丹参酮ⅡA通过miR-155-5p激活SIRT1-AMPK通路改善H9c2心肌细胞缺血/再灌注损伤

TanshinoneⅡA ameliorates ischemia/reperfusion injury in H9c2 cardiomyocytes by activating SIRT1-AMPK pathway via miR-155-5p

目的:探究丹参酮ⅡA(TⅡA)通过miR-155-5p激活沉默信息调节因子1(SIRT1)-腺苷酸活化蛋白激酶(AMPK)通路改善H9c2心肌细胞缺血/再灌注(I/R)损伤的机制。方法:体外培养H9c2细胞并建立I/R损伤模型,造模完成后将H9c2细胞随机分为模型组、TⅡA组、TⅡA+miR-NC组、TⅡA+miR-155-5p mimics组,转染后分别加入10μmol/L TⅡA进行干预,并以添加DMSO的H9c2细胞作为对照组。qRT-PCR检测miR-155-5p表达水平;MTT法分析细胞增殖能力;流式细胞术评估细胞凋亡情况;ELISA测定TNF-α、IL-4、IL-10、IL-17、乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平;Western blot检测SIRT1、AMPK、p-AMPK蛋白水平。结果:与对照组相比,模型组miR-155-5p表达升高,细胞活力下降,凋亡率及TNF-α、IL-17、LDH、MDA表达升高,IL-4、IL-10、SOD、SIRT1、p-AMPK表达减少(P<0.05);与模型组比较,TⅡA组miR-155-5p表达降低,细胞活力增强,凋亡率及TNF-α、IL-17、LDH、MDA表达下降,IL-4、IL-10、SOD、SIRT1、p-AMPK表达升高(P<0.05);与TⅡA组和TⅡA+miR-NC组比较,TⅡA+miR-155-5p mimics组miR-155-5p表达升高,细胞活力降低,凋亡率上升,TNF-α、IL-17、LDH、MDA表达上升,IL-4、IL-10、SOD、SIRT1、p-AMPK表达降低(P<0.05)。结论:TⅡA可通过下调miR-155-5p改善H9c2心肌细胞I/R损伤,其机制可能与激活SIRT1-AMPK通路有关。

Objective:To explore the mechanism of TanshinoneⅡA(TⅡA)in improving ischemia/reperfusion(I/R)injury of H9c2 cardiomyocytes by activating Sirtuin 1(SIRT1)-adenosine 5'-monophosphateactivated protein kinase(AMPK)pathway through miR-155-5p.Methods:H9c2 cells were cultured in vitro and I/R damage model was established.After modeling,H9c2 cells were randomly divided into model group,TⅡA group,TⅡA+miR-NC group,TⅡA+miR-155-5p mimics group,10μmol/L TⅡA was added for intervention after transfection,and the H9c2 cells supplemented with DMSO were used as control group.qRT-PCR was used to detect expression level of miR-155-5p;MTT method was used to analyze cell proliferation ability;flow cytometry was used to evaluate cell apoptosis;ELISA was used to determine the levels of TNF-α,IL-4,IL-10,IL-17,lactate dehydrogenase(LDH),malo⁃ndialdehyde(MDA)and superoxide dismutase(SOD);Western blot was used to detect relative expressions of SIRT1,AMPK and p-AMPK proteins.Results:Compared with control group,expression of miR-155-5p in model group was increased,cell viability was decreased,apoptosis rate and expressions of TNF-α,IL-17,LDH and MDA were increased,while expressions of IL-4,IL-10,SOD,SIRT1 and p-AMPK were decreased(P<0.05);compared with model group,expression of miR-155-5p in TⅡA group was reduced,cell viability was increased,apoptosis rate and expressions of TNF-α,IL-17,LDH and MDA were decreased,while expressions of IL-4,IL-10,SOD,SIRT1 and p-AMPK were increased(P<0.05);compared with TⅡA group and TⅡA+miR-NC group,expression of miR-155-5p in TⅡA+miR-155-5p mimics group was increased,cell viability was decreased,apoptosis rate and expressions of TNF-α,IL-17,LDH and MDA were increased,while expressions of IL-4,IL-10,SOD,SIRT1 and p-AMPK were decreased(P<0.05).Conclusion:TⅡA can improve I/R injury of H9c2 cardiomyocytes by down-regulating miR-155-5p,and its mechanism may be related to the activation of SIRT1-AMPK pathway.