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目的:观察左归丸对叔丁基过氧化氢诱导的MC3T3-E1细胞凋亡的保护效应,探讨其作用机制是否与其干预线粒体途径有关。方法:以成骨细胞MC3T3-E1为研究对象,制备左归丸含药血清,建立叔丁基过氧化氢(t-BHP)成骨细胞MC3T3-E1氧化应激模型,实验分为空白组,t-BHP组,左归丸+t-BHP组,补佳乐+t-BHP组。孵育24,48,72 h后,采用噻唑蓝(MTT)法检测左归丸含药血清对t-BHP诱导MC3T3-E1细胞存活的影响;孵育48 h后,采用吖啶橙溴乙啶(AO/EB)染色法检测细胞凋亡,采用Hoechst 33342染色法观察凋亡细胞核变化,采用罗丹明123荧光染色法观察线粒体膜电位的改变,采用蛋白质免疫印迹(Western blot)法分析线粒体蛋白家族B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平。结果:与空白组比较,t-BHP组显著抑制细胞增殖(P<0.05,P<0.01),细胞凋亡增加,线粒体膜电位降低,Bcl-2蛋白表达明显下调(P<0.01);与t-BHP组比较,左归丸加t-BHP组促进暴露于t-BHP诱导的MC3T3-E1氧化损伤细胞的存活(P<0.01),48 h者尤佳,逆转细胞凋亡情况,提高细胞线粒体膜电位,上调Bcl-2蛋白表达(P<0.05),抑制Bax和Caspase-3蛋白表达(P<0.01)。结论:左归丸含药血清能够抗t-BHP诱导的MC3T3-E1细胞凋亡,其作用机制可能与其干预线粒体途径有关。
OBJECTIVE: To observe the protective effect of Zuogui Pill on the apoptosis of MC3T3-E1 cells induced by tert-butyl hydroperoxide and to explore whether its mechanism of action is related to its intervention in mitochondrial pathway. Methods: Osteoblasts MC3T3-E1 was used as the research object to prepare the Zuogui pill drug-containing serum and to establish a model of oxidative stress of t-BHP osteoblast MC3T3-E1. The experiment was divided into blank group, t-BHP group, Zuoguiwan + t-BHP group and nadrogu group + t-BHP group. After 24h, 48h and 72h incubation, the effect of Zuogui pill-containing serum on the survival of MC3T3-E1 cells induced by t-BHP was detected by MTT assay. After incubation for 48 h, the cells were stained with acridine orange ethidium bromide / EB) staining was used to detect apoptosis. Hoechst 33342 staining was used to observe the changes of apoptotic nuclei. The changes of mitochondrial membrane potential were observed by rhodamine 123 fluorescence staining. Mitochondrial protein family B cells were analyzed by Western blot Bcl-2, Bcl-2 and Caspase-3 protein expression levels were detected by Western blot. Results: Compared with the blank group, t-BHP significantly inhibited cell proliferation (P <0.05, P <0.01), increased apoptosis, reduced mitochondrial membrane potential and decreased Bcl-2 protein expression (P <0.01) Zuoguiyin plus t-BHP group promoted the survival of MC3T3-E1-oxidative injury cells exposed to t-BHP (P <0.01), especially 48 h, reversing the apoptosis of cells and increasing the mitochondrial (P <0.05), and inhibited the expression of Bax and Caspase-3 protein (P <0.01). Conclusion: Zuogui pill containing serum can resist t-BHP-induced apoptosis of MC3T3-E1 cells, and its mechanism may be related to its intervention in the mitochondrial pathway.