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目的:基于p38信号通路探讨左、右归丸含药血清对BMSCs成骨诱导的机制。方法:运用全骨髓贴壁法分离和培养大鼠BMSCs;分别以左归丸、右归丸、两方共同药、滋肾阴药、补肾阳药、阳性对照药补佳乐制备的大鼠含药血清加诱导剂(地塞米松、维生素C、β-甘油磷酸钠)、诱导剂和空白含药血清组共8组对BMSCs进行干预,采用改良钙钴染色法检测碱性磷酸酶(ALP)表达,采用茜素红染色法检测钙化结节,采用Western blotting法检测核结合因子α1(Cbfα1)和Ⅰ型胶原(ColⅠ)、p38、p-p38蛋白表达,采用real time PCR法检测Cbfα1、ColⅠmRNA表达。结果:左、右归丸及滋阴药组可以上调ALP表达,促进BMSCs矿化结节形成,增强Cbfα1、ColⅠmRNA和蛋白的表达并且可以促进p38蛋白的磷酸化;给予p38特异性阻滞剂SB203580后,各组BMSCs ALP表达下调,矿化结节形成减少,p38蛋白磷酸化水平降低,并且Cbfα1、ColⅠmRNA和蛋白的表达下降。结论:左、右归丸及其拆方含药血清可能部分通过p38 MAPK信号通路对BMSCs成骨分化产生调控作用的。
OBJECTIVE: To investigate the osteogenic mechanism of BMSCs induced by Zuogui Gui Wan containing serum on the basis of p38 signaling pathway. Methods: BMSCs were isolated and cultured by using whole bone marrow adherent method. The rats were treated with Zuogui Pill, Yougui Wan, common medicine of both sides, Kidney-Yin recipe, Bushenyangyin, Eight groups of BMSCs were treated with serum plus inducers (dexamethasone, vitamin C, β-glycerophosphate), inducers and blank drug-containing serum groups. Alkaline phosphatase (ALP) The expression of Cbfα1, Col Ⅰ, p38 and p-p38 protein were detected by Western blotting. The expression of Cbfα1, ColⅠmRNA was detected by real time PCR expression. Results: Zuogui Gui Wan and Ziyin Decoction could up-regulate the expression of ALP, promote the formation of mineralized nodules, enhance the expression of Cbfα1, ColⅠmRNA and protein, and promote the phosphorylation of p38 protein. The p38-specific inhibitor SB203580 After treatment, the expression of ALP in BMSCs was down-regulated, the formation of mineralized nodules decreased, the phosphorylation of p38 protein decreased, and the expression of Cbfα1, ColⅠmRNA and protein decreased. CONCLUSION: Zuogui Gui Wan and its disassembled medicated serum may partly regulate the osteogenic differentiation of BMSCs via the p38 MAPK signaling pathway.