活血通络胶囊促进骨髓间充质干细胞成骨中的ERα-Wnt/β-catenin信号通路

Huo Xue Tong Luo capsule improves osteoblastogenesis of bone marrow mesenchymal stem cells through the ERα-Wnt/β-catenin signaling pathway

背景:活血通络胶囊在临床上可预防无症状股骨头坏死的进展,但其相关机制尚不明确。目的:探讨活血通络胶囊对人骨髓间充质干细胞成骨分化的影响及ERα-Wnt/β-catenin通路的作用。方法:将第3代人骨髓间充质干细胞加入含不同质量浓度活血通络胶囊(0,1,5,10 mg/L)的成骨诱导基培养14 d,采用茜素红染色和碱性磷酸酶活性检测确定活血通络胶囊促进人骨髓间充质干细胞成骨分化的最佳质量浓度。然后将第3代人骨髓间充质干细胞进一步分成4组:对照组、活血通络胶囊组、活血通络胶囊+ICI 182780组、活血通络胶囊+ICI 182780+DKK1组,采用qRT-PCR、Western blot及免疫荧光染色等方法观察活血通络胶囊、雌激素受体抑制剂ICI 182780和重组人DKK1蛋白对人骨髓间充质干细胞成骨的影响。结果与结论:①活血通络胶囊(10 mg/L)能显著促进人骨髓间充质干细胞的增殖和成骨分化,并促进成骨基因Runx2、OPN、DLX5、BGLAP和ERα的mRNA表达,以及Wnt/β-catenin通路相关靶点β-catenin、TCF7、Lef1、C-MYC、CYCLIN D和C-JUN的mRNA表达;②活血通络胶囊的成骨作用受到ERα抑制剂ICI 182780和Wnt/β-catenin通路抑制剂DKK1的抑制;③结果表明,ERα-Wnt/β-catenin信号通路参与了活血通络胶囊对人骨髓间充质干细胞增殖和成骨分化的促进作用。

BACKGROUND:The Huo Xue Tong Luo capsule has been found to prevent the progression of asymptomatic osteonecrosis of the femoral head,but the mechanism is not clear.OBJECTIVE:To investigate the effect of Huo Xue Tong Luo on the osteoblast differentiation of human bone marrow mesenchymal stem cells,and the effect of ERα-Wnt/β-catenin pathway.METHODS:The third generation of human bone marrow mesenchymal stem cells was cultured in different concentrations of Huo Xue Tong Luo capsule(0,1,5,10 mg/L)for 14 days.Alizarin red staining and alkaline phosphatase activity assay were used to determine the optimal concentration of Huo Xue Tong Luo in promoting osteogenic differentiation of human bone marrow mesenchymal stem cells.Human bone marrow mesenchymal stem cells were further divided into four groups:control group,Huo Xue Tong Luo group,Huo Xue Tong Luo+ICI 182780 group,and Huo Xue Tong Luo+ICI 182780+DKK1 group.qRT-PCR,western blot assay,and immunofluorescence staining were used to investigate the effect of Huo Xue Tong Luo,ICI 182780 and recombinant human DKK1 protein on osteogenesis of human bone marrow mesenchymal stem cells.RESULTS AND CONCLUSION:(1)Huo Xue Tong Luo capsule(10 mg/L)could significantly promote the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells,and promote the mRNA expression of osteogenic genes Runx2,OPN,DLX5,BGLAP,and ERα,as well as the mRNA expression of Wnt/β-catenin pathway related targetsβ-catenin,TCF7,Lef1,C-MYC,CYCLIN and C-JUN.(2)The osteogenesis of Huo Xue Tong Luo capsule was inhibited by ICI 182780 and DKK1.(3)The results showed that ERα-Wnt/β-catenin signaling pathway was involved in the promoting effect of Huo Xue Tong Luo capsule on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells.