基于AMPK/mTOR通路探讨注射用益气复脉对TBHP诱导H9c2细胞损伤的保护作用  

Protective Effect of Yiqi Fumai Injection(注射用益气复脉)on H9c2 Cell Damage Induced by TBHP Via AMPK/mTOR Pathway

作　　者：王瑜[1] 苗婷[1] 马胜男 WANG Yu;MIAO Ting;MA Shengnan(Tianjin 4th Centre Hospital,Tianjin 300140)

机构地区：[1]天津市第四中心医院,天津300140

出　　处：《中国中医药科技》2024年第4期602-606,共5页Chinese Journal of Traditional Medical Science and Technology

基　　金：天津市卫生健康委员会中西医结合课题(2021193);天津市第四中心医院优秀青年人才培养基金项目(tjdszxyy20220020)。

摘　　要：目的:探讨注射用益气复脉(YQFM)通过AMPK/mTOR信号通路调控自噬减轻叔丁基过氧化氢(TBHP)所致H9c2心肌细胞损伤的机制。方法:体外培养H9c2心肌细胞,分为正常对照组、TBHP组、TBHP+YQFM组。CCK-8法检测各组H9c2心肌细胞存活率;免疫荧光检测细胞自噬情况;免疫印迹法检测各组细胞自噬相关蛋白LC3Ⅱ/LC3Ⅰ及AMPK/mTOR通路相关蛋白表达。结果:用2、4、8、16 g/L的YQFM预处理后,可显著恢复TBHP诱导的H9c2细胞损伤;免疫荧光染色结果显示,与正常对照组相比,TBHP组细胞LC3荧光强度显著升高(P<0.01);与TBHP组相比,TBHP+YQFM组LC3荧光强度降低,其中2 g/L YQFM组具有显著性(P<0.05)。Western blot检测结果显示:TBHP组p-AMPK/AMPK、LC3-Ⅱ/LC3-Ⅰ表达显著增加,p-mTOR/mTOR比率显著降低;YQFM预处理使自噬相关蛋白p-AMPK/AMPK、LC3-Ⅱ/LC3-Ⅰ比率明显降低,4 g/L YQFM组p-mTOR/mTOR比率明显升高。结论:注射用益气复脉能有效拮抗TBHP引起的H9c2细胞损伤,其机制与通过AMPK/mTOR信号通路调控自噬途径有关。Objective:To investigate the mechanism of Yiqi Fumai Injection(YQFM)in alleviating H9c2 cells injury induced by tert-butyl peroxide(TBHP)through regulating autophagy via AMPK/mTOR signaling pathway.Methods:H9c2 cells were cultured in vitro and divided into control group,TBHP group and TBHP+YQFM group.The survival rate of H9c2 cells was detected by CCK-8,and autophagy was detected by immunofluorescence The expression of autophagy-related proteins LC3II/LC3I and AMPK/mTOR pathway-related proteins were detected by Western blotting.Results:Precultured with 2,4,8,16 g/L YQFM could significantly restore the damage of H9c2 cells induced by TBHP.The immunofluorescence staining results showed that compared with the control group,the LC3 fluorescence intensity of cells was significantly increased in the TBHP group(P<0.01);Compared with the TBHP group,the fluorescence intensity of LC3 in the TBHP+YQFM group was decreased,with a significant decrease observed in the 2 g/L YQFM group(P<0.05).Western blot showed that the expressions of p-AMPK/AMPK,LC3-II/LC3-I and the ratio of p-mTOR/mTOR were significantly increased in TBHP group,while the ratio of p-AMPK/AMPK,LC3-II/LC3-I were significantly decreased in YQFM group,p-mTOR/mTOR ratio was significantly increased in YQFM group.Conclusion:Yiqi Fumai Injection can effectively antagonize the injury of H9c2 cells induced by TBHP,which may be related to the regulation of autophagy by AMPK/mTOR signaling pathway.

关 键 词：注射用益气复脉 叔丁基过氧化氢 H9C2心肌细胞 AMPK/mTOR信号通路 自噬 体外实验 

分 类 号：R285[医药卫生—中药学]