The therapeutic effect of Jiawei Danshen Decoction on myocardial ischemia-reperfusion injury by inhibiting H_(2)S-mediated autophagy signaling pathway

Objective To investigate the protective effects of Jiawei Danshen Decoction(加味丹参饮,JWDSD)on myocardial ischemia-reperfusion injury(MIRI)via the regulation of serum Hydrogen sulfide(H2S)and cardiac Beclin1,light Chain 3 A/B(LC3 A/B),p62,and autophagy protein5(ATG5).Methods Seventy specific pathogen free(SPF)Sprague-Dawley(SD)rats were randomly assigned to seven groups(n=10 in each group),including normal control,sham operation,MIRI model(model),ischemic preconditioning,Na HS,JWDSD,and JWDSD+CSE inhibitor(JWDSD+PPG)groups,and orally administered the indicated drugs for 14 d.Two hours after the last administration,the left anterior decreased branch of the coronary artery of each rat in model,Na HS,JWDSD,and JWDSD+PPG groups was ligated for 30 min and subsequently reperfused for 90 min to establish the MIRI model,and the rats in the sham operation group were only exposed to the thorax after surgery without coronary ligation.Blood samples were collected to detect H2S levels using an enzyme-linked immunosorbent assay(ELISA).Heart tissues were harvested for histopathological and immunohistochemical examination and quantitative reverse transcription polymerase chain reaction analysis of Beclin1 and ATG5 m RNA expression and Western blot analysis of Beclin1,LC3 A/B,and p62 protein expression.Results(1)The serum H2S content in model group rats was significantly reduced(P<0.01),JWDSD significantly increased the serum H2S content of model group rats(P<0.01),and the CSE inhibitor(PPG)significantly reduced H2S levels in the JWDSD group rats(P<0.01).(2)Compared with the normal control group,the myocardial tissue necrosis and cell destruction occurred in the MIRI model group,and JWDSD could alleviate the myocardial tissue necrosis of model rats,but the ameliorative effect of JWDSD could be reversed by PPG.(3)Beclin1,LC3 A/B,and p62 expression levels in the heart tissues of the model group were significantly increased(P<0.001),whereas decreased by JWDSD(P<0.05,P<0.01,and P<0.001,respectively),and the inhibitory effects of JWDSD on Beclin1,LC3 A/B,and p62 expression were partially reversed by PPG(P<0.01,P<0.05,and P<0.01,respectively).(4)The expression levels of autophagy-related genes Beclin1 and ATG5 were significantly increased in the model group(P<0.001).JWDSD clearly downregulated the expression levels of Beclin1 and ATG5(P<0.05 and P<0.001,respectively),which were reversed by PPG(P<0.001).Conclusion Our experimental data show that JWDSD can exhibit an anti-MIRI role by increasing endogenous H2S generation,and downregulating the expression of Beclin1,LC3 A/B,p62 and ATG5,which are related to inhibiting autophagy signaling.

加味丹参饮含药血清对缺氧/复氧H9C2心肌细胞自噬相关蛋白LC3Ⅱ及Beclin1表达的影响

目的探讨加味丹参饮治疗心肌缺血再灌注损伤的可能作用机制。方法将体外培养的H9C2心肌细胞随机分为6组,每组6个复孔。空白血清组在含15%空白血清培养液中常规培养29 h;缺氧预处理组更换缺氧培养液缺氧20 min,复氧20 min,再缺氧20 min后换为空白血清培养液常规培养24 h,缺氧2 h,再给氧3 h;缺氧/复氧组在含15%空白血清培养液中常规培养24 h,更换缺氧培养液,缺氧2 h,再给氧3 h;含药血清组在含15%药物血清中常规培养24 h,缺氧2 h,再给氧3 h;含药血清+自噬抑制剂组用3-甲基腺嘌呤10 mmol/L预处理30 min,余同含药血清组;含药血清+自噬激动剂组用雷帕霉素100 nmol/L预处理30 min,余同含药血清组。倒置显微镜观察各组心肌细胞生长及形态变化,比色法检测定各组细胞培养上清液中乳酸脱氢酶(LDH)漏出率,透射电子显微镜观察心肌细胞自噬体形态及数量变化,Western blot法检测各组自噬相关蛋白LC3Ⅱ及Beclin1表达。结果与空白血清组比较,缺氧/复氧组镜下心肌细胞变性坏死程度增加,电镜下有少量自噬体,培养液中LDH漏出率增加,Beclin1蛋白水平增加(P<0.05或P<0.01);与缺氧/复氧组比较,含药血清组及含药血清+自噬激动剂组镜下心肌细胞变性坏死程度减轻,电镜下自噬体数量增多,培养液中LDH漏出率显著降低,LC3Ⅱ及Beclin1蛋白表达上升(P<0.05)。结论加味丹参饮可能通过上调自噬相关蛋白LC3Ⅱ及Beclin1表达,增强细胞自噬,从而保护损伤的心肌细胞。