基于GAS6/Axl通路探讨茯苓酸预处理对过氧化氢诱导的心肌细胞损伤的影响

Exploring the effect of pachymic acid pretreatment on hydrogen peroxide-induced cardiomyocyte damage based on the GAS6/Axl pathway

关键词:  茯苓酸  GAS6/Axl通路  过氧化氢  心肌细胞损伤

Key words:  pachymic acid  GAS6/Axl pathway  hydrogen peroxide  cardiomyocyte damage

摘要:

目的 探讨茯苓酸预处理（PA）对过氧化氢（H₂O₂）诱导的大鼠心肌细胞H9c2氧化损伤的作用，并基于生长停滞特异性蛋白6/酪氨酸蛋白激酶受体（GAS6/Axl）信号通路阐明其潜在机制。方法 使用不同浓度的茯苓酸（0~40 μmol/L）处理H9c2细胞及H₂O₂诱导的H9c2细胞，采用CCK-8法筛选茯苓酸最佳实验药物浓度。将H9c2细胞分为：对照组（CN组），H₂O₂组，茯苓酸低、中、高浓度组（PA-L组、PA-M组、PA-H组，2.50、5.00、10.00 μmol/L），茯苓酸高浓度+GAS6/Axl通路抑制剂组（PA-H+R428组）。通过TUNEL染色检测细胞凋亡；采用Western blot法检测凋亡相关蛋白[B淋巴细胞瘤-2（Bcl-2）、BCL2相关X蛋白（Bax）、活化的半胱氨酸天冬氨酸特异性蛋白酶-3（Cleaved-Caspase-3）、半胱氨酸天冬氨酸特异性蛋白酶-3（Caspase-3）]及GAS6/Axl通路蛋白（GAS6、p-Axl、Axl）的表达。检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性及丙二醛（MDA）、活性氧（ROS）水平。结果 与CN组比较，H₂O₂处理可诱导H9c2细胞严重氧化损伤，表现为细胞存活率和形态恶化、凋亡率显著升高（P < 0.05），Bcl-2/Bax比值及SOD、GSH-Px活性降低（P < 0.05），Cleaved-Caspase-3/Caspase-3比值及MDA、ROS水平升高（P < 0.05），同时GAS6/Axl通路被显著抑制（P < 0.05）。茯苓酸预处理能浓度依赖性地逆转上述损伤，其中PA-H组效果最为显著，各项指标均接近正常水平。然而，加入GAS6/Axl通路特异性抑制剂R428后，茯苓酸的保护作用被显著削弱（P < 0.05）。结论 茯苓酸预处理能够有效减轻H₂O₂诱导的H9c2心肌细胞氧化损伤和凋亡，其作用机制可能与激活GAS6/Axl信号通路，进而增强细胞抗氧化能力、调节凋亡相关蛋白表达有关。

Abstract:

Objective To investigate the effect of pachymic acid (PA) pretreatment on oxidative damage of rat cardiomyocytes H9c2 induced by hydrogen peroxide (H₂O₂), and to clarify its potential mechanism based on the growth arrest specific protein 6/tyrosine protein kinase receptor (GAS6/Axl) signaling pathway. Methods H9c2 cells and H9c2 cells induced b H₂O₂ were treated with different concentrations of pachymic acid (0 to 40 μmol/L), and the optimal experimental drug concentration of pachymic acid was screened by the CCK-8 method. H9c2 cells were assigned into: the control group (CN group), the H₂O₂ group, the low, medium and high concentration pachymic acid groups (PA-L group, PA-M group, PA-H group, 2.50, 5.00, 10.00 μmol/L), and the high concentration pachymic acid+GAS6/Axl pathway inhibitor group (PA-H+R428 group). Cell apoptosis was detected by TUNEL staining. The expressions of apoptosis-related proteins [B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Cleaved-Caspase-3, Caspase-3) ]and GAS6/Axl pathway proteins (GAS6, p-Axl, Axl) were measured by Western blot. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were detected using the corresponding kits. Results Compared with the CN group, H₂O₂ treatment could induce severe oxidative damage to H9c2 cells, manifested as deterioration of cell survival rate and morphology, a prominent increase in apoptosis rate (P < 0.05), the decreases of Bcl-2/Bax ratio and the activities of SOD and GSH-Px (P < 0.05), and the increases of Cleaved-Caspase-3/Caspase-3 ratio and the levels of MDA and ROS (P < 0.05), while the GAS6/Axl pathway was prominently inhibited (P < 0.05). Pachymic acid pretreatment could reverse the above-mentioned injuries in a concentration-dependent manner, and among them, the PA-H group had the most prominent effect, and all indicators were close to the normal level. However, after the addition of the GAS6/Axl pathway-specific inhibitor R428, the protective effect of pachymic acid was prominently weakened (P < 0.05). Conclusion Pachymic acid pretreatment can effectively alleviate oxidative damage and apoptosis of H9c2 cardiomyocytes induced b H₂O₂. Its mechanism of action may be related to the activation of the GAS6/Axl signaling pathway, thereby enhancing the antioxidant capacity of cells and regulating the expression of apoptosis-related proteins.