冬凌草甲素对脑缺血再灌注模型AMPK/SIRT1/PGC-1α信号通路调控研究

Regulation of AMPK/SIRT1/PGC-1a signaling pathway in cerebral ischemia-reperfusion model

目的探讨冬凌草甲素(ORI)通过调控AMPK/SIRT1/PGC-1α信号通路介导的线粒体功能,减轻局灶性脑缺血再灌注(CIR)损伤。方法随机将PC12细胞分为5组:Control组,细胞正常培养不做处理;氧-葡萄糖剥夺和再灌注组(OGD/R组),使用PC12细胞建立氧-葡萄糖剥夺和再灌注模型;氧-葡萄糖剥夺和再灌注组+ORI(OGD/R+Orbicin),PC12细胞建立氧-葡萄糖剥夺和再灌注模型前使用ORI预处理PC12细胞4h;氧-葡萄糖剥夺和再灌注组+ORI+化合物C组(OGD/R+Orbicin+CC),氧-葡萄糖剥夺和再灌注前加入化合物C(3μmol·L^(-1)6h)以抑制AMPK信号传导,同时使用ORI预处理PC12细胞4h;氧-葡萄糖剥夺和再灌注组+ORI+si-SIRT1组(OGD/R+Orbicin+si-SIRT1),在氧-葡萄糖剥夺和再灌注前,使用ORI预处理PC12细胞4h,并将si-SIRT1转染至PC12细胞中,四甲基偶氮唑盐比色法(MTT)测定法评估PC12细胞的细胞活力,酶联免疫法定量乳酸脱氢酶(LDH)释放量,丙二醛(MDA)、一氧化氮(NO)、超氧化物歧化酶(SOD)、腺嘌呤核苷三磷酸(ATP)和过氧化氢酶(CAT)活性,实时定量聚合酶链式反应(PCR)测定AMP依赖的蛋白激酶(AMPK)、沉寂信息调节因子(SIRT1)和过氧化物酶体增殖物激活受体γ辅助因子1-α(PGC-1α)mRNA水平,蛋白印迹(Western blot)分析胱天蛋白酶3(caspase-3)、活化半胱胺酸蛋白酶蛋白-3(cleave-caspase-3)、AMPK、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、SIRT1、PGC-1α、核呼吸因子1(NRF1)、线粒体转录因子A(TFAM)、叉头框蛋白O1(FOXO1)的蛋白表达情况。结果在PC12细胞的OGD/R模型中,ORI降低NO、MDA水平和LDH泄漏释放水平,通过上调SOD、ATP和CAT酶活性提高抗氧化能力,上调了AMPK、SIRT1和PGC-1αmRNA的表达,抑制cleave-caspase-3的蛋白表达,上调p-AMPK、SIRT1、PGC-1α、NRF1、TFAM和FOXO1蛋白表达。然而,这些作用分别被化合物C(一种特异性AMPK抑制剂)和si-SIRT1消除。结论ORI可以通过AMPK/SIRT1/PGC-1α信号通路改善线粒体能量代谢和减少氧化应激来减轻局灶性CIR损伤。

Objective To investigate whether orbicin(ORI)can reduce focal cerebral ischemia-reperfusion(ORI)injury by regulating mitochondrial function mediated by AMPK/SIRT1/PGC-1αsignaling pathway.Methods PC12 was randomly divided into 5 groups:Control group,cells were cultured normally without treatment;Oxygenglucose deprivation and reperfusion group(OGD/R group),oxygen-glucose deprivation and reperfusion models were established using PC12 cells;Oxygen-glucose deprivation and reperfusion group plus Orbicin(OGD/R+Orbicin),PC12 cells were pretreated with Orbicin for 4 h before establishing oxygen-glucose deprivation and reperfusion model;Oxygen-glucose deprivation and reperfusion+Orbicin+compound C group(OGD/R+Orbicin+CC),compound C(3μmol·L^(-1))was added to inhibit AMPK signaling for 6 h before oxygen-glucose deprivation and reperfusion,and PC12 cells were pretreated with Orbicin for 4 h;Oxygen-glucose deprivation and reperfusion group+Orbicin+si-SIRT1 group(OGD/R+Orbicin+si-SIRT1),PC12 cells were pretreated with Orbicin for 4 h before oxygen-glucose deprivation and reperfusion,and si-SIRT1 was transfected into PC12 cells.The cell viability of PC12 cells was evaluated by Methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay,and Enzyme-linked immunosorbent assay(ELISA)was used to the quantify lactate dehydrogenase(LDH)release,Malondialdehyde(MDA),Nitrogen monoxide(NO),Superoxide dismutase(SOD),Adenosine triphosphate(ATP)and Catalase(CAT)enzyme activities,Real-time quantitative polymerase chain reaction(PCR)assay Adenosine 5‘-monophosphate(AMP)-activated protein kinase(AMPK),Sirtuin 1(SIRT1)and Peroxisome proliferator-activated receptor coactivator 1α(PCC-1α)mRNA levels,Western blot analysis of caspase-3,cleave-caspase-3,AMPK,Phosphorylated adenosine monophosphate activated protein kinaseelisa Kit(p-AMPK),SIRT1,PCC-1α,Recombinant Nuclear Respiratory Factor 1(NRF1),Recombinant Transcription Factor A(TFAM),Recombinant Forkhead Box Protein O1(FOXO1)protein expression.Results In the OGD/R model of PC12 cells,Orbicin decreased the levels of NO,MDA and LDH leakage and release,increased the antioxidant capacity by upregulating the activities of SOD,ATP and CAT,and up-regulated the mRNA expression of AMPK,SIRT1 and PGC-1α.The protein expression of cleave-caspase-3 was inhibited,and the protein expression of p-AMPK,SIRT1,PGC-1α,NRF1,TFAM and FOXO1 was up-regulated.However,these effects were eliminated by compound C,a specific AMPK inhibitor,and si-SIRT1,respectively.Conclusion Orbicin can ameliorate mitochondrial energy metabolism and reduce oxidative stress through AMPK/SIRT1/PGC-1αsignaling pathway to alleviate focal cerebral ischemia-reperfusion injury.