加味丹参饮通过抑制H2S介导的自噬信号通路对心肌缺血再灌注损伤的治疗作用

The therapeutic effect of Jiawei Danshen Decoction on myocardial ischemia-reperfusion injury by inhibiting H2S-mediated autophagy signaling pathway

    摘要
  • 摘要:
    目的探讨加味丹参饮(JWDSD)通过调节血清H2S和心肌Beclin1、LC3A/B、p62、ATG5对心肌缺血再灌注损伤(MIRI)的保护作用。
    方法SPF级SD大鼠70只，随机分为7组，每组10只，分别为正常对照组、假手术组、MIRI模型组(模型组)、缺血预适应组、NaHS组、JWDSD组、JWDSD + CSE抑制剂（JWDSD + PPG）组，各组大鼠均口服给药 14 d。最后一次给药两小时后，模型组、外源性H2S、JWDSD、和JWDSD + PPG组大鼠结扎冠状动脉左前降支30分钟，随后剪开结扎线再灌注90分钟建立模型，假手术组大鼠术后仅暴露胸腔，不进行冠状动脉结扎。采集血液样本，采用酶联免疫吸附法(ELISA)检测H2S水平。采集心脏组织，进行组织病理学和免疫组化检测，实时荧光定量聚合酶链式反应分析Beclin1、自噬蛋白5 (ATG5) mRNA表达，Western blot分析Beclin1、轻链3A/B (LC3A/B)、p62蛋白表达。
    结果(1)模型组大鼠血清H2S含量明显降低(P < 0.01)， JWDSD可明显提高模型组大鼠血清H2S含量(P < 0.01)，CSE抑制剂(PPG)明显降低JWDSD组大鼠H2S水平(P < 0.01)。(2)与正常对照组比较，MIRI模型组发生心肌组织坏死和细胞破坏，JWDSD可缓解模型大鼠心肌组织坏死，但PPG可逆转JWDSD的改善作用。(3)模型组心脏组织Beclin1、LC3A/B、p62表达水平明显升高(P < 0.001)，但可被JWDSD降低(P < 0.05, P < 0.01, P < 0.001)， JWDSD对Beclin1、LC3A/B、p62表达的抑制作用可被PPG部分逆转(P < 0.05, P < 0.01)。(4)模型组大鼠自噬相关基因Beclin1、ATG5表达水平明显升高(P < 0.001)，JWDSD可明显下调Beclin1和ATG5的表达(P < 0.05, P< 0.001)，而JWDSD的抑制作用可被PPG部分逆转（P< 0.001）。
    结论我们的实验结果显示，JWDSD可以通过增加内源性H2S的生成，下调Beclin1、LC3A/B、p62和ATG5的表达来抑制自噬信号通路，从而发挥抗MIRI作用。

     

    Abstract:
    ObjectiveTo 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 3A/B (LC3A/B), p62, and autophagy protein5 (ATG5).
    MethodsSeventy 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, NaHS, 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, NaHS, 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 mRNA expression and Western blot analysis of Beclin1, LC3A/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, LC3A/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, LC3A/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).
    ConclusionOur experimental data show that JWDSD can exhibit an anti-MIRI role by increasing endogenous H2S generation, and downregulating the expression of Beclin1, LC3A/B, p62 and ATG5, which are related to inhibiting autophagy signaling.

     

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