左归丸通过线粒体途径抗叔丁基过氧化氢诱导的MC3T3-E1细胞凋亡

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(P0.05,P0.01),细胞凋亡增加,线粒体膜电位降低,Bcl-2蛋白表达明显下调(P0.01);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(P0.01),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(P0.05),抑制Bax和Caspase-3蛋白表达(P0.01)。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。

Protective Effect of Zuoguiwan on Tert-butyl Hydroperoxide-induced MC3T3-E1 Cell Apoptosis via Mitochondria-dependent Pathways

Objective: To observe the protective effects of Zuoguiwan (ZGW) on tertiary butyl hydroperoxide-induced MC3T3-E1 cell apoptosis, in order to study the possible mechanisms of ZGW via mitochondria-dependent pathways. Method: ZGW drug-contained serum was prepared. With murine osteoblastic MC3T3 cells as the research object, the oxidative stress model was established by tertiary butyl hydroperoxide (t-BHP), and then divided into 3 groups, blank control group, t-BHP group, ZGW+t-BHP group and Estradiol Valerate+t-BHP group. After incubation for 24, 48, 72 hours, the effects of ZGW on viability and apoptosis of t-BHP-induced MC3T3-E1 cells were investigated by MTT assay. After incubation for 48 hours, cell apoptosis was measured by acidine orange/ethidium bromide (AO/EB) staining. The changes of nucleus were observed by Hoechst 33342 staining. The changes of mitochondrial membrane potential were analyzed with rhodamine 123 (Rh123) staining. Western blot analysis was used to evaluate expressions of Bcl-2, Bax and Caspase-3 protein expressions. Result: Compared with blank model group, the proliferation of MC3T3-E1 cell was significantly inhibited in t-BHP group (P < 0.05, P < 0.01), cell apoptosis increased, mitochondrial membrane potential decreased, and the protein expression of Bcl-2 was significantly down-regulated (P<0.01). Compared with t-BHP group, the survival of MC3T3-E1 cells exposed to the oxidative damage induced by t-BHP was promoted in ZGW+t-BHP group (P<0.01), particularly at 48 h, cell apoptosis reduced, mitochondrial membrane potential significantly improved, and the protein expression of Bcl-2 was increased (P<0.05), while Bax and caspase-3 expression were decreased (P<0.01). Conclusion: Drug-contained serum of ZGW can inhibite t-BHP-induced MC3T3-E1 cell apoptosis. The possible mechanism may be related to mitochondria-dependent pathways.