小檗碱联合绿原酸抗骨质疏松的作用机制

Study on the mechanism of the anti-osteoporosis effect of berberine combined with chlorogenic acid

目的 研究小檗碱联合绿原酸对MC3T3-E1细胞分化作用及对绝经后骨质疏松小鼠的干预作用,并探讨其可能的机制。方法 观察小檗碱、绿原酸及两药联用对MC3T3-E1细胞增殖和分化能力的影响,并检测细胞中Wnt3a、β-catenin及成骨分化相关基因mRNA的表达水平。在体构建绝经后骨质疏松症小鼠模型,随机分为模型组、小檗碱组、绿原酸组及两药联用组,同时设置假手术组为对照,尾静脉给药8周后处死小鼠,检测其血清BALP、BGP含量,Micro-CT扫描观察小鼠股骨微结构变化,免疫组织化学法检测小鼠股骨Wnt3a和β-catenin蛋白表达,HE染色观察小鼠重要器官的组织病理结构变化。结果 与正常组相比,小檗碱组、绿原酸组及两药联用组对MC3T3-E1细胞增殖率无明显影响,但均可促进MC3T3-E1细胞成骨分化能力和矿化水平,以两药联用组最明显;并且两药联用组能显著升高Wnt3a、β-catenin、ALP、Runx2、OPN和OCN mRNA的表达(P<0.01)。体内实验表明,与模型组相比,两药联用组小鼠血清BALP、BGP含量明显降低,股骨微观结构得到明显改善,股骨Wnt3a和β-catenin蛋白表达明显升高(P<0.05),并对小鼠重要器官组织切片无明显损伤性改变,表现出良好的生物安全性。结论 小檗碱联合绿原酸可促进MC3T3-E1细胞成骨分化和改善去势小鼠部分骨质疏松表型特征,其机制可能与激活Wnt3a/β-catenin信号通路有关。

Objective To study the effect of berberine(Ber)combined with chlorogenic acid(CA)on the differentiation of MC3T3-E1 cells and the intervention effect on postmenopausal osteoporosis mice,and to explore the possible mechanism.Methods The effects of Ber,CA,and Ber+CA on the proliferation and differentiation capacity of MC3T3-E1 cells were observed.The mRNA expression levels of Wnt3a,β-catenin and osteogenic differentiation-related genes in MC3T3-E1 cells were detected.Forty female mice were randomly divided into sham operation group,osteoporosis model group,Ber group,CA group,and Ber+CA group.They were sacrificed 8 weeks after caudal intravenous administration.Serum BALP and BGP contents of the mice were detected.The microstructure changes of the femur were observed with micro-CT scanning.The protein expressions of Wnt3a andβ-catenin in the femur was detected with immunohistochemistry.The histopathological structure changes of the important organs were observed by HE staining.Results The proliferation rate of MC3T3-E1 cells were not influenced in Ber group,CA group,and Ber+CA group,bu the osteogenic differentiation ability and mineralization level of MC3T3-E1 cells were promoted,which was the most obvious in Ber+CA group.Moreover,the mRNA expressions of Wnt3a,β-catenin,ALP,Runx2,OPN,and OCN significantly increased in the Ber+CA group(P<0.01).In vivo experiments showed that compared with those in the model group,serum contents of BALP and BGP in the Ber+CA group significantly reduced,the protein expressions of Wnt3a andβ-catenin significantly increased,and the microstructure of femoral tissue was significantly improved in the Ber+CA group(P<0.05).Moreover,Ber+CA had no obvious damage to the important organ tissue sections of mice,indicating good biosafety.Conclusion Ber combined with CA promotes osteogenic differentiation of MC3T3-E1 cells and improves partial osteoporosis phenotypic characteristics in ovariectomized mice.The mechanism may be related to the activation of Wnt3a/β-catenin signaling pathway.