基于“转录组-成分靶标-表型基因”多维网络全局式探索化湿败毒方抗心肌损伤潜在作用机制

Underlying Mechanisms of Huashi Baidu Prescription Against Myocardial Injury Based on "Transcriptome-Putative Target-Phenotype Gene" Interactions

摘要:

目的 基于“转录组-成分靶标-表型基因”多维网络全局式探索化湿败毒方抗心肌损伤的作用机制。方法 基于中医药百科全书（ETCM 2.0，http：//www.tcmip.cn/ETCM2/front/）及中医药整合药理学研究平台（TCMIP v2.0，http：//www.tcmip.cn/TCMIP/index.php/Home/Login/login.html）收集化湿败毒方成分预测靶标集；基于CNKI及PubMed数据库挖掘化湿败毒方抗新型冠状病毒感染（COVID-19）的临床优势症状表型，基于CTD数据库收集多柔比星临床不良反应症状，并利用HPO数据库及SoFDA数据平台（http：//www.tcmip.cn/Syndrome/front/#/）收集表型相关基因。构建“转录组-成分靶标-表型基因”多维分子网络，并计算网络节点拓扑特征值，筛选网络核心节点，并开展功能挖掘阐释方药作用机制。在此基础之上，基于多柔比星诱导心肌损伤动物模型，开展药效学评价和机制验证。65只SPF级C57BL/6J雄性小鼠，随机分为5组：正常组、多柔比星诱导模型组、化湿败毒方低、中、高给药组，各组13只。用于转录组测序采用苏木素-伊红（HE）及马松（Masson）染色进行组织病理学检测、实时荧光定量聚合酶链式反应（Real-time PCR）检测心脏组织中纤维化指标、蛋白免疫印迹法（Western blot）检测蛋白表达检测。结果 基于ETCM 2.0及TCMIP v2.0共得到1 044个化湿败毒方候选靶标；基于CTD数据库、HPO数据集及SoFDA数据平台，共得到1 223个方药潜在效应靶标集；心脏转录组测序（RNA-seq），以P<0.05，FC>1.5/<0.667为筛选标准，共获得214个多柔比星诱导心肌损伤相关基因，58个化湿败毒方效应基因。多维分子网络构建与分析后，发现化湿败毒方干预心肌损伤的相关作用机制主要与机体免疫-炎症失衡相关，其次参与细胞代谢，心肌细胞功能及纤维化，血管生成、收缩与血小板活化，DNA合成、代谢与修复，以及细胞死亡与氧化应激反应，改善射血分数降低、心肌纤维化、心肌炎、高油酸三脂血症、动/静脉血栓形成等心血管系统异常性症状表型。动物实验表明，化湿败毒方可通过抑制多柔比星诱导后心肌组织NOD样受体热蛋白结构域蛋白3（NLRP3）、胱天蛋白酶-1（Caspase-1）、凋亡相关斑点样蛋白（ASC）、消皮素D（GSDMD）异常高表达（P<0.05），改善转化生长因子-β₁（TGF-β₁）、Smad3及α-平滑肌肌动蛋白（α-SMA）纤维化指标mRNA水平的异常升高（P<0.05），发挥抗心肌纤维化作用。结论 化湿败毒方可通过多成分、多靶点、多效应改善心肌损伤，其中抑制NLRP3炎症小体激活途径为其优势药效环节，为后续靶向治疗药物的开发提供新思路。

Abstract:

Objective To systematically explore the underlying mechanisms of Huashi Baidu prescription （HBP） against myocardial injury through a multidimensional network analysis of "transcriptome-putative target-phenotype gene".Method Putative targets of compounds in HBP were predicted using the Encyclopedia of Traditional Chinese Medicine （ETCM 2.0，http：//www.tcmip.cn/ETCM2/front/） and the Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine （TCMIP v2.0，http：//www.tcmip.cn/TCMIP/index.php/Home/Login/login.html）. Clinical predominant symptom phenotypes for HBP against coronavirus disease 2019 （COVID-19） were collected from CNKI and PubMed databases， while clinical side effects of doxorubicin were gathered from the CTD database. Phenotype-related genes were collected from the HPO database and SoFDA data platform （http：//www.tcmip.cn/Syndrome/front/#/）. An interaction network of "transcriptome-putative target-phenotype gene" was constructed. Key nodes were identified through topological feature calculations， and functional enrichment analysis was conducted to explain the underlying mechanism. Pharmacodynamic evaluation and mechanism verification were performed using a doxorubicin-induced myocardial injury mouse model. Sixty-five SPF-grade C57BL/6J male mice were randomly divided into a control group， a doxorubicin model group， and low-， medium-， and high-dose HBP groups （HBP-L/M/H）， with 13 mice per group. Histopathological severity was assessed using hematoxylin-eosin （HE） and Masson staining. Fibrosis markers were measured by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and protein expression was detected by Western blot.Result A total of 1 044 putative targets for HBP were obtained from ETCM 2.0 and TCMIP v2.0. From the CTD， HPO， and SoFDA databases， 1 223 potential effect-related targets were identified. Cardiac transcriptome sequencing （RNA-seq） yielded 214 genes related to doxorubicin-induced myocardial injury and 58 genes associated with the effects of HBP， using criteria of P-value<0.05 and FC > 1.5/< 0.667. Analysis of the multidimensional molecular network revealed that the mechanisms of HBP on myocardial injury were mainly related to immune-inflammation imbalance， cellular metabolism， myocardial cell function and fibrosis， angiogenesis， contraction and platelet activation， DNA synthesis， metabolism and repair， as well as cell death and oxidative stress. HBP improved cardiovascular abnormalities such as reduced ejection fraction， myocardial fibrosis， myocarditis， hypertriglyceridemia， and arterial/venous thrombosis. Animal experiments demonstrated that HBP reduced the abnormal high expression of Nod-like receptor heat protein domain Protein 3 （NLRP3）， Caspase-1， apoptosis-associated speck-like protein （ASC）， and Gasdermin D （GSDMD） in myocardial tissue induced by doxorubicin （P<0.05）， and improved the elevated mRNA levels of fibrosis markers transforming growth factor-β₁ （TGF-β₁）， Smad3， and α-smooth muscle actin （α-SMA） （P<0.05）.Conclusion HBP can improve myocardial injury through multiple components， targets， and effects， with the inhibition of the NLRP3 inflammasome activation pathway being a key pharmacodynamic mechanism. This study is expected to provide new insights for the development of targeted therapeutic drugs.