PDTC调节线粒体功能障碍与Klotho蛋白表达对大鼠脓毒症合并急性肾损伤的影响

The effects of PDTC regulating mitochondrial dysfunction and Klotho protein expression on acute kidney injury in rat with sepsis

目的:探究吡咯烷二硫代氨基甲酸酯(PDTC)对脓毒症合并急性肾损伤(AKI)大鼠线粒体功能与Klotho表达的影响。方法:将60只SD大鼠随机分为对照组、模型组、PDTC组,每组20只,模型组和PDTC组大鼠通过盲肠结扎穿刺(CLP)制备脓毒症AKI模型,造模后1 h,PDTC组大鼠腹腔注射50 mg/kg PDTC,模型组与对照组注射同等剂量生理盐水,每日1次,持续1周。ELISA法检测大鼠血清肌酐(SCr)、尿素氮(BUN)和24 h尿蛋白(24 h UP)水平,硫代巴比妥酸法检测肾组织丙二醛(MDA)水平,微板法检测肾组织超氧化物歧化酶(SOD)水平,光谱光度测量工具检测肾组织总抗氧化能力(T-AOC)水平,苏木精—伊红(HE)染色和Masson染色观察肾组织病理形态学改变与胶原沉积现象,TUNEL染色检测肾组织细胞凋亡,免疫组织化学染色和实时荧光定量PCR(RT-qPCR)检测肾组织Klotho蛋白和mRAN表达水平,免疫荧光染色检测肾组织细胞色素C氧化酶Ⅳ(COXⅣ)和自噬标记轻链3(LC3)的共定位与表达以来评估线粒体水平,western blotting法检测肾皮质匀浆细胞色素-C(Cyto-C)、抗增殖蛋白(Prohibitin)蛋白表达。结果:相较于模型组,PDTC组血清SCr、BUN含量及24 UP水平均显著下降(均P<0.05),肾组织SOD、T-AOC含量显著增加,MDA含量显著减少(P<0.05),肾组织病理改变得到明显改善,胶原沉积减少,TUNEL阳性细胞率显著降低(P<0.05),Klotho蛋白与mRNA表达水平均显著上调(P<0.05),同时,LC3水平下降,肾皮质线粒体Cyto-C、Prohibitin蛋白表达显著升高(P<0.05),肾皮质胞浆Cyto-C、Prohibitin蛋白表达显著降低(P<0.05)。结论:PDTC可通过调节线粒体功能和Klotho表达来改善大鼠脓毒症合并AKI,并有效减轻肾组织的氧化损伤。

Objective:To explore the effects of pyrrolidine dithiocarbamate(PDTC) on mitochondrial function and Klotho expression in rats with sepsis and acute kidney injury(AKI).Methods:Sixty SD rats were randomly divided into control group,model group and PDTC group,with 20 rats in each group.The sepsis AKI models of rats were established by cecal ligation and puncture(CLP) in model group and PDTC group.After 1 h post-modeling,rats in the PDTC group were injected with 50 mg/kg PDTC intraperitoneally,and rats in the model group and the control group were injected with equal volume of normal saline,once a day for 1 week.The levels of serum creatinine(SCr),urea nitrogen(BUN) and 24 h urine protein(24 h UP) were measured by ELISA.The malondialdehyde(MDA) content in kidney tissues was detected by thibabituric acid method.The level of superoxide dismutase(SOD) in kidney tissue was detected by microplate method,and the level of total antioxidant capacity(T-AOC) was detected by spectrophotometry.Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological changes and collagen deposition of kidney tissues.Cell apoptosis was evaluated by TUNEL staining.The mRNA and protein expressions of Klotho were determined by q PCR and immunohistochemistry,respectively.The co-localization of cytochrome C oxidase IV(COX IV) and autophagy labeled light chain 3(LC3) in renal tissues were detected by immunofluorescence in order to assess mitochondrial levels.The expressions of cytochrome-C(Cyto-C) and anti-proliferation protein(Prohibitin) in renal cortex homogenate were determined by western blotting.Results:Compared with the model group,the SCr,BUN,24 UP and MDA levels,LC3 protein level,collagen deposition,the rate of TUNEL positive cells,and the expression of Cyto-C and Prohibitin protein in renal cortex cytoplasm were notably reduced in the PDTC group,while the SOD and TAOC contents,the m RNA and protein expressions of Klotho,and the protein levels of Prohibitin and Cyto-C in renal cortex mitochondria were markedly elevated(P<0.05).The pathological changes of kidney tissues were significantly improved.Conclusion:PDTC can improve sepsis rats with AKI by regulating mitochondrial function and Klotho expression,and it effectively reduces the oxidative damage of kidney tissues.