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OA
Exploring the effect of pachymic acid pretreatment on hydrogen peroxide-induced cardiomyocyte damage based on the GAS6/Axl pathway
[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.
[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 by 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 by 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.
张晓娟;刘沙沙;刘影;程丽;宋燕齐;李春玲
邯郸市中西医结合医院药材科,邯郸 056000保定市第一中心医院心血管内五科,保定 071000河北省第八人民医院中西医结合科,石家庄 050000邯郸市中西医结合医院药材科,邯郸 056000邯郸市中西医结合医院药材科,邯郸 056000邯郸市中西医结合医院药材科,邯郸 056000
医药卫生
茯苓酸GAS6/Axl通路过氧化氢心肌细胞损伤
pachymic acidGAS6/Axl pathwayhydrogen peroxidecardiomyocyte damage
《天津中医药》 2026 (8)
1009-1016,8
河北省医学科学研究课题(20261384).
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