牡荆素调节自噬与自噬流介导缺氧复氧大鼠原代心肌细胞的保护作用

Protective effect and mechanism of vitexin on rat primary cardiomyocytes induced by hypoxia/reoxygenntion injury via regulating autophagy and autophagic flow

目的探讨牡荆素调节自噬与自噬流介导缺氧复氧大鼠原代心肌细胞的保护作用。方法采用原代心肌细胞缺氧/复氧(H/R)模拟缺血再灌注损伤。CCK-8检测心肌细胞的活力。试剂盒检测心肌细胞上清液中乳酸脱氢酶(LDH)水平。2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)检测原代心肌细胞中活性氧(ROS)的水平。自噬双标记腺病毒(mRFP-GFP-LC3)转染原代心肌细胞,观察自噬小体和自噬溶酶体数量。Western blot检测LC3BⅡ/Ⅰ、Beclin1、P62蛋白的表达。结果大鼠原代心肌细胞缺氧复氧后,H/R组心肌细胞活力显著降低,LDH漏出增多,心肌细胞内ROS水平明显升高,而牡荆素各剂量组可提高缺氧复氧心肌细胞活力,抑制心肌细胞的LDH释放,降低心肌细胞中ROS水平。心肌细胞转染mRFP-GFP-LC3病毒后,检测结果显示,与正常对照组比较,H/R组心肌细胞出现明显的自噬和自噬流增强,而牡荆素各剂量组可抑制心肌细胞H/R后的自噬和自噬流增强,减少心肌细胞内自噬小体的累积。另外,Western blot结果显示,与正常对照组比较,H/R组原代心肌细胞中自噬蛋白Beclin 1、LC3Ⅱ蛋白表达增加,LC3Ⅱ/Ⅰ比值增大,而P62蛋白表达下调。与H/R组比较,牡荆素各剂量组均不同程度抑制Beclin 1上调,降低LC3Ⅱ蛋白表达,LC3Ⅱ/Ⅰ的比值减小,同时上调P62蛋白表达。结论牡荆素对大鼠原代心肌细胞H/R损伤有明显的保护作用,其机制可能与其抑制心肌细胞H/R后ROS产生,抑制自噬和自噬流增强,减少心肌细胞自噬体累积,防止过度自噬有关。

Objective To investigate protective effect and mechanism of vitexin on rat primary cardiomyocytes induced by hypoxia/reoxygenntion injury via regulating autophagy and autophagic flow.Methods Hypoxia/reoxygenation(H/R)of primary cardiomyocytes was used to simulate ischemia-reperfusion injury.The viability of cardiomyocytes was tested by CCK8.LDH levels in cardiomyocyte supernatant were detected by kit.ROS levels were detected by DCFH-DA fluorescent probe.Primary cardiomyocytes were infected with mRFP-GFP-LC3,and the number of Autophagolysosomes and autophagosomes was evaluated.Beclin1,LC3Ⅱ/Ⅰand P62 protein expression were detected by Western blot.Results After hypoxia and reoxygenation(H/R)of primary cardiomyocytes,the LDH value of primary cardiomyocytes in the H/R group was significantly higher than that in the control group,and the ROS level significantly increased,the doses of vitexin could significantly inhibit the release of LDH from H/R cardiomyocytes and reduce the ROS level in cardiomyocytes.The primary cardiomyocytes were transfected with mRFP-GFP-LC3 virus,and the results showed that compared with the control group,the cardiomyocytes in the H/R group showed significant enhancement of autophagy and autophagy flow.suggesting that autophagosomes accumulated in myocardial cells after H/R,and autophagolysosomes increased accordingly;The vitexin dose groups could inhibit autophagy and increase autophagy flow in cardiomyocytes after H/R,and reduce the accumulation of autophagosomes in cardiomyocytes.In addition,Western blot results showed that compared with the normal control group,the expression of autophagy proteins Beclin 1,and LC3Ⅱin primary myocardial cells of the H/R group increased,and the ratio of LC3Ⅱ/Ⅰincreased,P62 protein in cardiomyocytes was down regulated;Compared with the H/R group,vitexin inhibited Beclin 1 up-regulation and decreased the expression of LC3Ⅱprotein,the ratio of LC3Ⅱ/Ⅰreduced,and the expression of P62 protein also increased in cardiomyocytes.Conclusion Vitexin has a significant protective effect on H/R injury in rat primary myocardial cells,and its mechanism may be related to the inhibition of ROS production,autophagy and autophagic flow enhancement,and the accumulation of autophagosomes after H/R in myocardial cells.