丹参对阻塞性黄疸大鼠肝脏、肾脏细胞凋亡及相关机制的研究
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摘要
实验目的:
阻塞性黄疸(Obstructive jaundice,OJ)是临床上常见并发症,胆道系统的良、恶性梗阻、手术损伤等均可导致OJ并随即出现严重的多脏器损伤。目前认为阻塞性黄疸(OJ)可诱导肝脏、脾脏、淋巴细胞、胃肠黏膜以及心脏血流动力学障碍等功能损害。但是,OJ时引起多脏器损害的内在机制还不清楚。普遍认为,内毒素、炎症介质、以及胆汁酸升高诱发OJ时肝或其他器官损伤。此外,高胆管内压力和门静脉压力,也是造成这一伤害的重要因素。近年来随着细胞凋亡研究的深入,已发现许多肝胆系统疾病与细胞凋亡规律失常有关,细胞凋亡在维持肝胆系统正常生理状态和内环境稳定中起着十分重要的作用。研究认为细胞凋亡参与OJ多脏器功能损伤的发病机制有两种:一是受基因调控,另一种是受非基因调控;基因调控又分为直接调控和间接调控,前者包括p53、Bax、Fas-Fasl、Bcl-2等,后者如TGF、TNF、NF-κB等。其中,Bax是一种可溶性蛋白质,正常情况下以单体形式定位于细胞浆,当收到凋亡刺激时构象发生变化,从细胞浆转位至线粒体并与线粒体外膜结合,直接降低线粒体外膜稳定性或与Bcl-2结合成异源二聚体的结构,发挥促进细胞凋亡的作用。NF-κB是一种广泛存在于多种细胞细胞质中的具有细胞核基因转录调节作用的蛋白质因子,是转录调节蛋白Rel家族成员之一,参与多种炎性因子基因表达的调控及细胞凋亡等多种病理生理过程。当机体细胞处于氧自由基水平升高、缺血-再灌注损伤、内毒素血症等应激状态下,I-κB可使NF-κB活化易位进入细胞核并结合于靶基因特异的启动子区域的κB基因序列结合,从而启动促炎症分子基因的转录核蛋白质的合成,引起TNF-α、IL-6mRNA等的强烈表达,产生大量的炎性细胞因子,同时间接促使凋亡细胞增加,加速对脏器细胞的毒性作用并最终出现多器官功能损伤。有实验证实Bax蛋白、NF-κB蛋白明显高表达时,梗阻性黄疸大鼠肝肾组织细胞凋亡明显增多。现今研究发现,NF-κB亚单位的种类及数量在细胞凋亡中起着决定性的作用,当c-RelA亚型表达增加时,则促进凋亡的发生。药物治疗OJ多脏器功能损害能否通过调控脏器细胞凋亡从而获得疗效正引起研究者的兴趣。丹参是一种传统中药,通常用于活血治疗。丹参是野生丹参根的提取物,主要活性成分包括丹参素,丹酚酸以及丹参酮等,能够起到保护内皮细胞,对抗炎症,防止脂质过氧化和钙超载的作用。研究表明,丹参治疗OJ大鼠时通过降低内毒素水平、减少炎症介质等已经得到普遍认同的机制,发挥保护肝细胞,维护肝功能,以及减少对肾皮质和肾功能损害的作用。但是丹参是否通过抗细胞凋亡的分子机制起到治疗作用,尚无进一步研究。本研究的目的就是为了弥补这个不足。通过观察丹参对阻塞性黄疸大鼠(OJ)肝脏细胞、肾脏细胞凋亡和Bax、NF-κB蛋白表达的影响,探讨丹参对肝脏、肾脏的保护机制。
方法:
1.取雄性清洁级健康SD大鼠180只进行实验(体重270～330g)。购自上海斯莱克实验动物有限责任公司[实验动物许可证号:SCXK(沪)2007-0005]。于浙江中医药大学实验动物中心屏障系统实验室饲养[实验动物许可证号:SYXK(浙)2007-0003]。大鼠实验前适应性饲养1周,术前禁食12h,自由饮水。
2.将大鼠随机均分为假手术组(n=60)、模型组(n=60)、丹参治疗组(n=60)。假手术组仅游离大鼠胆总管而不结扎,然后分层关腹。模型组和丹参治疗组游离大鼠胆总管后用丝线双重结扎胆总管近端,并切断胆总管,分层关腹。丹参治疗组组按每只0.2ml/100g/d腹腔内注射丹参注射液,而假手术组、模型组每日注射等量的生理盐水。按术后不同时间段各组分为OJ 7d、14d、21d、28d组(n=15)。
3.记录术后各相应时点大鼠的一般情况,包括大鼠活动度、精神状况、大小便颜色及是否改变、眼白及皮毛染色有无改变,死亡率等。
4.检测各组、各时间点血清TBIL、DBIL、γ-GT,BUN、CREA含量,通过对大体标本肉眼观察、光镜法及电镜法观察各组、各时间点肝脏、肾脏组织病理改变特征,判断大鼠阻塞性黄疸模型建立是否成功,以及运用丹参治疗后与模型组一般情况、血清指标、病理变化来判断治疗效果。
5.运用组织芯片技术制作肝、肾组织微阵列切片。采用组织微阵列片制作机(Beecher Instruments,USA)制备肝、肾的组织微阵列片,将100个直径2毫米的小组织样本,以阵列方式包埋于同一块20mm×45 mm的石蜡块中,再进行切片,用于同一实验指标的研究。
6.在制作完成的肝肾组织微阵列切片上通过DNA缺口原位末端标记法(TUNEL法)检测细胞凋亡指数。在微阵列切片上进行TUNEL染色,高倍镜下(400倍)观察凋亡细胞的分布。每张切片随机选择5-10个高倍视野,计数1000个细胞中的凋亡细胞个数,用百分数表示,计算凋亡指数(AI),AI=凋亡细胞数/总细胞数×100%。本部分工作在陕西超英生物科技有限公司完成。
7.在制作完成的肝肾组织微阵列切片上通过免疫组化检测组织切片中Bax、NF-κB c-RelA蛋白表达水平。在微阵列切片上采用二步EnVision法,计分标准如下:1)按着色细胞染色强度计分为:细胞呈阴性染色为-;染色弱,阳性细胞呈淡黄色为+;染色中等,阳性细胞呈棕黄色为++;染色强,阳性细胞呈棕褐色为+++。2)阳性细胞数(面积):无阳性细胞数为-;阳性细胞数＜25%为+;阳性细胞数25～50%为++;阳性细胞数＞50%为+++。
结果:
1.假手术组21d死亡1只,其余时间点无死亡;模型组7d死亡2只,14d死亡4只,21d死亡4只,28d死亡7只;丹参治疗组7d无死亡,14d死亡3只,21d死亡2只,28d死亡4只。7d三组间死亡率无统计学差异,14d、21d假手术组与模型组间死亡率均存在统计学差异(P=0.032),28d假手术组与模型组间死亡率存在统计学差异(P=0.006),假手术组与治疗组间死亡率存在统计学差异(P=0.032)。
2.各时间点假手术组TBIL、DBIL、γ-GT和CREA含量明显小于模型组与治疗组(P＜0.01);OJ 21、28d治疗组大鼠血清TBIL、CREA含量均明显低于模型组(P＜0.05 or P＜0.01);OJ 28d治疗组大鼠血清DBIL含量均明显低于模型组(P＜0.01),OJ 21d治疗组大鼠血清γ-GT含量均明显低于模型组(P＜0.01)。
3.大体、光镜及电镜观察下,OJ丹参治疗组大鼠的肝脏、肾脏病理改变较模型组均不同程度地减轻。
4.肝脏凋亡指数在21d丹参治疗组明显低于模型组(P＜0.01),其余各时间点各组间无显著性差异。肾脏凋亡指数14d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组无明显差异(P＞0.05),14d丹参治疗组明显小于模型组(P＜0.01)。
5.肝脏Bax蛋白阳性强度以及阳性强度和面积的乘积在各时间点模型组明显大于假手术组(P＜0.01),7,21,28天时间点丹参治疗组明显大于假手术组(P＜0.01),各时间点丹参治疗组与模型组无显著性差异(P＞0.05)。肝脏NF-κB c-RelA蛋白阳性强度以及阳性强度和面积的乘积在7d、14d、21d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组、模型组无显著性差异(P＞0.05)。肾脏Bax蛋白阳性强度21d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组、模型组无明显差异(P＞0.05)。肾脏Bax蛋白阳性强度和面积乘积21d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组无明显差异(P＞0.05),21d丹参治疗组明显小于模型组(P＜0.01)。肾脏NF-κB c-RelA蛋白阳性强度以及阳性强度和面积的乘积在各时间点假手术组与模型组、丹参治疗组以及模型组与丹参治疗组之间均无明显差异(P＞0.05)。
结论:
1.结扎大鼠胆总管后,模型组及丹参治疗组大鼠血清TBIL、DBIL及γ-GT浓度均较未结扎胆总管的假手术组明显升高,且血清胆红素以DBIL为主;同时模型组及丹参治疗组大鼠血清BUN、CREA浓度也均较假手术组明显升高,且肝肾组织有显著的病理损伤表现,说明阻塞性黄疸在肝功能受损同时也可导致肾功能损伤。
2.阻塞性黄疸大鼠可出现肝、肾组织Bax蛋白、肝脏NF-κB c-RelA蛋白表达明显升高,肝、肾组织凋亡细胞指数明显增加。
3.丹参能明显地降低OJ大鼠血清TBIL、DBIL、γ-GT及CREA含量,减少肝肾细胞凋亡数目,减轻肝脏、肾脏病理损伤,降低大鼠死亡率。
4.丹参对OJ大鼠肝、肾组织NF-κB c-RelA蛋白表达无明显的调节作用。丹参可能通过抑制OJ大鼠肾脏Bax蛋白的表达,减轻肾脏细胞的凋亡,对肾功能起到积极的保护作用。
Purpose:
Obstructive jaundice is a common clinical complication,which could be caused by biliary system in benign and malignant obstruction,surgery injury and so on usually. And OJ could result in serious injuries such as multiple organ injuries quickly.It is currently believed that obstructive jaundice(OJ) can induce functional damage to the liver,spleen,lymphocytes,and gastrointestinal mucosa as well as cardiac hemodynamic dysfunction.However,the mechanisms underlying OJ-induced multiple organ damage are still unclear,and there are many controversial hypotheses to explain this phenomenon.It is generally believed that endotoxin,inflammatory mediators,and increased bile acids contribute to OJ induced liver or other organs injuries.Moreover, elevated intrabiliary duct pressure and portal vein pressure are also important factors contributing to this injury.In recent years,along with in-depth study of apoptosis has been found that many of hepatobiliary system diseases and disorders related to apoptosis of cells.Apoptosis plays a very important role in the maintenance of a stable internal environment,normal physiological state and the hepatobiliary system.Studies suggest that apoptosis involved in OJ multiple organ dysfunctions by two ways:one is subject to gene regulation;the other is by non-genetic regulation.Gene regulation is divided into the direct control and indirect control.The former includes the p53,Bax, Fas-Fasl,Bcl-2 and so on,the latter,such as TGF,TNF,NF-κB and so on.Bax is a soluble protein and located in the cytoplasm as the form of monomer under normal circumstances.It emerges conformational changes under stimulus of apoptosis and translocates from the cytoplasm to the mitochondria.Then,it is combined with the outer mitochondrial membrane and directly reduces the stability of mitochondrial outer membrane or with Bcl-2 together to form the structure of heterodimers to play a role in promoting apoptosis.NF-κB is a kind of nuclear protein factors with effects of gene transcription regulation,widely present in the cytoplasm in a variety of cells.As one of Rel family of transcription protein,it takes part in the mediation of expression of inflammatory factors gene and apoptosis.When the body cells were involved in elevated levels of oxygen free radicals,ischemia-reperfusion injury,endotoxemia and other stress state,I-κB makes NF-κB activating and translocating into the nucleus in the target genes with specific promoter regionκB binding sequence.NF-κB can initiates synthesis of the transcription nuclear protein of pro-inflammatory molecules gene,and produce the expression of TNF-α,IL-6mRNA strongly,produce a large number of inflammatory cytokines,and indirectly promote an increase of apoptotic cells to accelerate the toxic effects on cells,organs,and eventually become a multi-organ dysfunction.There experiments confirmed that apoptosis was increased in liver and kidney tissues markedly,when the Bax protein,NF-κB protein expression was significantly higher in OJ rats.Salvia miltiorrhizae(S.miltiorrhizae) is a kind of traditional Chinese drug commonly used for activating blood circulation,the research on the effect of it in the treatment of OJ has been reported.S.miltiorrhizae injection is the extraction of wild Salvia roots,the main active ingredients include danshensu, salvianolic acid as well as tanshinone,dihydrotanshinone,and cryptotanshinone,which are able to protect endothelial cells,fight against inflammation,and prevent lipid peroxidation and calcium overload.Some studies have shown that S.miltiorrhizae can exert some therapeutic effects against this damage through protecting hepatic cells, maintaining liver function,as well as reducing the damage to renal cortex and renal function in OJ rats.There is no further study on whether it is played the therapeutic effect through anti-apoptosis.For this reason,the purpose of this study is to make up for this deficiency.Through observing the impact of S.miltiorrhizae injection on the pathological alterations,cell apoptosis and Bax/NF-κB protein expression in liver and kidney of OJ rats,we explored the protection mechanisms of S.miltiorrhizae in these organs.
Methods:
A total 180 healthy male SD rats of clean grade,were utilized for OJ associated experiments and randomly divided into sham-operated,model-control,and treated group,which were further randomly subdivided into 7d,14d,21 d and 28 d groups(n=15) according to time duration after operation.Rats in sham-operated group were separated common bile ducts only and without ligation,then closed abdomen layer by layer.Model group and treatment group were cut off the proximal common bile ducts after separation and ligation with double silk thread,and closed abdomen layer by layer.Intraperitoneal injection was adopted,S.miltiorrhizae injection(0.2ml/100g/d) in treatment group,while the same amount of saline in sham operation group and model group every day.Then,we recorded the mortality rate,serum TBIL,DBIL,γ-GT,BUN, CREA content,observed the characteristics of pathological changes of liver and kidney tissue,detected of Bax,NF-κB protein expression level and apoptosis index changes on the corresponding time point.
Results:
The numbers of dead OJ rats in the treated groups declined as compared with those in the model control group,but not significantly(P＞0.05).The contents of TBIL、DBIL、γ-GT and CREA(7,14,21,and 28 d in OJ rats,respectively) in the sham-operated group were significantly lower than those in the model control and treated groups(P＜0.01,respectively).The contents of TBIL and CREA(21 and 28 d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01 or P＜0.05,respectively).The contents of DBIL(28 d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01,respectively).The contents ofγ-GT,apoptosis index of liver and Bax protein Positive intensity(21 d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01, respectively).The apoptosis index of kidney(14d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01, respectively).Others,those pathological damages were reducing with vary degrees in the treated groups.
Conclusion:
1.With cutting off the proximal common bile ducts in rats after separation and ligation,serum TBIL,DBIL,andγ-GT levels in the model-control and the treated group were significantly higher than those in sham-operated group in this experiment.Serum BUN,CREA levels in the model-control and the treated group were significantly higher than those in sham-operated group,and livers and kidneys had experienced significant pathological damage in these two groups,as expected.
2.Obstructive jaundice can cause Bax protein and apoptosis index increased significantly in livers and kidneys of OJ rats.And NF-κB protein expression increases significantly in livers.
3.S.miltiorrhizae can significantly reduce the serum TBIL,DBIL,γ-GT,and CREA levels,and the number of liver/kidney cell apoptosis,reduce pathological damage of the livers and kidneys,and get lower mortality rate in OJ rats.
4.S.miltiorrhizae had no obvious regulatory effects on the expression of NF-κB c-RelA in livers and kidneys of OJ rats.It may be through inhibiting the protein expression to reduce renal cell apoptosis,and play a positive protective effect on renal function.
阻塞性黄疸(Obstructive jaundice,OJ)是临床上常见并发症,胆道系统的良、恶性梗阻、手术损伤等均可导致OJ并随即出现严重的多脏器损伤。目前认为阻塞性黄疸(OJ)可诱导肝脏、脾脏、淋巴细胞、胃肠黏膜以及心脏血流动力学障碍等功能损害。但是,OJ时引起多脏器损害的内在机制还不清楚。普遍认为,内毒素、炎症介质、以及胆汁酸升高诱发OJ时肝或其他器官损伤。此外,高胆管内压力和门静脉压力,也是造成这一伤害的重要因素。近年来随着细胞凋亡研究的深入,已发现许多肝胆系统疾病与细胞凋亡规律失常有关,细胞凋亡在维持肝胆系统正常生理状态和内环境稳定中起着十分重要的作用。研究认为细胞凋亡参与OJ多脏器功能损伤的发病机制有两种:一是受基因调控,另一种是受非基因调控;基因调控又分为直接调控和间接调控,前者包括p53、Bax、Fas-Fasl、Bcl-2等,后者如TGF、TNF、NF-κB等。其中,Bax是一种可溶性蛋白质,正常情况下以单体形式定位于细胞浆,当收到凋亡刺激时构象发生变化,从细胞浆转位至线粒体并与线粒体外膜结合,直接降低线粒体外膜稳定性或与Bcl-2结合成异源二聚体的结构,发挥促进细胞凋亡的作用。NF-κB是一种广泛存在于多种细胞细胞质中的具有细胞核基因转录调节作用的蛋白质因子,是转录调节蛋白Rel家族成员之一,参与多种炎性因子基因表达的调控及细胞凋亡等多种病理生理过程。当机体细胞处于氧自由基水平升高、缺血-再灌注损伤、内毒素血症等应激状态下,I-κB可使NF-κB活化易位进入细胞核并结合于靶基因特异的启动子区域的κB基因序列结合,从而启动促炎症分子基因的转录核蛋白质的合成,引起TNF-α、IL-6mRNA等的强烈表达,产生大量的炎性细胞因子,同时间接促使凋亡细胞增加,加速对脏器细胞的毒性作用并最终出现多器官功能损伤。有实验证实Bax蛋白、NF-κB蛋白明显高表达时,梗阻性黄疸大鼠肝肾组织细胞凋亡明显增多。现今研究发现,NF-κB亚单位的种类及数量在细胞凋亡中起着决定性的作用,当c-RelA亚型表达增加时,则促进凋亡的发生。药物治疗OJ多脏器功能损害能否通过调控脏器细胞凋亡从而获得疗效正引起研究者的兴趣。丹参是一种传统中药,通常用于活血治疗。丹参是野生丹参根的提取物,主要活性成分包括丹参素,丹酚酸以及丹参酮等,能够起到保护内皮细胞,对抗炎症,防止脂质过氧化和钙超载的作用。研究表明,丹参治疗OJ大鼠时通过降低内毒素水平、减少炎症介质等已经得到普遍认同的机制,发挥保护肝细胞,维护肝功能,以及减少对肾皮质和肾功能损害的作用。但是丹参是否通过抗细胞凋亡的分子机制起到治疗作用,尚无进一步研究。本研究的目的就是为了弥补这个不足。通过观察丹参对阻塞性黄疸大鼠(OJ)肝脏细胞、肾脏细胞凋亡和Bax、NF-κB蛋白表达的影响,探讨丹参对肝脏、肾脏的保护机制。
方法:
1.取雄性清洁级健康SD大鼠180只进行实验(体重270～330g)。购自上海斯莱克实验动物有限责任公司[实验动物许可证号:SCXK(沪)2007-0005]。于浙江中医药大学实验动物中心屏障系统实验室饲养[实验动物许可证号:SYXK(浙)2007-0003]。大鼠实验前适应性饲养1周,术前禁食12h,自由饮水。
2.将大鼠随机均分为假手术组(n=60)、模型组(n=60)、丹参治疗组(n=60)。假手术组仅游离大鼠胆总管而不结扎,然后分层关腹。模型组和丹参治疗组游离大鼠胆总管后用丝线双重结扎胆总管近端,并切断胆总管,分层关腹。丹参治疗组组按每只0.2ml/100g/d腹腔内注射丹参注射液,而假手术组、模型组每日注射等量的生理盐水。按术后不同时间段各组分为OJ 7d、14d、21d、28d组(n=15)。
3.记录术后各相应时点大鼠的一般情况,包括大鼠活动度、精神状况、大小便颜色及是否改变、眼白及皮毛染色有无改变,死亡率等。
4.检测各组、各时间点血清TBIL、DBIL、γ-GT,BUN、CREA含量,通过对大体标本肉眼观察、光镜法及电镜法观察各组、各时间点肝脏、肾脏组织病理改变特征,判断大鼠阻塞性黄疸模型建立是否成功,以及运用丹参治疗后与模型组一般情况、血清指标、病理变化来判断治疗效果。
5.运用组织芯片技术制作肝、肾组织微阵列切片。采用组织微阵列片制作机(Beecher Instruments,USA)制备肝、肾的组织微阵列片,将100个直径2毫米的小组织样本,以阵列方式包埋于同一块20mm×45 mm的石蜡块中,再进行切片,用于同一实验指标的研究。
6.在制作完成的肝肾组织微阵列切片上通过DNA缺口原位末端标记法(TUNEL法)检测细胞凋亡指数。在微阵列切片上进行TUNEL染色,高倍镜下(400倍)观察凋亡细胞的分布。每张切片随机选择5-10个高倍视野,计数1000个细胞中的凋亡细胞个数,用百分数表示,计算凋亡指数(AI),AI=凋亡细胞数/总细胞数×100%。本部分工作在陕西超英生物科技有限公司完成。
7.在制作完成的肝肾组织微阵列切片上通过免疫组化检测组织切片中Bax、NF-κB c-RelA蛋白表达水平。在微阵列切片上采用二步EnVision法,计分标准如下:1)按着色细胞染色强度计分为:细胞呈阴性染色为-;染色弱,阳性细胞呈淡黄色为+;染色中等,阳性细胞呈棕黄色为++;染色强,阳性细胞呈棕褐色为+++。2)阳性细胞数(面积):无阳性细胞数为-;阳性细胞数＜25%为+;阳性细胞数25～50%为++;阳性细胞数＞50%为+++。
结果:
1.假手术组21d死亡1只,其余时间点无死亡;模型组7d死亡2只,14d死亡4只,21d死亡4只,28d死亡7只;丹参治疗组7d无死亡,14d死亡3只,21d死亡2只,28d死亡4只。7d三组间死亡率无统计学差异,14d、21d假手术组与模型组间死亡率均存在统计学差异(P=0.032),28d假手术组与模型组间死亡率存在统计学差异(P=0.006),假手术组与治疗组间死亡率存在统计学差异(P=0.032)。
2.各时间点假手术组TBIL、DBIL、γ-GT和CREA含量明显小于模型组与治疗组(P＜0.01);OJ 21、28d治疗组大鼠血清TBIL、CREA含量均明显低于模型组(P＜0.05 or P＜0.01);OJ 28d治疗组大鼠血清DBIL含量均明显低于模型组(P＜0.01),OJ 21d治疗组大鼠血清γ-GT含量均明显低于模型组(P＜0.01)。
3.大体、光镜及电镜观察下,OJ丹参治疗组大鼠的肝脏、肾脏病理改变较模型组均不同程度地减轻。
4.肝脏凋亡指数在21d丹参治疗组明显低于模型组(P＜0.01),其余各时间点各组间无显著性差异。肾脏凋亡指数14d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组无明显差异(P＞0.05),14d丹参治疗组明显小于模型组(P＜0.01)。
5.肝脏Bax蛋白阳性强度以及阳性强度和面积的乘积在各时间点模型组明显大于假手术组(P＜0.01),7,21,28天时间点丹参治疗组明显大于假手术组(P＜0.01),各时间点丹参治疗组与模型组无显著性差异(P＞0.05)。肝脏NF-κB c-RelA蛋白阳性强度以及阳性强度和面积的乘积在7d、14d、21d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组、模型组无显著性差异(P＞0.05)。肾脏Bax蛋白阳性强度21d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组、模型组无明显差异(P＞0.05)。肾脏Bax蛋白阳性强度和面积乘积21d模型组明显大于假手术组(P＜0.01),各时间点丹参治疗组与假手术组无明显差异(P＞0.05),21d丹参治疗组明显小于模型组(P＜0.01)。肾脏NF-κB c-RelA蛋白阳性强度以及阳性强度和面积的乘积在各时间点假手术组与模型组、丹参治疗组以及模型组与丹参治疗组之间均无明显差异(P＞0.05)。
结论:
1.结扎大鼠胆总管后,模型组及丹参治疗组大鼠血清TBIL、DBIL及γ-GT浓度均较未结扎胆总管的假手术组明显升高,且血清胆红素以DBIL为主;同时模型组及丹参治疗组大鼠血清BUN、CREA浓度也均较假手术组明显升高,且肝肾组织有显著的病理损伤表现,说明阻塞性黄疸在肝功能受损同时也可导致肾功能损伤。
2.阻塞性黄疸大鼠可出现肝、肾组织Bax蛋白、肝脏NF-κB c-RelA蛋白表达明显升高,肝、肾组织凋亡细胞指数明显增加。
3.丹参能明显地降低OJ大鼠血清TBIL、DBIL、γ-GT及CREA含量,减少肝肾细胞凋亡数目,减轻肝脏、肾脏病理损伤,降低大鼠死亡率。
4.丹参对OJ大鼠肝、肾组织NF-κB c-RelA蛋白表达无明显的调节作用。丹参可能通过抑制OJ大鼠肾脏Bax蛋白的表达,减轻肾脏细胞的凋亡,对肾功能起到积极的保护作用。
Purpose:
Obstructive jaundice is a common clinical complication,which could be caused by biliary system in benign and malignant obstruction,surgery injury and so on usually. And OJ could result in serious injuries such as multiple organ injuries quickly.It is currently believed that obstructive jaundice(OJ) can induce functional damage to the liver,spleen,lymphocytes,and gastrointestinal mucosa as well as cardiac hemodynamic dysfunction.However,the mechanisms underlying OJ-induced multiple organ damage are still unclear,and there are many controversial hypotheses to explain this phenomenon.It is generally believed that endotoxin,inflammatory mediators,and increased bile acids contribute to OJ induced liver or other organs injuries.Moreover, elevated intrabiliary duct pressure and portal vein pressure are also important factors contributing to this injury.In recent years,along with in-depth study of apoptosis has been found that many of hepatobiliary system diseases and disorders related to apoptosis of cells.Apoptosis plays a very important role in the maintenance of a stable internal environment,normal physiological state and the hepatobiliary system.Studies suggest that apoptosis involved in OJ multiple organ dysfunctions by two ways:one is subject to gene regulation;the other is by non-genetic regulation.Gene regulation is divided into the direct control and indirect control.The former includes the p53,Bax, Fas-Fasl,Bcl-2 and so on,the latter,such as TGF,TNF,NF-κB and so on.Bax is a soluble protein and located in the cytoplasm as the form of monomer under normal circumstances.It emerges conformational changes under stimulus of apoptosis and translocates from the cytoplasm to the mitochondria.Then,it is combined with the outer mitochondrial membrane and directly reduces the stability of mitochondrial outer membrane or with Bcl-2 together to form the structure of heterodimers to play a role in promoting apoptosis.NF-κB is a kind of nuclear protein factors with effects of gene transcription regulation,widely present in the cytoplasm in a variety of cells.As one of Rel family of transcription protein,it takes part in the mediation of expression of inflammatory factors gene and apoptosis.When the body cells were involved in elevated levels of oxygen free radicals,ischemia-reperfusion injury,endotoxemia and other stress state,I-κB makes NF-κB activating and translocating into the nucleus in the target genes with specific promoter regionκB binding sequence.NF-κB can initiates synthesis of the transcription nuclear protein of pro-inflammatory molecules gene,and produce the expression of TNF-α,IL-6mRNA strongly,produce a large number of inflammatory cytokines,and indirectly promote an increase of apoptotic cells to accelerate the toxic effects on cells,organs,and eventually become a multi-organ dysfunction.There experiments confirmed that apoptosis was increased in liver and kidney tissues markedly,when the Bax protein,NF-κB protein expression was significantly higher in OJ rats.Salvia miltiorrhizae(S.miltiorrhizae) is a kind of traditional Chinese drug commonly used for activating blood circulation,the research on the effect of it in the treatment of OJ has been reported.S.miltiorrhizae injection is the extraction of wild Salvia roots,the main active ingredients include danshensu, salvianolic acid as well as tanshinone,dihydrotanshinone,and cryptotanshinone,which are able to protect endothelial cells,fight against inflammation,and prevent lipid peroxidation and calcium overload.Some studies have shown that S.miltiorrhizae can exert some therapeutic effects against this damage through protecting hepatic cells, maintaining liver function,as well as reducing the damage to renal cortex and renal function in OJ rats.There is no further study on whether it is played the therapeutic effect through anti-apoptosis.For this reason,the purpose of this study is to make up for this deficiency.Through observing the impact of S.miltiorrhizae injection on the pathological alterations,cell apoptosis and Bax/NF-κB protein expression in liver and kidney of OJ rats,we explored the protection mechanisms of S.miltiorrhizae in these organs.
Methods:
A total 180 healthy male SD rats of clean grade,were utilized for OJ associated experiments and randomly divided into sham-operated,model-control,and treated group,which were further randomly subdivided into 7d,14d,21 d and 28 d groups(n=15) according to time duration after operation.Rats in sham-operated group were separated common bile ducts only and without ligation,then closed abdomen layer by layer.Model group and treatment group were cut off the proximal common bile ducts after separation and ligation with double silk thread,and closed abdomen layer by layer.Intraperitoneal injection was adopted,S.miltiorrhizae injection(0.2ml/100g/d) in treatment group,while the same amount of saline in sham operation group and model group every day.Then,we recorded the mortality rate,serum TBIL,DBIL,γ-GT,BUN, CREA content,observed the characteristics of pathological changes of liver and kidney tissue,detected of Bax,NF-κB protein expression level and apoptosis index changes on the corresponding time point.
Results:
The numbers of dead OJ rats in the treated groups declined as compared with those in the model control group,but not significantly(P＞0.05).The contents of TBIL、DBIL、γ-GT and CREA(7,14,21,and 28 d in OJ rats,respectively) in the sham-operated group were significantly lower than those in the model control and treated groups(P＜0.01,respectively).The contents of TBIL and CREA(21 and 28 d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01 or P＜0.05,respectively).The contents of DBIL(28 d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01,respectively).The contents ofγ-GT,apoptosis index of liver and Bax protein Positive intensity(21 d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01, respectively).The apoptosis index of kidney(14d in OJ rats,respectively) in the treated group were significantly lower than those in the model control groups(P＜0.01, respectively).Others,those pathological damages were reducing with vary degrees in the treated groups.
Conclusion:
1.With cutting off the proximal common bile ducts in rats after separation and ligation,serum TBIL,DBIL,andγ-GT levels in the model-control and the treated group were significantly higher than those in sham-operated group in this experiment.Serum BUN,CREA levels in the model-control and the treated group were significantly higher than those in sham-operated group,and livers and kidneys had experienced significant pathological damage in these two groups,as expected.
2.Obstructive jaundice can cause Bax protein and apoptosis index increased significantly in livers and kidneys of OJ rats.And NF-κB protein expression increases significantly in livers.
3.S.miltiorrhizae can significantly reduce the serum TBIL,DBIL,γ-GT,and CREA levels,and the number of liver/kidney cell apoptosis,reduce pathological damage of the livers and kidneys,and get lower mortality rate in OJ rats.
4.S.miltiorrhizae had no obvious regulatory effects on the expression of NF-κB c-RelA in livers and kidneys of OJ rats.It may be through inhibiting the protein expression to reduce renal cell apoptosis,and play a positive protective effect on renal function.
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[30]Spirey J,Bronk S,Gores GJ.GCDC induced lethal hepatocellular injury in rat hepatecytes.Role of ATP depletion and cytosolic free calcium[J].J Clin Invest,1993,92(1):17.
[31]Peng B,Qiao AY,Luo YL,Li ZY He S,Wu YT.The mechanism and significance of cellular signal transduction in kidney glomerular mesangial cells of rat with obstructive jaundice.Sichuan Da Xue Xue Bao Yi Xue Ban,2007,38(1):116-8.Chinese.
[32]Tan Jingwang,Zhang Shaogeng,Wang Lei,et al.Apoptosis as a Mechanism of Cell Death in the Liver of Obstructive Jaundice.J Surg Concepts Prac.2003,8(2):129-32.
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[34]Ortiz A,Justo P,Sanz A,Melero R,et al.Tubular apoptosis and cidofovir-induced acute renal failure.Antivir Ther,2005;10(1):185-90.
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[36]印彤,童善庆,谢玉才,等.超抗原SEB和D2氨基半乳糖对小鼠肝细胞的作用观察.免疫学杂志,1998,14:36-39.
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[41]Chia SJ,Tang WY,Elnatan J,et al.Prostate tumors from an Asian population:examination of bax,bcl-2,p53 and ras and identification of bax as a prognostic marker.Br J Cancer,2000,83(6):761-8.
[42]汤礼军,田伏洲,王雨等.肝脏低温保存2再灌注期间肝细胞凋亡及其与Bax 基因蛋白相关性的研究.中华实验外科杂志,2001,18(2):144-46.
[43]Padillo F J,Cruz A,Segura-Jimenez I,et al.Anti-TNF-alpha treatment and bile duct drainage restore cellular immunity and prevent tissue injury in experimental obstructive jaundice.Int J Immunopathol Pharmacol,2007,20(4):855-60.
[44]刘素荣,叶孟.NF-kB和凋亡相关的调控途径.现代实用医学,2009,21(1):86-88.
[45]Chen X,Kandasamy K,Srivastava RK.Differential roles of RelA(P65)and c-Rel subunits of nuclear factor Kappa-B in tumor necrosis factor-related apoptosisinducing ligend signaling.Cancer Res,2003,63(5):1059-1066.
[46]Liu Hai,Wang bing-huang,Zhang Bing-yan,et al.Study of hepatic injury induced by lipidperoxidation and protective effects of salvia miltiorrhizea in the bile duct ligated rabbits.Medi & Phar OF Yunnan.1995,16(5):332-34.Chinese.
[47]Chen Xi-hua,Wang Feng,Han De-wu.Effects of salvia miltiorrhiza solution on endotoxemia and hepatic CD14 expression after obstructive jaundice.Chin J Exp Surg,2003,20(9):802-04.
[1]Agarwal MK,Lazar G.Metabolic basis of endotoxicosis.Microbios.1977;20(81-82):183-214.
[2]Nolan JP.The role ofendotoxin in liver injury.Gastroenterology.1975 Dec;69(6):13,46-56.
[3]俞毅君,王毓璈,徐德征.内毒素血症和高胆红素血症对外科梗阻性黄疽患者的毒性作用.中国临床综合,2001,17(5):32-38.
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[6]Han DW.Intestinal endotoxemia as a pathogenetic mechanism in liver failure.World J Gastroenterol.2002 Dec;8(6):961-5.
[7]Spirey J,Bronk S,Gores GJ.GCDC induced lethal hepatocellular injury in rat hepatecytes.Role of ATP depletion and cytosolic free calcium[J].J Clin Invest,1993,92(1):17.
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[13]李小刚,刘青光,潘承恩,等.丹参注射液对梗阻性黄疸大鼠肝内NO、MDA、ET的影响及其意义.西安医科大学学报.2002,23(2):93.
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[19]Kramer HJ.Impaired renal function in obstructive jaundice:roles of the thromboxane and endothelin systems.Nephron.1998;78(1):124.
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[18]张金池,林永垫,郭平凡,林雁娟.丹参对梗阻性黄疸肾损害保护作用的实验研究.中国现代普通外科进展,2002,5(1):25-28.
[19]张建新,瞿建国,李龙,谢嵘,程国祚.急性坏死性胰腺炎并发肾损害的机制及对丹参的效应.中华急诊医学杂志,2003,12(2):97102.
[20]李小刚,刘青光,潘承恩,等.丹参注射液对梗阻性黄疸大鼠肝内NO、MDA、ET的影响及其意义.西安医科大学学报.2002,23(2):93
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[23]Bansal V,Schuchert VD.Jaundice in the intensive care unit.Surg Clin North Am,2006,86(6):1495-502.Review.
[24]Uslu A,Cayci M,Nart A,et al.Renal failure in obstructive jaundice.Hepatogastroenterology,2005,52(61):52-4.
[25]Shi Liu-bin,Peng Cheng-hong,Wu Yu-lian.Effect of systemic inflammatory response syndrome and multiple organ dysfunction syndromes on patients with malignant obstructive jaundice(report of 152 cases).Chin J of Critical Care Med.2001,21(6):332-33.Chinese.
[26]张文,施维锦,姜广杰等.影响阻塞性黄疸预后的多因素分析.肝胆胰外科杂志.1997,9(3):109-11.
[27]Greve JW,Gouma DJ,Buurman WA.Complications in obstructive jaundice:role of endotoxin.Scand J Gastroenterol Suppl,1992,194:8-12.
[28]Yuceyar S,Gumustas K,Erttirk S,Hamzaoglu IH,Uygun N,Ayaz M,Cengiz A,Kafadar Y.The role of oxygen free radicals in acute renal failure complicating obstructive jaundice:an experimental study.HPB Surg,1998,10(6):387-93.
[29]Han DW.Intestinal endotoxemia as a pathogenetic mechanism in liver failure.World J Gastroenterol.2002 Dec;8(6):961-5.
[30]Spirey J,Bronk S,Gores GJ.GCDC induced lethal hepatocellular injury in rat hepatecytes.Role of ATP depletion and cytosolic free calcium[J].J Clin Invest,1993,92(1):17.
[31]Peng B,Qiao AY,Luo YL,Li ZY He S,Wu YT.The mechanism and significance of cellular signal transduction in kidney glomerular mesangial cells of rat with obstructive jaundice.Sichuan Da Xue Xue Bao Yi Xue Ban,2007,38(1):116-8.Chinese.
[32]Tan Jingwang,Zhang Shaogeng,Wang Lei,et al.Apoptosis as a Mechanism of Cell Death in the Liver of Obstructive Jaundice.J Surg Concepts Prac.2003,8(2):129-32.
[33]Bonegio R,Lieberthal W.Role of apoptosis in the pathogenesis of acute renal failure.Curr Opin Nephrol Hypertens,2002,11(3):301-8.Review.
[34]Ortiz A,Justo P,Sanz A,Melero R,et al.Tubular apoptosis and cidofovir-induced acute renal failure.Antivir Ther,2005;10(1):185-90.
[35]Gantner F,Leist M,Jilg S,et al.Tumor necrosis factor2induced hep2atic DNA fragmentation as an early marker of T cell2dependent liver injury in mice.Gastroenterology,1995,109:166-176.
[36]印彤,童善庆,谢玉才,等.超抗原SEB和D2氨基半乳糖对小鼠肝细胞的作用观察.免疫学杂志,1998,14:36-39.
[37]Joza N,Kroemer G,Penninger JM.Genetic analysis of the mammalian cell death machinery.Trends Genet.2002,18(3):142-9.Review.
[38]Maurer M,Tsai M,Metz M,et al.A role for Bax in the regulation of apoptosis in mouse mast cells.J Invest Dermatol.2000,114(6);1205-1206.
[39]Wolter KG,Hsu YT,Smith CL,et al.Movement of Bax from the cytosol to mitochondria during apoptosis.J Cell Biol.1997,139(5);1281-92.
[40]Thomas A,Pepper C,Hoy T,Bentley P.Bcl-2 and bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients.Leuk Res,2000,24(10):813-21.
[41]Chia SJ,Tang WY,Elnatan J,et al.Prostate tumors from an Asian population:examination of bax,bcl-2,p53 and ras and identification of bax as a prognostic marker.Br J Cancer,2000,83(6):761-8.
[42]汤礼军,田伏洲,王雨等.肝脏低温保存2再灌注期间肝细胞凋亡及其与Bax 基因蛋白相关性的研究.中华实验外科杂志,2001,18(2):144-46.
[43]Padillo F J,Cruz A,Segura-Jimenez I,et al.Anti-TNF-alpha treatment and bile duct drainage restore cellular immunity and prevent tissue injury in experimental obstructive jaundice.Int J Immunopathol Pharmacol,2007,20(4):855-60.
[44]刘素荣,叶孟.NF-kB和凋亡相关的调控途径.现代实用医学,2009,21(1):86-88.
[45]Chen X,Kandasamy K,Srivastava RK.Differential roles of RelA(P65)and c-Rel subunits of nuclear factor Kappa-B in tumor necrosis factor-related apoptosisinducing ligend signaling.Cancer Res,2003,63(5):1059-1066.
[46]Liu Hai,Wang bing-huang,Zhang Bing-yan,et al.Study of hepatic injury induced by lipidperoxidation and protective effects of salvia miltiorrhizea in the bile duct ligated rabbits.Medi & Phar OF Yunnan.1995,16(5):332-34.Chinese.
[47]Chen Xi-hua,Wang Feng,Han De-wu.Effects of salvia miltiorrhiza solution on endotoxemia and hepatic CD14 expression after obstructive jaundice.Chin J Exp Surg,2003,20(9):802-04.
[1]Agarwal MK,Lazar G.Metabolic basis of endotoxicosis.Microbios.1977;20(81-82):183-214.
[2]Nolan JP.The role ofendotoxin in liver injury.Gastroenterology.1975 Dec;69(6):13,46-56.
[3]俞毅君,王毓璈,徐德征.内毒素血症和高胆红素血症对外科梗阻性黄疽患者的毒性作用.中国临床综合,2001,17(5):32-38.
[4]胡铭荣,徐德征,王雄.梗阻性黄疸手术前后内毒素水平和肾功能变化.中国普通外科杂志,2002,11(2):94-97.
[5]Assimakopoulos SF,Scopa CD,Vagianos CE.Pathophysiology of increased intestinal permeability in obstructive jaundice.World J Gastroenterol.2007 Dec 28;13(48):64,58-64.
[6]Han DW.Intestinal endotoxemia as a pathogenetic mechanism in liver failure.World J Gastroenterol.2002 Dec;8(6):961-5.
[7]Spirey J,Bronk S,Gores GJ.GCDC induced lethal hepatocellular injury in rat hepatecytes.Role of ATP depletion and cytosolic free calcium[J].J Clin Invest,1993,92(1):17.
[8]季福.梗阻性黄疽时内毒素血症.肝胆胰外科杂志,1997,9(3):141-142.
[9]Inan M,Seyak I,Tel BC,et al.Role of endotoxin and nitric oxide inthe pathogenesis of renal failure in obstructive jaundice.Br J Surg,1997,84(7):943-947.
[10]Greve JW,Gouma D J,Soeters PB,Buurman WA.Suppression of cellular immunity in obstructive jaundice is caused by endotoxins:a study with germ-free rots.Gastroenterology.1990;98(2):478-485.
[11]Scott-Conner CE,Grogan JB.The pathophysiology of biliary obstruction and its effect on phagocytic and immune function.J Surg Res.1994;57(2):316-336.
[12]O'Neil S,Hunt J,Filkins J,Gamelli R.Obstructive jaundice in rats results in exaggerated hepatic production of tumor necrosis factor-alpha and systemic and tissue tumor necrosis factor-alpha levels after endotoxin.Surgery.1997;122(2):281-286.
[13]李小刚,刘青光,潘承恩,等.丹参注射液对梗阻性黄疸大鼠肝内NO、MDA、ET的影响及其意义.西安医科大学学报.2002,23(2):93.
[14]陈达民.内皮素对肝脏的多种作用.国外医学·消化系统疾病分册.1997,17(3):167.
[15]Pinzani M,Milani S,De Franco R,Grappone C,Caligiuri A,Gentilini A,Tosti-Guerra C,Maggi M,Failli P,Ruocco C,Gentilini P.Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells.Gastroenterology.1996;110(2):534-548.
[16]Rockey DC,Weisiger RA.Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver:implications for regulation of portal pressure and resistance.Hepatology.1996;24(1):233-240.
[17]Padillo FJ,Cruz A,Segura-Jimenez I.Anti-TNF-alpha treatment and bile duct drainage restore cellular immunity and prevent tissue injury in experimental obstructive jaundice.Int J Immunopathol Pharmacol.2007;20(4):855-60.
[18]Tsai LY,Lee KT,Liu TZ.Evidence for accelerated generation of hydroxyl radicals in experimental obstructive jaundice of rats.Free Radic Biol Med.1998;24(5):732-737
[19]Kramer HJ.Impaired renal function in obstructive jaundice:roles of the thromboxane and endothelin systems.Nephron.1998;78(1):124.
[20]Bodes PN,Campillo B,Azaou L,Scherman E.Long-lasting NO overproduction in cirrhotic patients with spontaneous bacterial peritonitis.Hepatology.1997;25(6):1328-1333.
[21]Padillo F J,Cruz A,Navarrete C.Melatonin prevents oxidative stress and hepatocyte cell death induced by experimental cholestasis.Free Radio Res.2004;38(7):697-704.
[22]张金池,林永,郭平凡,等.丹参对梗阻性黄疸肾损害保护作用的实验研究.中国现代普通外科进展.2002,5(1):25-28.
[23]袁爱力.梗阻性黄疸对肾脏的影响.中国实用内科杂志.1999;19(7):391-393.
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