芎归汤通过抑制氧化应激诱导的心肌凋亡减轻小鼠心梗后心衰引起的心肌损伤

doi:10.12122/j.issn.1673-4254.2024.07.22

摘要/Abstract

摘要：

目的基于网络药理学结合实验验证探索芎归汤对心梗后心衰小鼠心脏功能、心脏病理的改善及作用机制。方法通过TCMSP、GeneCards、CTD等数据库搜索药物活性成分作用靶点与疾病相关靶点，并将二者取交集。采用DAVID数据库对交集靶点进行GO、KEGG通路富集分析。体内实验采用冠脉结扎构建心梗后心衰小鼠模型，分为假手术组（Sham，n=8）、模型组（Model，n=8）和芎归汤治疗组（XGT，3 g/kg，n=8）。体外实验采用叔丁基过氧化氢诱导H9C2细胞凋亡，分为常规培养的对照组（Control）、叔丁基过氧化氢组（TBHP）、芎归汤低剂量组（XGT，50 μg/mL）和芎归汤高剂量组（XGT，100 μg/mL）。通过HE、Masson、qPCR、免疫组化、CCK8、ROS、MDA、SOD、Hoechst 33342/PI和JC-1检测，探索芎归汤抗心衰的作用机制。结果网络药理学分析结果显示，芎归汤治疗心衰的潜在作用靶点共62个，其核心靶点包括PTGS2、ESR1、Caspase3、PPARG、HSP90AA1、BCL2、JUN、GSK3B等，富集分析显示这些靶点可能涉及细胞凋亡、AGE-RAGE信号通路、P53信号通路、PI3K-Akt信号通路、VEGF信号通路等。体内结果显示，芎归汤能够减少心衰小鼠心肌细胞损伤、减轻心室重构，从而改善心衰小鼠心功能（P<0.05）。芎归汤能够抑制心肌凋亡相关蛋白Caspase3和BAX的表达，促进BCL2的表达（P<0.05）。体外实验表明，芎归汤通过抑制氧化应激，从而减轻叔丁基过氧化氢诱导的心肌细胞凋亡（P<0.05）。结论芎归汤能减轻心梗后心衰引起的心肌损伤，改善心衰小鼠心功能，芎归汤可能通过抑制氧化应激诱导的心肌凋亡发挥抗心衰的作用。

关键词: 芎归汤, 心力衰竭, 网络药理学, 细胞凋亡, 氧化应激

Abstract:

Objective To explore the protective effect of Xionggui Decoction against cardiac myopathy in a mouse model of heart failure following myocardial infarction (MI) and explore the underlying mechanism. Methods We searched TCMSP, GeneCards, and CTD databases for the targets of active ingredients Xionggui Decoction and heart failure, and the intersecting targets were analyzed with GO and KEGG pathway enrichment analysis using DAVID database. In a mouse model of heart failure following acute MI induced by coronary artery ligation, the cardiac protective effects of 3 g/kg Xionggui Decoction were evaluated by assessing cardiac function, cardiac myopathy and ventricular remodeling of the mice using HE staining, Masson staining, RT-qPCR, and immunohistochemistry. We also tested the effect of Xionggui Decoction at 50 and 100 μg/mL on tert-butylhydrogen peroxide (TBHP)-induced apoptosis of H9C2 cells using CCK8 assay, detection kits for ROS, MDA, SOD, JC-1 and Hoechst 33342/PI staining. Results Network pharmacological analysis identified 62 potential targets of Xionggui Decoction for treatment of heart failure, and the core targets included PTGS2, ESR1, caspase-3, PPARG, HSP90AA1, BCL2, JUN, and GSK3B, which were involved in cell apoptosis and the AGE-RAGE, P53, PI3K-Akt, and VEGF signaling pathways. In the mouse models of heart failure, treatment with Xionggui Decoction significantly alleviated cardiac myopathy and ventricular remodeling, obviously improved heart function of the mice, lowered myocardial expressions of caspase-3 and BAX, and enhanced the expression of BCL2. In H9C2 cells, Xionggui Decoction significantly alleviated TBHP-induced cell apoptosis by inhibiting oxidative stress in the cells. Conclusion Xionggui Decoction can alleviate myocardial injury and improve cardiac function in mice with heart failure following acute MI possibly by inhibiting cardiomyocyte apoptosis induced by oxidative stress.

Key words: Xionggui Decoction, heart failure, network pharmacology, apoptosis, oxidative stress

任志军, 刁建新, 王奕婷. 芎归汤通过抑制氧化应激诱导的心肌凋亡减轻小鼠心梗后心衰引起的心肌损伤[J]. 南方医科大学学报, 2024, 44(7): 1416-1424.

Zhijun REN, Jianxin DIAO, Yiting WANG. Xionggui Decoction alleviates heart failure in mice with myocardial infarction by inhibiting oxidative stress-induced cardiomyocyte apoptosis[J]. Journal of Southern Medical University, 2024, 44(7): 1416-1424.

图/表 9

图2 GO和KEGG富集分析

Fig.2 GO and KEGG pathway enrichment analysis of the potential targets.

图3 芎归汤改善心衰小鼠的心功能

Fig.3 Xionggui Decoction improves heart function of the mice with heart failure. A: Left ventricular end-systolic dimension (LVEDs). B: Left ventricular end-diastolic dimension (LVEDd). C: Left ventricular ejection fraction (LVEF). D: Left ventricular fractional shortening (LVFS). ##P<0.01 vs Sham group; *P<0.05, **P<0.01 vs Model group.

图4 芎归汤减轻心衰小鼠的心肌损伤

Fig.4 Xionggui Decoction alleviates myocardial damage in mice with heart failure. A: Heart weight/body weight ratio. B-D: Serum LDH, CK, and AST levels. ##P<0.01 vs Sham group; *P<0.05, **P<0.01 vs Model group.

图5 芎归汤减轻心衰小鼠的心肌细胞坏死和炎症

Fig.5 Xionggui Decoction alleviates myocardial necrosis and inflammation in mice with heart failure. The green arrows indicate the inflammatory cells (scale bar: 100 μm).

图6 芎归汤减轻心衰小鼠的心肌纤维化

Fig.6 Xionggui Decoction alleviates myocardial fibrosis in mice with heart failure. A: Masson staining of the myocardial tissue. B: Collagen volume fraction. C: Relative expression of Collagen I. D: Relative expression of Collagen III. ##P<0.01 vs Sham group; **P<0.01 vs Model group.

图7 芎归汤减轻心肌细胞凋亡

Fig.7 Xionggui Decoction alleviates myocardial cell apoptosis in mice with heart failure. A: Immunohistochemical analysis. B: Relative expression of BAX. C: Relative expression of BCL2. D: Relative expression of caspase-3. #P<0.05, ##P<0.01 vs Sham group; **P<0.01 vs Model group.

图8 芎归汤减轻氧化应激诱导心肌细胞损伤

Fig.8 Xionggui Decoction alleviate H9C2 cell injury induced by oxidative stress. A: Viability of H9C2 cells incubated with different concentrations of Xionggui Decoction (XGT). B: Viability of H9C2 cells incubated with different concentrations of TBHP. C: Viability of H9C2 cells incubated with different concentrations of XGT (50 and 100 µg/mL), followed by treatment with TBHP (200 μmol/L). ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs TBHP group.

图9 芎归汤保护心肌细胞免受氧化应激

Fig.9 Xionggui Decoction protects H9C2 cells from TBHP-induced oxidative stress. A: Reactive oxygen species (ROS) levels. B: Malondialdehyde (MDA) content. C: Superoxide dismutase (SOD) activity. ##P<0.01 vs Control; *P<0.05 vs TBHP; **P<0.01 vs TBHP.

图10 芎归汤减轻氧化应激诱导的心肌细胞凋亡

Fig.10 Xionggui Decoction alleviates TBHP-induced H9C2 cell apoptosis. A: Hoechst 33342/PI fluorescent staining. B: JC-1 staining. ##P<0.01 vs Control; **P<0.01 vs TBHP.

参考文献 20

1	Virani SS, Alonso A, Aparicio HJ, et al. Heart disease and stroke statistics-2021 update: a report from the American heart association[J]. Circulation, 2021, 143(8): e254-e743.
2	Zhao W, Zhao J, Rong J. Pharmacological modulation of cardiac remodeling after myocardial infarction[J]. Oxid Med Cell Longev, 2020, 2020: 8815349.
3	Chen ZJ, Zhang C, Gao F, et al. A systematic review on the rhizome of Ligusticum chuanxiong Hort. (Chuanxiong)[J]. Food Chem Toxicol, 2018, 119: 309-25.
4	Lv XF, Wen RQ, Liu K, et al. Role and molecular mechanism of traditional Chinese medicine in preventing cardiotoxicity associated with chemoradiotherapy[J]. Front Cardiovasc Med, 2022, 9: 1047700.
5	Huang CY, Kuo WW, Kuo CH, et al. Protective effect of Danggui (Radix Angelicae Sinensis) on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells[J]. BMC Complement Altern Med, 2014, 14: 358.
6	王心意, 鞠建庆, 樊懿萱, 等. 基于数据挖掘的国家级名老中医治疗冠心病用药规律研究[J]. 辽宁中医药大学学报, 2021, 23(3): 159-63. DOI: 10.13194/j.issn.1673-842x.2021.03.036
7	谢凌鹏, 周楚莹, 吕志平. 基于网络药理学探讨黄连抗肝细胞癌的作用机制[J]. 中医肿瘤学杂志, 2022, 4(1): 29-35. DOI: 10.19811/j.cnki.ISSN2096-6628.2022.01.006
8	Chen GH, Xu HL, Xu T, et al. Calycosin reduces myocardial fibrosis and improves cardiac function in post-myocardial infarction mice by suppressing TGFBR1 signaling pathways[J]. Phytomedicine, 2022, 104: 154277.
9	Branch of Cardiovascular Physicians, Chinese Medical Doctor Association;China Cardiovascular Health Alliance;The Expert Consensus Working Group on the Prevention and Treatment of Heart Failure After Myocardial Infarction. 2020心肌梗死后心力衰竭防治专家共识[J]. 中国循环杂志, 2020, 35(12): 1166-80.
10	Teringova E, Tousek P. Apoptosis in ischemic heart disease[J]. J Transl Med, 2017, 15(1): 87.
11	Li WX, Tang YP, Chen YY, et al. Advances in the chemical analysis and biological activities of Chuanxiong[J]. Molecules, 2012, 17(9): 10614-51.
12	董培良, 李慧, 韩华. 当归及其药对的研究进展[J]. 中医药信息, 2019, 36(2): 127-30. DOI: 10.19656/j.cnki.1002-2406.190063
13	Zhou Z, Chen B, Chen S, et al. Applications of network pharmacology in traditional Chinese medicine research[J]. Evid Based Complement Alternat Med, 2020: 1646905.
14	Eskandari E, Eaves CJ. Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis[J]. J Cell Biol, 2022, 221(6): e202201159.
15	Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch[J]. Nat Rev Cancer, 2002, 2(9): 647-56.
16	Elmore S. Apoptosis: a review of programmed cell death[J]. Toxicol Pathol, 2007, 35(4): 495-516.
17	Perelman A, Wachtel C, Cohen M, et al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry[J]. Cell Death Dis, 2012, 3(11): e430.
18	Panth N, Paudel KR, Parajuli K. Reactive oxygen species: a key hallmark of cardiovascular disease[J]. Adv Med, 2016: 9152732.
19	Brieger K, Schiavone S, Miller FJ, et al. Reactive oxygen species: from health to disease[J]. Swiss Med Wkly, 2012, 142: w13659.
20	Krylatov AV, Maslov LN, Voronkov NS, et al. Reactive oxygen species as intracellular signaling molecules in the cardiovascular system[J]. Curr Cardiol Rev, 2018, 14(4): 290-300.

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