花旗松素基于过氧化物酶体增殖物激活受体γ共激活因子1α介导的抗氧化通路对顺铂致急性肾损伤小鼠的保护作用及机制

Study on the effect and mechanism of taxifolin on ameliorating cisplatin-induced acute kidney injury in mice through peroxisome proliferator-activated receptor γ coactivator-1α-mediated antioxidant pathway

目的探讨花旗松素(taxifolin,TAX)改善顺铂(cisplatin)致急性肾损伤的作用及机制。方法(1)40只C57BL/6小鼠随机分为4组:对照组、TAX组、顺铂组、顺铂+TAX组。测量各组小鼠体重情况。检测各组小鼠血清肌酐(Scr)、尿素氮(BUN)水平;通过HE染色观察小鼠肾组织病理学改变;通过酶联免疫吸附测定(ELISA)法测定血清炎性因子白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α水平;通过试剂盒测定各组小鼠肾组织氧化应激指标[活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)];实时荧光定量PCR法测定炎性因子IL-6、TNF-α和IL-1β,抗氧化基因解耦联蛋白2(UCP2)、SOD2、CAT以及过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)mRNA表达情况;通过测定肾组织ATP水平和线粒体DNA(mtDNA)含量评价线粒体功能;通过Western印迹法测定腺苷酸活化蛋白激酶(AMPK)和磷酸化(p)-AMPK蛋白表达情况。(2)通过建立Lewis肺癌移植瘤C57BL/6小鼠模型评价TAX对顺铂化疗效果的影响。结果与对照组比较,TAX组小鼠体重未明显降低,且未出现明显肾损伤(均P>0.05),提示口服TAX具有良好的安全性。与顺铂组比较,TAX能够显著延缓顺铂引起的小鼠体重下降,降低小鼠血清Scr和BUN水平,并缓解肾组织病理学改变(均P<0.05)。TAX能够显著下调顺铂诱导的小鼠血清炎性因子IL-6和TNF-α水平,以及肾脏炎性因子IL-6、TNF-α、IL-1βmRNA表达(均P<0.05)。TAX能够显著降低顺铂致急性肾损伤小鼠肾组织ROS和MDA水平,并提高SOD、CAT和GSH活性(均P<0.01)。同时,TAX能够显著上调顺铂致急性肾损伤小鼠肾脏抗氧化基因UCP2、SOD2、CAT mRNA以及PGC-1αmRNA表达,并提高肾组织ATP和mtDNA水平(均P<0.01)。Western印迹结果显示,TAX显著促进顺铂致急性肾损伤小鼠肾脏AMPK磷酸化蛋白表达(P<0.01)。此外,通过Lewis肺癌移植瘤C57BL/6小鼠模型证实,TAX对顺铂抗肿瘤疗效无显著影响。结论TAX能够改善顺铂引起的肾脏氧化损伤,其机制可能与激活AMPK/PGC-1α通路有关。

Objective To explore the effect and mechanism of taxifolin(TAX)on ameliorating cisplatin-induced renal oxidative damage.Methods(1)Forty male C57BL/6 mice were divided into 4 groups:control group(n=10),TAX group(n=10),cisplatin group(n=10)and cisplatin+TAX group(n=10).The weight of mice in each group was measured.The level of serum creatinine(Scr)and blood urea nitrogen(BUN)was analyzed.Kidney histopathological change in mice was analyzed by HE staining.The pro-inflammatory cytokines levels of interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-α)were measured by enzyme linked immunosorbent assay.The levels of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione(GSH)were measured by multifunctional microplate reader.The expression of inflammatory factors,antioxidant genes,and peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1)mRNA were measured by real-time PCR.Evaluation of mitochondrial function by measuring ATP level and mtDNA content.Determination of AMP-activated protein kinase(AMPK)and phosphorylated AMPK protein expression by Western blotting.(2)Evaluate the effect of taxifolin on chemotherapy of cisplatin by establishing Lewis lung cancer transplantation tumor C57BL/6 mice model.Results Compared with the control group,the weight of the mice in the TAX group was not significantly reduced(P>0.05),and there was no obvious kidney damage(P>0.05),indicating that oral TAX had good safety.Compared with the cisplatin group,TAX could significantly delay cisplatin-induced the weight loss of mice,reduce the levels of Scr and BUN,and alleviate the pathological changes of kidney tissue(all P<0.05).TAX could reduce the levels of serum inflammatory factors IL-6 and TNF-α and the expression of renal inflammatory factors IL-6,TNF-α and IL-1β mRNA induced by cisplatin in mice(all P<0.05).TAX could significantly reduce the levels of ROS and MDA,and increase the activities of SOD,CAT and GSH in cisplatin-induced acute kidney injury mice(all P<0.01).Meanwhile,TAX could up-regulate the mRNA expression of UCP2,SOD2,CAT antioxidant genes and PGC-1αin the kidneys of mice with acute kidney injury induced by cisplatin,and increase the levels of ATP and mtDNA in cisplatin-induced acute kidney injury mice(all P<0.01).Western blotting results showed that TAX significantly promoted the expression of phosphorylated AMPK protein in cisplatin-induced acute kidney injury mice(P<0.01).In addition,through the establishment of Lewis lung cancer transplantation tumor C57BL/6 mice model,it was found that TAX had no significant effect on the anti-tumor efficacy of cisplatin.Conclusions TAX can ameliorate cisplatin-induced renal oxidative damage,and its mechanism may be related to the activation of AMPK/PGC-1α pathway.