线粒体靶向抗氧化剂SKQ1对脂多糖所致急性肾损伤小鼠的保护作用及机制

Protective effects of mitochondria-targeted antioxidant SKQ1 on mice with lipopolysaccharide-induced acute renal injury

目的观察线粒体靶向抗氧化剂SKQ1对急性肾损伤小鼠的保护作用及机制。方法将24只8周龄健康雄性C57BL/6小鼠随机分为对照组、SKQ1组、LPS组、LPS+SKQ1组,每组6只。采用腹腔注射脂多糖(LPS)10 mg/kg制备小鼠急性肾损伤模型,以生理盐水作为对照,SKQ1组腹腔注射0.2 mg/kg的SKQ1(溶解于10%DMSO、40%PEG300、5%Tween-80和45%生理盐水),LPS+SKQ1组于造模前24 h予SKQ1预处理,LPS腹腔注射24 h后处死小鼠并保留各组血液及肾脏。苏木精-伊红(HE)染色法观察肾脏损伤程度,并通过Paller评分法量化比较;采用全自动生化分析仪测量血清肌酐(SCr)和尿素氮(BUN);RT-qPCR法检测肾脏组织中炎症因子mRNA变化,如白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α;酶联免疫吸附试验(ELISA)检测肾脏组织氧化应激标志物水平,包括谷胱甘肽过氧化物酶(GPx)、超氧化物歧化酶(SOD)和丙二醛(MDA);免疫印迹法检测凋亡途径相关蛋白Bcl-2、Bax、Cleaved Caspase-3蛋白表达;采用电子显微镜观察各组小鼠肾小管线粒体结构。结果与对照组比较,LPS组小鼠肾脏病理损伤加重,肾脏损伤评分、SCr、BUN水平升高,肾组织中IL-1β、IL-6、TNF-αmRNA水平升高,Bax、Cleaved Caspase3蛋白表达升高,Bcl-2表达降低,GPx及SOD活性降低,MDA含量升高(P均<0.05);线粒体肿胀增大、线粒体嵴断裂及空泡形成。与LPS组比较,LPS+SKQ1组小鼠肾脏病理损伤减轻,肾脏损伤评分、SCr、BUN水平降低,肾脏组织IL-1β、IL-6、TNF-αmRNA表达量降低,Bax、Cleaved Caspase-3蛋白表达降低,Bcl-2表达升高,GPx、SOD活性升高,MDA含量降低(P均<0.05);线粒体肿胀、线粒体嵴断裂及空泡化减轻。结论SKQ1预处理可保护脂多糖所致小鼠急性肾损伤,其机制可能是通过减少炎症反应、调节细胞异常凋亡、降低氧化应激水平以及维持线粒体正常形态来实现的。

Objective To observe the protective effect of mitochondria-targeted antioxidant SKQ1 on mice with acute kidney injury(AKI)and to analyze its mechanism.Methods Twenty-four healthy male C57BL/6 mice were randomly divided into four groups:the control group,SKQ1-treated group(SKQ1 group),lipopolysaccharide-treated group(LPS group),and LPS-induced SKQ1-treated group(LPS+SKQ1 group),with six mice in each.AKI models were established by intraperitoneal injection(i.p.)of LPS(10 mg/kg).Mice in the control group received i.p.of normal saline.In the SKQ1 group,mice were intraperitoneally injected with 0.2 mg/kg SKQ1 dissolved in vehicle(10%DMSO,40%PEG300,5%Tween-80,and 45%normal saline);mice in the LPS+SKQ1 group were intraperitoneally injected with SKQ1 at 24 h before modeling;mice were sacrificed at 24 h after AKI induction,and the blood and kidneys were collected.Renal pathologic changes were assessed by HE staining and histopathological changes were quantified using the Paller score.Serum creatinine(SCr)and blood urea nitrogen(BUN)were measured using an automated biochemical analyzer.The mRNA expression levels of inflammatory factors,such as interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)in the renal tissues were measured by qRT-PCR.The levels of oxidative stress biomarkers,such as glutathione perocidase(GPx),superoxide dismutase(SOD)and malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay(ELISA).The protein expression levels of apoptosis-related markers,including B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax)and Cleaved caspase-3 were analyzed by Western blotting.The mitochondrial structures of the renal tubules in mice of each group were observed by electron microscopy.Results Compared with the control group,mice in the LPS group exhibited more severe kidney pathological damage;specifically,serum levels of SCr and BUN,the scores of kidney tissue damage,and the mRNA levels of inflammatory cytokines IL-1β,IL-6 and TNF-α,as well as the protein expression levels of Bax,Cleaved caspase-3 significantly increased,the protein expression of Bcl-2 and the activities of GPx and SOD decreased,and the content of MDA increased;irregular swelling of mitochondria along with fractured and vacuolization was observed(all P<0.05).Compared with the LPS group,the LPS+SKQ1 group showed a marked reduction in kidney pathological damage,and the serum levels of SCr and BUN,the scores of kidney tissue damage,and the mRNA levels of inflammatory cytokines IL-1β,IL-6 and TNF-α,as well as the protein expression levels of Bax,Cleaved caspase-3,and the content of MDA decreased,the protein expression of Bcl-2 and the activity of GPx and SOD increased(all P<0.05);mitochondrial swelling,fractured and vacuolization were alleviated in the LPS+SKQ1 group.Conclusion SKQ1 pretreatment protected against LPS-induced acute kidney injury via reducing inflammation,modulating apoptosis,decreasing levels of oxidative stress and maintaining the normal mitochondrial morphology.