基于AMPK-mTOR信号通路探讨通心络胶囊通过调节自噬改善高糖环境下H9c2心肌细胞损伤的机制

Exploration the Mechanism of Tongxinluo Capsules Ameliorating H9c2 Cardiomyocyte Injury in A High Glucose Environment Through Regulation of Autophagy Based on AMPK-mTOR Signaling Pathway

目的 采用高糖诱导H9c2心肌细胞损伤建立糖尿病心肌病(DCM)细胞模型,观察通心络胶囊对H9c2细胞的保护作用并探讨其可能机制。方法 以葡萄糖不同浓度(45、65、75、100 mmol·L^(-1))及75 mmol·L^(-1)葡萄糖+通心络胶囊不同浓度(20、40、80、160、300μg·mL^(-1))分别处理H9c2细胞24 h,采用CCK-8法测定细胞存活率。将H9c2细胞分为对照组、模型组(75 mmol·L^(-1)葡萄糖)、通心络胶囊组(75 mmol·L^(-1)葡萄糖+160μg·mL^(-1)通心络胶囊),给药干预24 h。采用DCFH-DA荧光探针法检测细胞活性氧(ROS)水平;JC-1荧光探针法检测细胞线粒体膜电位;ELⅠSA法检测细胞乳酸脱氢酶(LDH)及丙二醛(MDA)水平;Western Blot法检测细胞自噬蛋白(P62、Beclin-1、LC3Ⅱ/Ⅰ)及AMPK-mTOR信号通路蛋白的表达。结果 75 mmol·L^(-1)葡萄糖浓度组的H9c2细胞存活率约为70%,采用该浓度建立高糖损伤H9c2细胞模型。与对照组相比,模型组H9c2细胞存活率显著下降(P<0.01),ROS绿色荧光强度显著增强(P<0.01),线粒体膜电位红/绿荧光强度显著降低(P<0.01),LDH、MDA水平显著升高(P<0.01), P62、 p-mTOR/mTOR蛋白表达显著上调(P<0.01), Beclin-1、LC3-Ⅱ/LC3-Ⅰ及p-AMPK/AMPK蛋白表达显著下调(P<0.01)。与模型组比较,通心络胶囊各浓度组的H9c2心肌细胞存活率明显提高(P<0.05,P<0.01),ROS绿色荧光强度显著减弱(P<0.01),线粒体膜电位红/绿荧光强度显著升高(P<0.01),LDH、MDA水平显著降低(P<0.01),P62、p-mTOR/mTOR蛋白表达显著下调(P<0.01),Beclin-1、LC3-Ⅱ/LC3-Ⅰ及p-AMPK/AMPK蛋白表达显著上调(P<0.01)。结论 通心络胶囊对高糖环境下的H9c2心肌细胞具有保护作用,可能与其降低氧化应激,通过AMPK-mTOR通路上调细胞自噬水平有关。

Objective A diabetic cardiomyopathy(DCM)cell model was established using high-glucose-induced H9c2 cardiomyocyte injury to observe the protective effect of Tongxinluo Capsules on H9c2 cells and to explore the possible mechanism.Methods H9c2 cells were treated with different concentrations of glucose(45,65,75,100 mmol·L^(-1))and Tongxinluo Capsules(20,40,80,160,300μg·mL^(-1))for 24 hours.The cell survival rate was determined by CCK-8 method.H9c2 cells were divided into control group,model group(75 mmol·L^(-1)glucose)and Tongxinluo Capsules group(75 mmol·L^(-1)glucose+160μg·mL^(-1))and administered for 24 hours.Cellular reactive oxygen species(ROS)levels were detected by DCFH-DA fluorescent probe;cellular mitochondrial membrane potential was detected by JC-1 fluorescent probe;cellular LDH and MDA levels were detected by ELISA;the expressions of autophagic proteins(P62,Beclin-1,LC3Ⅱ/Ⅰ)and AMPK-mTOR signaling pathway proteins were detected by Western Blot.Results The survival rate of H9c2 cells in the 75 mmol·L^(-1)glucose concentration group was approximately 70%,and this concentration was used to establish a model of high glucose damage to H9c2 cells.Compared with the control group,the survival rate of H9c2 cells in the model group was significantly decreased(P<0.01),the green fluorescence intensity of ROS was significantly increased(P<0.01),the red/green fluorescence intensity mitochondrial membrane potential was significantly decreased(P<0.01),LDH and MDA levels were significantly increased(P<0.01),the protein expressions of P62 and p-mTOR/mTOR were significantly up-regulated(P<0.01),and protein expressions of Beclin-1,LC3-Ⅱ/LC3-Ⅰand p-AMPK/AMPK were significantly down-regulated(P<0.01).Compared with the model group,the survival rate of H9c2cardiomyocytes in the Tongxinluo Capsules concentration group was significantly increased(P<0.05,P<0.01),the green fluorescence intensity of ROS was significantly decreased(P<0.01),the red/green fluorescence intensity of mitochondrial membrane potential was significantly increased(P<0.01),LDH and MDA levels were significantly decreased(P<0.01),and protein expressions of P62 and p-mTOR/mTOR were significantly downregulated(P<0.01),and protein expressions of Beclin-1,LC3-Ⅱ/LC3-Ⅰand p-AMPK/AMPK was significantly up-regulated.Conclusion The protective effect of Tongxinluo Capsules on H9c2 cardiomyocytes in a high glucose environment may be related to its reduction of oxidative stress and up-regulation of autophagy levels through the AMPK-mTOR pathway.