党参改善H2O2诱导心肌细胞损伤的机制研究

Study on the mechanism of Codonopsis Radix ameliorating H2O2-induced myocardial cell injury

关键词:  党参  心肌损伤  氧化应激  凋亡  网络药理学

Key words:  Codonopsis Radix  myocardial injury  oxidative stress  apoptosis  network pharmacology

摘要:

[目的] 运用网络药理学结合细胞实验探讨党参（CR）抗心肌损伤的作用及潜在机制。[方法] 通过中药系统药理学数据库与分析平台（TCMSP）、化合物靶点预测数据库（SwissTargetPrediction）获取党参的活性成分及靶点，利用基因组数据库（GeneCards）获取心肌损伤相关靶点，Cytoscape软件构建“成分-靶点-疾病”网络及蛋白互作（PPI）网络图；通过注释、可视化和综合发现数据库（DAVID）对交集靶点进行基因本体论（GO）分析和基因组百科全书通路（KEGG）富集分析，预测党参改善心肌损伤的潜在作用靶点。体外使用不同浓度党参（50、100 μg/mL）处理过氧化氢（H2O2，300 μmol/L）刺激的H9c2心肌细胞，活性氧荧光探针检测细胞内线粒体活性氧（ROS）含量；生化试剂盒检测心肌细胞丙二醛（MDA）和超氧化物歧化酶（SOD）水平；免疫印迹法（Western blot）检测凋亡相关蛋白B细胞淋巴瘤-2相关X蛋白（Bax）、半胱氨酸蛋白酶-3（Caspase-3）和细胞色素C（Cytochrome C）表达，以探讨党参抗心肌损伤的潜在作用机制。[结果] 网络药理学分析得到党参21个活性成分，潜在靶点278个。心肌损伤相关靶点835个，疾病与药物交集靶点106个。GO和KEGG富集分析显示，党参对心肌细胞损伤的保护主要涉及磷脂酰肌醇3激酶-蛋白激酶B信号通路、化学致癌-活性氧和凋亡信号通路等。细胞实验结果显示，50和100 μg/mL党参均能显著降低心肌细胞MDA含量和线粒体ROS含量，升高SOD水平。Western blot结果显示，党参抑制了促凋亡蛋白Bax、Cytochrome C和Caspase-3的表达。[结论] 党参可能通过抑制氧化应激引起的细胞凋亡发挥对H₂O 刺激的心肌细胞的保护作用。

Abstract:

[Objective] To investigate the effect and potential mechanism of Codonopsis Radix(CR) in preventing myocardial injury by network pharmacology combined with in vitro experiments.[Methods] The active ingredients and targets of CR were obtained from Traditional Chinese Medicine System Pharmacology(TCMSP) and SwissTargetPrediction databases, and the targets related to myocardial injury were obtained by using GeneCards database, and the “component-target-disease” network and protein-protein interaction(PPI) network diagram were constructed by using Cytoscape. Gene ontology(GO) analysis and kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis were performed on the intersection targets by using the Annotation, Visualization, and Integrated Discovery(DAVID) database, to predict CR potential targets for the treatment of myocardial injury. H9c2 cardiomyocytes stimulated by hydrogen peroxide(H₂O₂, 300 μmol/L) were treated with different concentrations of CR(50 and 100 μg/mL) in vitro, and the content of mitochondrial reactive oxygen species(ROS) was detected by ROS fluorescent probe. The biochemical kit was used to detect the levels of malondialdehyde(MDA) and superoxide dismutase(SOD) in cardiomyocytes. Western Blot was used to detect the expressions of apoptosis-related proteins bcl-2-associated X protein(Bax), cysteine protease-3(Caspase-3) and cytochrome C(Cytochrome C), to investigate the potential mechanism of CR in anti-myocardial injury.[Results] Network pharmacology analysis revealed 21 active ingredients and 278 potential targets of CR. There were 835 myocardial injury-related targets and 106 intersecting targets. GO and KEGG enrichment analysis showed that the protection of CR on cardiomyocyte injury mainly involved PI3K-Akt signaling pathway, chemical carcinogenesis-reactive oxygen species and apoptosis signaling pathway. The results of cell experiments showed that 50 and 100 μg/mL CR could significantly reduce the content of MDA and mitochondrial ROS in cardiomyocytes, and increase the level of SOD. Western Blot results showed that CR inhibited the expression of pro-apoptotic proteins Bax, Cytochrome C and Caspase-3.[Conclusion] CR may exert a protective effect on cardiomyocytes by inhibiting apoptosis caused by oxidative stress.