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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 of Tongluo Huatan Capsule on the apoptosis of rat hepatoma cell line (PC12) induced by hydrogen peroxide (H2O2) and its mechanism. Methods: After routine culture of PC12 cells, the cell viability was assayed by MTT method to explore the model of H2O2 injury and the drug concentration of Tongluo Huatan capsule; to determine the concentration of H2O2 that caused PC12 cell injury and the safety of Tongluo Huatan capsule PC12 cells in logarithmic growth phase were divided into control group, model group and Tongluo Huatan capsule 6.25,12.5,25,50,100 mg.L-15 treatment groups after the action concentration. PC12 cells were stimulated with 200μmol.L-1H2O2 to induce apoptosis. The survival rate of PC12 cells was detected by MTT assay. The apoptosis of PC12 cells was observed by fluorescent staining with Hoechst33342. The apoptosis rate of PC12 cells was detected by flow cytometry and Western blot Changes in the expression of cytochrome C (CytC) and caspase-3, which are closely related to the apoptosis mitochondrial pathway. Results: The injury of PC12 cells induced by 200μmol.L-1H2O2 was obvious, and the concentration of Tongluanshutan capsule with no more than 100 mg.L-1 had no effect on the survival rate of normal PC12 cells. Compared with the model group, the survival rate of PC12 cells increased (P <0.01), the apoptosis rate decreased (P <0.01) and the expression of Cytc and Caspase-3 decreased Dose-related. Conclusion: Tongluo Huatan Capsule can inhibit the apoptosis of PC12 cells induced by H2O2. The mechanism may be related to the inhibition of apoptosis related CytC and caspase-3 expression in mitochondrial pathway.