丹酚酸B通过调控Cx43抑制铁死亡对心肌梗死大鼠模型的保护机制研究

Salvianolic acid B inhibits ferroptosis through Cx43 in myocardial infarc⁃tion rat model

目的:基于铁死亡探讨丹酚酸B(Sal B)对心肌梗死(MI)大鼠模型的保护作用,并分析缝隙连接蛋白43(Cx43)在其中的作用。方法:经结扎左冠状动脉法构建MI大鼠模型,随机分组为假手术(sham)组、MI组、Sal B组、Sal B+AAV9-GFP组、Sal B+AAV9-GFP-Cx43-siRNA组和Sal B+AAV9-GFP-Cx43-siRNA+铁死亡抑制剂ferro⁃statin-1(Fer-1)组,造模2 d后给予相应处理。4周后,HE染色和Perl blue染色分别观察心肌形态和铁累积;免疫荧光染色观察Cx43在心肌中分布;透射电镜观察心肌超微结构;试剂盒检测血清肌酸激酶MB(CK-MB)、心肌钙蛋白I(cTnI)和乳酸脱氢酶(LDH)水平,以及心肌丙二醛(MDA)、活性氧(ROS)、谷胱甘肽(GSH)和铁含量;Western blot检测心肌p-Cx43和Cx43蛋白水平。结果:相较于sham组,MI组心肌纤维出现坏死、断裂,排列紊乱,心肌线粒体出现损伤,Cx43蛋白在心肌细胞侧侧连接处弥散分布,血清CK-MB、cTnI和LDH水平,以及心肌MDA、ROS、铁离子含量及Perl blue阳性面积均增加(P<0.05),心肌GSH、p-Cx43和Cx43水平均降低(P<0.05)。Sal B治疗后心肌损伤明显缓解,心肌闰盘内Cx43蛋白增加,血清CK-MB、cTnI和LDH水平,以及心肌MDA、ROS、铁离子含量及Perl blue阳性面积均降低(P<0.05),心肌GSH、p-Cx43和Cx43水平均升高(P<0.05)。AAV9-GFP-Cx43-siRNA能减弱Sal B的上述保护作用,且这种减弱作用可被Fer-1抑制。结论:Sal B通过上调Cx43蛋白表达及磷酸化,改善其在心肌中分布,抵抗细胞铁死亡,从而对MI大鼠的心脏进行保护。

AIM:To investigate the protective effect of salvianolic acid B(Sal B)on myocardial infarction(MI)rat model based on ferroptosis,and to analyze the role of connexin 43(Cx43)in this process.METHODS:The MI rat model was established by ligating the left coronary artery.The rats were randomly divided into sham group,MI group,Sal B group,Sal B+AAV9-GFP group,Sal B+AAV9-GFP-Cx43-siRNA group and Sal B+AAV9-GFP-Cx43-siRNA+ferrop⁃tosis inhibitor ferrostatin-1(Fer-1)group,and the corresponding treatment was given 2 d after modeling.After 4 weeks,the myocardial morphological changes and iron accumulation were observed by HE staining and Perl blue staining,respec⁃tively.The distribution of Cx43 in the myocardium was observed by immunofluorescence staining.The myocardial ultra⁃structure was observed by transmission electron microscopy.The serum creatine kinase-MB(CK-MB),cardiac troponin I(cTnI)and lactate dehydrogenase(LDH)levels,and myocardial malondialdehyde(MDA),reactive oxygen species(ROS),glutathione(GSH),iron ion levels were detected by kits.The myocardial p-Cx43 and Cx43 protein levels were measured by Western blot.RESULTS:Compared with sham group,the myocardial fibers in MI group were necrotic,fractured and disarranged,and the myocardial mitochondria were damaged.The Cx43 protein was expressed in the side-toside junction of cardiomyocytes.Serum CK-MB,cTnI and LDH levels,myocardial MDA,ROS and iron ion levels,and Perl blue positive area were all increased(P<0.05),while myocardial GSH,p-Cx43 and Cx43 levels were all decreased(P<0.05).Myocardial injury was significantly relieved after treatment with Sal B.The Cx43 expression was increased in myocardial intercalated discs.Serum CK-MB,cTnI and LDH,myocardial MDA,ROS and iron ion levels,and Perl blue positive area were all decreased(P<0.05),while myocardial GSH,p-Cx43 and Cx43 levels were all increased(P<0.05).AAV9-GFP-Cx43-siRNA attenuated the protective effects of Sal B through Fer-1.CONCLUSION:Sal B protects the hearts of MI rats by up-regulating Cx43 expression and phosphorylation,and attenuating cell ferroptosis.