唑来膦酸盐通过p38 MAPK信号通路调控高糖微环境下成骨细胞分化

Zoledronate regulates osteoblast differentiation in high glucose microenvironment via p38 MAPK signaling pathway

目的 探讨唑来膦酸盐(ZOL)对高糖微环境下小鼠前成骨细胞MC3T3-E1成骨分化的影响及p38丝裂原活化蛋白激酶(p38 MAPK)通路的调节作用。方法 体外培养MC3T3-E1细胞并分为低糖(LG)组、LG+ZOL组、高糖(HG)组、HG+ZOL组。ZOL为0.1μmol/L,LG、HG的葡萄糖浓度分别为5.5 mmol/L和16.5 mmol/L。采用四甲基偶氮唑蓝(MTT)法检测细胞增殖水平;鬼笔环肽染色观察细胞骨架;试剂盒检测细胞碱性磷酸酶(ALP)活性;茜素红染色检测细胞矿化结节生成情况;免疫荧光法检测Wnt5a、p38 MAPK的荧光表达强度。另设HG+ZOL+p38 MAPK通路抑制剂(SB203580)组,SB203580为10μmol/L。Western blot检测5组细胞中Wnt5a、p38 MAPK、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)、骨形态发生蛋白2(BMP2)和Ⅰ型胶原蛋白(COLⅠ)的表达水平。结果 4组细胞增殖水平差异无统计学意义(P>0.05)。与LG组比较,LG+ZOL组细胞骨架清晰程度,ALP活性,茜素红矿化结节生成,Wnt5a、p38 MAPK蛋白荧光表达强度,Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COLⅠ蛋白表达水平明显升高;HG组上述指标变化相反(P<0.05)。与HG组相比,HG+ZOL组细胞骨架清晰程度,ALP活性,茜素红矿化结节生成,Wnt5a、p38 MAPK蛋白荧光表达强度,Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COLⅠ蛋白表达水平明显升高(P<0.05)。与HG+ZOL组相比,HG+ZOL+SB203580组Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COLⅠ的蛋白表达水平降低(P<0.05)。结论 高糖对细胞成骨分化起抑制作用,ZOL可有效改善其抑制作用,其机制可能与p38 MAPK通路相关。

Objective To investigate the effect of zoledronate(ZOL) on the differentiation of mouse pre-osteoblast MC3T3-E1 cells under high glucose microenvironment and the regulatory effect of p38 mitogen-activated protein kinases(p38 MAPK) signaling pathway.Methods MC3T3-E1 cells were cultured in vitro and divided into the low glucose(LG)group,the LG+ZOL group,the high glucose(HG) group and the HG+ZOL group.ZOL concentration was 0.1 μmol/L.The gluse concentrations of LG and HG were 5.5 mmol/L and 16.5 mmol/L.Methyl thiazolyl tetrazolium(MTT) assay was used to detect cell proliferation.The cell cytoskeleton was detected by phalloidin staining.The alkaline phosphatase(ALP) activity was detected by kit.The mineralized nodule was observed by alizarin red staining.The fluorescence expression intensity of Wnt5 a and p38 MAPK was detected by immunofluorescence staining.The HG+ZOL+p38 MAPK inhibitor(SB203580)group was also set in the study.SB203580 concentration was 10 μmol/L.The protein expressions of Wnt5 a,p38 MAPK,phosphorylation p38 mitogen-activated protein kinases(p-p38 MAPK),bone morphogenetic protein 2(BMP2) and collagen type I(COLⅠ) were detected with Western blot assay.Results There was no significant difference in cell proliferation between the four groups(P> 0.05).Compared with the LG group,the clarity of cytoskeleton,ALP activity,the generation of mineralized nodules,the fluorescence expression intensity of Wnt5 a and p38 MAPK,the expression levels of Wnt5 a,p38 MAPK,p-p38 MAPK,BMP2 and COLⅠ protein were significantly higher in the LG+ZOL group(P <0.05),while the HG group showed opposite changes in the above indexes(P <0.05).Compared with the HG group,the clarity of cytoskeleton,ALP activity,the generation of mineralized nodules,the fluorescence expression intensity of Wnt5 a and p38 MAPK,the expression levels of Wnt5 a,p38 MAPK,p-p38 MAPK,BMP2 and COLⅠ protein were significantly higher in the HG+ZOL group(P<0.05).Compared with the HG+ZOL group,the expression levels of Wnt5 a,p38 MAPK,p-p38 MAPK,BMP2 and COLⅠ protein were lower in the HG+ZOL+SB203580 group(P <0.05).Conclusion High glucose can inhibit the osteogenic differentiation of cells,and ZOL can effectively improve the inhibitory effect,which may be related to p38 MAPK signaling pathway.