丹参酮ⅡA改善脂多糖诱导的心肌细胞损伤机制研究

TanshinoneⅡA improves lipopolysaccharide-induced myocardial cell injury

目的探讨丹参酮ⅡA(TanⅡA)改善脂多糖(LPS)诱导心肌细胞损伤的机制。方法采用LPS构建脓毒症心肌损伤体外实验模型,将心肌H9C2细胞分为Control组、LPS组(25 mg/L LPS处理)、TanⅡA-L组(LPS+5μmol/L TanⅡA处理)、TanⅡA-M组(LPS+10μmol/L TanⅡA处理)、TanⅡA-H组(LPS+20μmol/L TanⅡA处理)、TanⅡA-H-CC组(LPS+20μmol/L TanⅡA+10μmol/L AMPK信号通路抑制剂Compound C)。CCK-8法测定细胞存活率。试剂盒检测丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)水平,ELISA法测定细胞培养上清液中的白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)水平。Western blot检测腺苷酸活化蛋白激酶(AMPK)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、细胞因子信号传导抑制因子3(SOCS3)蛋白表达。结果与Control组相比,5、10、20μmol/L的TanⅡA处理后的心肌细胞存活率差异无统计学意义(P>0.05);与Control组相比,LPS组细胞存活率降低,MDA、SOD、LDH、IL-1β、IL-6和TNF-α水平升高,p-AMPK、SOCS3蛋白表达水平降低(P<0.05)。与LPS组相比,TanⅡA-L组、TanⅡA-M组、TanⅡA-H组细胞存活率依次升高,MDA、SOD、LDH、IL-1β、IL-6和TNF-α水平依次降低,p-AMPK、SOCS3蛋白表达水平依次升高(P<0.05)。AMPK信号通路抑制剂能够逆转TanⅡA对LPS诱导的心肌细胞存活率和MDA、SOD、LDH、IL-1β、IL-6、TNF-α水平的影响以及下调p-AMPK、SOCS3的蛋白表达。结论TanⅡA改善LPS诱导的心肌细胞损伤,其作用机制可能与上调AMPK/SOCS3信号通路激活水平,减轻氧化应激损伤及炎症反应有关。

Objective To explore the mechanism by which tanshinoneⅡA(TanⅡA)improves lipopolysaccharide(LPS)-induced myocardial cell injury.Methods An in vitro model of septic myocardial injury was established using LPS.H9C2 myocardial cells were divided into the following groups:a Control group,a LPS group(treatment with LPS at 25 mg/L),a TanⅡA-L group(LPS+5μmol/L TanⅡA),a TanⅡA-M group(LPS+10μmol/L TanⅡA),a TanⅡA-H group(LPS+20μmol/L TanⅡA),and a TanⅡA-H-CC group(LPS+20μmol/L TanⅡA+10μmol/L AMPK pathway inhibitor Compound C).The cell survival rate was measured by CCK-8 assay.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),and lactate dehydrogenase(LDH)were determined using assay kits.The levels of interleukin(IL)-1β,IL-6,and tumor necrosis factor-α(TNF-α)in cell culture supernatant were detected by ELISA.The levels of AMP-activated protein kinase(AMPK),phosphorylated AMP-activated protein kinase(p-AMPK),and suppressor of cytokine signaling 3(SOCS3)proteins were measured by Western blot.Results Compared with the Control group,there were no statistical differences in myocardial cell survival rate among the TanⅡA treatment groups(5,10,and 20μmol/L TanⅡA)(P>0.05).Compared with the Control group,the LPS group showed decreases in cell survival rate and increases in MDA,SOD,LDH,IL-1β,IL-6,and TNF-αlevels,with a reduction in the expression of p-AMPK and SOCS3 proteins(P<0.05).Compared with the LPS group,the TanⅡA-L,TanⅡA-M,and TanⅡA-H groups exhibited increased cell survival rate,decreased levels of MDA,SOD,LDH,IL-1β,IL-6,and TNF-αand elevated expression of p-AMPK and SOCS3 proteins in a dose-dependent manner(P<0.05).The AMPK pathway inhibitor reversed the effect of TanⅡA on LPS-induced myocardial cell survival rate,as well as the levels of MDA,SOD,LDH,IL-1β,IL-6,and TNF-α,and down-regulated the expression of p-AMPK and SOCS3 proteins.Conclusions TanⅡA improves LPS-induced myocardial cell injury,which may be related to the upregulation of the AMPK/SOCS3 signaling pathway,thereby reducing oxidative stress damage and inflammatory responses.