基于“化痰祛浊”治法探索瓜蒌薤白汤改善糖尿病心肌纤维化的机制研究

Diabetes mellitus may produce extensive myocardial fibrosis (CF), which causes heart failure and death, and there is no effective controlling measures for it at present. Traditional chinese medicine believes that diabetes developed Yin deficiency and body fluid metabolism in the early period. If delayed healing, it may cause organ dysfunction, which then produce phlegm and turbidity stasis. If the disease aggravate, symptoms of heart failure may appear，such as “chest congestion with phlegm stasis, shortness of breath with orthopnoea ”. Phlegm and turbidity endogeny is an important pathogenesis of CF. Therefore, we envisage whether the treatment of "liminating phlegm and removing turbidity" is effective. Our previous research using trichosanthes kirilowii and allium macrostemon extract (TAE), confirmed that it could inhibit activation of myocardial fibroblasts (CFs), significantly improve myocardial fibrosis in diabetic mice. Combined with other studies, we put forward the following hypothesis: "TAE inhibits CFs activation and improves diabetic myocardial fibrosis through regulating AP-1/miR-125-5p/Apelin signaling pathway". Based on two levels of cell and animal models, we used DARTS, luciferase reporter gene and mimics/inhibitor plasmid intervention to analyze the effection and molecular mechanism of TAE on diabetic CF. The study not only provides new perspectives and new targets for the combination of traditional Chinese and Western medicine in the treatment of CF, but also helps to develop new drugs for the treatment of CF.

糖尿病导致广泛心肌纤维化（CF），引起心力衰竭和死亡，目前尚无有效防治措施。中医认为消渴症初期以阴虚为本、津液代谢失常，若迁延不愈，则脏腑功能失调、聚湿生痰、精化为浊、不得散布，进一步加重可出现“痰多喘闷、气短不得卧”的心衰表现。痰浊内生是CF的重要病机。据此我们设想“化痰祛浊”治法是否对其有效。前期研究选用瓜蒌薤白汤（TAE），证实其能抑制心肌成纤维细胞（CFs）激活，显著改善糖尿病小鼠心肌纤维化。结合其他预研究结果，我们提出以下假设：“TAE通过调控AP-1/miR-125-5p/Apelin信号通路，抑制CFs激活，改善糖尿病心肌纤维化”。本研究拟通过细胞和动物模型两个层次，采用DARTS、荧光素酶报告基因、mimics/inhibitor质粒干预等多种生物学技术，分析TAE对糖尿病CF的作用及其分子机制。研究不但为中西医结合治疗CF提供新视角、新靶点，更有助于开发治疗CF的新药物。

糖尿病导致广泛心肌纤维化（CF），引起心力衰竭和死亡，目前尚无有效防治措施。中医认为消渴症初期以阴虚为本、津液代谢失常，若迁延不愈，则脏腑功能失调、聚湿生痰、精化为浊、不得散布，进一步加重可出现“痰多喘闷、气短不得卧”的心衰表现。痰浊内生是CF的重要病机。据此我们基于“化痰祛浊”治法探讨瓜蒌薤白汤（TAE）对心肌纤维化及糖尿病心肌病（DCM）的疗效。.研究内容主要包括探讨TAE对DCM和CF的作用、miR-125b-5p在TAE抑制CFs转化中的作用、TAE对AP-1的作用、TAE抑制miR-125b-5p和miR-125b-5p对Apelin基因负调控的机制。结果显示TAE治疗可抑制AGEs诱导的CFs增殖和胶原表达，显著降低DCM小鼠CFs中miR125b-5p的水平并增加Apelin蛋白，这与核转录因子AP-1有关。通过沉默Apelin基因、过表达miR125b-5p、免疫沉淀、芯片PCR、荧光素酶分析、生物信息学分析和荧光素酶报告基因法证实TAE预处理能有效抑制AP-1亚基c-Jun和c-Fos之间转录复合物的形成， AP-1可以通过TFBS位点与miR125b-5p启动子结合并正向调控其转录水平，miR125b-5p在Apelin mRNA的3'-UTR存在种子区结合位点，且通过结合该靶位点抑制Apelin蛋白表达。.研究证实TAE通过与AP-1转录复合物两个亚基之一的c-Jun结合而抑制核转录因子AP-1的转录活性，并通过AP-1/miR125b-5p/Apelin途径上调CFs中抗纤维化蛋白Apelin表达并最终抑制AGEs诱导的CFs活化。该研究不但为TAE抗糖尿病心肌纤维化的作用提供了理论基础，还首次发现并阐述了AP-1/miR125b-5p/Apelin途径在DCM患者心肌纤维化调控中的作用。本研究对于将我国传统中药应用于DCM患者治疗具有重要的意义和推动作用。