基于网络药理学探究白藜芦醇抗大鼠心肌缺血再灌注损伤的分子机制

doi:10.3969/j.issn.1674-5817.2020.06.006

摘要/Abstract

摘要： 目的基于网络药理学信息筛选方法,探究白藜芦醇（resveratrol,RSV）抗大鼠心肌缺血再灌注（myocardial ischemia-reperfusion,MI/R）损伤的作用,以及其在大鼠体内的作用机制。方法利用TCMSP数据库筛选RSV作用基因靶点,并利用GeneCards数据库筛选MI/R损伤相关靶基因。运用R语言软件对RSV和MI/R相关靶基因取交集,筛选得到RSV治疗MI/R损伤可能的靶基因,并通过Cytoscape软件绘制基因网络,筛选出核心靶基因;最后运用R语言软件对这些靶基因进行GO及KEGG通路分析。将30只SD大鼠随机分为假手术组（Sham）、缺血再灌注组（MI/R）和白藜芦醇给药组（RSV）。通过结扎左冠状动脉前降支诱导MI/R损伤大鼠模型,然后测定大鼠血浆中乳酸脱氢酶（lactate dehydrogenase,LDH）、肌酸激酶（creatine kinase,CK）、心肌肌钙蛋白Ⅰ（cardiac troponinⅠ,cTnⅠ）含量以及心肌梗死面积。HE染色观察心肌结构,TUNEL染色检测心肌细胞凋亡,免疫组织化学和蛋白质印迹法检测心肌组织中凋亡相关蛋白caspase-3、Bcl-2和肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand,TRAIL）表达。结果网络药理学信息筛选发现RSV治疗MI/R损伤的核心靶基因有86个,主要涉及细胞因子受体、磷酸酶结合和死亡受体等生物学过程,以及参与调控糖尿病并发症的AGE-RAGE信号通路和细胞凋亡信号通路。大鼠体内实验结果显示,相比于Sham组,MI/R组心肌组织中TRAIL蛋白表达水平明显升高（P<0.01）,血浆LDH、CK及cTnⅠ含量明显增加（均P<0.01）,心肌梗死面积增大,同时心肌组织中肌纤维排列紊乱,细胞凋亡率和caspase-3蛋白表达水平明显升高（P<0.05）,Bcl-2表达明显下调（P<0.05）;而RSV组心肌纤维排列相对规则,LDH、CK、cTnⅠ含量及TRAIL蛋白表达量减少,心肌梗死面积减少,细胞凋亡率降低（均P<0.05）。结论 RSV可能通过调节TRAIL蛋白表达抑制细胞凋亡,从而逆转MI/R损伤。

关键词: 网络药理学, 白藜芦醇, 心肌缺血再灌注损伤, 肿瘤坏死因子相关凋亡诱导配体, 细胞凋亡, 大鼠

Abstract: Objective To investigate the protective effect of resveratrol (RSV) against myocardial ischemia-reperfusion (MI/R) injury and its mechanism in rats based on the network pharmacological information screening method. Methods TCMSP database was used to screen the target genes of RSV, and GeneCards database was used to screen MI/R injury-related target genes. R programming language was used to achieve the intersection of the two sets, indicating the possible target genes for RSV treating MI/R injury, then a gene network was drawn by Cytoscape software to screen out the core target genes. Finally, the GO function and KEGG pathway of the screened gene targets were analyzed by R language. Thirty SD rate were randomly divided into a sham operation group (Sham group), an ischemia-reperfusion group (MI/R group) and a RSV administration group (RSV group). The MI/R injury rat model was induced by ligating the anterior descending coronary artery. The serum contents of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin Ⅰ (cTn Ⅰ) as well as the size of myocardial infarction area in the MI/R injuny model rats induced by ligature of left anterior descending of coronary artery were determined. Hematoxylin-eosin staining (HE) was used to observe the structure of myocardium, TUNEL staining was used to detect cardiomyocyte apoptosis, immunohistochemistry and Western blotting were used to detect the expressions of apoptosis-related proteins (caspase-3 and Bcl-2) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Results According to the screening of pharmacology information, there were 86 core genes in RSV treatment of MI/R injury, which mainly involved the biological processes such as cytokine receptors, phosphatase binding, and death receptors, as well as the AGE-RAGE signaling pathway, apoptosis and other signaling pathways regulating diabetic complications. Compared with the Sham group, the expression of TRAIL protein, the contents of LDH, CK and cTn Ⅰ, and the infarct size increased (P<0.01, or P<0.05), while the arrangement of myocardial muscle fibers was disordered, and the apoptosis rate and caspase-3 protein expression level increased (P<0.05), but the expression of Bcl-2 was down-regulated (P<0.05). However, in the RSV group, the arrangement of myocardial muscle fibers was relatively regular, while the expression of TRAIL protein, the contens of LDH, CK and cTn Ⅰ, as well as the infarct size and apoptosis rate were significantly reduced (all P<0.05). Conclusion RSV may inhibit apoptosis and reverse MI/R injury by regulating TRAIL protein experssion.

Key words: Network pharmacology, Resveratrol, Myocardial ischemia-reperfusion, Tumor necrosis factor-related apoptosis-inducing ligand, Apoptosis, Rats

中图分类号:

Q95-33

张晓节, 蒋磊. 基于网络药理学探究白藜芦醇抗大鼠心肌缺血再灌注损伤的分子机制[J]. 实验动物与比较医学, 2020, 40(6): 496-505.

ZHANG Xiaojie, JIANG Lei. Molecular Mechanism of Resveratrol Against Myocardial Ischemia-Reperfusion Injury in Rats Based on Network Pharmacology[J]. Laboratory Animal and Comparative Medicine, 2020, 40(6): 496-505.

图/表 7

参考文献

[1] Bugger H, Pfeil K.Mitochondrial ROS in myocardial ischemia reperfusion and remodeling[J]. Biochim Biophys Acta Mol Basis Dis, 2020, 1866(7):165768.
[2] Aslan G, Atessahin A, Sahna E.The inhibition of apoptosis through myocardial postconditioning by affecting Fas/FasIg signaling through miR139-3p and miR181a-1[J]. J Card Surg, 2020, 35(3):564-570.
[3] 刘丽凤, 王禹. 细胞程序性死亡与心肌再灌注损伤的研究进展[J]. 中华老年心脑血管病杂志, 2015, 17(5):549-550.
[4] 符武岛, 曾敏, 陈娟, 等. 表没食子儿茶素没食子酸酯通过抑制心肌细胞凋亡缓解心肌缺血再灌注损伤的机制研究[J]. 中国药房, 2019, 30(16):2187-2192.
[5] Xu RY, Xu XW, Deng YZ, et al.Resveratrol attenuates myocardial hypoxia/reoxygenation-induced cell apoptosis through DJ-1-mediated SIRT1-p53 pathway[J]. Biochem Biophys Res Commun, 2019, 514(2):401-406.
[6] Guo Y, Zhang L, Li F, et al.Restoration of sirt1 function by pterostilbene attenuates hypoxia-reoxygenation injury in cardiomyocytes[J]. Eur J Pharmacol, 2016(776):26-33.
[7] 尹佳, 杨若晗, 赵晓东, 等. 蒙药广枣叶总黄酮对心肌缺血再灌注损伤模型大鼠的保护作用研究[J]. 中国药房, 2019, 30(16):2253-2257.
[8] Hung LM, Su MJ, Chu WK, et al.The protective effect of resveratrols on ischaemia-reperfusion injuries of rat hearts is correlated with antioxidant efficacy[J]. Br J Pharmacol, 2002, 135(7):1627-1633.
[9] 李先宽, 李赫宇, 李帅, 等. 白藜芦醇研究进展[J]. 中草药, 2016, 47(14):2568-2578.
[10] Xu G, Zhao X, Fu J, et al.Resveratrol increase myocardial Nrf2 expression in type 2 diabetic rats and alleviate myocardial ischemia/reperfusion injury (MIRI)[J]. Ann Palliat Med, 2019, 8(5):565-575.
[11] Li J, Xie C, Zhuang J, et al.Resveratrol attenuates inflammation in the rat heart subjected to ischemia-reperfusion: Role of the TLR4/NF-κB signaling pathway[J]. Mol Med Rep, 2015, 11(2):1120-1126.
[12] Cheng L, Jin Z, Zhao R, et al.Resveratrol attenuates inflammation and oxidative stress induced by myocardial ischemia-reperfusion injury: role of Nrf2/ARE pathway[J]. Int J Clin Exp Med, 2015, 8(7):10420-10428.
[13] Xu H, Cheng J, Wang X, et al.Resveratrol pretreatment alleviates myocardial ischemia/reperfusion injury by inhibiting STIM1-mediated intracellular calcium accumulation[J]. J Physiol Biochem, 2019, 75(4):607-618.
[14] Wang B, Yang Q, Sun YY, et al.Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice[J]. J Cell Mol Med, 2014, 18(8):1599-1611.
[15] Beyer K, Baukloh AK, Stoyanova A, et al.Interactions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the immune system: implications for inflammation and cancer[J]. Cancers (Basel), 2019, 11(8):1161.
[16] Nash M, McGrath JP, Cartland SP, et al. Tumour necrosis factor superfamily members in ischaemic vascular diseases[J]. Cardiovasc Res, 2019, 115(4):713-720.
[17] 田锐, 秦仁义, 杜志勇, 等. 肿瘤坏死因子相关凋亡诱导配体的抗胰腺癌细胞的作用[J]. 中国普通外科杂志, 2006, 7(11):821-825.
[18] 陈诚, 马钰, 李义德, 等. 枸杞多糖单独或联合TRAIL对MLL基因重排急性淋巴细胞白血病细胞凋亡的影响[J]. 中国实验血液学杂志, 2019, 27(4):1104-1110.