补骨生髓方调控ERK磷酸化介导Smad/ERK通路治疗骨质疏松症的效应机制研究

In the previous studies, the clinical effect and safety of Bugushengsui decoction (BGSSD) treating for osteoporosis had been confirmed. BGSSD also increased the lumbar and femoral bone mineral density, and improved the bone biomechanical indexes in the castrated model rats. However, the molecular mechanism of BGSSD for osteoblast function is not clear yet. During the bone formation regulation, ERK phosphorylated protein in the ERK signaling passway influences the osteoblast differentiation through inhabiting Smad protein expression and function. According to the literature analysis, the drug target of BGSSD may regulate ERK protein phosphorylation and key factors in the Smad, ERK1/2 signaling passway, then promote the osteoblast differentiation. Based on this hypothesis, firstly, the SAMP6 spontaneous senile osteoporosis model will be established and pharmacological effect of Chinese herbal compound is verified. Secondly, the role of ERK phosphorylation protein (p-ERK) for osteoblast differentiation and the expression of p-ERK, ERK1/2, Smad4, Runx2 regulated by BGSSD are explored in the aspect of bone tissue. Thirdly, we will apply ERK agonists (epidermal growth factor, EGF) and BGSSD that induce bone mesenchymal stem cells to the osteoblast differentiation repectively, in order to further observe the regulation role of BGSSD for the key factors in the vitro studies. This study deepens the knowledge to pathogenesis mechanism of OP, providing the new experimental evidence for Chinese herbal compound in the treatment of OP.

课题组前期证实补骨生髓方治疗骨质疏松症（OP）临床有效、安全性好，能够改善去势模型大鼠腰椎、股骨骨密度和骨生物力学指标，但其对于成骨细胞的效应机制尚不明确。在骨形成调控过程中，ERK通路中的ERK蛋白磷酸化通过抑制Smad蛋白的表达，影响了成骨细胞的分化。根据文献分析，补骨生髓方作用靶点可能是通过调控ERK蛋白磷酸化及Smad、ERK1/2通路中关键因子的表达，从而促进了成骨细胞分化。围绕此假说，本研究拟建立SAMP6骨质疏松模型，验证药效作用的同时，在骨组织层面探索ERK磷酸化蛋白（p-ERK）对成骨细胞分化的作用，以及复方对p-ERK、ERK1/2、Smad4、Runx2蛋白及基因水平表达的影响；体外实验方面，进一步采用ERK激动剂（EGF）、补骨生髓方干预BMSCs诱导向成骨细胞分化，观察复方对关键因子的表达调控作用。本研究深化了对OP发病机制的理解，为中药复方治疗OP提供了新证据。

前期证实中药复方补骨生髓方治疗骨质疏松症（OP）具有较好疗效，但尚缺乏从促成骨角度阐释其效应机制的基础研究。本研究体内实验通过建立SAMP6自发型老年性骨质疏松模型，检测骨密度、骨组织形态计量学、骨代谢标志物以及苏木素-伊红染色的方法，验证了补骨生髓方治疗OP药效作用的同时，选择高剂量为最佳给药剂量，与空白对照组、模型组同时应用Western blot方法、Real time-PCR技术，检测了p-ERK、ERK1/2、Smad4、Runx2等关键因子的表达情况，结果显示在p-ERK表达降低的同时，ERK1/2、Smad4、Runx2表达升高，初步探讨了这些关键因子之间相互作用的关系。体外实验通过建立MC3T3-E1细胞模型，采用CCK-8方法，确证了补骨生髓方含药血清对细胞的促增殖作用；同时，利用ELISA法、Western Blot等方法研究了ALP、BGP、ERK1/2、p-ERK、Smad4、Runx2等关键蛋白的表达情况，并通过Real time-PCR技术检测了ERK1/2、Smad4、Runx2等关键因子的转录水平，得出了与动物实验较为一致的结果，进一步说明了补骨生髓方治疗OP过程中相关因子的相互作用关系。综上得出结论：补骨生髓方治疗OP发挥效应的靶点可能是通过降低ERK蛋白磷酸化调控Smad/ERK通路促进骨形成。该研究阐明了补骨生髓方治疗OP的部分作用机制及生物学途径，为今后OP临床治疗寻找相应治疗靶点提供了科学依据。