复方贞术调脂胶囊调控MEKK2-Wnt偶联拮抗β-catenin泛素化改善大鼠糖皮质激素骨质疏松症

Ameliorating effect of the Chinese traditional medicine Fufang Zhenzhu Tiaozhi Capsules on glucocorticoid-induced osteoporosis by antagonizing β-catenin ubiquitination and its mechanism of action

目的探究复方贞术调脂胶囊(FTZ)干预下调控促分裂原活化蛋白激酶激酶激酶2(mitogen-activated protein kinase kinase kinase 2,MEKK2)-Wnt偶联拮抗β-catenin泛素化,及对骨量变化、细胞成骨能力的影响。方法 SPF级雄性大鼠随机均分为正常对照组、甲强龙组(模型组)、甲强龙+生理盐水组(空白对照组)和甲强龙+FTZ组(实验组)。分别对股骨近端松质骨进行Micro-CT检查、组织病理染色,测定Wnt3a、MEKK2、β-catenin蛋白表达;提取模型BMSCs,经FTZ含药血清干预后,进行碱性磷酸酶和茜素红染色;测定成骨分化相关基因ALP、Runx2、OCN表达;测定MEKK2、β-catenin蛋白表达;以及β-catenin/TCF转录水平。结果 1)在Micro-CT中,实验组与模型组相比,前者BV/TV、Tb.Th、Tb/N等值降低,Tb/sp升高(P<0.05)。ROI三维重建可见实验组骨小梁骨质改善,局部性修复;2)经苏木精-伊红染色中,实验组可见骨小梁密度较模型组高、骨小梁形态较好;3)实验组骨组织中Wnt3a、MEKK2、β-catenin表达较模型组增加(P<0.05);4)GIOP大鼠模型提取BMSCs并予BMP2诱导,经FTZ含药血清(实验组)干预后,碱性磷酸酶和茜素红染色结果提示实验组成骨反应增强(P<0.05);5)BMSCs经FTZ含药血清干预,可提高β-catenin/TCF转录活性(P<0.05);促进β-catenin、MEKK2蛋白表达(P<0.05)。结论FTZ通过调控MEKK2-Wnt偶联拮抗β-catenin泛素化防治GIOP,从而改善微观骨性结构。

Objective To establish a rat model of glucocorticoid-induced osteoporosis（ GIOP） and to explore the interventional effect of the Chinese medicine Fufang Zhenzhu Tiaozhi（ FTZ） capsules on regulation of mitogen-activated protein kinase kinase kinase 2（ MEKK2）-Wnt coupling and inhibiting β-catenin ubiquitination,and to investigate the effect of FTZ on the bone mineral density and cell osteogenic ability. Methods SPF male rats were randomly divided into normal control group,methylprednisolone group（ model group）,methylprednisolone ＋ saline group（ blank control group）and methylprednisolone ＋ FTZ group（ experimental group）. The proximal femoral cancellous bone was examined by micro-CT and histopathology,and assessment of expressions of Wnt3a,MEKK2,and β-catenin proteins. Bone mesenchymal stem cells（ BMSCs were isolated and treated with serum containing FTZ,stained by alkaline phosphatase and alizarin red.The expressions of osteogenic differentiation-related genes ALP,Runx2 and OCN,the expressions of MEKK2 and β-catenin proteins,and the transcription level of β-catenin/TCF were determined. Results 1） The micro-CT imaging showed that compared with the control group,the BV/TV,Tb. Th and Tb/N expressions were significntly decreased,and Tb/sp increased in the experimental group（ P〈0. 05）. Region of interest（ ROI） three-dimensional reconstruction of trabecular bone in the experimental group showed improvement of bone trabeculae and local bone repair. 2） The pathology using hematoxylin and eosin staining showed that in the experimental group,the bone trabecular density was higher than that of the model group,and observed a better trabecula morphology. 3） The Wnt3a,MEKK2 and β-catenin expressions in the experimental group were significantly increased compared with the model model（ P〈0. 05）. 4） After treated with FTZ and BMP2,the result of alkaline phosphatase and alizarin red staining indicated an enhanced osteogenic response（ P〈0. 05）in the GIOP rat models. 5） After treatment with seum containing FTZ,The BMSCs isolated from the GIOP rats enhanced the transcriptional activity of β-catenin/TCF/LEF（ P〈0. 05） and promoted the expression of β-catenin and MEKK2 proteins（ P〈0. 05）. Conclusions FTZ can ameliorate GIOP by regulating the MEKK2-Wnt coupling and inhibiting the β-catenin ubiquitination,and improve the bone microstructure.