIA gene subfamily. Drug Metab. Dispos, 1989; 17: 197–207
46陈新谦主编.新编药物学.第13版.人民卫生出版社,北京, 1992:324
47 Durand F, Bernuau J, Pessayre D. Hepatotoxicity of antituberculosis drugs: practical implications for monitoring.Gastroenterol Clin Biol, 1998; 22(2):117~20
48 Steele MA, Burk RF, DesPrez RM. Toxic hepatitis with isoniazid and rifampin: a meta-analysis. Chest, 1991; 99: 465–471
49 Skakun NP, Shman'ko VV. Synergistic effect of rifampicin on hepatotoxicity Marof isoniazid. Antibiot Med Biotekhnol, 1985; 30(3): 185~9
50 Toulet J, Viteau JM. Isoniazid-rifampicin: a model for hepatotoxicity and for bibliographic Anglomania.Gastroenterol Clin Biol, 1978; 2(8-9): 756~7
51 Askgaard DS, Wilcke T, Dossing M. Hepatotoxicity caused by the combined action of isoniazid and rifampicin.Thorax. 1995, 50(2): 213~4
52 Ozawa S. Drug-drug interaction in pharmacogenetics and pharmacogenomics . Rinsho Byori, 2002; 50(2): 146~50
53 Levy RH, Hachad H, Yao C, et al. Relationship between extent of inhibition and inhibitor dose: literature evaluation based on the metabolism and transport drug interaction database.Curr Drug Metab. 2003; 4(5): 371~80
54 Venkatakrishnan K, von Moltke LL, Obach RS, ea tl. Drug metabolism and drug interactions: application and clinical value of in vitro models.Curr Drug Metab, 2003; 4(5): 423~59
55 Erhardt PW. A human drug metabolism database : potential roles in the quantitative predictions of drug metabolism and metabolism-related drug-drug interactions.Curr Drug Metab, 2003; 4(5): 411~22
56 Yamazaki H. Roles of human cytochrome P450 enzymes involved in drug metabolism and toxicological studies. Yakugaku Zasshi. 2000; 120(12): 1347~57
57 Glue P, Clement RP.Cytochrome P450 enzymes and drug metabolism- basic concepts and methods of assessment. Cell Mol Neurobiol, 1999;19(3):309~23
58 Sarma GR, Immanuel C, Kailasam S, et al. Rifamp in induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment oftuberculosis with regimens containing isoniazid and rifampin. Am Rev Resp ir D is, 1986,133: 1072~7
59 Sarma GR, Immanuel C, Kailasam S, et al. Rifamp induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifamp in. Am Rev Respir Dis, 1986,133: 1072
60 Mitchell JR, Tho rgeirsson U P, Black M , et al. Increased incidence of isoniazid hepatitis in rapid acetylato rs: possible relation to hydrazine metabo lites. Clin Pharmacol Ther, 1975,18: 70
61 Ellard GA , M itch ison DA , Girling DJ, et al. The hepatic toxicity of isoniazid among rapid and slow acetyators of the drug. Am Rev Respir Dis, 1978, 118: 628
62 Timbrell JA , Park BK, Harland SJ. A study of the effects of rifamp icin on isoniazidmetabo lism in human volunteer subjects. Hum Toxicol, 1985, 4 (3): 279
63 Singh J, Garg PK, Thakur V S , et al. A nti tubercular treatment induced hepato toxicity: do se acetylator status matter? Indian J Physiol Pharmacol, 1995, 39 (1) : 43
64 Ohno M , Yamaguch i I, Yamamo to I, et al. Slow N acetyltransferase 2 genotype affects the incidence of isoniazid and rifampic induced hepatotoxicity. Int J Tuberc Lung Dis,2000, 4 (3) : 256
65 FernandezVA, Sopefia B, FernandezVJ, et al. The influence of risk factors on the severity of antituberculosis drug induced hepatotoxicity. Tuberc lung dis, 2004, 8 (12): 1499~505
66 Sharma SK, Balamurugan A, Saha PK, et al. Evaluation of clinical and immunogenetic risk factors for the development of hepatotoxicity during anti tuberculosis treatment. Am. J. Resp ir. Crit. Care Med, 2002, 166: 916~9
67 Tost JR,Vidal T, Cayl J, et al. Severe hepatotoxicity due to antituber culosis drugs in Spain. Tuberc Lung Dis, 2005, 9 (5) : 534~40
68 Wu JC, Lee SD, Yeh PF, et al. Isoniazid and rifamp induced hepatitis inhepatitisB carriers. Gastroenterology, 1990, 98 (2) : 502~4
69 Wong WM,Wu PC, Yuen MF, et al. Antituberculosis drug– related liver dysfunction in chronic hepatitis B infection. Hepatology, 2000, 31 (1) : 201~6
70郭虹,李环,李淑芬,等.抗结核药物治疗对肝功能的影响.北华大学学报(自然科学版), 2005, 6: 425~7
71 Shakya R, Rao BS, Shrestha B. Incidence of hepatotoxicity due to antitubercular medicines and assessment of risk factors. Ann Pharmacother, 2004, 38: 1074~9
72 DossingM,Wilcke JT,Askgaard DS, et al. Liver injury during antituberculosis treatment: an 11 years study. Tuber Lung Dis, 1996,77: 335~40
73 Schaberg T, Rebhan K,Lode H. Risk factors for sideeffects of isoniazid, rifampin and pyrazinamide in patients hospitalized for pulmonary tuberculosis. Eur Resp ir J, 1996, 9: 2026~30
74路西明,王建刚,侯湘波等.白头翁对利福平异烟肼肝毒性研究.中国中医药信息杂志, 1998, 5 (8) : 21~7
75侯艳宁,朱秀媛,程桂芳.黄芩苷对小鼠肝细胞色素P450及其亚家族的诱导.解放军药学学报, 2000, 16 (4) : 58~63
76薛洪源,侯艳宁,刘会臣水飞蓟宾胶囊对异烟肼和利福平肝损害小鼠的保护作用.中成药, 2003, 25(4): 307~310
77 Kinzig-Schippers M, Tomalik-Scharte D, Jetter A et al. Should we use N-acetyltransferase type 2 genotyping to personalize isoniazid doses? Antimicrob. Agents Chemother. 2005; 49:1733~8
78 Tostmann A, boeree MJ, Aarnoutse RE, et al. Antituberculosis drug-induced hepatotoxicity: concise up-to-date review. Journal of Gastroenterology and Hepatology, 2008, 23:192~202
1 Tayal V, Kalra BS, Agarwal S, et al. Hepatoprotective effect of tocopherol against isoniazid and rifampicin induced hepatotoxicity in albino rabbits. Indian J Exp Biol. 2007, 45(12): 1031~1036
2 Lieber CS. Pathogenesis and treatment of alcoholic liver disease:progress over the last 50 years. Rocz Akad Med Bialymst, 2005, 50:7~ 20
3 Lieber CS. Alcoholic fatty liver : its pathogenesis and mechanismof progression toinflammation and fibrosis . Alcoholic, 2004, 34(1):9~ 19
4 Bai J, Cederbaum AI. Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletionofglutathione. J Biol Chem, 2006, 281(8): 5128~ 5136
5胡国平,刘凯王,唐红等.多烯磷脂酰胆碱(易善复)治疗慢性肝炎的系统评价.中国循证医学杂志,2005, 5(7):543~548
6 TORRIELLI MV,GABRIEL L ,DIANZZMI M U. Ethanol-inducedHepatotoxicity : Experimental Observations on the Role of Lipid Peroxidation. Journal of Pathology,1978 ,26:11
7闻勤生,郝云龙,赵海峰,等.氧自由基在肝缺血再灌注损伤中的作用及凯西莱的影响.中国现代医学杂志, 2002, 12(11): 5~7
8全冰.硫普罗宁对抗痨药物肝损作用的预防效果观察.第四军医大学学报, 2006, 27(15): 1417~1420
9张海,韩志启,朱增红,等.凯西莱治疗抗结核药肝损害的疗效.医药论坛杂志, 2004, 25(1):23~24
10毕国春,王传力.还原型谷胱甘肽治疗抗结核药物性损害62例疗效分析.中国医师杂志, 2004: 233~234
11 Atkuri KR,Mantovani JJ,Herzenberg LA,et al. N-Acetylcysteine-a safe antidote for cysteine/glutathione deficiency. Current Opinion Pharmacology. 2007, 7(4): 355~359
12 Kelly GS. Clinical applications of N-acetylcysteine. Altern Med Rev, 1998,3(2): 114~127
13 ZHAO J X,YANG Y M,HUANG Y T. Role of reactive species and N-acetylcysteine in hyperacutere jection of the rats liver in the isolated perfusion model. Chin J Exp Surg, 2004, 21(2): 141~143
14 PATRIARCAA S, FURFAROA A L, DOMENICOTTI C, et al. Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics butprotected from oxidative stress. Biochim BiophysActa, 2005, 1741(1-2): 48~54
15 Pastor A,Collado PS,AlmarM, et al. Antioxidant enzyme status in biliary obstructed rats: effects of N-acetylcysteine. J Hepatol, 1997,27(2): 363~370
16 LORENZO M,KRANTIS A. Nitric oxide synthase isoenzyme activities in a premature pigletmodel of necrotizing enterocolitis:effects of nitrergic manipulation[ J].Ped Surg Inter, 2002, 18:624~629
17 Semin Liver Dis,Fontana Robert J RJ,et al. Acute liver failure due to drugs.Semin Liver Dis 2008, 28(2):175~87
18 Warnet JM,Christen MO,Thevenin M, et al. Protective effect of anethol dithiolthione against acetaminophen hepatotoxicity in mice. Pharmacology & Toxicology, 1989, 65(1):63~64
19王龙虎,吴国良,程翼宇.壳聚糖及其衍生物在中药制药工业中的应用.中国中药杂志, 2004,29(4): 289~291
20 CHEN XG, WANG Z, LIU WS, et al. The effect of carboxymethyl- chltosan on proliferation and collagen secretion of normal and keloid skin fibroblasts. Bionmterials, 2002, 23 (23): 4609~4614
21向轶,张志荣.巯基壳聚糖载胰岛素亚微球的制备及影响包封率的因素.华西药学杂志, 2005, 2(3): 195~199
22蒋新宇,周春山,张俊山.应用三聚磷酸钠为交联剂制备载药物纳米粒的研究.中国现代医学杂志, 2003, 13(22): 69~72
23 Ho YP, Chen HH, Leong KW, et a1. Evaluating the intracellular slability and unpacking of DNA nanocomplexes by quantum dots-FRET . J Control Release, 2006, 116(1): 83~89
24 24.S. Santhosh, T.K. Sini, R. Anandan, et al. Effect of chitosan supplementation on antitubercular drugs-induced hepatotoxicity in rats. Toxicology, 2006, 219, 53~59
25 S. Santhosh, T.K. Sini, R. Anandan, et al. Hepatoprotective activity of chitosan against isoniazid and rifampicin-induced toxicity in experimental rats. European Journal of Pharmacology, 2007, 572: 69~73
26 Yoshikawa M, Matsui Y, Kawamoto H, et al. Effects of glycyrrhizin on immune mediated cytotoxicity. Gastroenterol. Hepatol. 1997, 12: 243~248
27 Miyaji C, Miyakawa R, Watanabe H, et al. Mechanisms underlying the activation of cytotoxic function mediated by hepatic lymphocytes following the administration of glycyrrhizin. Int. Immunopharmacol. 2002, 2: 1079~1086
28 Shiki Y, Shinai K, Satto Y, et al. Effect of glycyrrhizin on lysis of hepatocyte membranes induced by anti-liver cell membrane antibody. J. Gastroenterol. Hepatol,1992, 7:12~16
29 Abe N, Ebina T, Ishida N. Interferon induction by glycyrrhizin andglycyrrhetinic acid in mice. Microbiol, Immunol. 1982, 26: 535~539
30 Dhiman RK, Chawla YK. Herbal medicines for liver diseases.Dig. Dis. Sci, 2005, 10:1807~1812
31 Miyazawa Naoki N,Takahashi Hiroshi H,Yoshiike Yasuhiro Y.Effect of glycyrrhizin on anti-tuberculosis drug-induced hepatitis.Kekkaku 2003 ,78(1):15~19
32孙鹏,李娥,邱杰文.复方甘草酸苷治疗抗结核药物性肝炎临床观察.现代医院, 2006, 6(8): 52~53
33 Muriel P. S-adenosyl-L-methionine prevents and reverses erythrocyte membrane alterations in cirrhosis. Appl. Toxicol. 1993, 13: 179~182
34 Muriel P, Mourelle M. Characterization of membrane fraction lipid composition and function of cirrhotic rat liver.Role of S-adenosyl-L-methionine. J. Hepatol. 1992, 14: 16~21
35 Benz C, Angermuller S, Kloters-PlachkyP, et al. Effect of adenosylmethionine versus tauroursodexycholic acid obile acidinduced app potosis and cytolysis in rat hepato-cytes. EurJclin Invest, 1998, 28(7): 577~583
36王少华,李宁,任建安,等. S-腺苷-L-蛋氨酸治疗全肠营养导致的胆汁淤积.中华普通外科杂志, 2000, 15(12): 746~747
37 Belli DC, Fournier LA, Lepage G, et al. S-adenosylmethionine prevents totol parenteral nutrition induced cholestasis in rat.J Hepatol, 1994, 21(1): 18~23
38 Trauner M, Graziadei IW. Review article: mechanisms of action and therapeutic applications of ursodeoxycholic acid in chronic liver diseases. Aliment phamacol Ther, 1999, 13(8): 979~996
39 Xie Q, Khaoustov VI, Chung C C, et al. Effect of tauro- ursodeoxycholic acidon endoplasmic reticulum stress-induced caspase-12 activation. Hepatology, 2002, 36(3): 592~601
40 Angulo P. Use of ursodeoxycholic acid in patients with liver disease. Curr Gastroenterol Rep, 2002, 4(1): 42~44
41 Schoemaker MH, conoedela RosaL, Bulsi-Homan M, et al.Taurourso-deoxycholio acid protecs rat hepatocyies from Bile acid-induced apoptosis via acthailon of survival pathways. Hepatology, 2004, 39(6): 1563~1573
42 Sohn J, Knaoustov VI, Xie O, et al. The effect of ursodeoxy-cholic acid on the survivin in thapsigargin-induced apoptosis. Cancer letter, 2003, 191(1): 83~92
43 Guicciardi ME, Gores GJ. Ursodeoxycholio acid cytoprotection: dancing with death receptors and survival pathways. Hepatology, 2002, 35(4): 971~973
44 Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998, 281(5381): 1309~1312
45 Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectemied rats. Biochem Biophys Res Commun, 1984, 122 (3): 1450~1459
46刘琨,吴碧彤,蒋蓬,等.促肝细胞生长因子对抗结核药物性肝损的治疗观察.广州医学2006, 37(1):25~28
47魏世俊,张杰,韩连风.护肝片五味子乙素的薄层光密度法测定.黑龙江医药, 1998, 11(6): 338~340
48 Luper S. A review of plants used in the treatment of liver disease. Altern. Med. Rev. 1998. 3: 410~421
49 Pascual C, González R, Armesto J, et al. Effect of silymarin and silybinin on oxygen radicals. Drug Develop Res, 1993, 29: 73~77
50 Mourelle M, Meza MA. Colchicine prevents D-galactosamineinduced hepatitis. Hepatol. 1989, 8(2): 165~172
51 Pradhan SC, Girish C. Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian J. Med. Res. 2006, 124(5): 491~504
52 Muriel P, Ali S, Friedman SL, et al. Role of nitric oxide in liver disorders, In LiverDiseases: Biochemical Mechanisms and New Therapeutic Insights. Science Publishers: Enfield (NH) Jersey. 2006, 115~131
53 Wang M, Grange LL, Tao J. Hepatoprotective properties of Silybummarianum herbal preparation on ethanol-induced liver damage. Fitoterapia, 1996, 67: 167~171
54 Kropacova K, Misurova E, Hakova H. Protective and therapeutic effect of silymarin on the development of latent liver damage. Radiats Biol Radioecol, 1998, 38(3): 411~415
55 Luper S. A review of plants used in the treatment of liver disease: Part 1. Altern. Med. Rev. 1998, 3: 410~421
56薛洪源,侯艳宁,刘会臣等.水飞蓟宾胶囊对异烟肼和利福平肝损害小鼠的保护作用.中成药2003, 25 (4):307~310
57 Ammon HP, Walh MA. Pharmacology of Curcuma longa. Planta Med, 1991, 57(1): 1~7
58 Maheswari RK, Singh AK, Gaddipati J, et al. Multiplebiological activities of curcumin: A short review. Life Sci, 2006, 78(5): 2081~2087
59 Aggarwal BB, Bhatt ID, Ichikawa H, et al. Curcumin: biological and medicinal properties. In Turmeric: The Genus Curcuma, Ravindran PN, Nirmal-Babu K, Sivaraman K (eds). Series: Medicinal and Aromatic Plants-Industrial Profiles. CRC Press: New York, 2007, 45(7): 297~368
60 Rao CV, Rivenson A, Simi B, et al. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 1995, 55(4): 259~266
61 Muriel P. Cytokines in liver diseases. In Hepatotoxicity: From Genomics to In Vitro and In Vivo Models. Pharmacology 2007, 48(9): 63~68
62 Abe Y, Hashimoto S, Horie T, et al. Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages. Pharmacol. Res. 1999, 39(5): 41~47
63 Reyes-Gordillo K, Segovia J, Shibayama M, et al. Curcumin protects against acute liver damage in the rat by inhibiting NF-κB, proinflammatory cytokines production and oxidative stress. Biochim. Biophys. Act . 2007, 1770: 989~996
64 Bruck R, Ashkenazi M, Weiss S, Goldiner I, et al. Prevention of liver cirrhosis in rats by curcumin. Liver Int. 2007, 27: 373~383
65 AaBurkitt MJ, Duncan J. Effects of trans-resveratrol on copper- dependent hydroxyl radical scavenging and a novel, glutathione-sparing mechanism of action. Arch Biochem Biophys, 2000, 381(2): 253~263
66 Das S, Das DK . Resveratrol: a therapeutic promise for cardiovasculardiseases. Recent Patent Cardiovasc Drug discov, 2007, 2(2):133~138
67 Araki T, Sasaki Y, Milbrandt J. Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science, 2004, 305(5686): 1010~1013.
68 Fulda S, Debatin KM. Sensitization for tumor necrosis factor related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol . Cancer Res, 2004, 64(1): 337~346
69 Zhang Q, Tang X, Lu QY, et a1. Resveratrol inhibits hypoxia-induced accumulation of hypoxia-inducible factor-I A and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells. Mol Cancer Ther, 2005, 4(10): 1465~1474
70 Vivancos M, Moreno JJ. Effect of resveratrol, tyrosol and beta sitosterol on oxidised low-density lipoprotein-stimulated oxidative stress, arachidonic acid release and prostaglandin E2 synthesis by RAW 264.7 macrophages. Br J Nutr, 2008, 99(6): 1199~1207
71 Yang S, Meyskens FL Jr. Alterations in activating protein I composition correlate with phenotypic differentiation changes induced by resveratrol in human melanoma. Mol Pharmacol, 2005, 67(1): 298~308
72 Chiou W F, Lin LC, Chen CF. Acteoside protects endothelial cells against free radical-induced oxidative stress. J Pharm Pharmacol, 2004, 56(6): 743~748
73 Miura T, Muraoka S, Fujimoto Y. Inactivation of creatine kinase induced by stilbene derivatives. Pharmacol Toxicol, 2002, 90(2): 66~72
74杜文明,徐克涵.银杏的营养和药用价值.河北林业科技, 2002, 3: 276~287
75张卫明,赵泊涛,王红.银杏种仁对小白鼠SOD的影响.中国野生植物资源, 1995, l: 23~24
76张卫明,赵泊涛.银杏对果蝇寿命的影响.中国野生植物资源, 1995, l: 25~26
77戴建国,陈景衡.白果提取液对小白鼠心脑脂褐质水平的影响.南京医科大学学报, 1994, 14: 411~413
78 Wang H, Ng TB, Ginkbilobin, et al. a nove1 antifungal protein from Ginkgo biloba seeds with sequence simi1arity to embryo-abundant protein . Biochem Biophys Res Commun, 2000, 279: 407~411
79邓乾春,黄文,谢笔钧等.白果活性蛋白的抗生物氧化作用.营养学报, 2006, 28(3): 259~262
80 Naik SR, Panda VS. Hepatoprotective effect of Ginkgoselect Phytosome in rifampicin induced liver injury in rats: evidence of antioxidant activit y . Fitoterapia, 2008, 79(6): 439~445
81王建刚,周海梅,侯湘波等.银杏叶醇提取物对异烟肼和利福平肝毒性保护作用的实验研究.中国现代应用药学杂志, 2000, 17 (3), 15~18
82 Jiangsu New Medical College(江苏新医学院). Zhong Yao Da Ci Dian(中药大辞典). Shanghai: Shanghai Science and Technology Press, 1998, 1174~1177
83杨永康,格桑索朗,吴家坤.诃子、毛诃子和余甘子的植物分类研究和药学特性综述.中国医学生物技术应用杂志, 2004, 3(1): 14~25
84潘燕,张述禹,侯金凤等.诃子对大鼠心肌酶的影响.中国中药杂志, 2004, 29(4): 382~383
85 Malekzadeh F, Ehsanifar H, Shahamat M, et a1. Antibacterial activity of black myrobalan(Terminalia chebula Retz.)against Helicobacter priori . Int J Antimicrob Agents, 2001, 18(1): 85~88
86 Shin T Y, Jeong H J, Kim D K, et a1. Inhibitory action of water soluble fraction of Terminalia chebula on systemic and local anaphylaxis . J Ethnopharmacol, 2001, 74(2): 133~140
87 Saleem A, Husheem M, H~kSnen P, et a1. Inhibition of cancer cell growth by crude extract and the phenohcs of Terminalia chebula Retz. Fruit . J Ethnopharmacol, 2002, 81(3): 327~336
88 Jagetia G C, Baliga M S, Mal K J, et a1. The evaluation of the radioprotective effect of Trlphala(an ayurvedlc rejuvenating drug)in the mice exposed toγ-radiation . Phytomedicine, 2002, 9(2): 99~108
89 Aim M J, Kim C Y, Lee J S, et a1. Inhibition of HIV-1 integrase by galloyl glucoses from Terminalia chebula andflavonol glycoside gallates from Euphorbiapekinensis. Planta Med, 2002, 68(5): 457~459
90 Naik G H, Priyadarsini K I, Satav J C, et al. Comparative antioxidam activity of individual herbal components used in Ayttrvedic medicine. 2003 ;63(1):97~104
91 Saleem A, Ahotupa M, Pihlaja K. Tota1 phenollcs concentnation and antioxidant potential of extracts ofmedicinal plants of Pakistan. Naturforsch, 2001, 56(11-12): 973~978
92傅乃武,全兰萍,黄磊等.诃子提取物对活性氧的清除和对抗TPA对人白细胞DNA的损伤.中草药, 1992, 23 (1): 26~29
93 SA Tasduq, K Singh, NK Satti, et al. Terminalia chebula (fruit) prevents liver toxicity caused by sub-chronic administration of rifampicin, isoniazid and pyrazinamide in combination. Hum Exp Toxicol, 2006, 25 (3): 111~118
94 Dhiman RK, Chawla YK. Herbal medicines for liver diseases. Dig. Dis. Sci. 2005, 10: 1807~1812
95 Huseini HF, Alavian SM, Heshmat R, et al. The efficacy of Liv-52 on liver cirrhotic patients: a randomized double-blind, placebo-controlled first approach. Phytomedicine. 2005, 12: 619~624
96 S. D. Saraswathy, C. S. Shyamala Devi. Modulating Effect of Liv.100, an Ayurvedic Formulation on Antituberculosis Drug-induced Alterations in Rat Liver Microsomes. Indian J. Phytother. Res, 2001, 15, 501~505
97 Sandhir R, Gill KD. Hepatoprotective effects of Liv 52 on ethanol induced liver damage in rats. Indian J. Exp. Biol. 1999, 37: 762~766
98 Roy A, Soni GR, Kolhapure RM, et al. Down regulation of tumor necrosis factor activity in experimental hepatitis by a herbal formulation, Liv 52. Indian J. Exp. Biol. 1994, 32: 694~697
99中华人民共和国卫生部药典委员会.中华人民共和国药典.一部,化学工业出版社, 2005: 124
100周涛,邱德文.民族药余甘子本草药学概况.贵阳中医学院学报, 2002, 24 (3): 3~6
101王小李,詹琳.余甘子甲醇萃取物对色拉油抗氧化效果的研究.武汉工业学院学报, 2000: 13~15
102Sabu MC, Kuttan R. Anti2diabetic activity of medicinal plants and itsrelationship with their antioxidant property. J Ethnopharmacol, 2002, 81: 155~160
103Bhattacharya A, Chatterjee A, Ghoshal S, et al. Antioxidant activity of tannoid principles of Emblica officinalis (amla) . Ind J Bio, 1999, 37: 676~680
104肖湘,俞丽君,邱玉莹等.油柑多糖的提取与清除氧自由基作用研究.中国药学杂志, 1998, 23 (5): 279~282
105S.A.Tasduq, P.Kaisar, D.K.Gupta, et al. Protective Effect of a 50% Hydroalcoholic Fruit Extract of Emblica officinalis against Anti-tubercul osis Drugs induced Liver Toxicity. Phytother. Res, 2005, 19, 193~197
106Dhuley JN, Naik SR. Effect of rhinax on bacterial lipopolysaccharide induced endotoxemia in rats. Indian J Exp Biol, 1998, 36(3): 315~317
107Dhuley J. Healing-promoting action of rhinax with dual action on chronic gastric and duodenal ulcers induced by acetic acid in rats. Hindustan Antibiot Bull, 2003, Feb, 45~46; 2004, Nov: 34~40
108Dhuley JN, Naik SR. Protection by Rhinax in various models of ulceration in rats. J Ethnopharmacol, 1998, 63(3): 219~225
109Dhuley JN, Naik SR. Protective effect of Rhinax, a herbal formulation, against CCl4-induced liver injury and survival in rats. J Ethnopharmacol, 1997, 56(2): 159~64
110Dhuley JN. Hepatoprotective effect of rhinax on antitubercular drug-induced hepato-toxicity in rats. Hindustan Antibiot Bull, 2002 Feb-Nov; 44(1-4): 53~59
2 Noda A, Hsu KY, Noda H, et al. Is isoniazid-hepatotoxicity induced by the metabolite, hydrazine? J UOEH, 1983; 5: 183~90
3 Gent WL, Seifart HI, Parkin DP, et al. Factors in hydrazine formation from isoniazid by paediatric and adult tuberculosis patients. Eur J Clin Pharmacol, 1992; 43: 131~6
4 Menzies D, Dion MJ, Rabinovitch B, et al. Treatment completion and costs of a randomized trial of rifampin for 4 months versus isoniazid for 9 months. Am J Respir Crit Care Med, 2004; 170: 445–449
5 Hong Kong Chest Service, Tuberculosis Research Centre, Madras, British Medical Research Council.Adouble-blind placebo-controlled clinical trial of three anti-tuberculosis chemoprophylaxis regimens in patients with silicosis in Hong Kong. Am Rev Respir Dis, 1992; 145: 36~41
6乐江,彭仁修,刘娟. RP–HPLC法测定血浆中异烟肼代谢物的含量.中国药科大学学报, 2004, 35(1): 47
7殷琦,车宁,赫广威, et al. HPLC法测定人体血浆中利福平和异烟肼的浓度.药物分析杂志, 2002, 22(5): 358
8 Bertino J Jr, Fish D. The safety profile of the fluoroquinolones. Clin Ther, 2000; 22: 798~817
9 Wolfson JS, Hooper DC. Overview of fluoroquinolone safety. Am J Med, 1991; 91: 153S~161S
10 Coleman CI, Spencer JV, Chung JO, Reddy P. Possible gatifloxacin induced fulminant hepatic failure. Ann Pharmacother, 2002; 36: 1162~1167
11 Kahn JB. Latest industry information on the safety profile of levofloxacin in the US. Chemotherapy, 2001; 47: 32~37
12 Steele MA, Burk RF, Desprez RM. Toxic Hepatitis with Isoniazid and Rifampin. A meta2analysis. Chest, 1991, 99: 465
13 Sarma GR, Immanuel C, Kailasam S, et al. Rifampin induced release of hydrazine from isoniazid: A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifampin. Am Rev Respir Dis, 1986, 133(6): 1072
14 Zhang RL, Wang ZY, Li D, et al. Effect of rifampicin on pharmacoki- netics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacologica Sinica, 1992, 13(6): 494
15 Rae JM, Johnson MD, Lippman ME, et al. Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrayds. J Pharmacol Exp Ther, 2001, 299(3): 849~857
1 World Health Organization. Tuberculosis. WHO Fact Sheet No. 104. Revised. March 2006
2 Kaona FA, Tuba M, Siziya S, Sikaona L. An assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment. BMC Public Health 2004; 4: 68. 5
3 Knowles SR, Uetrecht J, Shear NH. Idiosyncratic drug reactions: the reactive metabolite syndromes. Lancet 2000; 356: 1587–91
4 Tostmann A, Boeree MJ, Aarnoutse RE, et al. Antituberculosis drug-induced hepatotoxicity: concise up-to-date review. Journal of Gastroenterology and Hepatology, 2008, 23: 192~202
5 Sarich TC, Youssefi M, Zhou T, et al. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. Arch Toxicol, 1996, 70: 835~840
6 Zhang RL, Wang ZY, Li D, et al. Effect of rifampicin on pharmacokinetics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacol Sin, 1992
7 Rae JM, Johnson MD, Lippman ME, et al. Rifampin is a selective pleiotropic inducer of drug metabolism genes in human hepatocytes: sutdies with cDNA and oligonucleotide expression arrayds. J PharmacolExp Ther, 2001, 299(3): 849~857
8 Panchagnula R, Sood A, Sharda N, et al. Determination of rifampicin and its main metabolite in plasma and urine in presence of pyrazinamide and isoniazid by HPLC method. Journal of Pharmaceutical and Biomedical Analysis18 (1999) 1013–1020
9 Zhang RL , Wang ZY, L i D, et al. Effect of rifampicin on pharmacokinetics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacologica Sinica, 1992, 13 (6): 494
10 Noda A, H su KY, Noda H, et al. Is isoniazid hepatotoxicity.induced by the metabolite, hydrazine? Sangyo Ika DaigakuZasshi, 1983, 5 (2) : 183
11 Noda A , Sendo T, Ohno K, et al. Effect of rifamp icin andPhenobarbital on the fate of isoniazid and hydrazine in vivo in rats. Toxicol Lett, 1985, 25 (3): 313
12 Sendo T, Noda A , Noda H, et al. Metabolic hydrolysis of isoniazid by subcellular fractions of rat liver. J UOEH, 1984, 6(3): 249
13 Nilesh Mehta, MD. Emmanuel Gbadehan, MD, Lisa Ozick, MD, Drug-Induced Hepatotoxicity Updated: Mar 28, 2008
14 Lee WM. Drug-induced hepatotoxicity. N Eng J Med, 2003; 349(5): 474~85
15 Lee WM. Impact of Drug Induced Liver Toxicity on Hepatology and Practice of Medicine. US Food and Drug Administration, Center for Drug Evaluation and Research; 2001
16 Chalasani N. Statins and hepatotoxicity: focus on patients with fatty liver. Hepatology, 2005; 41(4): 690~5
1 Nelson DR, Koymans L, Kamataki T, et al. P450 superfamily: update on new sequences, gene mapping, accessionnumbers and nomenclature . Pharmacogenetics, 1996, 6(1): 1~42
2 Ingelman SM. Pharmacogenetics of cytochrome P450 and its aplications in drug therapy: the past, present and future . Trends Pharmacol Sci, 2004, 25(4): 193~200
3 Watkins PB. Role of cytochromes p450 in drug metabolism and hepatotoxicity. Semin Liver Dis. 1990 10(4): 235~250
4 Dickson RC. Glycyne cytoprotection durying lethal hepatocellular injury from adenosinetfiphate depletion. Gastrerol 2001, 6(1): 428~431
5 Kaplowit N. Biochem ical and cellular mechanisms of toxic liver injury. Sem in Liver Dis 2002, 22(2): 137~144
6 Wen X, Wang JS, Neuvonen PJ, Backman JT. Eur J Clin Pharmacol, 2002, 57(11): 799~804
7 Zeruesenay D, Siegmund W, Zschiesche M, Franke G, et al. J Pharm Pharmacol, 1992, 44(11): 893~897
8 Catania JR, McGarrigle BP, Rittenhouse-Olson K, Olson JR. Toxicol In Vitro, 2007, 21(1): 109~115
9 Chung HJ, Choi YH, Kim SH, Lee MG. J Pharm Pharmacol, 2006, 58(4): 449~457
10 Ozawa S, Ohta K, Miyajima N, Matsurnoto Y, Fukuoka M, Ohno Y. Xenobiotica, 2000, 30(10): 1005~1017
11 Ramaiah SK, Apte U, Mehendale HM. Drug Metab Dispos, 2001, 29(8): 1088~1095
12 Madan A, Graham RA, Carroll KM, Mudra DR, Burton LA, et al. Drug Metab Dispos, 2003, 31(4): 421~431
13 Graham RA, Downey A, Mudra D, Krueger L, et al. Drug Metab Dispos, 2002, 30(11): 1206~1213
14 Lee DY, Lee SJ, Lee MG. Int J Pharm, 2005, 298(1): 38~46
15 Wang T, Shankar K, Bucci TJ, Warbritton A, Mehendale HM. Toxicol Appl Pharmacol, 2001, 173(1): 27~37
16 Sarich TC, Adams SP, Petricca G, Wright JM. J Pharmacol Exp Ther, 1999, 289(2): 695~702
17 Kinzig-Schippers M, Tomalik-Scharte D, Jetter A, Scheidel B, Jakob V, Rodamer M, Cascorbi I, Doroshyenko O, Sorgel F, Fuhr U. Antimicrob Agents Chemother, 2005, 49(5): 1733~1738
18 Anundi I, Lahteenmaki T, Rundgren M, Lindros KO. Biochem Pharmacol, 1993, 45(6): 1251~1259
19 Wen X,Wang JS, Neuvonen PJ,Backman JT. Isoniazid is a mechanism-based inhibitor of cytochrome P450 1A2,2A6,2C19 and 3A4 isoforms in human liver microsomes.Eur.J.Clin. Pharmacol. 2002; 57: 799~804
20 Zand R, Nelson SD, Slattery JT, Thummel KE, Kalhorn TF, et al. ClinPharmacol Ther, 1993, 54(2): 142~149
21 Self TH, Chrisman CR, Baciewicz AM, Bronze MS. Am J Med Sci, 1999, 317(5): 304~311
22 Yue J, Peng RX, Yang J, Kong R, Liu J. Acta Sin, 2004, 25(5): 699~704
23 Huang YS, Chern HD, Su WJ et al. Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis. Hepatology 2003; 37: 924~930
24 Vuilleumier N, Rossier MF, Chiappe A et al. CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis.Eur.J.Clin.Pharmacol. 2006; 62: 423~9
1 Guengerich, FP. Human Cytochrome P450 Enzymes. Life Sci, 1992, 50, 1471~1478
2 Rendic S, di Carlo F. Human Cytochrome P450 Enzymes: a Status Report Summarizing Their Reactions, Substrates, Inducers and Inhibitors. Drug Metab Rev, 1997, 29, 413~580
3 From the Centers for Disease Control and Prevention. Fatal and severe hepatitis associated with rifampin and pyrazinamide for the treatment of latent tuberculosis infection -New York and Georgia, 2000. JAMA, 2001; 285: 2572~3
4薛洪源,侯艳宁,刘会臣, et al.水飞蓟宾胶囊对异烟肼和利福平肝损害小鼠的保护作用.中成药, 2003, 25(4)
5侯艳宁,朱秀媛,程桂芳.黄芩甙对小鼠肝细胞色素P450及其亚家族的诱导.解放军药学学报, 2000, 16(2)
6汪晖,彭仁琇.阿魏酸钠减轻对乙酰氨基酚致小鼠肝损伤.中国药理学报, 1994, 15(1): 81~83
7薛洪源,侯艳宁,刘会臣, et al.阿魏酸钠对异烟肼和利福平肝损害小鼠的保护作用.中国医院药学杂志, 2003, 23(10): 584~586
8张娴文,李凤贤,李辉.三七皂苷抗肝纤维化作用的现状.中国新药杂志, 2007, 16(4): 260~263
9 WEBER A, JAGER R, BORNER A, et al. Can Grapefruit Juice Influence Ethinylestradiol Bioavailability. Contraception, 1996, 53(1): 41~47.
10 Bailey DG. Food-durg interactions: enphasis on grapefruit juice effects. Can J Clin Pharmacol. 1995, 2: 10~14
11 Guengerich FP. Role of cytochrome P450 enzymes in drug-durg interactions. Adv Pharmacol, 1997, 43: 7~35
12 PARI L, GNANASOUNDARI M. Influence of naringenin on oxytetracyline mediated oxidative damage in rat liver. Basic Clin Pharmacol Toxicol, 2006, 98(5): 456~461
13 LEE MH, YOON S, MOON JO. The flavonoid naringenin inhibits dinethylnitrosamine-induced liver damage in rats. Bio Pharma Bull, 2004, 27(1): 72~76
1 World Health Organization. Tuberculosis. WHO Fact Sheet No.104. Revised. March 2006.
2卫生部,2008年我国卫生改革与发展情况,中国新闻网,2009.2.17
3 Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C.Tuberculosis. Lancet, 2003; 362: 887~99
4 Kaona FA, Tuba M, Siziya S, Sikaona L. An assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment. BMC Public Health, 2004; 4: 68
5 Wares DF, Singh S, Acharya AK, Dangi R. Non-adherence to tuberculosis treatment in the eastern Tarai of Nepal. Int J Tuberc Lung Dis, 2003; 7: 327~35
6 World Health Organization/IUATLD Global project on anti-tuberculous drug Resistance Surveillance. Anti-tuberculous Drug Resistance in the World. Third global report.WHO/HTM/TB/2004.343. Geneva: World Health Organization, 2004
7 Andres E. Drug-induced hepatotoxicity.N Engl J Med, 2003, 13; 349(20): 1974~6
8曾民德,萧树东.肝脏与内分泌.北京:人民卫生出版社, 1995: 132~145
9 Lee J, Boyer JL. Molecular alterations in hepatocyte transport. Semin Liver Dis, 2000; 20: 373~384
10 Hilsden RJ, Shaffer E. Liver structure and function. In: Thomson A, Shaffer E, editors. First principles of gastroenterology: the basis of disease and an approach to management, 4th ed. Edmonton, AB,Canada: Astra; 2000: 462~564
11 Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: its clinical and toxicological role. J Clin Pharm Ther, 2000; 25: 165~175
12 Lieber CS. Susceptibility to alcohol-related liver injury. Alcohol Alcohol Suppl, 1994; 2: 315~326
13 Kanathur N, Mathai MG, Byrd RP, Fields CL, Roy TM. Simvastatindiltiazem drug interaction resulting in rhabdomyolysis and hepatitis. Tenn Med, 2001; 94: 339~341
14 Lee WM. Drug-induced hepatotoxicity. N Engl J Med, 2003; 349: 474~85
15 Fountain FF, Tolley E, Chrisman CR, Self TH. Isoniazid hepatotoxicity associated with treatment of latent tuberculosisinfection: a 7-year evaluation from a public health tuberculosisclinic. Chest, 2005; 128: 116~23
16 Stuart RL, Grayson ML. A review of isoniazid-related hepatotoxicity during chemoprophylaxis. Aust N Z J Med, 1999; 29(3): 362~7
17 Walubo A, Smith P, Folb PI. The role of oxygen free radicals in isoniazid-induced hepatotoxicity. Methods Find Exp Clin Pharmacol, 1998; 20(8): 649~55
18李键,王修齐,杨桂仙,等.药源性消化系统疾病.北京:科学出版社, 2001: 265~67
19 SteeleMA,Burk RF,DesPrez RM. Toxic hepatitiswith isoniazid and rifamp in: a meta2analysis. Chest, 1991, 99: 465~71
20 Andrade RJ, Lucena MI, Melgarejo F.Hepatotoxicity caused by isoniazid or by paracetamol Gastroenterol Hepatol, 1998; 21(6): 314~5
21赵凤芹.抗结核药物诱导肝损伤的评价.国外医学呼吸系统分册, 2003, 23(6): 322~4
22 Girling DJ. The hepatic toxicity of antituberculosis regimens containing isoniazid, rifampicin and pyrazinamide. Tubercle, 1978; 59: 13~32
23 Villarino ME, Ridzon R, Weismuller PC et al. Rifampin preventive therapy for tuberculosis infection: experience with 157 adolescents. Am J Respir Crit Care Med, 1997; 155: 1735~8
24 Slivka IuI. Comparative characterization of the hepatotoxicity of isoniazid, rifampicin and pyrazinamide. Farmakol Toksikol, 1989; 52(4):82~5
25 Yew WW, Leung CC. Antituberculosis drugs and hepatotoxicity. Respirology, 2006, 11: 699~707
26 Jussi J, Saukkonen,David L, et al. An official ATS statement:hepatotoxicity of antituberculosis therapy. Am J Respir Crit Care Med, 2006, 174: 935~52
27 Yee D, Valiquette C, Pelletier M, Parisien I, Rocher I, Menzies D.Incidence of serious side effects from first-line antituberculosis drugs among patients treated for active tuberculosis. Am J Respir Crit Care Med, 2003; 167: 1472~7
28 Schaberg T, Rebhan K, Lode H. Risk factors for side-effects of isoniazid, rifampin and pyrazinamide in patients hospitalized for pulmonary tuberculosis. Eur Respir J, 1996; 9: 2026~30
29 Danan G, Pessayre D, Larrey D. Pyrazinamide fulminant hepatitis: an old hepatotoxin strikes again.Lancet. 1981, 7; 2(4): 1056~7
30 Corbella X, Vadillo M, Cabellos C. Hypersensitivity hepatitis due to pyrazinamide.Scand J Infect Dis, 1995; 27(1):93~4
31 Sarich TC, Youssefi M, Zhou T, Adams SP, Wall RA, Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. Arch. Toxicol. 1996; 70: 835~40
32 Steele MA ,Burk RF ,Desprez RM. Toxic Hepatitis with Isoniazid and Rifampin. A meta analysis. Chest, 1991, 99: 465
33 Sarma GR ,Immanuel C , Kailasam S , et al . Rifampin induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifampin. Am Rev Respir Dis, 1986, 133(6): 1072
34 Zhang RL ,Wang ZY,Li D , et al . Effect of rifampicin on pharmacoki netics of isoniazid and its metabolite acetylhydrazine in rats. Acta Pharmacologica Sinica, 1992, 13(6): 494
35 Jenner AM, Timbrell JA. In vitro microsomal metabolism of hydrazine. Xenobiotica, 1995; 25: 599~609
36 Jenner AM, Timbrell JA. Influence of inducers and inhibitors of cytochrome P450 on the hepatotoxicity of hydrazine in vivo. Arch Toxicol, 1994; 68: 349~57
37 Jenner AM, Timbrell JA. Effect of acute and repeated exposure to lowdoses of hydrazine on hepatic microsomal enzymes and biochemical parameters in vivo. Arch Toxicol 1994; 68: 240~5
38 Sodhi CP, Rana SV, Mehta SK, Vaiphei K, Attari S, Mehta S. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats. Drug Chem Toxicol, 1997; 20: 255~69
39 Sodhi CP, Rana SV, Mehta SK et al. Study of oxidative stress in isoniazid-induced hepatic injury in young rats with and without protein-energy malnutrition. J Biochem Toxicol, 1996; 11: 139~46
40 Chowdhury A, Santra A, Kundu S et al. Induction of oxidative stress in antitubercular drug-induced hepatotoxicity. Indian J Gastroenterol, 2001; 20: 97~100
41孙友乐译.临床药理学,第1版.科学技术文献出版社重庆分社,重庆: 1982: 240~247
42 Wen X, Wang JS, Neuvonen PJ, Backman JT. Isoniazid is a mechanism-based inhibitor of cytochrome P450 1A2, 2A6, 2C19 and 3A4 isoforms in human liver microsomes. Eur J Clin Pharmacol, 2002; 57: 799~804
43 Rae JM, Johnson MD, Lippman ME, et al. Rifamp in is a selective, pleiotropic inducer of drugmetabolism genes in human hepatocytes: studies with cDNA and oligonucleotide exp ression arrays. J Pharmacol Exp Ther, 2001, 299: 849~57
44 Kolars JC, Schmiedlin-Ren P, Schuetz JD, Fang C, Watkins PB. Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes. J Clin Invest, 1992; 90: 1871~8
45 Combalbert J, Fabre I, Fabre G et al. Metabolism of cyclosporin A. IV. Purification and identification of the rifampicin-inducible human liver cytochrome P-450 (cyclosporin A oxidase) as a product of P450IIIA gene subfamily. Drug Metab. Dispos, 1989; 17: 197–207
46陈新谦主编.新编药物学.第13版.人民卫生出版社,北京, 1992:324
47 Durand F, Bernuau J, Pessayre D. Hepatotoxicity of antituberculosis drugs: practical implications for monitoring.Gastroenterol Clin Biol, 1998; 22(2):117~20
48 Steele MA, Burk RF, DesPrez RM. Toxic hepatitis with isoniazid and rifampin: a meta-analysis. Chest, 1991; 99: 465–471
49 Skakun NP, Shman'ko VV. Synergistic effect of rifampicin on hepatotoxicity Marof isoniazid. Antibiot Med Biotekhnol, 1985; 30(3): 185~9
50 Toulet J, Viteau JM. Isoniazid-rifampicin: a model for hepatotoxicity and for bibliographic Anglomania.Gastroenterol Clin Biol, 1978; 2(8-9): 756~7
51 Askgaard DS, Wilcke T, Dossing M. Hepatotoxicity caused by the combined action of isoniazid and rifampicin.Thorax. 1995, 50(2): 213~4
52 Ozawa S. Drug-drug interaction in pharmacogenetics and pharmacogenomics . Rinsho Byori, 2002; 50(2): 146~50
53 Levy RH, Hachad H, Yao C, et al. Relationship between extent of inhibition and inhibitor dose: literature evaluation based on the metabolism and transport drug interaction database.Curr Drug Metab. 2003; 4(5): 371~80
54 Venkatakrishnan K, von Moltke LL, Obach RS, ea tl. Drug metabolism and drug interactions: application and clinical value of in vitro models.Curr Drug Metab, 2003; 4(5): 423~59
55 Erhardt PW. A human drug metabolism database : potential roles in the quantitative predictions of drug metabolism and metabolism-related drug-drug interactions.Curr Drug Metab, 2003; 4(5): 411~22
56 Yamazaki H. Roles of human cytochrome P450 enzymes involved in drug metabolism and toxicological studies. Yakugaku Zasshi. 2000; 120(12): 1347~57
57 Glue P, Clement RP.Cytochrome P450 enzymes and drug metabolism- basic concepts and methods of assessment. Cell Mol Neurobiol, 1999;19(3):309~23
58 Sarma GR, Immanuel C, Kailasam S, et al. Rifamp in induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment oftuberculosis with regimens containing isoniazid and rifampin. Am Rev Resp ir D is, 1986,133: 1072~7
59 Sarma GR, Immanuel C, Kailasam S, et al. Rifamp induced release of hydrazine from isoniazid. A possible cause of hepatitis during treatment of tuberculosis with regimens containing isoniazid and rifamp in. Am Rev Respir Dis, 1986,133: 1072
60 Mitchell JR, Tho rgeirsson U P, Black M , et al. Increased incidence of isoniazid hepatitis in rapid acetylato rs: possible relation to hydrazine metabo lites. Clin Pharmacol Ther, 1975,18: 70
61 Ellard GA , M itch ison DA , Girling DJ, et al. The hepatic toxicity of isoniazid among rapid and slow acetyators of the drug. Am Rev Respir Dis, 1978, 118: 628
62 Timbrell JA , Park BK, Harland SJ. A study of the effects of rifamp icin on isoniazidmetabo lism in human volunteer subjects. Hum Toxicol, 1985, 4 (3): 279
63 Singh J, Garg PK, Thakur V S , et al. A nti tubercular treatment induced hepato toxicity: do se acetylator status matter? Indian J Physiol Pharmacol, 1995, 39 (1) : 43
64 Ohno M , Yamaguch i I, Yamamo to I, et al. Slow N acetyltransferase 2 genotype affects the incidence of isoniazid and rifampic induced hepatotoxicity. Int J Tuberc Lung Dis,2000, 4 (3) : 256
65 FernandezVA, Sopefia B, FernandezVJ, et al. The influence of risk factors on the severity of antituberculosis drug induced hepatotoxicity. Tuberc lung dis, 2004, 8 (12): 1499~505
66 Sharma SK, Balamurugan A, Saha PK, et al. Evaluation of clinical and immunogenetic risk factors for the development of hepatotoxicity during anti tuberculosis treatment. Am. J. Resp ir. Crit. Care Med, 2002, 166: 916~9
67 Tost JR,Vidal T, Cayl J, et al. Severe hepatotoxicity due to antituber culosis drugs in Spain. Tuberc Lung Dis, 2005, 9 (5) : 534~40
68 Wu JC, Lee SD, Yeh PF, et al. Isoniazid and rifamp induced hepatitis inhepatitisB carriers. Gastroenterology, 1990, 98 (2) : 502~4
69 Wong WM,Wu PC, Yuen MF, et al. Antituberculosis drug– related liver dysfunction in chronic hepatitis B infection. Hepatology, 2000, 31 (1) : 201~6
70郭虹,李环,李淑芬,等.抗结核药物治疗对肝功能的影响.北华大学学报(自然科学版), 2005, 6: 425~7
71 Shakya R, Rao BS, Shrestha B. Incidence of hepatotoxicity due to antitubercular medicines and assessment of risk factors. Ann Pharmacother, 2004, 38: 1074~9
72 DossingM,Wilcke JT,Askgaard DS, et al. Liver injury during antituberculosis treatment: an 11 years study. Tuber Lung Dis, 1996,77: 335~40
73 Schaberg T, Rebhan K,Lode H. Risk factors for sideeffects of isoniazid, rifampin and pyrazinamide in patients hospitalized for pulmonary tuberculosis. Eur Resp ir J, 1996, 9: 2026~30
74路西明,王建刚,侯湘波等.白头翁对利福平异烟肼肝毒性研究.中国中医药信息杂志, 1998, 5 (8) : 21~7
75侯艳宁,朱秀媛,程桂芳.黄芩苷对小鼠肝细胞色素P450及其亚家族的诱导.解放军药学学报, 2000, 16 (4) : 58~63
76薛洪源,侯艳宁,刘会臣水飞蓟宾胶囊对异烟肼和利福平肝损害小鼠的保护作用.中成药, 2003, 25(4): 307~310
77 Kinzig-Schippers M, Tomalik-Scharte D, Jetter A et al. Should we use N-acetyltransferase type 2 genotyping to personalize isoniazid doses? Antimicrob. Agents Chemother. 2005; 49:1733~8
78 Tostmann A, boeree MJ, Aarnoutse RE, et al. Antituberculosis drug-induced hepatotoxicity: concise up-to-date review. Journal of Gastroenterology and Hepatology, 2008, 23:192~202
1 Tayal V, Kalra BS, Agarwal S, et al. Hepatoprotective effect of tocopherol against isoniazid and rifampicin induced hepatotoxicity in albino rabbits. Indian J Exp Biol. 2007, 45(12): 1031~1036
2 Lieber CS. Pathogenesis and treatment of alcoholic liver disease:progress over the last 50 years. Rocz Akad Med Bialymst, 2005, 50:7~ 20
3 Lieber CS. Alcoholic fatty liver : its pathogenesis and mechanismof progression toinflammation and fibrosis . Alcoholic, 2004, 34(1):9~ 19
4 Bai J, Cederbaum AI. Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletionofglutathione. J Biol Chem, 2006, 281(8): 5128~ 5136
5胡国平,刘凯王,唐红等.多烯磷脂酰胆碱(易善复)治疗慢性肝炎的系统评价.中国循证医学杂志,2005, 5(7):543~548
6 TORRIELLI MV,GABRIEL L ,DIANZZMI M U. Ethanol-inducedHepatotoxicity : Experimental Observations on the Role of Lipid Peroxidation. Journal of Pathology,1978 ,26:11
7闻勤生,郝云龙,赵海峰,等.氧自由基在肝缺血再灌注损伤中的作用及凯西莱的影响.中国现代医学杂志, 2002, 12(11): 5~7
8全冰.硫普罗宁对抗痨药物肝损作用的预防效果观察.第四军医大学学报, 2006, 27(15): 1417~1420
9张海,韩志启,朱增红,等.凯西莱治疗抗结核药肝损害的疗效.医药论坛杂志, 2004, 25(1):23~24
10毕国春,王传力.还原型谷胱甘肽治疗抗结核药物性损害62例疗效分析.中国医师杂志, 2004: 233~234
11 Atkuri KR,Mantovani JJ,Herzenberg LA,et al. N-Acetylcysteine-a safe antidote for cysteine/glutathione deficiency. Current Opinion Pharmacology. 2007, 7(4): 355~359
12 Kelly GS. Clinical applications of N-acetylcysteine. Altern Med Rev, 1998,3(2): 114~127
13 ZHAO J X,YANG Y M,HUANG Y T. Role of reactive species and N-acetylcysteine in hyperacutere jection of the rats liver in the isolated perfusion model. Chin J Exp Surg, 2004, 21(2): 141~143
14 PATRIARCAA S, FURFAROA A L, DOMENICOTTI C, et al. Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics butprotected from oxidative stress. Biochim BiophysActa, 2005, 1741(1-2): 48~54
15 Pastor A,Collado PS,AlmarM, et al. Antioxidant enzyme status in biliary obstructed rats: effects of N-acetylcysteine. J Hepatol, 1997,27(2): 363~370
16 LORENZO M,KRANTIS A. Nitric oxide synthase isoenzyme activities in a premature pigletmodel of necrotizing enterocolitis:effects of nitrergic manipulation[ J].Ped Surg Inter, 2002, 18:624~629
17 Semin Liver Dis,Fontana Robert J RJ,et al. Acute liver failure due to drugs.Semin Liver Dis 2008, 28(2):175~87
18 Warnet JM,Christen MO,Thevenin M, et al. Protective effect of anethol dithiolthione against acetaminophen hepatotoxicity in mice. Pharmacology & Toxicology, 1989, 65(1):63~64
19王龙虎,吴国良,程翼宇.壳聚糖及其衍生物在中药制药工业中的应用.中国中药杂志, 2004,29(4): 289~291
20 CHEN XG, WANG Z, LIU WS, et al. The effect of carboxymethyl- chltosan on proliferation and collagen secretion of normal and keloid skin fibroblasts. Bionmterials, 2002, 23 (23): 4609~4614
21向轶,张志荣.巯基壳聚糖载胰岛素亚微球的制备及影响包封率的因素.华西药学杂志, 2005, 2(3): 195~199
22蒋新宇,周春山,张俊山.应用三聚磷酸钠为交联剂制备载药物纳米粒的研究.中国现代医学杂志, 2003, 13(22): 69~72
23 Ho YP, Chen HH, Leong KW, et a1. Evaluating the intracellular slability and unpacking of DNA nanocomplexes by quantum dots-FRET . J Control Release, 2006, 116(1): 83~89
24 24.S. Santhosh, T.K. Sini, R. Anandan, et al. Effect of chitosan supplementation on antitubercular drugs-induced hepatotoxicity in rats. Toxicology, 2006, 219, 53~59
25 S. Santhosh, T.K. Sini, R. Anandan, et al. Hepatoprotective activity of chitosan against isoniazid and rifampicin-induced toxicity in experimental rats. European Journal of Pharmacology, 2007, 572: 69~73
26 Yoshikawa M, Matsui Y, Kawamoto H, et al. Effects of glycyrrhizin on immune mediated cytotoxicity. Gastroenterol. Hepatol. 1997, 12: 243~248
27 Miyaji C, Miyakawa R, Watanabe H, et al. Mechanisms underlying the activation of cytotoxic function mediated by hepatic lymphocytes following the administration of glycyrrhizin. Int. Immunopharmacol. 2002, 2: 1079~1086
28 Shiki Y, Shinai K, Satto Y, et al. Effect of glycyrrhizin on lysis of hepatocyte membranes induced by anti-liver cell membrane antibody. J. Gastroenterol. Hepatol,1992, 7:12~16
29 Abe N, Ebina T, Ishida N. Interferon induction by glycyrrhizin andglycyrrhetinic acid in mice. Microbiol, Immunol. 1982, 26: 535~539
30 Dhiman RK, Chawla YK. Herbal medicines for liver diseases.Dig. Dis. Sci, 2005, 10:1807~1812
31 Miyazawa Naoki N,Takahashi Hiroshi H,Yoshiike Yasuhiro Y.Effect of glycyrrhizin on anti-tuberculosis drug-induced hepatitis.Kekkaku 2003 ,78(1):15~19
32孙鹏,李娥,邱杰文.复方甘草酸苷治疗抗结核药物性肝炎临床观察.现代医院, 2006, 6(8): 52~53
33 Muriel P. S-adenosyl-L-methionine prevents and reverses erythrocyte membrane alterations in cirrhosis. Appl. Toxicol. 1993, 13: 179~182
34 Muriel P, Mourelle M. Characterization of membrane fraction lipid composition and function of cirrhotic rat liver.Role of S-adenosyl-L-methionine. J. Hepatol. 1992, 14: 16~21
35 Benz C, Angermuller S, Kloters-PlachkyP, et al. Effect of adenosylmethionine versus tauroursodexycholic acid obile acidinduced app potosis and cytolysis in rat hepato-cytes. EurJclin Invest, 1998, 28(7): 577~583
36王少华,李宁,任建安,等. S-腺苷-L-蛋氨酸治疗全肠营养导致的胆汁淤积.中华普通外科杂志, 2000, 15(12): 746~747
37 Belli DC, Fournier LA, Lepage G, et al. S-adenosylmethionine prevents totol parenteral nutrition induced cholestasis in rat.J Hepatol, 1994, 21(1): 18~23
38 Trauner M, Graziadei IW. Review article: mechanisms of action and therapeutic applications of ursodeoxycholic acid in chronic liver diseases. Aliment phamacol Ther, 1999, 13(8): 979~996
39 Xie Q, Khaoustov VI, Chung C C, et al. Effect of tauro- ursodeoxycholic acidon endoplasmic reticulum stress-induced caspase-12 activation. Hepatology, 2002, 36(3): 592~601
40 Angulo P. Use of ursodeoxycholic acid in patients with liver disease. Curr Gastroenterol Rep, 2002, 4(1): 42~44
41 Schoemaker MH, conoedela RosaL, Bulsi-Homan M, et al.Taurourso-deoxycholio acid protecs rat hepatocyies from Bile acid-induced apoptosis via acthailon of survival pathways. Hepatology, 2004, 39(6): 1563~1573
42 Sohn J, Knaoustov VI, Xie O, et al. The effect of ursodeoxy-cholic acid on the survivin in thapsigargin-induced apoptosis. Cancer letter, 2003, 191(1): 83~92
43 Guicciardi ME, Gores GJ. Ursodeoxycholio acid cytoprotection: dancing with death receptors and survival pathways. Hepatology, 2002, 35(4): 971~973
44 Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998, 281(5381): 1309~1312
45 Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectemied rats. Biochem Biophys Res Commun, 1984, 122 (3): 1450~1459
46刘琨,吴碧彤,蒋蓬,等.促肝细胞生长因子对抗结核药物性肝损的治疗观察.广州医学2006, 37(1):25~28
47魏世俊,张杰,韩连风.护肝片五味子乙素的薄层光密度法测定.黑龙江医药, 1998, 11(6): 338~340
48 Luper S. A review of plants used in the treatment of liver disease. Altern. Med. Rev. 1998. 3: 410~421
49 Pascual C, González R, Armesto J, et al. Effect of silymarin and silybinin on oxygen radicals. Drug Develop Res, 1993, 29: 73~77
50 Mourelle M, Meza MA. Colchicine prevents D-galactosamineinduced hepatitis. Hepatol. 1989, 8(2): 165~172
51 Pradhan SC, Girish C. Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian J. Med. Res. 2006, 124(5): 491~504
52 Muriel P, Ali S, Friedman SL, et al. Role of nitric oxide in liver disorders, In LiverDiseases: Biochemical Mechanisms and New Therapeutic Insights. Science Publishers: Enfield (NH) Jersey. 2006, 115~131
53 Wang M, Grange LL, Tao J. Hepatoprotective properties of Silybummarianum herbal preparation on ethanol-induced liver damage. Fitoterapia, 1996, 67: 167~171
54 Kropacova K, Misurova E, Hakova H. Protective and therapeutic effect of silymarin on the development of latent liver damage. Radiats Biol Radioecol, 1998, 38(3): 411~415
55 Luper S. A review of plants used in the treatment of liver disease: Part 1. Altern. Med. Rev. 1998, 3: 410~421
56薛洪源,侯艳宁,刘会臣等.水飞蓟宾胶囊对异烟肼和利福平肝损害小鼠的保护作用.中成药2003, 25 (4):307~310
57 Ammon HP, Walh MA. Pharmacology of Curcuma longa. Planta Med, 1991, 57(1): 1~7
58 Maheswari RK, Singh AK, Gaddipati J, et al. Multiplebiological activities of curcumin: A short review. Life Sci, 2006, 78(5): 2081~2087
59 Aggarwal BB, Bhatt ID, Ichikawa H, et al. Curcumin: biological and medicinal properties. In Turmeric: The Genus Curcuma, Ravindran PN, Nirmal-Babu K, Sivaraman K (eds). Series: Medicinal and Aromatic Plants-Industrial Profiles. CRC Press: New York, 2007, 45(7): 297~368
60 Rao CV, Rivenson A, Simi B, et al. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 1995, 55(4): 259~266
61 Muriel P. Cytokines in liver diseases. In Hepatotoxicity: From Genomics to In Vitro and In Vivo Models. Pharmacology 2007, 48(9): 63~68
62 Abe Y, Hashimoto S, Horie T, et al. Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages. Pharmacol. Res. 1999, 39(5): 41~47
63 Reyes-Gordillo K, Segovia J, Shibayama M, et al. Curcumin protects against acute liver damage in the rat by inhibiting NF-κB, proinflammatory cytokines production and oxidative stress. Biochim. Biophys. Act . 2007, 1770: 989~996
64 Bruck R, Ashkenazi M, Weiss S, Goldiner I, et al. Prevention of liver cirrhosis in rats by curcumin. Liver Int. 2007, 27: 373~383
65 AaBurkitt MJ, Duncan J. Effects of trans-resveratrol on copper- dependent hydroxyl radical scavenging and a novel, glutathione-sparing mechanism of action. Arch Biochem Biophys, 2000, 381(2): 253~263
66 Das S, Das DK . Resveratrol: a therapeutic promise for cardiovasculardiseases. Recent Patent Cardiovasc Drug discov, 2007, 2(2):133~138
67 Araki T, Sasaki Y, Milbrandt J. Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science, 2004, 305(5686): 1010~1013.
68 Fulda S, Debatin KM. Sensitization for tumor necrosis factor related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol . Cancer Res, 2004, 64(1): 337~346
69 Zhang Q, Tang X, Lu QY, et a1. Resveratrol inhibits hypoxia-induced accumulation of hypoxia-inducible factor-I A and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells. Mol Cancer Ther, 2005, 4(10): 1465~1474
70 Vivancos M, Moreno JJ. Effect of resveratrol, tyrosol and beta sitosterol on oxidised low-density lipoprotein-stimulated oxidative stress, arachidonic acid release and prostaglandin E2 synthesis by RAW 264.7 macrophages. Br J Nutr, 2008, 99(6): 1199~1207
71 Yang S, Meyskens FL Jr. Alterations in activating protein I composition correlate with phenotypic differentiation changes induced by resveratrol in human melanoma. Mol Pharmacol, 2005, 67(1): 298~308
72 Chiou W F, Lin LC, Chen CF. Acteoside protects endothelial cells against free radical-induced oxidative stress. J Pharm Pharmacol, 2004, 56(6): 743~748
73 Miura T, Muraoka S, Fujimoto Y. Inactivation of creatine kinase induced by stilbene derivatives. Pharmacol Toxicol, 2002, 90(2): 66~72
74杜文明,徐克涵.银杏的营养和药用价值.河北林业科技, 2002, 3: 276~287
75张卫明,赵泊涛,王红.银杏种仁对小白鼠SOD的影响.中国野生植物资源, 1995, l: 23~24
76张卫明,赵泊涛.银杏对果蝇寿命的影响.中国野生植物资源, 1995, l: 25~26
77戴建国,陈景衡.白果提取液对小白鼠心脑脂褐质水平的影响.南京医科大学学报, 1994, 14: 411~413
78 Wang H, Ng TB, Ginkbilobin, et al. a nove1 antifungal protein from Ginkgo biloba seeds with sequence simi1arity to embryo-abundant protein . Biochem Biophys Res Commun, 2000, 279: 407~411
79邓乾春,黄文,谢笔钧等.白果活性蛋白的抗生物氧化作用.营养学报, 2006, 28(3): 259~262
80 Naik SR, Panda VS. Hepatoprotective effect of Ginkgoselect Phytosome in rifampicin induced liver injury in rats: evidence of antioxidant activit y . Fitoterapia, 2008, 79(6): 439~445
81王建刚,周海梅,侯湘波等.银杏叶醇提取物对异烟肼和利福平肝毒性保护作用的实验研究.中国现代应用药学杂志, 2000, 17 (3), 15~18
82 Jiangsu New Medical College(江苏新医学院). Zhong Yao Da Ci Dian(中药大辞典). Shanghai: Shanghai Science and Technology Press, 1998, 1174~1177
83杨永康,格桑索朗,吴家坤.诃子、毛诃子和余甘子的植物分类研究和药学特性综述.中国医学生物技术应用杂志, 2004, 3(1): 14~25
84潘燕,张述禹,侯金凤等.诃子对大鼠心肌酶的影响.中国中药杂志, 2004, 29(4): 382~383
85 Malekzadeh F, Ehsanifar H, Shahamat M, et a1. Antibacterial activity of black myrobalan(Terminalia chebula Retz.)against Helicobacter priori . Int J Antimicrob Agents, 2001, 18(1): 85~88
86 Shin T Y, Jeong H J, Kim D K, et a1. Inhibitory action of water soluble fraction of Terminalia chebula on systemic and local anaphylaxis . J Ethnopharmacol, 2001, 74(2): 133~140
87 Saleem A, Husheem M, H~kSnen P, et a1. Inhibition of cancer cell growth by crude extract and the phenohcs of Terminalia chebula Retz. Fruit . J Ethnopharmacol, 2002, 81(3): 327~336
88 Jagetia G C, Baliga M S, Mal K J, et a1. The evaluation of the radioprotective effect of Trlphala(an ayurvedlc rejuvenating drug)in the mice exposed toγ-radiation . Phytomedicine, 2002, 9(2): 99~108
89 Aim M J, Kim C Y, Lee J S, et a1. Inhibition of HIV-1 integrase by galloyl glucoses from Terminalia chebula andflavonol glycoside gallates from Euphorbiapekinensis. Planta Med, 2002, 68(5): 457~459
90 Naik G H, Priyadarsini K I, Satav J C, et al. Comparative antioxidam activity of individual herbal components used in Ayttrvedic medicine. 2003 ;63(1):97~104
91 Saleem A, Ahotupa M, Pihlaja K. Tota1 phenollcs concentnation and antioxidant potential of extracts ofmedicinal plants of Pakistan. Naturforsch, 2001, 56(11-12): 973~978
92傅乃武,全兰萍,黄磊等.诃子提取物对活性氧的清除和对抗TPA对人白细胞DNA的损伤.中草药, 1992, 23 (1): 26~29
93 SA Tasduq, K Singh, NK Satti, et al. Terminalia chebula (fruit) prevents liver toxicity caused by sub-chronic administration of rifampicin, isoniazid and pyrazinamide in combination. Hum Exp Toxicol, 2006, 25 (3): 111~118
94 Dhiman RK, Chawla YK. Herbal medicines for liver diseases. Dig. Dis. Sci. 2005, 10: 1807~1812
95 Huseini HF, Alavian SM, Heshmat R, et al. The efficacy of Liv-52 on liver cirrhotic patients: a randomized double-blind, placebo-controlled first approach. Phytomedicine. 2005, 12: 619~624
96 S. D. Saraswathy, C. S. Shyamala Devi. Modulating Effect of Liv.100, an Ayurvedic Formulation on Antituberculosis Drug-induced Alterations in Rat Liver Microsomes. Indian J. Phytother. Res, 2001, 15, 501~505
97 Sandhir R, Gill KD. Hepatoprotective effects of Liv 52 on ethanol induced liver damage in rats. Indian J. Exp. Biol. 1999, 37: 762~766
98 Roy A, Soni GR, Kolhapure RM, et al. Down regulation of tumor necrosis factor activity in experimental hepatitis by a herbal formulation, Liv 52. Indian J. Exp. Biol. 1994, 32: 694~697
99中华人民共和国卫生部药典委员会.中华人民共和国药典.一部,化学工业出版社, 2005: 124
100周涛,邱德文.民族药余甘子本草药学概况.贵阳中医学院学报, 2002, 24 (3): 3~6
101王小李,詹琳.余甘子甲醇萃取物对色拉油抗氧化效果的研究.武汉工业学院学报, 2000: 13~15
102Sabu MC, Kuttan R. Anti2diabetic activity of medicinal plants and itsrelationship with their antioxidant property. J Ethnopharmacol, 2002, 81: 155~160
103Bhattacharya A, Chatterjee A, Ghoshal S, et al. Antioxidant activity of tannoid principles of Emblica officinalis (amla) . Ind J Bio, 1999, 37: 676~680
104肖湘,俞丽君,邱玉莹等.油柑多糖的提取与清除氧自由基作用研究.中国药学杂志, 1998, 23 (5): 279~282
105S.A.Tasduq, P.Kaisar, D.K.Gupta, et al. Protective Effect of a 50% Hydroalcoholic Fruit Extract of Emblica officinalis against Anti-tubercul osis Drugs induced Liver Toxicity. Phytother. Res, 2005, 19, 193~197
106Dhuley JN, Naik SR. Effect of rhinax on bacterial lipopolysaccharide induced endotoxemia in rats. Indian J Exp Biol, 1998, 36(3): 315~317
107Dhuley J. Healing-promoting action of rhinax with dual action on chronic gastric and duodenal ulcers induced by acetic acid in rats. Hindustan Antibiot Bull, 2003, Feb, 45~46; 2004, Nov: 34~40
108Dhuley JN, Naik SR. Protection by Rhinax in various models of ulceration in rats. J Ethnopharmacol, 1998, 63(3): 219~225
109Dhuley JN, Naik SR. Protective effect of Rhinax, a herbal formulation, against CCl4-induced liver injury and survival in rats. J Ethnopharmacol, 1997, 56(2): 159~64
110Dhuley JN. Hepatoprotective effect of rhinax on antitubercular drug-induced hepato-toxicity in rats. Hindustan Antibiot Bull, 2002 Feb-Nov; 44(1-4): 53~59