通络化痰胶囊对H2O2所致的PC12细胞凋亡的保护作用及机制

Protective Effects of Tongluo Huatan Capsule on H_2O_2-induced Apoptosis in PC12 Cells

目的:探讨通络化痰胶囊对过氧化氢(H2O2)所致的大鼠嗜铬瘤细胞(PC12)细胞凋亡的保护作用及机制。方法:PC12细胞常规培养后,首先通过MTT检测细胞存活率的方法进行H2O2损伤模型的摸索和通络化痰胶囊药物浓度的筛选;确定造成PC12细胞损伤的H2O2浓度和通络化痰胶囊的安全作用浓度后,将处于对数生长期的PC12细胞分为对照组、模型组和通络化痰胶囊6.25,12.5,25,50,100mg.L-15个治疗组。用200μmol.L-1H2O2刺激PC12细胞使其发生凋亡,MTT法检测PC12细胞存活率、Hoechst33342荧光核染色观察细胞凋亡形态学改变、流式细胞仪检测PC12细胞凋亡率、Western blot方法检测与细胞凋亡线粒体途径密切相关的蛋白细胞色素C(CytC)和半胱氨酸天冬氨酸蛋白酶-3(caspase-3)表达的变化。结果:200μmol.L-1H2O2诱导PC12细胞损伤明显,浓度小于等于100 mg.L-1的通络化痰胶囊浓度对正常PC12细胞的存活率没有抑制作用。与模型组比较,各通络化痰治疗组PC12细胞存活率提高(P<0.01),凋亡率下降(P<0.01),Cytc和Caspase-3的表达减少(P<0.01),其作用与剂量有关。结论:通络化痰胶囊可抑制H2O2诱导的PC12细胞凋亡,其机制可能与抑制细胞凋亡线粒体途径中凋亡相关CytC和caspase-3的表达有关。

Objective： To investigate the protective effect and mechanism of Tongluo Huatan capsule on H2O2-induced apoptosis in PC12 cells.Method： PC12 cells were cultured,and the cell viability of PC12 cells injured by different H2O2 concentrations and proliferated by different Tongluo Huatan Capsule were observed by MTT assay to determine the concentration that will cause a H2O2-induced apoptosis model of PC12 cells and the security concentrations of Tongluo Huatan Capsule for PC12 cells.PC12 cells in the logarithmic growth phase were divided into control group,model group and low,medium and high dose Tongluo Huatan capsule treatment groups.Apoptosis of PC12 cells were induced by H2O2 at the concentration of 200 μmol.L-1,MTT assay was used to detect the cell viability of PC12 cells protected by Tongluo Huatan capsule at different concentarions,Hoechst33342 fluorescent staining was used to observe morphological changes of cell apoptosis,the flow cytometry were used to detect the rate of apoptosis in PC12 cells,and the Western blot technology was used to detect the expressios of protein CytC and Caspase-3.Result： PC12 cells were injured by H2O2 at 200 μmol.L-1 concentration,while Tongluo Huatan capsule at less than or equal to the concentration of 100 μmol.L-1did not inhibit the cell viability of PC12 cells.Compared with the model group,Tongluo Huatan capsule groups improved cell viability（P ＆lt; 0.01）,decreased apoptotic cells and apoptosis rate（P ＆lt; 0.01）,and decreased the expression of Cytc and Caspase-3（P ＆lt; 0.01） which was related to doses Tongluohuatan capsule.Conclusion： Tongluo Huatan capsule can inhibit H2O2-induced apoptosis in PC12 cells,while the mechanism may be related to the inhibition of the expression of apoptosis-related protein CytC and caspase-3 in the mitochondrial pathway.