黄芪总黄酮对病毒性心肌炎miRNA-1及缝隙连接蛋白CX43作用的实验研究

Viral myocarditis (VMC) is caused by a variety of viruses, leading to localized or diffuse lesions in the myocardial substantial or interstitial tissues. Arrythmia caused by the viral infection is the major mortality reason for the paritents with viral myocarditis. Cx43 is one of important components in ventricular myocardial gap junctions and it contributes to communications between cardiomycytes. MicroRNA (miRNA) is a class of non-coding RNA and miRNA-1 is one of miRNAs widely expressed the cardiovascular system. It has been reported that miRNA expression was elevatd in the virus infected cardiomyocytes, resulting in repression of Cx43 expression and arrythmia. The reduced Cx43 content has proven to be one of the causes of arrythmia. Clinically, Astragalus has been widely uterilzed in the treatment of arrythmia. However, it is still not clear that the mechanism by which astragalus prevents arrythmia and reduces the arrythmia incidence. Our preliminary data identified that total flavonoids of Astragalus (TFA), the effective component of astragalus,increased Cx43 expression and reduced the arrythmia incidence in the VMC model mice. Therefore, we hypothesize that miRNA-1 might be a mediator of TFA in enhancing Cx43 expression and playing a protective role from arrythmia in viral myocarditis. To test this hypothesis, we are planning to take advantage of the techniques of molecular biology and electrophysiology to study the effects of TFA on the expression of miRNA-1 and Cx43 in the VMC cells and animal models. This could propose a novel mechanism by which TFA treatment prevents arrythmia in the VMC patients. This could also provide a potential target for viral myocarditis arrhythmia.

病毒性心肌炎(VMC)是由病毒侵犯心脏引起的心肌实质或间质的局限性或弥漫性病变，心律失常是造成VMC病人猝死的主要原因。Cx43是心室缝隙连接主要构成成分，miRNA-1是一类非编码小分子RNA。研究表明，VMC时miRNA-1通过下调Cx43表达使Cx43合成减少。Cx43合成减少是发生心律失常的重要原因。大量研究证实黄芪对VMC并发心律失常疗效确实，但有效药物成分及作用机制尚不清楚。我们前期研究发现黄芪有效作用成分-黄芪总黄酮（TFA）可有效减少VMC小鼠心律失常发生，并能提高VMC小鼠Cx43mRNA表达及蛋白合成。据此我们推测，TFA是通过抑制MiRNA-1表达从而促进Cx43合成，抑制VMC并发心律失常。为了证实这一推断，本研究采用PCR、膜片钳等技术手段，对TFA干预下VMC细胞和动物模型MiRNA-1与Cx43表达及合成差异进行研究，阐明TFA对VMC并发心律失常的作用机制。

病毒性心肌炎（VMC）并发心律失常是VMC病人猝死的主要原因，前期工作显示：传统中药黄芪的有效成分——黄芪总黄酮(TFA)治疗VMC并发心律失常疗效确切，但机制尚未明确，为此进行本实验研究。研究内容：实验①②黄芪总黄酮(TFA)对病毒性心肌炎小鼠及CVB3感染心肌细胞miRNA-1及CX43作用。实验③TFA对CVB3感染心肌细胞CX43通道电流的影响。实验④TFA对CVB3感染心肌细胞CX43磷酸化信号传导通路的影响。实验⑤TFA对miRNA-1对CX43调控的影响。实验结果：①TFA使病毒性心肌炎（VMC）小鼠心肌细胞miRNA-1表达显著降低（p<0.001），同时，增加CX43表达（p<0.001）。②TFA能增加VMC小鼠心肌细胞磷酸化CX43表达（p<0.05）。③TFA可以减低CVB3感染心肌细胞miRNA-1表达（p<0.001）；TFA增加CVB3感染心肌细胞CX43表达（p<0.001）。④TFA对CVB3感染心肌细胞CX43亚细胞分布无明显变化。⑤TFA增加CVB3感染心肌细胞CX43电流（p<0.05）。⑥TFA可明显使CVB3感染心肌细胞磷酸化水平增高（p<0.001）。研究结论：①TFA可以减低病毒性心肌炎miRNA-1表达，进而增加CX43表达及磷酸化水平，可能减少心律失常发生。②病毒性心肌炎时，心肌细胞CX43表达明显减少，但CX43亚细胞分布无明显变化。③TFA减少CBV3感染心肌细胞miRNA-1表达并通过CX43启动子480区域增加CX43表达。④TFA可增加CX43通道电流，从而减少VMC心律失常发生。⑤TFA引起CX43表达增加及磷酸化水平增高是通过Erk及磷酸化Erk通路实现的。上述研究成果为黄芪总黄酮治疗VMC合并心律失常提供理论依据，开发出药物治疗的新靶点。