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左卡尼汀在肾小管上皮细胞氧化应激损伤中的保护作用研究
Mechanism of Protective Effect of L-carnitine on Human Kidney Tubular Epithelial Cell Line Injury Induced by Oxidative Stress
【作者】 叶俊生;
【导师】 于立新;
【作者基本信息】 南方医科大学 , 泌尿外科, 2010, 博士
【摘要】 肾脏缺血再灌注损伤可发生于多种疾病情况下,如休克、外科手术、移植、糖尿病肾病、慢性肾小管-间质损伤等,并伴随着氧化应激的增加。氧化应激由活性氧介导,包括自由基如超氧阴离子、羟基和非自由基如过氧化氢。活性氧在肾组织中的肾小球、间质肾小管、系膜细胞中产生,而组织中的抗氧化防御系统如低分子量抗氧化剂(抗坏血酸素C、谷胱甘肽、维生素E等)、ROS反应酶(超氧化物歧化酶、过氧化物酶、过氧化氢酶)以及氧化还原调节酶等会同时被激活,抑制活性氧生成并降解活性氧。若活性氧的产生量超过机体自身的清除能力,会同时破坏机体抗氧化酶,通过脂质氧化、分解DNA、和蛋白变性等损伤细胞。肾移植过程中移植肾不可避免遭受缺血再灌注损伤,其中主要为氧化应激损伤。肾脏冷保存时组织缺氧,移植恢复血液灌注后即激活瀑布式的炎症反应,产生大量活性氧。移植后活性氧的增加可能还参与了慢性移植肾肾病的发生。活性氧导致的线粒体肿胀,凋亡蛋白酶-3的激活,直接引起细胞坏死或凋亡。在肾脏缺血再灌注损伤后的第一个24小时内,抗凋亡蛋白Bcl-2家族如Bc1-2和Bcl-XL,以及凋亡前蛋白Bax、p53、FADD和Bak在近曲、远曲小管的表达均增加,并且与损伤严重程度相关。抗氧化剂治疗能减轻氧化应激,抑制细胞凋亡和坏死,改善移植物保存效果,减轻相关的损伤和炎症反应。左卡尼汀(L-carnitine, LC),一种L-赖氨酸的衍生物,是内源性的线粒体膜复合物。人体左卡尼汀的主要生理作用是辅助长链脂肪酸转运到线粒体内部进入β氧化循环,做为一种营养添加剂已应用30余年。左卡尼汀对肾组织的保护作用已经在多种氧化应激模型中得到证实,如:顺铂导致的肾或小肠损伤模型,庆大霉素引起肾毒性模型,肾的缺血再灌注模型以及慢性肾衰模型。左卡尼汀能抑制肾脏缺血再灌注损伤中血清和肾组织MDA的生成。有学者发现左卡尼汀有清除DPPH、超氧阴离子、过氧化氢的能力,提高抗氧化酶的活性,如SOD, CAT和GPx等,并能降低肾组织中的MDA浓度。根据以上的发现,本研究应用人体近曲小管上皮细胞HK-2作为细胞模型,研究左卡尼汀抗氧化作用的分子机制。作为活性氧的主要成员,过氧化氢在氧化还原过程中产生,被认为是引起细胞内信号传递的信使。过氧化氢能引起脂质过氧化和DNA损伤。因此,我们应用过氧化氢诱导HK-2细胞氧化应激,验证使用左卡尼汀预处理能减轻过氧化氢对HK-2的氧化作用。在此基础上,我们还进一步研究了左卡尼汀在氧化应激中ROS的生成,脂质过氧化、抗氧化系统、线粒体功能和细胞凋亡情况。第一章左卡尼汀对氧化应激损伤肾小管上皮细胞的保护作用目的建立过氧化氢诱导的人肾小管上皮细胞系(HK-2)氧化应激损伤模型,探讨左卡尼汀(L-carnitine, LC)对过氧化氢氧化应激损伤人肾小管上皮细胞的保护作用及其可能机制。方法用不同浓度的H202作用于人肾小管上皮细胞(HK-2),四甲基偶氮唑蓝(MTT)法检测细胞活力,建立造成肾小管上皮细胞氧化应激损伤模型;实验分为5组:正常细胞组、H202损伤组、单独LC组、LC保护组、N-乙酰半胱氨酸(NAC)组,MTT法检测各组细胞活力,采用酶化学法测定细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性、以及测定其总抗氧化能力(T-AOC)、丙二醛(MDA)水平;以氧化敏感的2,7-二氢二氯荧光素(DCFH-DA)染色,荧光显微镜观察及流式细胞仪测定细胞内活性氧(ROS)强度,碘化丙啶(PI)染色流式细胞仪测定HK-2细胞的凋亡率。实验组测量值用均数±标准差(x±s)表示,应用SPSS16.0统计分析软件对数据进行分析,各组比较应用单因素方差分析(One-Way ANOVA)、组间多重比较应用LSD-t检验,P<0.05表示差异有统计学意义。结果H2O2500μM×30min处理后HK-2细胞活性较正常细胞显著降低(P<0.001),单独应用LC 10μM、50μM、100μM处理12小时HK-2细胞活力显著高于正常对照组(P<0.001),H202损伤前使用LC预处理12小时能抑制H202损伤所导致的HK-2细胞活力降低(P<0.001);H2O2500μM×30min处理后,细胞中SOD,GSH-Px和CAT含量及T-AOC显著低于正常细胞组(P<0.001),而LC保护组,细胞中SOD, GSH-Px和CAT含量及T-AOC显著高于H202损伤组(P<0.001);H202损伤组MDA、ROS水平高于正常细胞组,细胞凋亡率高于正常细胞组,差别有统计学意义(P<0.001),而LC保护组MDA、ROS水平以及细胞凋亡率均显著低于H202损伤组(P<0.001)。表明肾小管上皮细胞在经左卡尼汀预处理之后,抗损伤能力加强,损伤程度减少。结论左卡尼汀对氧化应激所致的肾小管上皮细胞损伤具有保护作用,其作用机制可能与增强细胞抗氧化能力,减少自由基生成,抑制脂质过氧化反应,减少细胞凋亡有关。第二章线粒体通路在左卡尼汀抑制氧化应激诱导HK-2细胞凋亡中的作用目的探讨线粒体通路在左卡尼汀(L-carnitine, LC)对过氧化氢氧化应激致人肾小管上皮细胞凋亡中的作用。方法建立H202诱导的肾小管上皮细胞氧化应激损伤模型;实验分为五组:正常细胞组、H202损伤组、单独LC组、LC保护组、N-乙酰半胱氨酸(NAC)组;Hoechst 33258染色观察细胞凋亡,计算细胞凋亡率;流式细胞仪检测线粒体膜电位和半胱天冬酶3 (caspase-3)的活性;蛋白质印迹法检测凋亡相关活性蛋白Bcl-2和Bax的表达以及细胞色素C的释放。各实验组测量值用均数±标准差(x±s)表示,应用SPSS16.0统计分析软件对数据进行分析,各组比较应用单因素方差分析(One-Way ANOVA)、组间多重比较应用LSD-t检验,P<0.05为差异有统计学意义。结果Hoechst 33258染色结果显示H2O2损伤组出现典型的细胞致密浓染,核固缩、边缘化等凋亡形态学改变;H2O2损伤组细胞凋亡率显著高于正常对照组(P<0.001),不同浓度LC保护组细胞凋亡率显著低于H202损伤组(P<0.001),且该效应呈剂量依赖性;使用流式细胞议检测H202损伤组caspase-3活性显著高于正常对照组(P<0.001),而LC保护组caspase-3活性显著低于H202损伤组(P<0.001);过氧化氢作用可使HK-2细胞线粒体功能紊乱,H202损伤组较正常细胞组线粒体膜电位显著降低(P<0.001),Bax/Bcl-2显著升高(P<0.001),胞浆细胞色素C显著升高(P<0.001);而LC保护组较H2O2损伤组线粒体膜电位显著升高(P<0.001),Bax/Bc1-2显著降低(P<0.001),胞浆细胞色素C显著降低(P<0.001),提示左卡尼汀预处理能剂量依赖性的抑制上述细胞凋亡时的线粒体功能紊乱。结论左卡尼汀对氧化应激所致的肾小管上皮细胞凋亡具有抑制作用,其作用机制可能与改善线粒体功能有关。
【Abstract】 Increased oxidative stress have been implicated in a variety of kidney diseases, such as renal ischemia-reperfusion (I/R) injury caused by shock or during surgery or transplantation, diabetic nephropathy, and chronic tubulointerstitial injury. It is mediated by reactive oxygen species (ROS), including free radicals such as superoxide ions and hydroxyl radicals as well as non-free radical species such as hydrogen peroxide (H2O2). ROS can be generated within the nephron segments like the glomeruli and proximal tubule and mammalian cells have developed several protective mechanisms to prevent ROS formation or detoxify ROS, which include low-molecular-mass antioxidants (ascorbic acid, glutathione, tocopherols, and others), ROS-interacting enzymes (superoxide dismutase, peroxidases, and catalases), and redox regulation enzymes. I/R excessively produces reactive oxygen species (ROS) beyond this organ’s scavenging capacity for ROS, simultaneously impairs antioxidant enzymes, and causes cell damage by lipid peroxidation, DNA breakdown, and protein damage. Studies have shown that ischemia/reperfusion (I/R) inevitably accompanied with renal transplantation is well characterized oxidative stress-induced tissue injury immediately after kidney transplantation. Injury initiated by the lack of oxygen during cold presentation is augumented by ROS during subsequent warm reperfusion of grafts through activation of imflammatory cascade. Reactive oxygen species (ROS) is also markedly increased after kidney transplantation and may participate in the development and/or progression of chronic renal allograft nephropathy [3]. ROS-induced mitochondrial swelling, caspase-3 activation, which contributes to both necrotic and apoptotic forms of cell death have been documented after I/R injury in the kidney and in posthypoxic isolated proximal tubules occurred. The expression of both the antiapoptotic Bcl-2 family of proteins, Bcl-2 and Bcl-XL, and the proapoptotic proteins Bax, p53, FADD, and Bak in the distal and proximal tubules during the first 24 h were increased after I/R injury in the kidney, with the net effect determining the severity of injury and dysfunction. Antioxidant strategy may reduce oxidative stress and inhibit apoptotic signaling and cell death which will allow better preservation of graft function and ameliorate the associated injury and inflammation in kidney.L-Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid), an L-lysine derivative, is an endogenous mitochondrial membrane compound. The main physical function of L-carnitine in human body is facilitating the transport of long chain fatty acids into mitochondria in order to enter the P-oxidation cycle. Used as a safe and effective nutritional supplement for more than three decades, the protective effect of 1-carnitine on kidney tissue has been proved in various models involving oxidative stress, such as cisplatin-induced injury of the kidney and small intestine, gentamycin-induced nephrotoxicity, ischaemia- reperfusion injury of the kidney and chronic renal failure. It has been demonstrated that L-carnitine administration inhibits both serum and kidney tissue MDA formation in response to renal ischaemia/reperfusion injury. By using different antioxidant assays,Ⅰlhami Gulcin demonstrated that L-carnitine had an effective DPPH·scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, total reducing power and metal chelating on ferrous ions activities compared to a-tocopherol and trolox as references antioxidants. Carnitine can also act as a chelator by decreasing the concentration of cytosolic iron, which plays a very important role in free radical chemistry. And carnitine supplementation enhances the activities of anti-oxidant enzymes, such as SOD, CAT and GPx, and GSH levels and decreases the MDA concentration in kidney tissues of 24-month-old rats.In light of the findings described above, the present study employed the human proximal tubule epithelial cell line, HK-2 cells as a cell model system and aimed to elucidate the molecular mechanisms of L-carnitine on renal oxidative stress. As the major component of ROS, H2O2 is produced during the redox process and is considered as a messenger in intracellular signaling cascades. H2O2 could cause lipid peroxidation and DNA damage. So we used H2O2 as an inducer of oxidative stress for HK-2 cells and tested whether pretreatment cells with L-carnitine resulted in the resistance of HK-2 to H2O2 challenge. Furthermore, the effect of L-carnitine on oxidative stress conditions such as ROS production, lipid peroxidation, antioxidant defensive system, mitochondrial dysfunction and DNA damage associated with cell apoptosis were also studied.Chapter one Protective effect of L-carnitine on human kidney tubular epithelial cell line damaged by oxidative stressObjective To establish the HK-2 cell oxidative model induced by H2O2, and investigate the protective effect and its mechanism of L-carnitine on human kidney tubular epithelial cell line damaged by hydrogen peroxide (H2O2)-mediated oxidative stress.Methods Human kidney tubular epithelial cell line (HK-2 cells) were exposed to H2O2 of different concentration. The exact dose of H2O2 for the oxidative model was determined according to the cell viability evaluted by MTT assays. Then the HK-2 cells were divided into 5 group:normal control group, LC alone group, H2O2 group, LC protected group(pretreated with L-carnitine for 12hs and then injuryed by H2O2), and NAC group. The cell viability evaluted by MTT assays. Enzyme activities including superoxide dismutase (SOD), glutat hione peroxidase (GSH-Px), catalase (CAT), total antioxidative capacity (T-AOC) and malondialdehyde (MDA) were determined by biochemical methods. Intracellular ROS was detected by means of an oxidation sensitive fluorescent probe (DCF-DA) and the cell apoptosis were quantified by determining DNA content of cells by propidium iodide staining by flow cytometry. The Data was shown as mean±sd, analysed by One-way ANOVA and LSD-t test, SPSS 16.0, P<0.05 was statistically significant difference.Results H2O2 500μM X 30min decreased the cell viability significantly compared to the normal control group (P<0.001). L-carnitine(LC) 1OμM、50μM、100μM alone for 12h increased the cell viability compared to the normal control group (P<0.001) Pretreated by LC for 12h could inhibit H2O2-induced cell viability loss(P<0.001). The activities of intracellar superoxide dismutase(SOD), glutathione peroxidase(GPx), catalase(CAT) and total anti-oxidative capacity (T-AOC) decresed 12h after the cells exposed to H2O2 500μM for 30 min(P<0.001) compared to the normal control group. Pretreated by LC for 12h could enhance the activities of these antioxidant enzymes significantly in a concentration-dependent manner compared to the H2O2 group(P<0.001). Also, L-carnitine pretreatment increased total anti-oxidative capacity (T-AOC) and inhibited MDA formation. The intracellular reactive oxygen species generation and cell apoptosis triggered by H2O2 characterized with the DNA fragment were also inhibited by L-carnitine.Conclusions These results indicated that L-carnitine exhibited protective effects on HK-2 cells injuried by oxidative stress. It may be related to its antioxidative action which included enhancing endogenous antioxiant defense components, inhibiting the ROS production, MDA formation and cell apoptosis.Chapter two Role of the mitochondrial in L-carnitine inhibiting oxidative stress-induced human kidney tubular epithelial cell apoptosisObjective To investigate the role of the mitochondrial in the inhibiting effect of L-carnitine on hydrogen peroxide (H2O2)-induced human kidney tubular epithelial cell apoptosis.Methods Human kidney tubular epithelial cell line (HK-2 cells) were pretreated with L-carnitine for 12hs and then injuryed by H2O2. The cell apoptosis were evaluted by nuclear staining assay using chromatin dye Hoechst 33258 and flow cytometric detection of caspase-3 activity. Mitochondrial membrane potential was monitored using the fluorescent dye Rh123 by flow cytometry. Expression levels of Bcl-2, Bax and the release of cytochrome c were determined by Western blot analysis. The Data was shown as mean±sd, analysed by One-way ANOVA and LSD-t test, SPSS16.0, P<0.05 was statistically significant difference.Results The apoptosis rate of H2O2 injured cells was significantly higher than that of L-carnitine pretreatment cells(P<0.001). Activities of caspase-3 in H2O2 injured cells seemed higher than normal cells, and L-cartine pretreatment could prohibit the activation of caspase-3 in a concentration-dependent manner(P<0.001). The mitochondrial trans-membrane potential (ΔψM) was rapidly reduced when HK-2 cells were exposed to H2O2 and H2O-induced dissipation ofΔψM was significantly blocked by the pretreatment with L-carnitine. There was significant decrease in the mitochondrial cytochrome c level after H2O2 injury, which was accompanied by a simultaneous increase in cytochrome c level in the cytosol. Treatment of HK-2 cells with L-carnitine reduced cytochrome c in the cytosol and increased the mitochondrial cytochrome c. In H2O2 group, the expression rate of Bcl-2 was significantly reduced and the expression of Bax was increased compared to normal group(P<0.001). When HK-2 cells were treated with 50μM L-carnitine before H2O2 injury, the expression rate of Bcl-2 was markedly increased compared with H2O2 group.Conclusions These results suggested that L-carnitine could inhibit H2O2-induced kidney tubalar epithelial cell apoptosis through the mitochondrial pathway.
【Key words】 L-carnitine; kidney tubalar epithelial cells; mitochondrial; oxidative stress; apoptosis;