血红素加氧酶-1在糖尿病氧化应激中的作用研究
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摘要
【背景】
目前糖尿病在全球尤其是发展中国家的患病人数急剧增加,已成为严重危害人类健康的流行性疾病。由于糖尿病慢性并发症可引起失明、尿毒症、截肢等严重后果,已成为目前全世界共同关注的公共卫生问题,也是目前的防治重点和难点。
研究认为,糖尿病慢性并发症的发病机制主要包括四大代谢通路的激活,即晚期糖基化终产物(advanced glycation end products,AGEs)、己糖胺(hexosamine)、蛋白激酶C(protein kinase C,PKC)和多元醇(polyol)通路。近年来Brownlee研究发现,抑制线粒体活性氧簇(reactive oxygen species,ROS)的产生可阻断上述四大代谢通路,因而提出了统一机制学说,认为高糖导致的氧化应激是糖尿病慢性并发症发病机制的中心环节,可以将上述四条代谢通路统一起来(如图1所示)。在正常情况下,线粒体氧化磷酸化过程中约有0.4%～4%的氧转化为超氧阴离子自由基(·O_2~-),少量的·O_2~-能被机体抗氧化保护机制所消除。但在高糖情况下,线粒体电子传递链产生的电子明显增多,产生过多的活性氧簇(reactive oxygen species,ROS),造成大分子物质脱氧核糖核酸(deoxyribonucleic acid,DNA)损伤,后者激活聚二磷酸腺苷核糖聚合酶(polyADP-ribose polymerase,PARP)进行DNA修复,该过程消耗大量NAD+(oxidizedform of nicotinamide-adenine dinucleotide,氧化型烟酰胺腺嘌呤二核苷酸),使NADH/NAD+比例增加,抑制了磷酸甘油醛脱氢酶(glyceraldehyde phosphatedehydrogenase,GAPDH)活性,导致葡萄糖的正常代谢通路受阻,中间产物积聚转而进入上述四大代谢途径,后者均能进一步促使细胞内ROS产量增加,形成恶性循环;同时抗氧化剂大量消耗,使细胞处于氧化与抗氧化失衡的应激状态,严重影响细胞功能,并加速细胞凋亡,抗氧化治疗则能阻止上述氧化应激所致的细胞凋亡。因此,目前认为,氧化应激是糖尿病慢性并发症发病机制的中心环节,拮抗氧化应激对糖尿病慢性并发症的防治极具治疗潜力。虽然严格控制血糖对拮抗高糖引起的氧化应激具有重要的意义,但目前全球糖尿病的血糖控制水平还很差,仅约1/3左右的患者达到了血糖的理想控制,因此,必须从抗氧化防御系统寻求合适的拮抗氧化应激的手段。
除经典的抗氧化酶如过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽系统[不含硒谷胱甘肽过氧化物酶(glutathion peroxidase,GPx)-谷胱甘肽硫转移酶(glutathione S-transferase,GST)]等之外,近年来,血红素加氧酶-1(heme oxygenase,HO-1)在拮抗氧化应激方面的作用正日益受到关注。HO有三种同工酶,其中HO-2基因定位于人染色体16p13,存在于大多数组织细胞中,生理状况下组织中的HO活性主要由HO-2决定,HO-2属于结构型,几乎不受外界刺激因素的影响,目前仅知大脑组织中HO-2的转录受肾上腺皮质激素的调节。HO-3是新近发现的一种HO,与HO-2有90%的氨基酸同源性,HO-3催化血红素降解的能力很低,其主要作用可能为介导HO与血红素结合。HO-1是诱导型,与HO-2在基因结构、调节上仅有约40%的相似性,HO-1基因定位于人染色体22q12,广泛分布于哺乳动物多种组织细胞的微粒体内,正常情况下表达量很低(只在脾、肝脏和睾丸中有较高表达),但多种应激性因素如重金属、细胞因子、紫外线、氧化应激、炎症因子等均可诱导HO-1表达及活性增强。由于HO-1蛋白的分子量在31～33KD之间,因此有人将HO-1归属为热休克蛋白32,它是目前已知最容易被诱导的蛋白,诱导后活性可升高至100倍。
研究认为,HO-1是体内最重要的内源性抗氧化保护因子之一,在许多病理生理过程中发挥重要的调节作用,尤其是对组织器官的保护作用,已成为目前包括器官移植、缺血再灌注损伤、心脑血管病、支气管哮喘、先兆子痫等多个领域的研究热点。HO是体内唯一催化血红素分解代谢的限速酶,HO的催化作用需要微粒体NADPH(reduced form of nicotinamide-adenine dinucleotidephosphate,还原型烟酰胺腺嘌呤二核苷酸磷酸)—细胞色素P450还原酶协同参与电子转移,它为分子氧的激活和铁的还原提供还原等价物,即HO蛋白结合血红素,并与细胞色素P450还原酶一起形成一个短暂的电子传递链,使卟啉环异构体特异性地分离,激活的氧和血红素分子的α中间碳桥相互作用,使该位置的四吡咯环裂解,形成胆绿素,并使碳桥转变为一氧化碳(carbon monoxide,CO),当卟啉环打开时螯合铁也被释放出来。最终HO将血红素降解为CO、铁和胆绿素,后者继续在胆绿素还原酶(biliverdin reductase,BVR)的作用下生成胆红素(bilirubin,BR),这些代谢产物均有抗氧化、抗炎、抗凋亡、信号传导和免疫调节等作用(图2)。CO是一种小分子气态物质,大量研究证实CO与一氧化氮(nitric monoxide,NO)类似,也是一种重要的细胞信使分子,它可以通过扩散以自分泌或旁分泌方式与自身或邻近细胞相互作用,激活可溶性鸟苷酸环化酶(soluble guanylate cyclase,sGC),后者催化三磷酸鸟苷(guanosinetriphosphate,GTP)生成环磷酸鸟苷(cyclic guanylic acid,cGMP),激活cGMP依赖性蛋白激酶、磷酸二酯酶,或通过调节离子通道而传递生理信息,达到舒张血管平滑肌、抑制平滑肌细胞增殖并抑制血小板聚集等作用。此外,CO也可通过cGMP非依赖途径发挥血管舒张效应,包括对某种钾通道的影响,增加脂联素水平等。采用锌原卟啉(zinc protoporphyrin,ZnPP)抑制HO活性可增加颈动脉体感受器活性,而CO可逆转该反应,提示CO在该感受器可能作为神经递质而发挥作用。CO还可能调节或抑制NOS,并与高血压及内皮功能紊乱有关。尽管与NO相比,CO舒张血管的效应相对较小,但CO通过cGMP介导的血管舒张潜能可能被其它制剂所大大增加。胆绿素和胆红素是体内丰富的抗氧化物质,流行病学研究发现低血清胆红素水平与缺血性心脏病密切相关,在调整了年龄、吸烟、胆固醇、收缩压等已知的冠状动脉硬化性心脏病(coronary heart disease,CHD)的危险因素后,这种相关性仍十分显著,因而认为低胆红素血症是CHD的独立危险因素。近年的研究发现胆红素和胆绿素可以维持内皮细胞的完整性,防止内皮细胞凋亡,促进血管反应性和防止血管硬化。胆红素可以抑制NADPH氧化酶和PKC的活性,而后两者介导了血管紧张素Ⅱ诱导的血管损伤;胆红素还可通过增加NO的生物活性而降低糖尿病动物模型体内的氧化应激水平。一般认为,铁通过Fenton反应导致羟自由基形成,发挥致氧化应激的作用,但HO衍生而来的铁可诱导铁蛋白表达和合成增加,后者具有抗氧化、抗凋亡等细胞保护功能,抑制铁沉积,对抗氧化型LDL所致内皮细胞的氧化应激,并被证实是一种保护上皮细胞的抗氧化剂。上调HO-1可增加铁蛋白合成,后者可捕获HO-1途径释放的铁,并通过与细胞表面受体结合将铁转运至细胞内。
研究认为,HO-1在拮抗高糖所致氧化损伤中发挥了十分重要的保护作用。高糖诱导的ROS生成增加在糖尿病慢性血管并发症的发生和发展中具有重要作用。增加的·O_2~-形成和HO-1诱导不足使内皮功能紊乱甚至细胞凋亡,但可被抗氧化剂或通过诱导抗氧化酶而逆转,诱导HO-1表达可拮抗氧化应激从而提供血管保护作用。糖尿病鼠体内ROS含量明显增高、HO-1活性不足、内皮细胞凋亡增加,给予HO-1诱导剂后内皮受到了保护,相反给予HO-1抑制剂后上述损害加重。转染人HO-1基因可降低高糖介导的循环内皮细胞脱落数量、·O_2~-的形成及尿8-异前列烷的产生,并提供血管保护作用,而下调鼠HO-1表达可加速这些不良病理过程。高表达HO-1可使糖尿病肥胖小鼠的血清脂联素水平和胰岛素敏感性增加,使内脏和腹部脂肪减少,以及血浆肿瘤坏死因子α(tumornecrosis factorα,TNFα)、白介素-6(interleukin 6,IL-6)和IL-1β水平降低。采用钴原卟啉(cobalt protoporphyrin,CoPP)诱导胰岛β细胞高表达HO-1可拮抗细胞氧化损伤,使TNFα诱导的β细胞凋亡百分比从75%降至5%,并受p38MAPK(mitogen-activated protein kinase,MAPK)信号途径调节,该研究认为促进HO-1高表达是一种新型的保护胰岛β细胞的可行的手段。体内外实验均证实,采用CoPP上调HO-1可使骨髓以及体外培养的间充质干细胞的脂肪形成减少,培养基上清脂联素水平增加。采用各种手段促进HO-1表达还可有效增强组织和细胞抗缺血缺氧的能力,相反,抑制HO-1表达将加重心肌缺血再灌注损伤。HO-1基因转染大鼠平滑肌细胞使其高表达HO-1还可提高细胞在过氧化物所致氧化应激中的存活率,而HO-1抑制剂锌原卟啉(zinc protoporphyria,ZnPP)预处理可使该保护作用明显减弱,因此认为HO-1具有降低动脉硬化发生危险的潜力。STZ诱导的糖尿病大鼠在缺血/再灌注后心肌的HO-1表达受损,心梗面积增加,在缺血前给予高铁血红素诱导HO-1后其HO-1表达仍低于非糖尿病大鼠。AGEs刺激小鼠巨噬细胞RAW264.7可使其HO-1蛋白表达增加,并具有一定的浓度依赖性。探讨高糖和AGEs分别刺激及二者联合刺激对单核细胞HO-1表达的影响可为进一步分析HO-1在糖尿病氧化应激中的作用提供研究基础。目前认为,线粒体功能异常参与了糖尿病慢性并发症的发病机制。人HO-1基因转染糖尿病大鼠可促使其线粒体转运载体的功能恢复,研究发现这与AKT磷酸化水平增加相关,后者和Bcl-xL水平增加可防止糖尿病β细胞数量减少。体内外实验均证实线粒体功能改变与AKT和Bcl-2家族蛋白激活相关,AKT激活可促进ATP合成以及己糖激酶与电压依赖的离子通道的联系,电压依赖的离子通道关闭可阻断细胞色素c的释放;而Bcl家族成员减少参与了细胞色素c由线粒体向胞浆的转位。这些研究结果均提示氧化应激在糖尿病慢性并发症的发生和发展中具有重要作用,采用适当的手段诱导HO-1高表达为细胞提供保护作用可能是极具潜力的一种新的糖尿病慢性并发症的防治手段。
然而,目前对HO-1在糖尿病氧化应激中的作用及其机制尚未完全阐明,本课题拟从体内外实验两方面对HO-1在拮抗糖尿病氧化应激中的作用进行探讨,为寻求有效的拮抗糖尿病氧化应激的手段提供科学的依据。
第一章HO-1在高糖和AGEs致THP-1细胞氧化应激中的表达变化研究
【目的】
1.探讨不同浓度葡萄糖、晚期糖基化终产物(AGEs)在不同刺激时间对THP-1细胞ROS产量的影响;
2.了解高糖和AGEs联合刺激对THP-1细胞氧化应激的影响;
3.了解HO-1在高糖和AGEs所致THP-1细胞氧化应激中的表达变化;
4.探讨p38MAPK信号通路是否参与了高糖和AGEs诱导的THP-1细胞HO-1表达。
【方法】
1.细胞培养:采用含10%胎生血清的RPMI1640培养基,于37℃、5%CO_2培养箱内传代培养人单核细胞株THP-1。
2.体外制备AGEs:将牛血清白蛋白(bovine serum albumin,BSA)与葡萄糖混合于37℃孵育60天,4℃透析24tl以去除未结合的葡萄糖。
3.细胞ROS产量检测:分别用5、15、25mmol/L的GLU或25、50、100μg/mL的AGEs刺激细胞,分别于刺激0.5、2、6、24h后收集细胞,DCFH-DA荧光探针标记,流式细胞仪检测平均荧光强度(mean fluorescence intensity,MFI)。
4.细胞分组:分为4组,即15mmol/LGLU组(高糖组)、100μg/mLAGEs组(AGEs组)、15mmol/LGLU+100μg/mLAGEs组(高糖+AGEs联合组)及对照组。
5.细胞培养液上清TNFa水平检测:采用ELISA方法进行。
6.细胞培养液上清中MDA水平检测:采用硫代巴比妥酸法(thiobarbituric acid,TBA),严格按照试剂盒说明步骤检测。
7.RT-PCR法检测HO-1mRNA表达:提取总核糖核酸(ribonucleic acid,RNA),两步法进行RT-PCR,扩增产物于1.5%琼脂糖凝胶进行电泳。
8.免疫印记(western blot,WB)法检测HO-1蛋白表达:提取总蛋白,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectropheresis,SDS-PAGE),电转移至硝酸纤维素膜(nitrocellulose filter,NC)封闭非特异位点。利用多克隆兔抗人HO-1抗体及HRP标记的二抗与NC反应,DAB显色后拍照分析条带亮度。
9.SB203580(SB,p38MAPK抑制剂)干预实验:分为高糖组、SB+15mmol/LGLU(SB+高糖)组、AGEs组、SB+100μg/mLAGEs(SB+AGEs)组。10μmo/LSB干预30min后换以含15mmol/LGLU或100μg/mL AGEs细胞培养液继续孵育24h,收集细胞进行RT-PCR检测HO-1mRNA表达水平。
10.统计学处理:用SPSS13.0软件对所有数据进行统计学处理;实验数据计量资料以均数±标准差((?)±s)表示。多组间资料比较用析因设计的方差分析(ANOVA),任意两组间比较采用独立样本的t检验,相关分析采用积矩相关系数(Pearson相关系数)分析,检验标准为α=0.05。
【结果】
1.制备的AGEs浓度:AGEs的荧光浓度为101.29U/mg,对照BSA的荧光浓度为13.68 U/mg,前者为后者的7.40倍。
2.GLU和AGEs刺激THP-1细胞的ROS产量:均表现为0.5h的ROS产量急剧升高,随着时间延长轻度下降,但于24h再度上升至较高水平,在6h、24h时间点的ROS产量表现为GLU和AGEs浓度依赖性升高。高糖组(65.88±1.61)和AGEs组(67.46±3.78)的ROS产量均显著高于对照组(41.95±1.36)(P＜0.001),高糖+AGEs联合组(107.21±8.94)显著高于GLU组和AGEs组(P＜0.01),且高糖和AGEs对ROS产量的影响具有协同作用(P＜0.05)。
3.细胞培养液上清的MDA水平(nmol/mL):高糖组(1.59±0.53)显著高于对照组(0.65±0.23)(P＜0.05),AGEs组(1.56±0.97)虽然也高于对照组,但统计学差异不明显(P＞0.05),高糖+AGEs联合组(3.26±0.32)显著高于GLU组和AGEs组(P＜0.05)。
4.细胞培养液上清的TNFa水平(pg/mL):高糖组(25.38±1.95)和AGEs组(24.43±2.65)显著高于对照组(14.97±1.49)(P＜0.01),高糖+AGEs联合组(51.25±1.72)显著高于高糖组及AGEs组(P＜0.001),高糖和AGEs对TNFa的影响存在协同作用(P＜0.001)。
5.细胞HO-1mRNA的表达:高糖组(0.42±0.02)和AGEs组(0.48±0.03)显著高于对照组(0.15±0.01)(P＜0.001),高糖+AGEs联合组(0.89±0.12)显著高于高糖组及AGEs组(P＜0.05)。
6.THP-1细胞的HO-1蛋白表达:高糖组(0.39±0.01)和AGEs组(0.45±0.03)显著高于对照组(0.15±0.01)(P＜0.05),高糖+AGEs联合组(0.81±0.02)显著高于高糖组及AGEs组(P＜0.05),高糖和AGEs存在协同作用(P＜0.01)。
7.相关分析:HO-1蛋白表达水平与HO-1mRNA、ROS产量、MDA及TNFα均呈显著正相关(P＜0.001)。HO-1mRNA与ROS、MDA及TNFα均呈显著正相关(P＜0.001)。ROS产量与MDA、TNFα呈显著正相关(P＜0.001)。TNFα与MDA呈显著正相关(P＜0.001)。
8.SB203580(SB)(p38MAPK抑制剂)干预实验结果:各组之间HO-1mRNA表达均无显著的统计学差异(P＞0.05)。
【结论】
1.高糖和AGEs单独刺激能导致THP-1细胞氧化损伤加剧、分泌炎症因子TINFα水平及HO-1表达增高,二者联合刺激能使上述改变进一步加剧。
2.p38MAPK抑制剂对高糖和AGEs诱导的THP-1细胞HO-1mRNA表达无显著影响。
3.HO-1表达与细胞氧化损伤及炎症指标呈显著正相关,提示高糖和AGEs导致THP-1细胞氧化损伤及炎症反应,后者诱导HO-1表达适应性和代偿性增高,从而发挥细胞保护作用。
第二章抑制HO-1对高糖和AGEs致THP-1细胞氧化应激的影响
【目的】
探讨抑制HO-1表达对高糖和AGEs刺激下THP-1细胞氧化应激的影响。
【方法】
1.细胞分组:分为4组,即对照组、15mmol/LGLU+100μg/mLAGEs(GLU+AGEs)组、ZnPP组及ZnPP+15mmol/LGLU+100μg/mLAGEs(ZnPP+GLU+AGEs)组,其中对照组和ZnPP组THP-1细胞培养液的GLU浓度均为5mmol/L。
2.细胞ROS产量、细胞培养液上清中MDA及TNFa水平、HO-1mRNA及蛋白表达水平检测同第一章。
3.统计学处理:用SPSS13.0软件对所有数据进行统计学处理。实验数据计量资料以均数±标准差((?)±s)表示。多组间资料比较用析因设计的方差分析(ANOVA),任意两组间比较采用独立样本的t检验,检验标准为α=0.05。
【结果】
1.各组细胞ROS产量(MFI)的比较:ZnPP+GLU+AGEs组(128.81±5.53)、GLU+AGEs组(107.21±8.94)和ZnPP组(51.56±2.09)均显著高于对照组(41.95±1.36)(P＜0.05),而ZnPP+GLU+AGEs组显著高于GLU+AGEs组及ZnPP组(P＜0.05)。
2.各组细胞培养液上清MDA水平(nmol/mL)的比较:ZnPP组(1.61±0.10)、GLU+AGEs组(3.26±0.32)、ZnPP+GLU+AGEs组(3.75±1.25)的MDA水平均显著高于对照组(0.65±0.23)(P＜0.01),其中ZnPP+GLU+AGEs组显著高于ZnPP组(P＜0.05),但与GLU+AGEs组相比无显著性差异(P＞0.05)。
3.各组细胞培养液上清TNFa水平(pg/mL)的比较:ZnPP组(27.34±10.18)、GLU+AGEs组(38.09±14.97)、ZnPP+GLU+AGEs组(141.10±12.84)的TNFa水平均显著高于对照组(18.55±7.26)(P＜0.05),其中ZnPP+GLU+AGEs组显著高于GLU+AGEs组(P＜0.05)。
4.各组细胞HO-1mRNA表达的比较:无论对照组还是各刺激组均可见到与设计片段大小相符的HO-1基因扩增条带(637bp)及内参照β-actin(285bp)。ZnPP组(0.24±0.02)、GLU+AGEs组(0.89±0.09)、ZnPP+GLU+AGEs组(0.39±0.02)的HO-1基因表达水平均显著高于对照组(0.15±0.01)(P＜0.01),其中ZnPP+GLU+AGEs组显著低于GLU+AGEs组(P＜0.05),并显著高于ZnPP组(P＜0.01)。
5.各组细胞HO-1蛋白表达的比较:ZnPP组(0.23±0.03)、GLU+AGEs组(0.81±0.02)、ZnPP+GLU+AGEs组(0.38±0.00)的HO-1蛋白表达水平均显著高于对照组(0.15±0.01)(P＜0.05),其中ZnPP+GLU+AGEs组显著低于GLU+AGEs组(P＜0.01),并显著高于ZnPP组(P＜0.01)。
【结论】
HO-1抑制剂ZnPP本身也能对THP-1细胞造成一定的氧化损伤,并引起HO-1表达增高,ZnPP预处理能显著抑制高糖和AGEs引起的HO-1表达增高,进一步加剧高糖和AGEs引起的细胞氧化损伤及分泌炎症因子TNFa的水平。
第三章诱导HO-1对高糖和AGEs致THP-1细胞氧化应激的影响
【目的】
探讨诱导HO-1高表达是否可对抗高糖和AGEs所致THP-1细胞的氧化损伤。
【方法】
1.细胞分组:分为4组,即对照组、15mmol/LGLU+100μg/mLAGEs(GLU+AGEs组)、CoPP组、CoPP+15mmol/LGLU+100μg/mL AGEs(CoPP+GLU+AGEs组),其中对照组和CoPP组THP-1细胞培养液的GLU浓度均为5mmol/L。
2.细胞ROS产量、细胞培养液上清中MDA及TNFa水平、HO-1mRNA及蛋白表达水平检测同第一章。
3.统计学处理:用SPSS13.0软件对所有数据进行统计学处理;实验数据计量资料以均数±标准差((?)±s)表示。多组间资料比较用析因设计的方差分析(ANOVA),各组内均数比较采用独立样本的t检验或单向方差分析(One-way ANOVA),多重比较采用LSD法(Least-significant difference test)。检验标准为α=0.05。
【结果】
1.各组细胞ROS产量(MFI)的比较:GLU+AGEs组(107.21±8.94)和CoPP+GLU+AGEs组的ROS产量(95.42±2.36)显著高于对照组(41.95±1.36)(P＜0.05),而CoPP组(49.42±6.45)与对照组相比无显著性差异(P＞0.05)。
2.各组细胞培养液上清的MDA水平(nmol/mL)的比较:GLU+AGEs组的MDA水平(3.26±0.32)明显高于对照组(0.65±0.23)及CoPP+GLU+AGEs组(1.28±0.61)(P＜0.01),而CoPP组(0.73±0.45)及CoPP+GLU+AGEs组与对照组之间无显著性差异(P＞0.05)。
3.各组细胞培养液上清的TNFa水平(pg/mL)的比较:GLU+AGEs组的TNFa水平(43.60±12.40)显著高于对照组(14.97±1.49)(P＜0.05),CoPP组(16.60±7.99)、CoPP+GLU+AGEs组(31.90±12.56)与对照组之间无显著性差异(P＞0.05),且CoPP+GLU+AGEs组与GLU+AGEs组、CoPP组相比无显著性差异(P＞0.05)。
4.各组细胞HO-1mRNA表达的比较:CoPP组(0.24±0.01)、GLU+AGEs组(0.89±0.12)、CoPP+GLU+AGEs组(0.91±0.01)的HO-1基因表达水平均显著高于对照组(0.15±0.01)(P＜0.01),其中CoPP+GLU+AGEs组与GLU+AGEs组相比无显著性差异(P＞0.05),但显著高于CoPP组(P＜0.05)。
5.各组细胞HO-1蛋白表达的比较:CoPP组(0.22±0.02)、GLU+AGEs组(0.81±0.02)、CoPP+GLU+AGEs组(0.87±0.01)的HO-1蛋白表达水平均显著高于对照组(0.15±0.01)(P＜0.01),其中CoPP+GLU+AGEs组显著高于GLU+AGEs组和CoPP组(P＜0.05)。
【结论】
在高糖和AGEs存在的情况下,HO-1诱导剂CoPP诱导HO-1蛋白表达增加,并显著抑制细胞氧化应激,明显减轻高糖和AGEs所致氧化损伤,而CoPP本身并不引起THP-1细胞氧化损伤和分泌炎症因子水平增加,结果提示诱导HO-1高表达可能是极具潜力的新的糖尿病慢性并发症的治疗靶点之一。
第四章2型糖尿病合并慢性并发症患者的外周血单核细胞HO-1表达与氧化应激的相关性研究
【目的】
1.了解初诊2型糖尿病(type 2 diabetes mellitus,T2DM)合并慢性并发症患者外周血单核细胞HO-1的表达情况;
2.探讨初诊T2DM合并慢性并发症患者外周血单核细胞HO-1表达与氧化应激的相关性。
【方法】
1.样本选择:来自我院内分泌代谢科门诊及住院的初次诊断T2DM的患者36例,健康志愿者10例。分为正常对照组、糖尿病无并发症组和糖尿病并发症组。
2.细胞收集:采集清晨空腹静脉抗凝血,Percoll法分离单核细胞。
3.细胞ROS产量、血清MDA水平及HO-1mRNA表达检测同第一章。
4.HO-1蛋白表达检测:采用免疫荧光法检测,将外周血单核细胞进行免疫荧光染色后,在荧光显微镜下观察HO-1在细胞内的表达情况。
5.统计学处理:用SPSS13.0软件对所有数据进行统计学处理;实验数据计量资料以均数±标准差((?)±s)表示。两组间资料比较用独立样本的t检验,相关分析采用积矩相关系数(Pearson相关系数)分析,检验标准为α=0.05。
【结果】
1.各组一般指标的比较:正常对照组的年龄(51.03±11.14)与两个糖尿病组之间无显著性差异(P＞0.05),BMI(20.91±2.37)和FPG(5.24±0.70)均分别显著低于两个糖尿病组(P＜0.01)。无并发症组的年龄(50.56±10.70 vs56.78±8.03)和BMI(24.60±2.25 vs 25.69±3.73)与并发症组相比无显著的统计学差异(P＞0.05),但并发症组的FPG(16.26±6.67 vs 10.61±3.61)、2hPG(18.76±7.05 vs 13.57±4.22)和HbAlc水平(12.54±2.10 vs 10.51±1.86)均显著高于无并发症组(P＜0.05)。
2.各组氧化应激指标的比较:正常对照组的血清MDA水平(40.71±20.30)和外周血单核细胞ROS产量(7.98±6.39)均显著低于两个糖尿病组(P＜0.01),而并发症组ROS产量(419.60±216.31 vs 172.38±145.24)、血清MDA水平(66.04±13.67 vs 0.77±0.23)明显高于无并发症组(P＜0.05)。
3.各组HO-1表达的比较:正常对照组的外周血单核细胞HO-1mRNA(0.44±0.26)和蛋白(16.37±15.01)表达水平均低于两个糖尿病组(P＜0.01),而并发症组HO-1mRNA(1.02±0.27 vs 0.77±0.23)及其蛋白(85.74±10.89 vs61.45±21.81)表达水平均显著高于无并发症组(P＜0.05)。
4.相关分析显示,HO-1蛋白表达水平与HO-1mRNA(r=0.750,P=0.000)、ROS(r=0.608,P=0.000)、MDA(r=0.623,P=0.000)呈显著正相关。HO-1mRNA与MDA(r=0.449,P=0.010)、FPG(r=0.364,P=0.041)及2hPG(r=0.477,P=0.016)呈显著正相关。MDA与ROS呈显著正相关(r=0.788,P=0.000)。
5.偏相关分析显示,在控制BMI、FPG、2hPG、HbAlc影响因素后,HO-1蛋白表达仍与ROS产量(r=0.567,P=0.027)、MDA(r=0.613,P=0.015)呈显著正相关,MDA与ROS产量呈显著正相关(r=0.911,P=0.000)。
【结论】
1.初诊T2DM患者的血糖、血清MDA、外周血单核细胞ROS产量及HO-1表达均显著高于正常对照组,提示初诊T2DM可能由于机体抗氧化防御机制的代偿性增高仍不足以对抗高血糖引起的氧化损伤,导致机体处于氧化应激状态。
2.并发症组的血糖水平、氧化应激指标及HO-1表达均显著高于无并发症组,提示高血糖及其引起的氧化损伤可能参与了初诊T2DM患者慢性并发症的发病机制,在排除BMI及血糖水平等因素的影响后,氧化应激指标仍与HO-1表达呈显著正相关,提示HO-1作为一种应激反应蛋白,在糖尿病所致氧化应激情况下代偿性表达增高,但仍不足以对抗该氧化应激。提示除严格控制血糖外,通过适当的手段诱导HO-1高表达将是极具潜力的糖尿病慢性并发症防治的新靶点之一。
[Background]
The patients of type 2 diabetes mellitus have increased rapidly in the world especially in the developing countries,which has been a threat to human's health as an epidemically disease.Since chronic diabetic complications can result in sight loss, uraemia and amputation,it has become difficult and important to prevent and treat diabetic complications.
Accumulated evidences have revealed advanced glycation end products(AGEs) as an important mechanism of chronic diabetic complications.AGEs describes a heterogeneous group of proteins,lipids,and nucleic acids that are formed nonenzymatically.AGE formation is enhanced in the presence of hyperglycemia and oxidative stress.AGE bind to their cognate cell-surface receptor,RAGE,resulting in the activation of postreceptor signaling,generation of intracellular oxygen free radicals,and the activation of gene expression.Thus,AGE are not only markers,but act also as mediators of late diabetic complications.The main mechanisms of diabetic complications include the ignition of four metabolic pathways:advanced glycation end products(AGEs),hexosamine,protein kinase C(PKC) and polynol pathway. Brownlee have found impressant of generation of reactive oxygen species(ROS) could hinder the four metabolic pathways as above mechanisms of diabetic complications.His theory considers high glucose could induce oxidative stress which results in the ignition of the four metabolic pathways(as fig1).
During the course of normal oxidative phosphorylation,however,between 0.4 and 4%of all oxygen consumed is converted into the free radical superoxide(·O_2~-) and is then either detoxified to H_2O and O_2 by glutathione peroxidase(in the mitochondria),or diffuses into the cytosol and is detoxified by catalase in peroxisomes.However,in the presence of reduced transition metals such as Cu or Fe, H_2O_2 can be converted to the highly reactive·OH radical.High glucose can result in electronicas increased which can result in the injury of DNA.After series of reactions the four metabolic pathways were ingited,which was a malignant circle.At the same time anti-oxygenics were comsumed.Oxidative stress was developed thus which can induce cells apoptosis.Accumulated evidences have proved hyperglycemia could result in the elevation of ROS output and induce oxidative stress since there were not enough anti-oxidative chemicals in diabetes.Oxidative stress can ignite the pathways sensitive to oxidative stress and result in some gene products.Thus chronic diabetic complications were developed.Though we have known some knowledgements about the mechnisms of chronic diabetic complications,there are limited methods directed against the mechanisms.
Considerable researches have proved the HO system play an important role in anti-oxidative stress.But the role of the HO system in diabetes,inflammation,heart disease,hypertension,neurological disorders,transplantation,endotoxemia and other pathologies is still a burgeoning area of research.The myriad metabolic systems that have subsequently been shown to be responsive to the up-regulation of HO activity make it clear that enhancing HO activity by either pharmacological or genetic means offers potential for the development of new therapeutic modalities,which could moderate the course of various pathological processes in humans.Heme oxygenase is the rate-limiting enzyme in the catabolism of heme,a process that leads to formation of equimolar amounts of the bile pigment biliverdin,iron,and carbon monoxide(CO). Biliverdin formed in this reaction is rapidly converted to bilirubin.Both CO and bilirubin are bioactive molecules,and the iron generated by HO-1 and HO-2 is immediately sequestered by associated increases in ferritin.HO-2 is a constitutive enzyme,whereas HO-1 is inducible by heavy metals,cytokines,UV light,oxidative stress,inflammatory cytokines and many drugs.It is therefore possible that the induction of HO-1 may be an essential event for some types of acute reactions and for cellular protection after injury.Because the major source of CO in animals is the degradation of heme by HO,it is now becoming apparent that CO serves as an important cellular signal molecule.Nitric oxide(NO) is generated by nitric-oxide synthase(NOS),a heme containing enzyme.It has been proposed that certain NO effects can be duplicated by CO,specifically;the action of certain neurotransmitters and muscle relaxants can be regulated by both molecules.The biological actions of bilirubin may be especially relevant to the prevention of oxidant-mediated cell death. Bilirubin at a low concentration scavenges ROS in vitro,thereby reducing oxidant-mediated cellular damage and attenuating oxidant stress in vivo.Bilirubin inhibits NADPH oxidase and PKC activity.Both enzymes have been shown to mediate angiotensinⅡ-induced vascular injury.Recently,biliverdin and bilirubin have been shown to preserve endothelial cell integrity,prevent endothelial cell death and sloughing,enhance vascular reactivity,and prevent restenosis.Bilirubin is also implicated in reducing oxidative stress in experimental diabetes,in part,by increasing the bioavailability of NO needed for endothelial cell integrity.Plasma iron is bound to transferrin,which can transfer iron to the intracellular milieu of endothelial cells via cell surface receptor binding.The up-regulation of HO-1 increases ferritin synthesis to sequester the iron released from the HO-1 pathway.
Hyperglycemia-mediated local formation of ROS is considered to be a major contributing factor to endothelial dysfunction which play a key role in developemnt of chronic diabetic complicaitons.This includes endothelial cell apoptosis,abnormalities in cell cycling due to lack of HO-1 induction and increased(?)formation.Some of these perturbations can be reversed by antioxidant agents or by increased levels of antioxidant enzymes.Increased levels of HO-1 through gene transfer in hyperglycemic rats resulted in a decrease of endothelial cell sloughing.Delivery of the human HO-1 gene to endothelial cells attenuated glucose-mediated oxidative stress,DNA damage,and cell death.HO-1 induction has been shown to provide vascular cytoprotection against oxidative stress.Thus,HO-1 plays a pivotal role in mitigating the detrimental effects of hyperglycemia.Increases in HO-1 expression in diabetic obese mice were paralleled by increases in serum adiponectin levels,insulin sensitivity,decreases in visceral and abdominal fat content,and decreased plasma TNFα,IL-6,and IL-1βlevels.Induction of HO-1 also resulted in decreased adipocyte superoxide production.Up-regulation of HO-1 by CoPP treatment caused a decrease in adipogenesis in bone marrow,both in vivo and in vitro in cultured mesenchymal stem cells and in increases in secretion of adiponectin in the culture media.
Diabetic complications have been related to abnormalities in mitochondrial function as well as to increased endothelial cell death and detachment.Specific human HO-1 gene transfer to diabetic rats has also resulted in the restoration of mitochondrial carriers,including ADP/ATP and decarboxylate.The increase in HO-1 in diabetic rats is associated with increased pAKT.An increase in AKT phosphorylation is critical to cell survival in diabetes.Increases in AKT phosphorylation and Bcl-xL levels have been shown to prevent the loss ofβ-cells in diabetes.It is interesting to note that the alteration in mitochondrial function in vitro and in vivo correlates with the levels of activation of AKT and the Bcl-2 family of proteins.A decrease in Bcl-2 family members has been reported to contribute to apoptosis and the translocation of cytochrome c from the mitochondria to cytosol. Activation of AKT has been shown to augment ATP synthesis and promote the association of hexokinase with the voltage-dependent anion channel and,in so doing, promote voltage-dependent anion channel closure,thus blocking release of cytochrome c.
Though the last decade has witnessed an explosion in the elucidation of the role that the HO system plays in human physiology,effects of HO-1 on oxidative stress in diabetes mellitus were not completely clarified yet.The use of pharmacological agents and genetic probes for manipulating HO has led to new insights into the prevention of chronic diabetic complications.
Chapter 1
Expressions of heme oxygen ase-1 in THP-1 cells incubated with high glucose and AGEs
[Objectives]
1.To investigate into the effects of glucose and advanced glycation end products (AGEs) on ROS outputs in THP-1 cells.
2.To investigate into the effects of high glucose combinded with AGEs on oxidative stress in THP-1 cells.
3.To investigate into the effects of glucose and AGEs on expressions of HO-1 in THP-1 cells.
4.To investigate if p38MAPK signal pathway were involved in oxidative stress in THP-1 cells induced by high glucose and AGEs.
[Methods]
1.Cell culture:THP-1 cells were cultured at 37℃,5%CO_2 with RPMI-1640 containing 10%FCS,penicillin(100units/mL),and streptomycin(100μg/mL).
2.Preparation of AGEs:Briefly,BSA and AGEs were prepared by incubation of 3.55 mg/mL BSA in the presence or absence of glucose and 0.5 mmol/L sodium azide in PBS(pH 7.4) at 37℃for 8 weeks.
3.Determination of ROS outputs:Cells were resuspended in medium containing 10mmol/L of DCFH-DA(Molecular Probes),and incubated for 30min.And then flow cytometric analysis was used by FACScan(Becton-Dickinson,Mountain View,CA) at excitation 488 nm and emission 525nm to detect the mean fluorescence intensity(MFI) as ROS output.
4.MDA assay:By TBA assay method.
5.TNF-a ELISA assay:By enzyme linked immunosorbent assay(ELISA).
6.RT-PCR for HO-1mRNA:Total RNA was isolated using TRIzol reagents (Invitrogen).The electrophoresis of the amplified products was operated with 1.5%agarose gel.
7.Western blot analysis for HO-1 expression:Cytoplasmic extracts were separated from the nuclei.An equal amount of protein(30mg/well) for each sample was boiled for 5 min,loaded,and run for electrophoresis in a 4-20%Tris-Glycine gel. The gel was transferred electrophoretically to nitrocellulose membranes,and the blots were blocked with 5%milk in TBST.Membranes were then probed with a polyclonal rabbit antibody against HO-1 and with a horseradish peroxidaseconjugated anti-rabbit IgG.For quantification,bands in photo-graphs were scanned by a densitometer linked to a computer system.
8.p38MAPK signal pathway:There were 4 groups:15mmol/L-GLU group, SB+15mmol/L-GLU group,100μg/mL-AGEs group and SB+100μg/mL-AGEs group.SB group means the cells were stimulated by 10μmol/L SB203580 (inhibitor of p38MAPK signal pathway) before incubation with 15mmol/L GLU or 100μg/mL AGEs for 24h.
9.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.Pearson coefficient correlation was used too.α=0.05.
[Results]
1.Concentration of AGEs:The fluorescence intensity of AGEs showed 7.40-fold (excitation 360 nm,emission 430nm) higher than that of BSA control.The concentration of AGEs was 101.29U/mg protein,while BSA was 13.68U/mg.
2.ROS output(mean fluorescence intensity,MFI):GLU+AGEs group(107.21±8.94) has significantly higher ROS outputs than GLU group(65.88±1.61) and AGEs group(67.46±3.78)(P＜0.05),while that of AGEs group and GLU group were all significantly higher than control group(41.95±1.36)(P＜0.05).
3.MDA(nmol/mL):GLU+AGEs group(3.26±0.32) has significantly higher MDA than GLU group(1.59±0.53) and AGEs group(1.56±0.97)(P<0.05),and that of GLU group was significantly higher than control group(P＜0.05).But there was no significantly difference between AGEs group and control group(0.65±0.23) (P＞0.05).
4.TNFa(pg/mL):GLU+AGEs group(51.25±1.72) has significantly higher TNFa than GLU group(25.38±1.95) and AGEs group(24.43±2.65)(P＜0.05),and that of GLU group and AGEs group were significantly higher than that of control group(14.97±1.49)(P＜0.05).
5.HO-1mRNA:Expression of HO-1mRNA of GLU+AGEs group(0.89±0.12) was significantly higher than that of GLU group(0.42±0.02) and AGEs group (0.48±0.03)(P＜0.05).And GLU group and AGEs group have significantly higher HO-1mRNA than control group(0.15±0.01)(P＜0.05).
6.HO-1 protein:GLU+AGEs group(0.81±0.02) has significantly higher HO-1 protein than GLU group(0.39±0.01) and AGEs group(0.45±0.03)(P＜0.05).And GLU group and AGEs group have significantly higher level than control group (0.15±0.01)(P＜0.05).
7.Correlation of ROS,MDA,TNF-a and HO-1mRNA and its protein:There was significantly positive correlation between the output of the expression of HO-1 mRNA and ROS,MDA(r=0.858,P=0.000) and TNFα(r=0.952,P=0.000).There were alos correlation between ROS output and HO-1mRNA(r=0.990,P=0.000), expression of HO-1 protein,MDA(r=0.873,P=0.000) and TNFα(r=0.964, P=0.000).And there were correlation between TNFαand ROS,MDA(r=0.851, P=0.000),HO-1mRNA and its protein.
8.There were no significant difference in expressions of HO-1 mRNA between GLU group,SB+GLU group,AGEs group and SB+AGEs group.
[Conclusions]
1.ROS output of THP-1 cells has been exacerbated when incubated with high glucose or AGEs and presented as dose-,time-dependent increasing. Furthermore,AGEs combined with high glucose group has much more ROS output than high glucose or AGEs group.These suggest that there is a synergistic relationship between high glucose and AGEs in oxidative stress.AGEs combined with high glucose could induce more MDA and TNFαin THP-1 cells.
2.The expression of HO-1mRNA and protein in THP-1 cells treated with high glucsoe combined AGEs has been increased significantly,which hint HO-1 could be an effective protective factor for oxidative stress induced by high glucose and AGEs in THP-1 cells.And some methods which can induce high expression of HO-1 may help to resist oxidative injury induced by high glucose and AGEs.
3.p38MAPK signal pathway may not be involved in the transcription of HO-1 in THP-1 cells treated by high glucose and AGEs.
Chapter 2
Effects of ZnPP(inhibitor of HO-1) on oxidative stress in THP-1 cells induced by high glucose and AGEs
[Objective]
1.To investigate into the effects of ZnPP(inhibitor of HO-1) on oxidative stress induced by high glucose and AGEs in THP-1 cells.
[Methods]
1.Groups:There were 4 groups as control group,15mmol/LGLU+100μg/mL AGEs group,ZnPP group,ZnPP+15mmol/LGLU+100μg/mLAGEs group.The concentration of glucose in the cell culture solution of control group and ZnPP group were all 5mmol/L.
2.Determination of ROS output,MDA,TNF-a,HO-1mRNA and proteins:Just the same as Chapter 1.
3.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.α=0.05.
[Result]
1 ROS output(mean fluorescence intensity,MFI):ZnPP+GLU+AGEs gorup (128.81±5.53) has significantly higher ROS output than GLU+AGEs group (107.21±8.94) and ZnPP group(51.56±2.09)(P＜0.05).ROS output of GLU+AGEs gorup and ZnPP group were significantly higher than that of control group(41.95±1.36)(P＜0.05).
2 MDA(nmol/mL):ZnPP+GLU+AGEs group(3.75±1.25) has significantly higher MDA than ZnPP group(1.61±0.10)(P＜0.05),but there were no significantly difference between ZnPP+GLU+AGEs group and GLU+AGEs group(3.26±0.32) (P＞0.05).MDA of GLU+AGEs group and ZnPP group were significantly higher than that of control group(0.65±0.23)(P＜0.05).
3 TNFa(pg/mL):ZnPP+GLU+AGEs group(141.10±12.84) has significantly higher TNFa than GLU+AGEs group(38.09±14.97) and ZnPP group(27.34±10.18) (P＜0.05).TNFa of GLU+AGEs group and ZnPP group were significantly higher than that of control gorup(18.55±7.26)(P＜0.05).
4 HO-1mRNA:ZnPP+GLU+AGEs group(0.39±0.02) has significantly lower expression of HO-1mRNA than GLU+AGEs group(0.89±0.09)(P＜0.05),and significantly higher than ZnPP group(0.24±0.02)(P＜0.05).GLU+AGEs group and ZnPP group have higher HO-1mRNA than control group(0.15±0.01) (P＜0.05).
5 HO-1 protein:ZnPP+GLU+AGEs group(0.38±0.00) has significantly lower HO-1 protein than GLU+AGEs group(0.81±0.02)(P＜0.05),and significantly higher than ZnPP group(0.23±0.03)(P＜0.05).GLU+AGEs group and ZnPP group have higher HO-1 protein than control group(0.15±0.01)(P＜0.05).
[Conclusion]
1.ZnPP itself could induced oxidative stress,injury and inflammation in THP-1 cells.ZnPP,as an inhibitor of HO-1,could aggravate oxidative stress and inflammation in THP-1 induced by high glucose and AGEs.
Chapter 3
Effects of CoPP(inducer of HO-1) on oxidative stress in THP-1 cells induced by high glucose and AGEs
[Objectives]
1.To investigate into the effects of CoPP(inducer of HO-1) on oxidative stress induced by high glucose and AGEs in THP-1.
[Methods]
1.Groups:There were 4 groups as control group,15mmol/LGLU+100μg/mLAGEs group,CoPP group,CoPP+15mmol/L-GLU+100μg/mL-AGEs group.The concentration of glucose in the cell culture solution of control group and CoPP group were all 5mmol/L.
2.Determination of ROS output,MDA,TNF-a,HO-1mRNA and proteins:Just the same as Chapter 1.
3.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.α=0.05.
[Results]
1 ROS output(mean fluorescence intensity,MFI):GLU+AGEs has the main effect on ROS output(F=287.930,P=0.000),while CoPP has not the main effect (F=0.433,P=0.529),and there were interact between GLU+AGEs and CoPP (F=8.632,P=0.019),which means with the GLU+AGEs,CoPP could lower the ROS output.ROS output of GLU+AGEs group was significantly higher than control group(41.95±1.36)(P＜0.05),and there were no significantly difference between CoPP group and control group(P＞0.05).
2 MDA(nmol/mL):CoPP+GLU+AGEs group(1.28±0.61) has significantly lower MDA than GLU+AGEs group(3.26±0.32)(P＜0.05),and there were no significantly difference between CoPP group(0.73±0.45) and control group (0.65±0.23)(P＞0.05).GLU+AGEs group has significantly higher MDA than control group(P＜0.05),and there were no difference between CoPP group and control group(P＞0.05).
3 TNFa(pg/mL):There were no significantly difference between CoPP+GLU+ AGEs group(31.90±12.56),GLU+AGEs group(43.60±12.40) and CoPP group (16.60±7.99)(P＞0.05).GLU+AGEs group has higher TNFa than control group (14.97±1.49)(P＜0.05),while there were no difference between CoPP group and control group(P＞0.05).
4 HO-1mRNA:There were no significantly difference between CoPP+GLU+ AGEs group(0.91±0.01) and GLU+AGEs group(0.89±0.12) and CoPP group (16.60±7.99)(P>0.05).CoPP+GLU+AGEs group has higher HO-1mRNA than CoPP group(0.24±0.01)(P＜0.05).CoPP group and GLU+AGEs group have higher level than control group(P＜0.05).
5 HO-1 protein:CoPP+GLU+AGEs group(0.87±0.01) has higher HO-1 protein than GLU+AGEs group(0.81±0.02) and CoPP group(0.22±0.02)(P＜0.05). CoPP group and GLU+AGEs group has higher level than control group (0.15±0.01)(P＜0.05).
[Conclusions]
1.CoPP,an inducer of HO-1,could effectively decrease oxidative stress in THP-1 induced by high glucose and AGEs,while CoPP itself has not cause oxidative stress.Though CoPP has decreased the level of TNFa in THP-1 cells treated by high glucose and AGEs there was no significantly differece.Certainly CoPP significantly enhanced the expression of HO-1 protein but not HO-1mRNA.
2.HO-1 as a stress protein could protect THP-1 cells against oxidative injury induced by high glucose and AGEs.Methods which could increase the expression of HO-1 would help to resist oxidative injury induced by high glucose and AGEs.
Chapter 4
Relationship between Transcription of HO-1 in Peripheral Blood Mononuclear Cells of Newly Diagnostic Type 2 Diabetic Patients with Chronic Complications and Oxidative Stress
[Objectives]
1.To investigate into the relationship between transcription of HO-1 in peripheral blood mononulclear cells(PBMC) of newly diagnostic type 2 diabetic patients with chronic complications and oxidative stress
[Methods]
1.Subjects were all newly diagnostic type 2 diabetes from our department.The diagnosis and classification criteria of type 2 diabetes mellitus was 1999 WHO criteria.Any acute metabolic disturbance and any drug intake were excluded. Diabetic chronic complications were determined by retinal photograph,feeling check and 24h urine albumin excretion.There were three groups:normal control group,non-complications group and complications group.
2.Peripheral blood mononuclear cells(PBMC):All of subjects were taken blood samples after fasting for 8～14 hours.PBMC were seperated by ficoll-urografin density gradient centrifugation.Serum of samples were kept in-20℃for MDA analysis.
3.ROS output of PBMC,serum MDA and expression of HO-1mRNA:Just the same as Chapter 1.
4.Expression of HO-1 protein:Immunohistochemistry was used to detect the expression of HO-1 protein.Disposition of HO-1 in the kytoplasm was analyzed by fluorescence microscope.cytoplasm has green fluorescent which means positive which were calculated.
5.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.The difference between two groups was analyzed by two-independent t test.Pearson Correlation was used to analyse the correlation among multiple variables.The received level of significance isα=0.05.
[Results]
1.Compared with non-complications group,complications group has higher fasting blood glucose(16.26±6.67 vs 10.61±3.61),HbAlc(12.54±2.10 vs 10.51±1.86), ROS output(419.60±216.31 vs 172.38±145.24),serum MDA(66.04±13.67 vs 0.77±0.23),HO-1mRNA(1.02±0.27 vs 0.77±0.23) and protein(85.47±10.89 vs 61.45±21.81)(P＜0.05).
2.There was positive correlation between HO-1mRNA and protein(r=0.750, P=0.000),MDA(r=0.449,P=0.010),FPG(r=0.364,P=0.041) and 2hPG(r=0.477, P=0.016).Expression of HO-1 protein was correlated with ROS output(r=0.608, P=0.000),MDA(r=0.623,P=0.000).There were positive correlation between MDA and ROS output(r=0.788,P=0.000),HbAlc and 2hPG(r=0.412,P=0.041), 2hPG and FPG(r=0.811,P=0.000).
[Conclusions]
1.There are obvious oxidative stress in newly diagnostic type 2 diabetic patients with chronic complications and hyperglycemia may induce oxidative stress which then results in diabetic chronic complications.
2.Correlation analysis hint that oxidative stress induced by diabetes mellitus could upregulate HO-1.Up-regulation of HO-1 could help to resist against oxidative injury induced by diabetes mellitus though it may not be enough to entirely protect cells.It would be a new pathway to prevent diabetic chronic complications by upregulating HO-1.
目前糖尿病在全球尤其是发展中国家的患病人数急剧增加,已成为严重危害人类健康的流行性疾病。由于糖尿病慢性并发症可引起失明、尿毒症、截肢等严重后果,已成为目前全世界共同关注的公共卫生问题,也是目前的防治重点和难点。
研究认为,糖尿病慢性并发症的发病机制主要包括四大代谢通路的激活,即晚期糖基化终产物(advanced glycation end products,AGEs)、己糖胺(hexosamine)、蛋白激酶C(protein kinase C,PKC)和多元醇(polyol)通路。近年来Brownlee研究发现,抑制线粒体活性氧簇(reactive oxygen species,ROS)的产生可阻断上述四大代谢通路,因而提出了统一机制学说,认为高糖导致的氧化应激是糖尿病慢性并发症发病机制的中心环节,可以将上述四条代谢通路统一起来(如图1所示)。在正常情况下,线粒体氧化磷酸化过程中约有0.4%～4%的氧转化为超氧阴离子自由基(·O_2~-),少量的·O_2~-能被机体抗氧化保护机制所消除。但在高糖情况下,线粒体电子传递链产生的电子明显增多,产生过多的活性氧簇(reactive oxygen species,ROS),造成大分子物质脱氧核糖核酸(deoxyribonucleic acid,DNA)损伤,后者激活聚二磷酸腺苷核糖聚合酶(polyADP-ribose polymerase,PARP)进行DNA修复,该过程消耗大量NAD+(oxidizedform of nicotinamide-adenine dinucleotide,氧化型烟酰胺腺嘌呤二核苷酸),使NADH/NAD+比例增加,抑制了磷酸甘油醛脱氢酶(glyceraldehyde phosphatedehydrogenase,GAPDH)活性,导致葡萄糖的正常代谢通路受阻,中间产物积聚转而进入上述四大代谢途径,后者均能进一步促使细胞内ROS产量增加,形成恶性循环;同时抗氧化剂大量消耗,使细胞处于氧化与抗氧化失衡的应激状态,严重影响细胞功能,并加速细胞凋亡,抗氧化治疗则能阻止上述氧化应激所致的细胞凋亡。因此,目前认为,氧化应激是糖尿病慢性并发症发病机制的中心环节,拮抗氧化应激对糖尿病慢性并发症的防治极具治疗潜力。虽然严格控制血糖对拮抗高糖引起的氧化应激具有重要的意义,但目前全球糖尿病的血糖控制水平还很差,仅约1/3左右的患者达到了血糖的理想控制,因此,必须从抗氧化防御系统寻求合适的拮抗氧化应激的手段。
除经典的抗氧化酶如过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽系统[不含硒谷胱甘肽过氧化物酶(glutathion peroxidase,GPx)-谷胱甘肽硫转移酶(glutathione S-transferase,GST)]等之外,近年来,血红素加氧酶-1(heme oxygenase,HO-1)在拮抗氧化应激方面的作用正日益受到关注。HO有三种同工酶,其中HO-2基因定位于人染色体16p13,存在于大多数组织细胞中,生理状况下组织中的HO活性主要由HO-2决定,HO-2属于结构型,几乎不受外界刺激因素的影响,目前仅知大脑组织中HO-2的转录受肾上腺皮质激素的调节。HO-3是新近发现的一种HO,与HO-2有90%的氨基酸同源性,HO-3催化血红素降解的能力很低,其主要作用可能为介导HO与血红素结合。HO-1是诱导型,与HO-2在基因结构、调节上仅有约40%的相似性,HO-1基因定位于人染色体22q12,广泛分布于哺乳动物多种组织细胞的微粒体内,正常情况下表达量很低(只在脾、肝脏和睾丸中有较高表达),但多种应激性因素如重金属、细胞因子、紫外线、氧化应激、炎症因子等均可诱导HO-1表达及活性增强。由于HO-1蛋白的分子量在31～33KD之间,因此有人将HO-1归属为热休克蛋白32,它是目前已知最容易被诱导的蛋白,诱导后活性可升高至100倍。
研究认为,HO-1是体内最重要的内源性抗氧化保护因子之一,在许多病理生理过程中发挥重要的调节作用,尤其是对组织器官的保护作用,已成为目前包括器官移植、缺血再灌注损伤、心脑血管病、支气管哮喘、先兆子痫等多个领域的研究热点。HO是体内唯一催化血红素分解代谢的限速酶,HO的催化作用需要微粒体NADPH(reduced form of nicotinamide-adenine dinucleotidephosphate,还原型烟酰胺腺嘌呤二核苷酸磷酸)—细胞色素P450还原酶协同参与电子转移,它为分子氧的激活和铁的还原提供还原等价物,即HO蛋白结合血红素,并与细胞色素P450还原酶一起形成一个短暂的电子传递链,使卟啉环异构体特异性地分离,激活的氧和血红素分子的α中间碳桥相互作用,使该位置的四吡咯环裂解,形成胆绿素,并使碳桥转变为一氧化碳(carbon monoxide,CO),当卟啉环打开时螯合铁也被释放出来。最终HO将血红素降解为CO、铁和胆绿素,后者继续在胆绿素还原酶(biliverdin reductase,BVR)的作用下生成胆红素(bilirubin,BR),这些代谢产物均有抗氧化、抗炎、抗凋亡、信号传导和免疫调节等作用(图2)。CO是一种小分子气态物质,大量研究证实CO与一氧化氮(nitric monoxide,NO)类似,也是一种重要的细胞信使分子,它可以通过扩散以自分泌或旁分泌方式与自身或邻近细胞相互作用,激活可溶性鸟苷酸环化酶(soluble guanylate cyclase,sGC),后者催化三磷酸鸟苷(guanosinetriphosphate,GTP)生成环磷酸鸟苷(cyclic guanylic acid,cGMP),激活cGMP依赖性蛋白激酶、磷酸二酯酶,或通过调节离子通道而传递生理信息,达到舒张血管平滑肌、抑制平滑肌细胞增殖并抑制血小板聚集等作用。此外,CO也可通过cGMP非依赖途径发挥血管舒张效应,包括对某种钾通道的影响,增加脂联素水平等。采用锌原卟啉(zinc protoporphyrin,ZnPP)抑制HO活性可增加颈动脉体感受器活性,而CO可逆转该反应,提示CO在该感受器可能作为神经递质而发挥作用。CO还可能调节或抑制NOS,并与高血压及内皮功能紊乱有关。尽管与NO相比,CO舒张血管的效应相对较小,但CO通过cGMP介导的血管舒张潜能可能被其它制剂所大大增加。胆绿素和胆红素是体内丰富的抗氧化物质,流行病学研究发现低血清胆红素水平与缺血性心脏病密切相关,在调整了年龄、吸烟、胆固醇、收缩压等已知的冠状动脉硬化性心脏病(coronary heart disease,CHD)的危险因素后,这种相关性仍十分显著,因而认为低胆红素血症是CHD的独立危险因素。近年的研究发现胆红素和胆绿素可以维持内皮细胞的完整性,防止内皮细胞凋亡,促进血管反应性和防止血管硬化。胆红素可以抑制NADPH氧化酶和PKC的活性,而后两者介导了血管紧张素Ⅱ诱导的血管损伤;胆红素还可通过增加NO的生物活性而降低糖尿病动物模型体内的氧化应激水平。一般认为,铁通过Fenton反应导致羟自由基形成,发挥致氧化应激的作用,但HO衍生而来的铁可诱导铁蛋白表达和合成增加,后者具有抗氧化、抗凋亡等细胞保护功能,抑制铁沉积,对抗氧化型LDL所致内皮细胞的氧化应激,并被证实是一种保护上皮细胞的抗氧化剂。上调HO-1可增加铁蛋白合成,后者可捕获HO-1途径释放的铁,并通过与细胞表面受体结合将铁转运至细胞内。
研究认为,HO-1在拮抗高糖所致氧化损伤中发挥了十分重要的保护作用。高糖诱导的ROS生成增加在糖尿病慢性血管并发症的发生和发展中具有重要作用。增加的·O_2~-形成和HO-1诱导不足使内皮功能紊乱甚至细胞凋亡,但可被抗氧化剂或通过诱导抗氧化酶而逆转,诱导HO-1表达可拮抗氧化应激从而提供血管保护作用。糖尿病鼠体内ROS含量明显增高、HO-1活性不足、内皮细胞凋亡增加,给予HO-1诱导剂后内皮受到了保护,相反给予HO-1抑制剂后上述损害加重。转染人HO-1基因可降低高糖介导的循环内皮细胞脱落数量、·O_2~-的形成及尿8-异前列烷的产生,并提供血管保护作用,而下调鼠HO-1表达可加速这些不良病理过程。高表达HO-1可使糖尿病肥胖小鼠的血清脂联素水平和胰岛素敏感性增加,使内脏和腹部脂肪减少,以及血浆肿瘤坏死因子α(tumornecrosis factorα,TNFα)、白介素-6(interleukin 6,IL-6)和IL-1β水平降低。采用钴原卟啉(cobalt protoporphyrin,CoPP)诱导胰岛β细胞高表达HO-1可拮抗细胞氧化损伤,使TNFα诱导的β细胞凋亡百分比从75%降至5%,并受p38MAPK(mitogen-activated protein kinase,MAPK)信号途径调节,该研究认为促进HO-1高表达是一种新型的保护胰岛β细胞的可行的手段。体内外实验均证实,采用CoPP上调HO-1可使骨髓以及体外培养的间充质干细胞的脂肪形成减少,培养基上清脂联素水平增加。采用各种手段促进HO-1表达还可有效增强组织和细胞抗缺血缺氧的能力,相反,抑制HO-1表达将加重心肌缺血再灌注损伤。HO-1基因转染大鼠平滑肌细胞使其高表达HO-1还可提高细胞在过氧化物所致氧化应激中的存活率,而HO-1抑制剂锌原卟啉(zinc protoporphyria,ZnPP)预处理可使该保护作用明显减弱,因此认为HO-1具有降低动脉硬化发生危险的潜力。STZ诱导的糖尿病大鼠在缺血/再灌注后心肌的HO-1表达受损,心梗面积增加,在缺血前给予高铁血红素诱导HO-1后其HO-1表达仍低于非糖尿病大鼠。AGEs刺激小鼠巨噬细胞RAW264.7可使其HO-1蛋白表达增加,并具有一定的浓度依赖性。探讨高糖和AGEs分别刺激及二者联合刺激对单核细胞HO-1表达的影响可为进一步分析HO-1在糖尿病氧化应激中的作用提供研究基础。目前认为,线粒体功能异常参与了糖尿病慢性并发症的发病机制。人HO-1基因转染糖尿病大鼠可促使其线粒体转运载体的功能恢复,研究发现这与AKT磷酸化水平增加相关,后者和Bcl-xL水平增加可防止糖尿病β细胞数量减少。体内外实验均证实线粒体功能改变与AKT和Bcl-2家族蛋白激活相关,AKT激活可促进ATP合成以及己糖激酶与电压依赖的离子通道的联系,电压依赖的离子通道关闭可阻断细胞色素c的释放;而Bcl家族成员减少参与了细胞色素c由线粒体向胞浆的转位。这些研究结果均提示氧化应激在糖尿病慢性并发症的发生和发展中具有重要作用,采用适当的手段诱导HO-1高表达为细胞提供保护作用可能是极具潜力的一种新的糖尿病慢性并发症的防治手段。
然而,目前对HO-1在糖尿病氧化应激中的作用及其机制尚未完全阐明,本课题拟从体内外实验两方面对HO-1在拮抗糖尿病氧化应激中的作用进行探讨,为寻求有效的拮抗糖尿病氧化应激的手段提供科学的依据。
第一章HO-1在高糖和AGEs致THP-1细胞氧化应激中的表达变化研究
【目的】
1.探讨不同浓度葡萄糖、晚期糖基化终产物(AGEs)在不同刺激时间对THP-1细胞ROS产量的影响;
2.了解高糖和AGEs联合刺激对THP-1细胞氧化应激的影响;
3.了解HO-1在高糖和AGEs所致THP-1细胞氧化应激中的表达变化;
4.探讨p38MAPK信号通路是否参与了高糖和AGEs诱导的THP-1细胞HO-1表达。
【方法】
1.细胞培养:采用含10%胎生血清的RPMI1640培养基,于37℃、5%CO_2培养箱内传代培养人单核细胞株THP-1。
2.体外制备AGEs:将牛血清白蛋白(bovine serum albumin,BSA)与葡萄糖混合于37℃孵育60天,4℃透析24tl以去除未结合的葡萄糖。
3.细胞ROS产量检测:分别用5、15、25mmol/L的GLU或25、50、100μg/mL的AGEs刺激细胞,分别于刺激0.5、2、6、24h后收集细胞,DCFH-DA荧光探针标记,流式细胞仪检测平均荧光强度(mean fluorescence intensity,MFI)。
4.细胞分组:分为4组,即15mmol/LGLU组(高糖组)、100μg/mLAGEs组(AGEs组)、15mmol/LGLU+100μg/mLAGEs组(高糖+AGEs联合组)及对照组。
5.细胞培养液上清TNFa水平检测:采用ELISA方法进行。
6.细胞培养液上清中MDA水平检测:采用硫代巴比妥酸法(thiobarbituric acid,TBA),严格按照试剂盒说明步骤检测。
7.RT-PCR法检测HO-1mRNA表达:提取总核糖核酸(ribonucleic acid,RNA),两步法进行RT-PCR,扩增产物于1.5%琼脂糖凝胶进行电泳。
8.免疫印记(western blot,WB)法检测HO-1蛋白表达:提取总蛋白,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectropheresis,SDS-PAGE),电转移至硝酸纤维素膜(nitrocellulose filter,NC)封闭非特异位点。利用多克隆兔抗人HO-1抗体及HRP标记的二抗与NC反应,DAB显色后拍照分析条带亮度。
9.SB203580(SB,p38MAPK抑制剂)干预实验:分为高糖组、SB+15mmol/LGLU(SB+高糖)组、AGEs组、SB+100μg/mLAGEs(SB+AGEs)组。10μmo/LSB干预30min后换以含15mmol/LGLU或100μg/mL AGEs细胞培养液继续孵育24h,收集细胞进行RT-PCR检测HO-1mRNA表达水平。
10.统计学处理:用SPSS13.0软件对所有数据进行统计学处理;实验数据计量资料以均数±标准差((?)±s)表示。多组间资料比较用析因设计的方差分析(ANOVA),任意两组间比较采用独立样本的t检验,相关分析采用积矩相关系数(Pearson相关系数)分析,检验标准为α=0.05。
【结果】
1.制备的AGEs浓度:AGEs的荧光浓度为101.29U/mg,对照BSA的荧光浓度为13.68 U/mg,前者为后者的7.40倍。
2.GLU和AGEs刺激THP-1细胞的ROS产量:均表现为0.5h的ROS产量急剧升高,随着时间延长轻度下降,但于24h再度上升至较高水平,在6h、24h时间点的ROS产量表现为GLU和AGEs浓度依赖性升高。高糖组(65.88±1.61)和AGEs组(67.46±3.78)的ROS产量均显著高于对照组(41.95±1.36)(P＜0.001),高糖+AGEs联合组(107.21±8.94)显著高于GLU组和AGEs组(P＜0.01),且高糖和AGEs对ROS产量的影响具有协同作用(P＜0.05)。
3.细胞培养液上清的MDA水平(nmol/mL):高糖组(1.59±0.53)显著高于对照组(0.65±0.23)(P＜0.05),AGEs组(1.56±0.97)虽然也高于对照组,但统计学差异不明显(P＞0.05),高糖+AGEs联合组(3.26±0.32)显著高于GLU组和AGEs组(P＜0.05)。
4.细胞培养液上清的TNFa水平(pg/mL):高糖组(25.38±1.95)和AGEs组(24.43±2.65)显著高于对照组(14.97±1.49)(P＜0.01),高糖+AGEs联合组(51.25±1.72)显著高于高糖组及AGEs组(P＜0.001),高糖和AGEs对TNFa的影响存在协同作用(P＜0.001)。
5.细胞HO-1mRNA的表达:高糖组(0.42±0.02)和AGEs组(0.48±0.03)显著高于对照组(0.15±0.01)(P＜0.001),高糖+AGEs联合组(0.89±0.12)显著高于高糖组及AGEs组(P＜0.05)。
6.THP-1细胞的HO-1蛋白表达:高糖组(0.39±0.01)和AGEs组(0.45±0.03)显著高于对照组(0.15±0.01)(P＜0.05),高糖+AGEs联合组(0.81±0.02)显著高于高糖组及AGEs组(P＜0.05),高糖和AGEs存在协同作用(P＜0.01)。
7.相关分析:HO-1蛋白表达水平与HO-1mRNA、ROS产量、MDA及TNFα均呈显著正相关(P＜0.001)。HO-1mRNA与ROS、MDA及TNFα均呈显著正相关(P＜0.001)。ROS产量与MDA、TNFα呈显著正相关(P＜0.001)。TNFα与MDA呈显著正相关(P＜0.001)。
8.SB203580(SB)(p38MAPK抑制剂)干预实验结果:各组之间HO-1mRNA表达均无显著的统计学差异(P＞0.05)。
【结论】
1.高糖和AGEs单独刺激能导致THP-1细胞氧化损伤加剧、分泌炎症因子TINFα水平及HO-1表达增高,二者联合刺激能使上述改变进一步加剧。
2.p38MAPK抑制剂对高糖和AGEs诱导的THP-1细胞HO-1mRNA表达无显著影响。
3.HO-1表达与细胞氧化损伤及炎症指标呈显著正相关,提示高糖和AGEs导致THP-1细胞氧化损伤及炎症反应,后者诱导HO-1表达适应性和代偿性增高,从而发挥细胞保护作用。
第二章抑制HO-1对高糖和AGEs致THP-1细胞氧化应激的影响
【目的】
探讨抑制HO-1表达对高糖和AGEs刺激下THP-1细胞氧化应激的影响。
【方法】
1.细胞分组:分为4组,即对照组、15mmol/LGLU+100μg/mLAGEs(GLU+AGEs)组、ZnPP组及ZnPP+15mmol/LGLU+100μg/mLAGEs(ZnPP+GLU+AGEs)组,其中对照组和ZnPP组THP-1细胞培养液的GLU浓度均为5mmol/L。
2.细胞ROS产量、细胞培养液上清中MDA及TNFa水平、HO-1mRNA及蛋白表达水平检测同第一章。
3.统计学处理:用SPSS13.0软件对所有数据进行统计学处理。实验数据计量资料以均数±标准差((?)±s)表示。多组间资料比较用析因设计的方差分析(ANOVA),任意两组间比较采用独立样本的t检验,检验标准为α=0.05。
【结果】
1.各组细胞ROS产量(MFI)的比较:ZnPP+GLU+AGEs组(128.81±5.53)、GLU+AGEs组(107.21±8.94)和ZnPP组(51.56±2.09)均显著高于对照组(41.95±1.36)(P＜0.05),而ZnPP+GLU+AGEs组显著高于GLU+AGEs组及ZnPP组(P＜0.05)。
2.各组细胞培养液上清MDA水平(nmol/mL)的比较:ZnPP组(1.61±0.10)、GLU+AGEs组(3.26±0.32)、ZnPP+GLU+AGEs组(3.75±1.25)的MDA水平均显著高于对照组(0.65±0.23)(P＜0.01),其中ZnPP+GLU+AGEs组显著高于ZnPP组(P＜0.05),但与GLU+AGEs组相比无显著性差异(P＞0.05)。
3.各组细胞培养液上清TNFa水平(pg/mL)的比较:ZnPP组(27.34±10.18)、GLU+AGEs组(38.09±14.97)、ZnPP+GLU+AGEs组(141.10±12.84)的TNFa水平均显著高于对照组(18.55±7.26)(P＜0.05),其中ZnPP+GLU+AGEs组显著高于GLU+AGEs组(P＜0.05)。
4.各组细胞HO-1mRNA表达的比较:无论对照组还是各刺激组均可见到与设计片段大小相符的HO-1基因扩增条带(637bp)及内参照β-actin(285bp)。ZnPP组(0.24±0.02)、GLU+AGEs组(0.89±0.09)、ZnPP+GLU+AGEs组(0.39±0.02)的HO-1基因表达水平均显著高于对照组(0.15±0.01)(P＜0.01),其中ZnPP+GLU+AGEs组显著低于GLU+AGEs组(P＜0.05),并显著高于ZnPP组(P＜0.01)。
5.各组细胞HO-1蛋白表达的比较:ZnPP组(0.23±0.03)、GLU+AGEs组(0.81±0.02)、ZnPP+GLU+AGEs组(0.38±0.00)的HO-1蛋白表达水平均显著高于对照组(0.15±0.01)(P＜0.05),其中ZnPP+GLU+AGEs组显著低于GLU+AGEs组(P＜0.01),并显著高于ZnPP组(P＜0.01)。
【结论】
HO-1抑制剂ZnPP本身也能对THP-1细胞造成一定的氧化损伤,并引起HO-1表达增高,ZnPP预处理能显著抑制高糖和AGEs引起的HO-1表达增高,进一步加剧高糖和AGEs引起的细胞氧化损伤及分泌炎症因子TNFa的水平。
第三章诱导HO-1对高糖和AGEs致THP-1细胞氧化应激的影响
【目的】
探讨诱导HO-1高表达是否可对抗高糖和AGEs所致THP-1细胞的氧化损伤。
【方法】
1.细胞分组:分为4组,即对照组、15mmol/LGLU+100μg/mLAGEs(GLU+AGEs组)、CoPP组、CoPP+15mmol/LGLU+100μg/mL AGEs(CoPP+GLU+AGEs组),其中对照组和CoPP组THP-1细胞培养液的GLU浓度均为5mmol/L。
2.细胞ROS产量、细胞培养液上清中MDA及TNFa水平、HO-1mRNA及蛋白表达水平检测同第一章。
3.统计学处理:用SPSS13.0软件对所有数据进行统计学处理;实验数据计量资料以均数±标准差((?)±s)表示。多组间资料比较用析因设计的方差分析(ANOVA),各组内均数比较采用独立样本的t检验或单向方差分析(One-way ANOVA),多重比较采用LSD法(Least-significant difference test)。检验标准为α=0.05。
【结果】
1.各组细胞ROS产量(MFI)的比较:GLU+AGEs组(107.21±8.94)和CoPP+GLU+AGEs组的ROS产量(95.42±2.36)显著高于对照组(41.95±1.36)(P＜0.05),而CoPP组(49.42±6.45)与对照组相比无显著性差异(P＞0.05)。
2.各组细胞培养液上清的MDA水平(nmol/mL)的比较:GLU+AGEs组的MDA水平(3.26±0.32)明显高于对照组(0.65±0.23)及CoPP+GLU+AGEs组(1.28±0.61)(P＜0.01),而CoPP组(0.73±0.45)及CoPP+GLU+AGEs组与对照组之间无显著性差异(P＞0.05)。
3.各组细胞培养液上清的TNFa水平(pg/mL)的比较:GLU+AGEs组的TNFa水平(43.60±12.40)显著高于对照组(14.97±1.49)(P＜0.05),CoPP组(16.60±7.99)、CoPP+GLU+AGEs组(31.90±12.56)与对照组之间无显著性差异(P＞0.05),且CoPP+GLU+AGEs组与GLU+AGEs组、CoPP组相比无显著性差异(P＞0.05)。
4.各组细胞HO-1mRNA表达的比较:CoPP组(0.24±0.01)、GLU+AGEs组(0.89±0.12)、CoPP+GLU+AGEs组(0.91±0.01)的HO-1基因表达水平均显著高于对照组(0.15±0.01)(P＜0.01),其中CoPP+GLU+AGEs组与GLU+AGEs组相比无显著性差异(P＞0.05),但显著高于CoPP组(P＜0.05)。
5.各组细胞HO-1蛋白表达的比较:CoPP组(0.22±0.02)、GLU+AGEs组(0.81±0.02)、CoPP+GLU+AGEs组(0.87±0.01)的HO-1蛋白表达水平均显著高于对照组(0.15±0.01)(P＜0.01),其中CoPP+GLU+AGEs组显著高于GLU+AGEs组和CoPP组(P＜0.05)。
【结论】
在高糖和AGEs存在的情况下,HO-1诱导剂CoPP诱导HO-1蛋白表达增加,并显著抑制细胞氧化应激,明显减轻高糖和AGEs所致氧化损伤,而CoPP本身并不引起THP-1细胞氧化损伤和分泌炎症因子水平增加,结果提示诱导HO-1高表达可能是极具潜力的新的糖尿病慢性并发症的治疗靶点之一。
第四章2型糖尿病合并慢性并发症患者的外周血单核细胞HO-1表达与氧化应激的相关性研究
【目的】
1.了解初诊2型糖尿病(type 2 diabetes mellitus,T2DM)合并慢性并发症患者外周血单核细胞HO-1的表达情况;
2.探讨初诊T2DM合并慢性并发症患者外周血单核细胞HO-1表达与氧化应激的相关性。
【方法】
1.样本选择:来自我院内分泌代谢科门诊及住院的初次诊断T2DM的患者36例,健康志愿者10例。分为正常对照组、糖尿病无并发症组和糖尿病并发症组。
2.细胞收集:采集清晨空腹静脉抗凝血,Percoll法分离单核细胞。
3.细胞ROS产量、血清MDA水平及HO-1mRNA表达检测同第一章。
4.HO-1蛋白表达检测:采用免疫荧光法检测,将外周血单核细胞进行免疫荧光染色后,在荧光显微镜下观察HO-1在细胞内的表达情况。
5.统计学处理:用SPSS13.0软件对所有数据进行统计学处理;实验数据计量资料以均数±标准差((?)±s)表示。两组间资料比较用独立样本的t检验,相关分析采用积矩相关系数(Pearson相关系数)分析,检验标准为α=0.05。
【结果】
1.各组一般指标的比较:正常对照组的年龄(51.03±11.14)与两个糖尿病组之间无显著性差异(P＞0.05),BMI(20.91±2.37)和FPG(5.24±0.70)均分别显著低于两个糖尿病组(P＜0.01)。无并发症组的年龄(50.56±10.70 vs56.78±8.03)和BMI(24.60±2.25 vs 25.69±3.73)与并发症组相比无显著的统计学差异(P＞0.05),但并发症组的FPG(16.26±6.67 vs 10.61±3.61)、2hPG(18.76±7.05 vs 13.57±4.22)和HbAlc水平(12.54±2.10 vs 10.51±1.86)均显著高于无并发症组(P＜0.05)。
2.各组氧化应激指标的比较:正常对照组的血清MDA水平(40.71±20.30)和外周血单核细胞ROS产量(7.98±6.39)均显著低于两个糖尿病组(P＜0.01),而并发症组ROS产量(419.60±216.31 vs 172.38±145.24)、血清MDA水平(66.04±13.67 vs 0.77±0.23)明显高于无并发症组(P＜0.05)。
3.各组HO-1表达的比较:正常对照组的外周血单核细胞HO-1mRNA(0.44±0.26)和蛋白(16.37±15.01)表达水平均低于两个糖尿病组(P＜0.01),而并发症组HO-1mRNA(1.02±0.27 vs 0.77±0.23)及其蛋白(85.74±10.89 vs61.45±21.81)表达水平均显著高于无并发症组(P＜0.05)。
4.相关分析显示,HO-1蛋白表达水平与HO-1mRNA(r=0.750,P=0.000)、ROS(r=0.608,P=0.000)、MDA(r=0.623,P=0.000)呈显著正相关。HO-1mRNA与MDA(r=0.449,P=0.010)、FPG(r=0.364,P=0.041)及2hPG(r=0.477,P=0.016)呈显著正相关。MDA与ROS呈显著正相关(r=0.788,P=0.000)。
5.偏相关分析显示,在控制BMI、FPG、2hPG、HbAlc影响因素后,HO-1蛋白表达仍与ROS产量(r=0.567,P=0.027)、MDA(r=0.613,P=0.015)呈显著正相关,MDA与ROS产量呈显著正相关(r=0.911,P=0.000)。
【结论】
1.初诊T2DM患者的血糖、血清MDA、外周血单核细胞ROS产量及HO-1表达均显著高于正常对照组,提示初诊T2DM可能由于机体抗氧化防御机制的代偿性增高仍不足以对抗高血糖引起的氧化损伤,导致机体处于氧化应激状态。
2.并发症组的血糖水平、氧化应激指标及HO-1表达均显著高于无并发症组,提示高血糖及其引起的氧化损伤可能参与了初诊T2DM患者慢性并发症的发病机制,在排除BMI及血糖水平等因素的影响后,氧化应激指标仍与HO-1表达呈显著正相关,提示HO-1作为一种应激反应蛋白,在糖尿病所致氧化应激情况下代偿性表达增高,但仍不足以对抗该氧化应激。提示除严格控制血糖外,通过适当的手段诱导HO-1高表达将是极具潜力的糖尿病慢性并发症防治的新靶点之一。
[Background]
The patients of type 2 diabetes mellitus have increased rapidly in the world especially in the developing countries,which has been a threat to human's health as an epidemically disease.Since chronic diabetic complications can result in sight loss, uraemia and amputation,it has become difficult and important to prevent and treat diabetic complications.
Accumulated evidences have revealed advanced glycation end products(AGEs) as an important mechanism of chronic diabetic complications.AGEs describes a heterogeneous group of proteins,lipids,and nucleic acids that are formed nonenzymatically.AGE formation is enhanced in the presence of hyperglycemia and oxidative stress.AGE bind to their cognate cell-surface receptor,RAGE,resulting in the activation of postreceptor signaling,generation of intracellular oxygen free radicals,and the activation of gene expression.Thus,AGE are not only markers,but act also as mediators of late diabetic complications.The main mechanisms of diabetic complications include the ignition of four metabolic pathways:advanced glycation end products(AGEs),hexosamine,protein kinase C(PKC) and polynol pathway. Brownlee have found impressant of generation of reactive oxygen species(ROS) could hinder the four metabolic pathways as above mechanisms of diabetic complications.His theory considers high glucose could induce oxidative stress which results in the ignition of the four metabolic pathways(as fig1).
During the course of normal oxidative phosphorylation,however,between 0.4 and 4%of all oxygen consumed is converted into the free radical superoxide(·O_2~-) and is then either detoxified to H_2O and O_2 by glutathione peroxidase(in the mitochondria),or diffuses into the cytosol and is detoxified by catalase in peroxisomes.However,in the presence of reduced transition metals such as Cu or Fe, H_2O_2 can be converted to the highly reactive·OH radical.High glucose can result in electronicas increased which can result in the injury of DNA.After series of reactions the four metabolic pathways were ingited,which was a malignant circle.At the same time anti-oxygenics were comsumed.Oxidative stress was developed thus which can induce cells apoptosis.Accumulated evidences have proved hyperglycemia could result in the elevation of ROS output and induce oxidative stress since there were not enough anti-oxidative chemicals in diabetes.Oxidative stress can ignite the pathways sensitive to oxidative stress and result in some gene products.Thus chronic diabetic complications were developed.Though we have known some knowledgements about the mechnisms of chronic diabetic complications,there are limited methods directed against the mechanisms.
Considerable researches have proved the HO system play an important role in anti-oxidative stress.But the role of the HO system in diabetes,inflammation,heart disease,hypertension,neurological disorders,transplantation,endotoxemia and other pathologies is still a burgeoning area of research.The myriad metabolic systems that have subsequently been shown to be responsive to the up-regulation of HO activity make it clear that enhancing HO activity by either pharmacological or genetic means offers potential for the development of new therapeutic modalities,which could moderate the course of various pathological processes in humans.Heme oxygenase is the rate-limiting enzyme in the catabolism of heme,a process that leads to formation of equimolar amounts of the bile pigment biliverdin,iron,and carbon monoxide(CO). Biliverdin formed in this reaction is rapidly converted to bilirubin.Both CO and bilirubin are bioactive molecules,and the iron generated by HO-1 and HO-2 is immediately sequestered by associated increases in ferritin.HO-2 is a constitutive enzyme,whereas HO-1 is inducible by heavy metals,cytokines,UV light,oxidative stress,inflammatory cytokines and many drugs.It is therefore possible that the induction of HO-1 may be an essential event for some types of acute reactions and for cellular protection after injury.Because the major source of CO in animals is the degradation of heme by HO,it is now becoming apparent that CO serves as an important cellular signal molecule.Nitric oxide(NO) is generated by nitric-oxide synthase(NOS),a heme containing enzyme.It has been proposed that certain NO effects can be duplicated by CO,specifically;the action of certain neurotransmitters and muscle relaxants can be regulated by both molecules.The biological actions of bilirubin may be especially relevant to the prevention of oxidant-mediated cell death. Bilirubin at a low concentration scavenges ROS in vitro,thereby reducing oxidant-mediated cellular damage and attenuating oxidant stress in vivo.Bilirubin inhibits NADPH oxidase and PKC activity.Both enzymes have been shown to mediate angiotensinⅡ-induced vascular injury.Recently,biliverdin and bilirubin have been shown to preserve endothelial cell integrity,prevent endothelial cell death and sloughing,enhance vascular reactivity,and prevent restenosis.Bilirubin is also implicated in reducing oxidative stress in experimental diabetes,in part,by increasing the bioavailability of NO needed for endothelial cell integrity.Plasma iron is bound to transferrin,which can transfer iron to the intracellular milieu of endothelial cells via cell surface receptor binding.The up-regulation of HO-1 increases ferritin synthesis to sequester the iron released from the HO-1 pathway.
Hyperglycemia-mediated local formation of ROS is considered to be a major contributing factor to endothelial dysfunction which play a key role in developemnt of chronic diabetic complicaitons.This includes endothelial cell apoptosis,abnormalities in cell cycling due to lack of HO-1 induction and increased(?)formation.Some of these perturbations can be reversed by antioxidant agents or by increased levels of antioxidant enzymes.Increased levels of HO-1 through gene transfer in hyperglycemic rats resulted in a decrease of endothelial cell sloughing.Delivery of the human HO-1 gene to endothelial cells attenuated glucose-mediated oxidative stress,DNA damage,and cell death.HO-1 induction has been shown to provide vascular cytoprotection against oxidative stress.Thus,HO-1 plays a pivotal role in mitigating the detrimental effects of hyperglycemia.Increases in HO-1 expression in diabetic obese mice were paralleled by increases in serum adiponectin levels,insulin sensitivity,decreases in visceral and abdominal fat content,and decreased plasma TNFα,IL-6,and IL-1βlevels.Induction of HO-1 also resulted in decreased adipocyte superoxide production.Up-regulation of HO-1 by CoPP treatment caused a decrease in adipogenesis in bone marrow,both in vivo and in vitro in cultured mesenchymal stem cells and in increases in secretion of adiponectin in the culture media.
Diabetic complications have been related to abnormalities in mitochondrial function as well as to increased endothelial cell death and detachment.Specific human HO-1 gene transfer to diabetic rats has also resulted in the restoration of mitochondrial carriers,including ADP/ATP and decarboxylate.The increase in HO-1 in diabetic rats is associated with increased pAKT.An increase in AKT phosphorylation is critical to cell survival in diabetes.Increases in AKT phosphorylation and Bcl-xL levels have been shown to prevent the loss ofβ-cells in diabetes.It is interesting to note that the alteration in mitochondrial function in vitro and in vivo correlates with the levels of activation of AKT and the Bcl-2 family of proteins.A decrease in Bcl-2 family members has been reported to contribute to apoptosis and the translocation of cytochrome c from the mitochondria to cytosol. Activation of AKT has been shown to augment ATP synthesis and promote the association of hexokinase with the voltage-dependent anion channel and,in so doing, promote voltage-dependent anion channel closure,thus blocking release of cytochrome c.
Though the last decade has witnessed an explosion in the elucidation of the role that the HO system plays in human physiology,effects of HO-1 on oxidative stress in diabetes mellitus were not completely clarified yet.The use of pharmacological agents and genetic probes for manipulating HO has led to new insights into the prevention of chronic diabetic complications.
Chapter 1
Expressions of heme oxygen ase-1 in THP-1 cells incubated with high glucose and AGEs
[Objectives]
1.To investigate into the effects of glucose and advanced glycation end products (AGEs) on ROS outputs in THP-1 cells.
2.To investigate into the effects of high glucose combinded with AGEs on oxidative stress in THP-1 cells.
3.To investigate into the effects of glucose and AGEs on expressions of HO-1 in THP-1 cells.
4.To investigate if p38MAPK signal pathway were involved in oxidative stress in THP-1 cells induced by high glucose and AGEs.
[Methods]
1.Cell culture:THP-1 cells were cultured at 37℃,5%CO_2 with RPMI-1640 containing 10%FCS,penicillin(100units/mL),and streptomycin(100μg/mL).
2.Preparation of AGEs:Briefly,BSA and AGEs were prepared by incubation of 3.55 mg/mL BSA in the presence or absence of glucose and 0.5 mmol/L sodium azide in PBS(pH 7.4) at 37℃for 8 weeks.
3.Determination of ROS outputs:Cells were resuspended in medium containing 10mmol/L of DCFH-DA(Molecular Probes),and incubated for 30min.And then flow cytometric analysis was used by FACScan(Becton-Dickinson,Mountain View,CA) at excitation 488 nm and emission 525nm to detect the mean fluorescence intensity(MFI) as ROS output.
4.MDA assay:By TBA assay method.
5.TNF-a ELISA assay:By enzyme linked immunosorbent assay(ELISA).
6.RT-PCR for HO-1mRNA:Total RNA was isolated using TRIzol reagents (Invitrogen).The electrophoresis of the amplified products was operated with 1.5%agarose gel.
7.Western blot analysis for HO-1 expression:Cytoplasmic extracts were separated from the nuclei.An equal amount of protein(30mg/well) for each sample was boiled for 5 min,loaded,and run for electrophoresis in a 4-20%Tris-Glycine gel. The gel was transferred electrophoretically to nitrocellulose membranes,and the blots were blocked with 5%milk in TBST.Membranes were then probed with a polyclonal rabbit antibody against HO-1 and with a horseradish peroxidaseconjugated anti-rabbit IgG.For quantification,bands in photo-graphs were scanned by a densitometer linked to a computer system.
8.p38MAPK signal pathway:There were 4 groups:15mmol/L-GLU group, SB+15mmol/L-GLU group,100μg/mL-AGEs group and SB+100μg/mL-AGEs group.SB group means the cells were stimulated by 10μmol/L SB203580 (inhibitor of p38MAPK signal pathway) before incubation with 15mmol/L GLU or 100μg/mL AGEs for 24h.
9.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.Pearson coefficient correlation was used too.α=0.05.
[Results]
1.Concentration of AGEs:The fluorescence intensity of AGEs showed 7.40-fold (excitation 360 nm,emission 430nm) higher than that of BSA control.The concentration of AGEs was 101.29U/mg protein,while BSA was 13.68U/mg.
2.ROS output(mean fluorescence intensity,MFI):GLU+AGEs group(107.21±8.94) has significantly higher ROS outputs than GLU group(65.88±1.61) and AGEs group(67.46±3.78)(P＜0.05),while that of AGEs group and GLU group were all significantly higher than control group(41.95±1.36)(P＜0.05).
3.MDA(nmol/mL):GLU+AGEs group(3.26±0.32) has significantly higher MDA than GLU group(1.59±0.53) and AGEs group(1.56±0.97)(P<0.05),and that of GLU group was significantly higher than control group(P＜0.05).But there was no significantly difference between AGEs group and control group(0.65±0.23) (P＞0.05).
4.TNFa(pg/mL):GLU+AGEs group(51.25±1.72) has significantly higher TNFa than GLU group(25.38±1.95) and AGEs group(24.43±2.65)(P＜0.05),and that of GLU group and AGEs group were significantly higher than that of control group(14.97±1.49)(P＜0.05).
5.HO-1mRNA:Expression of HO-1mRNA of GLU+AGEs group(0.89±0.12) was significantly higher than that of GLU group(0.42±0.02) and AGEs group (0.48±0.03)(P＜0.05).And GLU group and AGEs group have significantly higher HO-1mRNA than control group(0.15±0.01)(P＜0.05).
6.HO-1 protein:GLU+AGEs group(0.81±0.02) has significantly higher HO-1 protein than GLU group(0.39±0.01) and AGEs group(0.45±0.03)(P＜0.05).And GLU group and AGEs group have significantly higher level than control group (0.15±0.01)(P＜0.05).
7.Correlation of ROS,MDA,TNF-a and HO-1mRNA and its protein:There was significantly positive correlation between the output of the expression of HO-1 mRNA and ROS,MDA(r=0.858,P=0.000) and TNFα(r=0.952,P=0.000).There were alos correlation between ROS output and HO-1mRNA(r=0.990,P=0.000), expression of HO-1 protein,MDA(r=0.873,P=0.000) and TNFα(r=0.964, P=0.000).And there were correlation between TNFαand ROS,MDA(r=0.851, P=0.000),HO-1mRNA and its protein.
8.There were no significant difference in expressions of HO-1 mRNA between GLU group,SB+GLU group,AGEs group and SB+AGEs group.
[Conclusions]
1.ROS output of THP-1 cells has been exacerbated when incubated with high glucose or AGEs and presented as dose-,time-dependent increasing. Furthermore,AGEs combined with high glucose group has much more ROS output than high glucose or AGEs group.These suggest that there is a synergistic relationship between high glucose and AGEs in oxidative stress.AGEs combined with high glucose could induce more MDA and TNFαin THP-1 cells.
2.The expression of HO-1mRNA and protein in THP-1 cells treated with high glucsoe combined AGEs has been increased significantly,which hint HO-1 could be an effective protective factor for oxidative stress induced by high glucose and AGEs in THP-1 cells.And some methods which can induce high expression of HO-1 may help to resist oxidative injury induced by high glucose and AGEs.
3.p38MAPK signal pathway may not be involved in the transcription of HO-1 in THP-1 cells treated by high glucose and AGEs.
Chapter 2
Effects of ZnPP(inhibitor of HO-1) on oxidative stress in THP-1 cells induced by high glucose and AGEs
[Objective]
1.To investigate into the effects of ZnPP(inhibitor of HO-1) on oxidative stress induced by high glucose and AGEs in THP-1 cells.
[Methods]
1.Groups:There were 4 groups as control group,15mmol/LGLU+100μg/mL AGEs group,ZnPP group,ZnPP+15mmol/LGLU+100μg/mLAGEs group.The concentration of glucose in the cell culture solution of control group and ZnPP group were all 5mmol/L.
2.Determination of ROS output,MDA,TNF-a,HO-1mRNA and proteins:Just the same as Chapter 1.
3.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.α=0.05.
[Result]
1 ROS output(mean fluorescence intensity,MFI):ZnPP+GLU+AGEs gorup (128.81±5.53) has significantly higher ROS output than GLU+AGEs group (107.21±8.94) and ZnPP group(51.56±2.09)(P＜0.05).ROS output of GLU+AGEs gorup and ZnPP group were significantly higher than that of control group(41.95±1.36)(P＜0.05).
2 MDA(nmol/mL):ZnPP+GLU+AGEs group(3.75±1.25) has significantly higher MDA than ZnPP group(1.61±0.10)(P＜0.05),but there were no significantly difference between ZnPP+GLU+AGEs group and GLU+AGEs group(3.26±0.32) (P＞0.05).MDA of GLU+AGEs group and ZnPP group were significantly higher than that of control group(0.65±0.23)(P＜0.05).
3 TNFa(pg/mL):ZnPP+GLU+AGEs group(141.10±12.84) has significantly higher TNFa than GLU+AGEs group(38.09±14.97) and ZnPP group(27.34±10.18) (P＜0.05).TNFa of GLU+AGEs group and ZnPP group were significantly higher than that of control gorup(18.55±7.26)(P＜0.05).
4 HO-1mRNA:ZnPP+GLU+AGEs group(0.39±0.02) has significantly lower expression of HO-1mRNA than GLU+AGEs group(0.89±0.09)(P＜0.05),and significantly higher than ZnPP group(0.24±0.02)(P＜0.05).GLU+AGEs group and ZnPP group have higher HO-1mRNA than control group(0.15±0.01) (P＜0.05).
5 HO-1 protein:ZnPP+GLU+AGEs group(0.38±0.00) has significantly lower HO-1 protein than GLU+AGEs group(0.81±0.02)(P＜0.05),and significantly higher than ZnPP group(0.23±0.03)(P＜0.05).GLU+AGEs group and ZnPP group have higher HO-1 protein than control group(0.15±0.01)(P＜0.05).
[Conclusion]
1.ZnPP itself could induced oxidative stress,injury and inflammation in THP-1 cells.ZnPP,as an inhibitor of HO-1,could aggravate oxidative stress and inflammation in THP-1 induced by high glucose and AGEs.
Chapter 3
Effects of CoPP(inducer of HO-1) on oxidative stress in THP-1 cells induced by high glucose and AGEs
[Objectives]
1.To investigate into the effects of CoPP(inducer of HO-1) on oxidative stress induced by high glucose and AGEs in THP-1.
[Methods]
1.Groups:There were 4 groups as control group,15mmol/LGLU+100μg/mLAGEs group,CoPP group,CoPP+15mmol/L-GLU+100μg/mL-AGEs group.The concentration of glucose in the cell culture solution of control group and CoPP group were all 5mmol/L.
2.Determination of ROS output,MDA,TNF-a,HO-1mRNA and proteins:Just the same as Chapter 1.
3.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.α=0.05.
[Results]
1 ROS output(mean fluorescence intensity,MFI):GLU+AGEs has the main effect on ROS output(F=287.930,P=0.000),while CoPP has not the main effect (F=0.433,P=0.529),and there were interact between GLU+AGEs and CoPP (F=8.632,P=0.019),which means with the GLU+AGEs,CoPP could lower the ROS output.ROS output of GLU+AGEs group was significantly higher than control group(41.95±1.36)(P＜0.05),and there were no significantly difference between CoPP group and control group(P＞0.05).
2 MDA(nmol/mL):CoPP+GLU+AGEs group(1.28±0.61) has significantly lower MDA than GLU+AGEs group(3.26±0.32)(P＜0.05),and there were no significantly difference between CoPP group(0.73±0.45) and control group (0.65±0.23)(P＞0.05).GLU+AGEs group has significantly higher MDA than control group(P＜0.05),and there were no difference between CoPP group and control group(P＞0.05).
3 TNFa(pg/mL):There were no significantly difference between CoPP+GLU+ AGEs group(31.90±12.56),GLU+AGEs group(43.60±12.40) and CoPP group (16.60±7.99)(P＞0.05).GLU+AGEs group has higher TNFa than control group (14.97±1.49)(P＜0.05),while there were no difference between CoPP group and control group(P＞0.05).
4 HO-1mRNA:There were no significantly difference between CoPP+GLU+ AGEs group(0.91±0.01) and GLU+AGEs group(0.89±0.12) and CoPP group (16.60±7.99)(P>0.05).CoPP+GLU+AGEs group has higher HO-1mRNA than CoPP group(0.24±0.01)(P＜0.05).CoPP group and GLU+AGEs group have higher level than control group(P＜0.05).
5 HO-1 protein:CoPP+GLU+AGEs group(0.87±0.01) has higher HO-1 protein than GLU+AGEs group(0.81±0.02) and CoPP group(0.22±0.02)(P＜0.05). CoPP group and GLU+AGEs group has higher level than control group (0.15±0.01)(P＜0.05).
[Conclusions]
1.CoPP,an inducer of HO-1,could effectively decrease oxidative stress in THP-1 induced by high glucose and AGEs,while CoPP itself has not cause oxidative stress.Though CoPP has decreased the level of TNFa in THP-1 cells treated by high glucose and AGEs there was no significantly differece.Certainly CoPP significantly enhanced the expression of HO-1 protein but not HO-1mRNA.
2.HO-1 as a stress protein could protect THP-1 cells against oxidative injury induced by high glucose and AGEs.Methods which could increase the expression of HO-1 would help to resist oxidative injury induced by high glucose and AGEs.
Chapter 4
Relationship between Transcription of HO-1 in Peripheral Blood Mononuclear Cells of Newly Diagnostic Type 2 Diabetic Patients with Chronic Complications and Oxidative Stress
[Objectives]
1.To investigate into the relationship between transcription of HO-1 in peripheral blood mononulclear cells(PBMC) of newly diagnostic type 2 diabetic patients with chronic complications and oxidative stress
[Methods]
1.Subjects were all newly diagnostic type 2 diabetes from our department.The diagnosis and classification criteria of type 2 diabetes mellitus was 1999 WHO criteria.Any acute metabolic disturbance and any drug intake were excluded. Diabetic chronic complications were determined by retinal photograph,feeling check and 24h urine albumin excretion.There were three groups:normal control group,non-complications group and complications group.
2.Peripheral blood mononuclear cells(PBMC):All of subjects were taken blood samples after fasting for 8～14 hours.PBMC were seperated by ficoll-urografin density gradient centrifugation.Serum of samples were kept in-20℃for MDA analysis.
3.ROS output of PBMC,serum MDA and expression of HO-1mRNA:Just the same as Chapter 1.
4.Expression of HO-1 protein:Immunohistochemistry was used to detect the expression of HO-1 protein.Disposition of HO-1 in the kytoplasm was analyzed by fluorescence microscope.cytoplasm has green fluorescent which means positive which were calculated.
5.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.The difference between two groups was analyzed by two-independent t test.Pearson Correlation was used to analyse the correlation among multiple variables.The received level of significance isα=0.05.
[Results]
1.Compared with non-complications group,complications group has higher fasting blood glucose(16.26±6.67 vs 10.61±3.61),HbAlc(12.54±2.10 vs 10.51±1.86), ROS output(419.60±216.31 vs 172.38±145.24),serum MDA(66.04±13.67 vs 0.77±0.23),HO-1mRNA(1.02±0.27 vs 0.77±0.23) and protein(85.47±10.89 vs 61.45±21.81)(P＜0.05).
2.There was positive correlation between HO-1mRNA and protein(r=0.750, P=0.000),MDA(r=0.449,P=0.010),FPG(r=0.364,P=0.041) and 2hPG(r=0.477, P=0.016).Expression of HO-1 protein was correlated with ROS output(r=0.608, P=0.000),MDA(r=0.623,P=0.000).There were positive correlation between MDA and ROS output(r=0.788,P=0.000),HbAlc and 2hPG(r=0.412,P=0.041), 2hPG and FPG(r=0.811,P=0.000).
[Conclusions]
1.There are obvious oxidative stress in newly diagnostic type 2 diabetic patients with chronic complications and hyperglycemia may induce oxidative stress which then results in diabetic chronic complications.
2.Correlation analysis hint that oxidative stress induced by diabetes mellitus could upregulate HO-1.Up-regulation of HO-1 could help to resist against oxidative injury induced by diabetes mellitus though it may not be enough to entirely protect cells.It would be a new pathway to prevent diabetic chronic complications by upregulating HO-1.
引文
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2.Brownlee M.Biochemistry and molecular cell biology of diabetic complication.Nature,2001;414:813-20.
3.Brownlee M.Negative consequences of glycation.Metabolism,2000;49:9-13.
4.West IC.Radicals and oxidative stress in diabetes.Diabet Med,2000;17:171-180.
5.Oseph L.Evans,Ira D.Goldfine,Betty A.Maddux,et al.Oxidative Stress and Stress-Activated Signaling Pathways:A Unifying Hypothesis of Type 2 Diabetes.Endocrine,2002;23(5):599-622.
6.Shibahara S,Yoshizawa M,Suzuki H,et al.Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation.J Biochem,1993;113:214-8.
7.Maines MD.The heme oxygenase system and its functions in the brain[J].Cell Mol Biol,2000;46(3):573-585.
8.Maines MD.The heme oxygenase system and its functions in the brain.Cell Mol Biol(Noisy-le-grand),2000;(3):573-85.
9.W,Schafer AI.Carbon monoxide and vascular cell function(Review)[J].Int J Mol Med,1998,2(3):255-62.
10.Ollinger R,Yamashita K,Bilban M,et al.Bilirubin and biliverdin treatment of atherosclerotic diseases.Cell Cycle,2007;6:39-43.
11.Li M,Kim DH,Tsenovoy P,et al.Treatment of obese diabetic mice with an heme oxygenase inducer reduces visceral and abdominal adiposity increases adiponectin levels and improves insulin sensitivity and glucose tolerance.Diabetes,2008.in press.
12.Suematsu M,Kashiwagi S,Sano T,et al.Carbon monoxide as an endogenous modulator of hepatic vascular perfusion.Biochem Biophys Res Commun,1994;205:1333-7.
13.Johnson RA , Kozma F , Colombari E.Carbon monoxide : from toxin to endogenous modulator of cardiovascular functions [J].Braz J Med Biol Res , 1999,32(1): 1-14.
14.Friebe A, Schultz G, and Koesling D.Sensitizing soluble guanylyl cyclase to become a highly CO-sensitive enzyme.EMBO J, 1996; 15: 6863-8.
15.Schwertner HA.Association of smoking and low serum bilirubin antioxidant concent rations [J].Atherosclerosis, 1998; 136(2): 383-7.
16.Matsumi M, Takahashi T, Fujii H, et al.Increased heme oxygenase-1 gene expression in the livers of patients with portal hypertension due to severe hepatic cirrhosis.J Int Med Res, 2002; 30: 282-8.
17.Ren H, Leib SL, Ferriero DM, et al.Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents ironinduced oxidative damage.J Neurochem, 2007; 100: 532-4.
18.Eisenstein RS, Garcia-Mayol D, Pettingell W, et al.Regulation of ferritin and heme oxygenase synthesis in rat fibroblasts by different forms of iron.Proc Natl Acad Sci USA, 1991; 88: 688-92.
19.Abraham NG, Kappas A.Heme oxygenase and the cardiovascular-renal system.Free Radic Biol Med, 2005; 39: 1-25.
20.Quan S, Kaminski PM, Yang L, et al.Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats[J].Biochem Biophys Res Commun, 2004; 315:509-16.
21.Abraham NG, Rezzani R, Rodella L, et al.Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes [J].Am J Physiol Heart Circ Physiol, 2004; 287:H2468-2477.
22.Sacerdoti D, Colombrita C, Ghattas MH, et al.Heme oxygenase-1 transduction in endothelial cells causes downregulation of monocyte chemoattractant protein-1 and of genes involved in inflammation and growth.Cell Mol Biol (Noisy-le-grand), 2005; 51(4): 363-70.
23.Nader G Abraham, Rita Rezzani, Luigi Rodella, et al.Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes.AM J Physical Heart Circ Physical,2004;287:2468-77.
24.Das S,Fraga CG,Das DK.Cardioprotective effect of resveratrol via HO-1expression involves p38 map kinase and PI-3-kinase signaling,but does not involve NFkappaB.Free Radic Res,2006;40(10):1066-75.
25.Di Filippo C,Marfella R,Cuzzocrea S,et al.Hyperglycemia in streptozotocin-induced diabetic rat increases infarct size associated with low levels of myocardial HO-1 during ischemia/reperfusion_2 2005;54(3):803-10.
26.Sumi D,Ignarro LJ.Regulation of inducible nitric oxide synthase expression in advanced glycation end product-stimulated raw 264.7 cells:the role of heme oxygenase-1 and endogenous nitric oxide.Diabetes,2004;53(7):1841-50.
27.Xiao-ming Liu,Gary B.Chapman,Hong Wang,et al.Adenovirus-Mediated Heine Oxygenase-1 Gene Expression Stimulates Apoptosis in Vascular Smooth Muscle Cells.Circulation,2002;105:79-84.
28.Di Noia MA,Van DS,Palmieri F,et al.Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome c oxidase activity in experimental diabetes.J Biol Chem,2006;281:15687-93.
29.Bojunga J,Nowak D,Mitrou PS,et al.Antioxidative treatment prevents activation of death-receptor-and mitochondri-on-dependent apoptosis in the hearts of diabetic rats.Diabetologia,2004;47:2072-80.
30.Srinivasan S,Stevens M,Wiley JW.Diabetic peripheral neuropathy:evidence for apoptosis and associated mitochondrial dysfunction.Diabetes,2000;49:1932-8.
31.Iori E,Pagnin E,Gallo A,et al.Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells.Life Sci,2008;82(7-8):383-92.
32.Abraham NG,Kushida T,McClung J,et al.Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells.Circ Res,2003;93(6):507-14.
33.Kanakiriya SK,Croatt AJ,Haggard JJ,et al.Heme:a novel inducer of MCP-1through HO-dependent and HO-independent mechanisms.Am J Physiol Renal Physiol, 2003; 284(3): 546-54.
34.Lang D, Reuter S, Buzescu T, et al.Heme-induced heme oxygenase-1 (HO-1) in human monocytes inhibits apoptosis despite caspase-3 up-regulation.Int Immunol, 2005; 17(2): 155-65.
35.Guha M, Bai W, Nadler JL, et al.Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways.J Biol Chem, 2000; 275(23):17728-39.
36.Kronke G, Kadl A, Ikonomu E, et al.Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors.Arterioscler Thromb Vasc Biol, 2007; 27:1276-82.
37.Sacerdoti D, Olszanecki R, Li Volti G, et al.Heme oxygenase overexpression attenuates glucose-mediated oxidative stress in quiescent cell phase: linking heme to hyperglycemia complications.Curr Neurovasc Res, 2005; 2(2): 103-11.
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