Table2_An Integrated Approach Based on Network Analysis Combined With Experimental Verification Reveals PI3K/Akt/Nrf2 Signaling Is an Important Way for the Anti-Myocardial Ischemia Activity of Yi-Qi-Tong-Luo Capsule.xlsx

Background:Yiqi-Tongluo Capsule (YTC) is a Chinese traditional patent medicine that has been used in the treatment of myocardial ischemia (MI). However, its molecular mechanisms against MI have not been clear.
Methods: Network analysis and experimental verification were used to explore the potential molecular mechanisms of YTC for MI treatment. Firstly, the main components in the capsules and the potential targets of these components were predicted by online databases. The MI related genes were collected from Genecards and Online Mendelian Inheritance in Man (OMIM) databases. The drug targets and disease targets were intersected, and then the protein-protein interaction (PPI) and Drug-Molecular-Target-Disease Network (DMTD) were constructed, and GO enrichment analysis and KEGG pathway enrichment analysis were performed. Based on the H2O2-stimulated H9c2 cells, flow cytometry, western blot (WB) and immunofluorescence experiments were performed to verify the network analysis prediction.
Results: A total of 100 active components and 165 targets of YTC were predicted, in which there were 109 targets intersected with the targets of MI. GO and KEGG analysis showed that these potential targets were related to a variety of biological processes and molecular mechanisms, including oxidative stress and PI3K/AKT pathway. Astragaloside IV (AS IV) and paeoniflorin (PAE) might be the main active components in YTC. The results of cell counting kit-8 (CCK-8) showed that YTC alleviated the damage of H2O2 to H9c2 cells. The results of flow cytometry, DAPI staining and JC-1 probe showed that YTC alleviated H2O2 induced apoptosis in H9c2 cells. In addition, YTC reduced the level of intracellular superoxide anion, increased the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and reduced the content of malondialdehyde (MDA) in H2O2-induced H9c2 cells. The results of immunofluorescence and WB showed that the phosphorylation of PI3K and Akt were increased, the expression of Bcl-2 was up-regulated and the expression of cleaved caspase-3 and Bax were down-regulated. Besides, the nuclear translocation of Nrf2 were increased.
Conclusion: In conclusion, the results of this study showed that YTC might alleviate MI by suppressing apoptosis induced by oxidative stress via the PI3K/Akt/Nrf2 signal pathway.

背景：益气通络胶囊（Yiqi-Tongluo Capsule，YTC）是一款中国传统中成药，已被用于心肌缺血（myocardial ischemia，MI）的临床治疗，但其对抗心肌缺血的分子机制至今仍未明确。
方法：本研究采用网络分析与实验验证相结合的策略，探究益气通络胶囊治疗心肌缺血的潜在分子机制。首先，通过在线数据库预测该胶囊中的主要活性成分及其潜在作用靶点；从GeneCards和人类孟德尔遗传在线（Online Mendelian Inheritance in Man，OMIM）数据库中收集心肌缺血相关基因；将药物靶点与疾病靶点取交集，随后构建蛋白质相互作用（protein-protein interaction，PPI）网络与药物-分子-靶点-疾病（Drug-Molecular-Target-Disease，DMTD）调控网络，并进行基因本体（Gene Ontology，GO）功能富集分析及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析。基于过氧化氢（H₂O₂）诱导的H9c2细胞模型，本研究通过流式细胞术、蛋白质印迹（western blot，WB）及免疫荧光实验，对网络分析的预测结果进行验证。
结果：本研究共预测得到益气通络胶囊的100种活性成分及165个作用靶点，其中109个靶点与心肌缺血相关靶点存在交集。GO与KEGG富集分析结果显示，这些潜在靶点参与多种生物学过程与分子机制，包括氧化应激及PI3K/AKT信号通路。黄芪甲苷IV（Astragaloside IV，AS IV）与芍药苷（paeoniflorin，PAE）可能是益气通络胶囊的核心活性成分。细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验结果表明，益气通络胶囊可缓解过氧化氢对H9c2细胞的损伤。流式细胞术、DAPI染色及JC-1探针实验结果显示，益气通络胶囊可减轻过氧化氢诱导的H9c2细胞凋亡。此外，在过氧化氢诱导的H9c2细胞中，益气通络胶囊可降低细胞内超氧阴离子水平，提升超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）及谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）的活性，并降低丙二醛（malondialdehyde，MDA）含量。免疫荧光及蛋白质印迹实验结果显示，PI3K与Akt的磷酸化水平升高，B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）的表达上调，而剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved caspase-3）及Bcl-2相关X蛋白（Bax）的表达下调；同时，核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）的核转位水平升高。
结论：综上，本研究结果表明，益气通络胶囊可能通过调控PI3K/Akt/Nrf2信号通路，抑制氧化应激诱导的细胞凋亡，从而发挥对抗心肌缺血的作用。