目的 基于转录组学及生物信息学探索心肌纤维化进程中线粒体自噬介导的胞葬机制,并进行潜在中药预测及实验验证。方法 皮下灌注血管紧张素Ⅱ(AngⅡ)构建心肌纤维化大鼠模型,进行心肌组织RNA提取、高通量测序,查找差异表达基因(DEGs)。从GeneCards数据库获取胞葬相关基因(ERGs),采用R 4.2.0软件中的“venneuler”包绘制DEGs与ERGs交集,找出两组间差异表达的胞葬相关基因(DEERGs),并对DEERGs进行KEGG和GO富集分析。进行DEERGs和线粒体自噬关键基因(PINK1、P62)蛋白相互作用(PPI)网络分析及核心基因鉴定,将核心基因导入Coremine数据库进行中药预测,其中黄芪、红花、葶苈子出现频率最高。结合心肌纤维化中医病机及既往研究基础,选择黄芪、红花、葶苈子组方进行动物实验验证。Masson染色分析纤维化程度,线粒体肿胀实验及线粒体膜电位检测评价线粒体功能障碍程度,RT-PCR和Western blot检测线粒体自噬基因mRNA及蛋白表达评价线粒体自噬水平,靶向代谢组学分析心肌组织糖酵解水平。分离心脏驻留巨噬细胞并使用AngⅡ干预,检测巨噬细胞对调亡内皮细胞胞葬率。结果 转录组学差异基因分析共428个DEGs,其中165个上调,263个下调。从GeneCards数据库识别出250个ERGs,绘制DEGs与ERGs交集,得出12个DEERGs。对DEERGs进行KEGG和GO富集分析显示过程涉及胞葬、线粒体自噬、内皮细胞调控等。对DEERGs和线粒体自噬关键基因(PINK1、P62)进行PPI网络分析及核心基因鉴定,其中前10名的核心基因为HIF1A、PINK1、GABARAP、ARG1、PPARG、UCP2、SQSTM1、CD36、SIRT1、RAB7A。将核心基因导入Coremine数据库,查找对应中药,其中出现频率最高的为黄芪、红花、葶苈子组方。实验验证显示,黄芪-红花-葶苈子组方抑制AngⅡ诱导的心肌纤维化形成、改善大鼠心功能,可以显著提高AngⅡ干预驻留巨噬细胞对凋亡内皮细胞的胞葬率,又以高剂量组效果最佳(P＜0.05)。采用黄芪-红花-葶苈子高剂量组进行机制研究则发现,对照与模型组,黄芪-红花-葶苈子组其糖酵解水平增加,线粒体自噬程度降低,线粒体功能障碍减轻(P＜0.05)。结论 黄芪-红花-葶苈子组方可能通过调节PINK1、P62表达抑制线粒体过度自噬,促进巨噬细胞胞葬作用,进而抑制心肌纤维化形成。

Objective To explore the regulatory mechanism of mitophagy mediated efferocytosis in the process of myocardial fibrosis based on transcriptomic sequencing and bioinformatics analysis, and to predict the potential treatment of traditional Chinese medicine (TCM) and verify the prediction by animal experiments. Methods A rat model of myocardial fibrosis was constructed by injecting angiotensin II subcutaneously. RNA extraction and high-throughput sequencing were performed on myocardial tissue of rats, and differentially expressed genes (DEGs) were detected. Efferocytosis-related genes (ERGs) were obtained from the GeneCards database, and the intersection of DEGs and ERGs was mapped using the "venneuler protein-to-protein interactions (PPI) and identification of key genes of DEERGs and mitochondrial autophagy (PINK1, P62) were conducted. The key genes were imported into Coremine database for TCM prediction. Combined with the TCM pathogenesis of myocardial fibrosis and the previous research progress, the highest frequency of Astragali Radix, Carthami Flos, and Lepidii Semen (A-C-L) was selected for animal experiments. Masson staining was used to analyze the degree of fibrosis. Mitochondrial swelling assay and mitochondrial membrane potential detection were performed to evaluate the extent of mitochondrial dysfunction. RT-PCR and Western blot were employed to measure the mRNA and protein expression of mitophagy-related genes to assess mitophagy levels. Targeted metabolomics was conducted to analyze glycolysis levels in myocardial tissue. Cardiac-resident macrophages were isolated, treated with AngⅡ, and subsequently assessed for their efferocytosis of apoptotic endothelial cells. Results There were 428 DEGs between in the control group and model group, including 165 up-regulated and 263 down-regulated DEGs. 250 ERGs were identified from GeneCards database, and 12 DEERGs were obtained by drawing the intersection of DEGs and ERGs. KEGG and GO enrichment analysis of DEERGs showed that the process involved cellular burial, mitochondrial autophagy and endothelial cell regulation. PPI network analysis and identification of key genes of DEERGs and mitochondrial autophagy (PINK1, P62) were performed. The top 10 key genes were HIF1A, PINK1, GABARAP, ARG1, PPARG, UCP2, SQSTM1, CD36, SIRT1 and RAB7A. The key genes were imported into Coremine database to search for corresponding TCM. The most frequent ones were Astragali Radix, Carthami Flos, and Lepidii Semen. Experimental validation demonstrated that the A-C-L formulation inhibited AngⅡ-induced myocardial fibrosis and improved cardiac function in rats. Notably, it significantly enhanced the efferocytosis of apoptotic endothelial cells by AngⅡ-treated resident macrophages, with the high-dose group showing the most pronounced effect (P < 0.05). Mechanistic studies using the high-dose A-C-L group revealed that, compared to control and model groups, the treatment group exhibited increased glycolysis levels, reduced mitophagy, and ameliorated mitochondrial dysfunction (P < 0.05). Conclusion The Astragali Radix-Carthami Flos-Lepidii Semen formulation may attenuate myocardial fibrosis formation by suppressing excessive mitophagy, upregulating glycolytic response, and subsequently activating macrophage efferocytosis.

]国家自然科学基金青年项目(82104797): 基于孙络“溢奇邪、通营卫”从外泌体携带整合素αvβ6介导血管内皮细胞与成纤维细胞间协作探讨红花-葶苈子药对抑制心肌纤维化机制,负责人：王咏；山东省自然科学基金面上项目(ZR2022MH307)：基于“风胜则动”从冠状动脉内皮细胞 STAT3/KCa3.1/eNOS 通路探讨川芎-地龙药对诱导冠状动脉舒张机制,负责人：王震；济南市科技计划(202225004)：QFR指导下MSSD术式加载麝香通心滴丸治疗冠状动脉分叉病变研究,负责人：王震。