二甲双胍对去卵巢大鼠骨质疏松症的影响及机制研究

The impact and mechanism of metformin on osteoporosis in ovariectomized rats

目的以雌二醇为对照,观察二甲双胍(metformin,MF)对去卵巢大鼠骨密度及骨矿含量的影响,并从细胞、分子水平探究MF可能的骨保护机制。方法将60只雌性SD大鼠随机均分4组:假手术(SHAM)组、去卵巢(OVX)组、去卵巢+二甲双胍(OVX+MF)组和去卵巢+雌二醇(OVX+E2)组。分组灌胃给药60 d后测量大鼠右侧胫骨骨密度和骨矿含量;分离培养各组大鼠骨髓间充质干细胞(BMSCs)并诱导其向成骨细胞分化,用MTT法测定细胞活性及增殖能力;测定各组碱性磷酸酶(ALP)活性、矿化结节数目、钙含量以及I型胶原(collagen type I)、骨钙素(OC)、骨保护素(OPG)、NFκB受体的配体(RANKL)、白细胞介素-6(IL-6)基因表达水平。结果与OVX组相比,OVX+MF组和OVX+E2组成骨细胞的增殖能力与ALP活性明显增强,骨密度、骨矿含量以及钙沉积量显著增加(P均<0.05),且两组collagen type I、OC、OPG mRNA的表达水平显著升高,而RANKL、IL-6mRNA表达明显受到抑制;但OVX+MF组去卵巢大鼠成骨细胞的增殖能力、ALP活性、钙沉积量、collagen type I、OC、OPG mRNA表达水平低于OVX+E2组,RANKL、IL-6mRNA表达高于OVX+E2组(P均<0.05);与SHAM组比较,OVX+MF组的collagen type I、OC、OPG mRNA的表达水平更高(P<0.05)。结论二甲双胍可能通过OPG/RANKL/RANK信号通路促进BMSCs向成骨细胞分化,有效逆转去卵巢大鼠骨质疏松的状态,这种潜在的骨保护作用可能会改善糖尿病引起的骨质疏松。

Objective To observe the effect of metformin on bone mineral density and bone mineral content in ovariectomized rats,and explore the mechanism of bone protection at the cellular and molecular levels compared with estradiol.Methods Sixty female SD rats were distributed randomly into four groups:SHAM group,OVX group,OVX+MF group and OVX+E2 group.Bone mineral density and bone mineral content of right tibia of rats were detected after 60 days of drug administration;separated and cultured the BMSCs and induced them to differentiate into osteoblasts,MTT Assay was used to determine the cell activity and proliferation ability;the ALP activity,mineralized nodule formation,calcium content and gene expression levels of collagen type I,OC,OPG,RANKL,IL-6mRNA were also measured.Results Compared with OVX group,the proliferation ability,ALP activity,bone mineral density,bone mineral content and calcium deposition of osteoblasts were significantly enhanced in OVX+MF group and OVX+E2 group(P<0.05),moreover,the expression levels of collagen type I,OC and OPG mRNA in the two groups were significantly increased,while the expressions of RANKL and IL-6 mRNA were significantly inhibited.However,the proliferation ability,ALP activity,calcium deposition,expression levels of collagen type I,OC,OPG mRNA in OVX+MF group were lower than OVX+E2 group,and the expression of RANKL,IL-6 mRNA were higher than OVX+E2 group(P<0.05).In addition,compared with SHAM group,the expression levels of collagen type I,OC,and OPG mRNA in the OVX+MF group were higher(P<0.05).Conclusion Metformin may promote the differentiation of BMSCs into osteoblasts via OPG/RANKL/RANK signaling pathway,and effectively reverse the state of osteoporosis in ovariectomized rats,this potential osteoprotective effect of metformin may improve the osteoporosis caused by diabetes.