基于Wnt/β-catenin信号通路的复方补肾活血颗粒对骨髓间充质干细胞成骨、成脂分化的影响

Effects of Compound Bushen Huoxue Granules on Osteogenic and Lipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Based on Wnt/β-catenin Signaling Pathway

目的基于Wnt/β-catenin信号通路探讨复方补肾活血颗粒含药血清对人骨髓间充质干细胞(BMSCs)成骨、成脂分化的影响。方法取骨质疏松症患者骨髓,体外培养BMSCs,将细胞分为空白组和含药血清低、中、高剂量组(2%、5%、8%),CCK-8法检测细胞活力,碱性磷酸酶染色、茜素红染色观察成骨分化潜能,油红O染色观察成脂分化潜能,RT-PCR检测成骨分化相关基因Runt相关转录因子2(RUNX2)、Osterix(Osx)和成脂分化相关基因CCAAT/增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物激活受体(PPAR)γmRNA表达,Western blot检测RUNX2、Osx、C/EBPα、PPARγ、低密度脂蛋白受体相关蛋白(LRP)5及β-连环蛋白(β-catenin)蛋白表达。结果与空白组比较,含药血清各剂量组细胞活力显著升高,其中以含药血清中剂量组效果最佳;含药血清各剂量组可促进BMSCs早期钙化和钙沉积形成,抑制脂滴形成;含药血清各剂量组BMSCsRUNX2、OsxmRNA和蛋白表达升高,C/EBPα、PPARγmRNA和蛋白表达降低,LRP5和β-catenin蛋白表达明显升高,其中含药血清高剂量组差异有统计学意义(P<0.05,P<0.01,P<0.001)。结论复方补肾活血颗粒含药血清对BMSCs成骨分化相关因子表达有明显促进作用,并可抑制BMSCs成脂分化,其机制与激活Wnt/β-catenin信号通路,进而调节成骨-成脂平衡有关。

Objective To investigate the effects of compound Bushen Huoxue Granules on osteogenic and lipogenic differentiation of human bone marrow mesenchymal stem cells(BMSCs)based on Wnt/β-catenin signaling pathway.Methods BMSCs were isolated and cultured from bone marrow fluid of patients with osteoporosis.The cells were divided into blank group and medicated serum low-,medium-,and high-dosage group(2%,5%,8%).Cell viability were detected by CCK-8 method,alkaline phosphatase staining and alizarin red staining were used to observe the osteogenic differentiation potential of BMSCs,lipogenic differentiation potential was observed by oil red O staining,the expressions of osteogenic differentiation-related genes RUNX2,Osx,lipogenic differentiation-related genes C/EBPαand PPARγmRNA were detected by RT-PCR,Western blot was used to detect RUNX2,Osx,C/EBPα,PPARγ,LRP5 andβ-catenin protein expression.Results Compared with the blank group,the cell viability of each dosage group of medicated serum significantly increased,among which the medicated serum medium-dosage group had the best effect;each dosage group of medicated serum can promote the formation of early calcification and calcium deposition in BMSCs,and inhibit the formation of lipid droplets;the mRNA and protein expressions of RUNX2 and Osx in BMSCs in each dosage group of medicated serum increased,the expression of C/EBPα,PPARγmRNA and protein decreased,and the expression of LRP5 andβ-catenin protein significantly increased,and the medicated serum high-dosage group with statistical significance(P<0.05,P<0.01,P<0.001).Conclusion The medicated serum of compound Bushen Huoxue Granules can significantly promote the expression of osteogenic differentiation related factors in BMSCs and inhibit the adipogenic differentiation of BMSCs.The mechanism is related to the activation of Wnt/β-catenin signaling pathway,and then regulates the osteogenesis-lipogenesis balance.