芝麻素通过Nrf2信号通路对氧化应激诱导的小鼠成骨细胞骨形成的影响机制

The effects of sesamin on osteogenesis of mouse osteoblastsinduced by oxidative stress through Nrf2 signaling pathway

目的探讨芝麻素对过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导的小鼠成骨细胞系MC3T3-E1骨形成、凋亡及NF-E2相关因子(nuclear factor erythriod2-related factor2,Nrf2)信号通路的影响。方法将MC3T3-E1细胞分为对照组、H_(2)O_(2)组和芝麻素组。对照组细胞不经特殊处理,其余两组培养规定的时间后使用400μmol·L^(-1)H_(2)O_(2)干预3 h。MC3T3-E1细胞培养48 h,CCK-8法检测1、2.5、5 mg·L^(-1)的芝麻素对细胞增殖的影响。MC3T3-E1细胞培养7 d,碱性磷酸酶(alkalinephosphatase,ALP)活性检测试剂盒检测ALP活性。MC3T3-E1细胞培养21 d,茜素红法检测成骨细胞钙结节的形成。MC3T3-E1细胞培养48 h,Western blotting检测骨形成蛋白2(bone morphogenetic protein 2,BMP2)、骨保护素(osteoprorotegerin,OPG)、I型胶原(type I collagen,COLI)、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Nrf2、过氧化氢酶(catalase,CAT)、超氧化物歧化酶-1(superoxide dismutase-1,SOD-1)和超氧化物歧化酶-2(superoxide dismutase-2,SOD-2)表达,流式细胞术检测细胞凋亡率,二氯荧光素双醋酸盐(2′,7′-dichlorodihydrofluorescin diacetate,DCFH2-DA)探针检测活性氧(reactive oxygen species,ROS)含量。结果与对照组比较,H_(2)O_(2)组细胞增殖、ALP活性、钙结节及BMP-2、OPG、COLI、Bcl-2、Nrf2、CAT、SOD-1和SOD-2表达明显降低,细胞凋亡率、ROS水平及Bax表达明显升高(P<0.05),而芝麻素可不同程度促进细胞增殖,提高ALP活性及钙结节形成,促进BMP-2、OPG、COLI、Bcl-2、Nrf2、CAT、SOD-1和SOD-2表达,抑制细胞凋亡及ROS产生,抑制Bax表达(P<0.05)。结论芝麻素可提高氧化应激下成骨细胞增殖、分化和矿化能力,上调骨形成相关蛋白表达,抑制细胞凋亡及ROS产生,机制可能与激活Nrf2信号通路有关。

Objective To investigate the effects of sesamin on bone formation,apoptosis and Nrf2 signaling pathway in mouse osteoblast cell line MC3 T3-E1 induced by H_(2)O_(2).Methods MC3 T3-E1 cells were divided into control group,H_(2)O_(2)group and sesamin group.The cells in the control group were not treated specially,the other two groups were treated with 400μmol·L^(-1)H_(2)O_(2)for 3 h after the specified culture time.MC3 T3-E1 cells were cultured for 48 h,CCK-8 assay was used to detect the effects of sesamin at 1,2.5 and 5 mg·L^(-1) on cell proliferation.MC3 T3-E1 cells were cultured for 7 days,and ALP activity was detected by ALP activity detection kit.MC3 T3-E1 cells were cultured for 21 days,and the formation of calcium nodules in osteoblasts was detected by alizarin red method.The expression of BMP-2,OPG,COLI,Bcl-2,Bax,Nrf2,CAT,SOD-1 and SOD-2 were detected by Western blotting.The apoptosis rate of MC3 T3-E1 cells was detected by flow cytometry and the content of ROS was detected by DCFH-DA probe.Results Compared with the control group,the cell proliferation,ALP activity,calcium nodule and the expression of BMP-2,OPG,COLI,Bcl-2,Nrf2,CAT,SOD-1 and SOD-2 in H_(2)O_(2)group were significantly decreased,while the apoptosis rate,ROS level and Bax expression were significantly increased(P<0.05).Sesamin could promote cell proliferation,increase ALP activity and calcium nodule formation,promote expression of BMP-2,OPG,COLI,Bcl-2,Nrf2,CAT,SOD-1 and SOD-2,inhibited apoptosis,ROS production and Bax expression(P<0.05).Conclusion Sesamin can improve the proliferation,differentiation and mineralization of osteoblasts under oxidative stress,up regulate the expression of bone formation related proteins,and inhibit apoptosis and ROS production.The mechanism may be related to the activation of Nrf2 signaling pathway.