黄芩素通过BMP-2/Smad通路促进MC3T3-E1成骨作用的实验研究

目的 探讨黄芩素（BAI）对小鼠胚胎成骨细胞前体细胞（MC3T3-E1）成骨分化的作用及其分子机制。方法 将MC3T3-E1分为对照组（正常培养）和BAI组（以Baicalein处理），在成骨分化条件培养下采用CCK-8检测BAI对MC3T3-E1细胞增殖的影响；分别以碱性磷酸酶染色（ALP）、茜素红染色（ARS）检测MC3T3-E1细胞成骨分化水平与矿化能力，实时荧光定量PCR检测成骨标志基因ALP、COL1A1、RUNX2、OSX的mRNA表达水平，通过免疫印迹法（Western-blot）检测MC3T3-E1细胞中BMP-2、Smad1、p-Smad1蛋白表达水平，通过免疫荧光技术（IF）检测RUNX2、COL1A1表达水平。结果 与对照组比较，BAI干预1 d后发现，BAI组COL1A1（P<0.001）、RUNX2（P <0.05）、OSX（P <0.05） mRNA表达水平在成骨分化中表达上升；干预3 d后发现，与对照组比较，BAI组ALP（P <0.05）、RUNX2（P <0.001）mRNA表达上升；干预7 d后发现，与对照组比较，BAI组COL1A1（P <0.05）mRNA表达水平较对照组上升，BMP-2、p-Smad1/Smad1蛋白表达水平上升（P <0.05）。免疫荧光中成骨标志蛋白RUNX2、COL1A1表达增多（P <0.05）。结论 BAI可通过激活BMP-2/Smad通路促进MC3T3-E1成骨分化。

Experimental study of Baicalein promoting osteogenic differentiation of MC3T3-E1 through BMP-2 / Smad pathway

Objective To investigate the effect of baicalein (BAI) on the osteogenic differentiation of mouse embryonic osteoblast precursor cells (MC3T3-E1) and its molecular mechanism. Methods MC3T3-E1 was divided into control group (normal culture) and BAI group (treated with baicalein),CCK-8 was used to detect the effect of BAI on the proliferation of MC3T3-E1 cells, alkaline phosphatase staining (ALP) and alizarin red staining (ARS) were used to detect the osteogenic differentiation level and mineralization ability of MC3T3-E1 cells, real time fluorescence quantitative PCR was used to detect the mRNA expression levels of osteogenic marker genes ALP, COL1A1, Runx2 and OSX, western blot was used to detect the protein expression levels of BMP-2, Smad1 and p-smad1 in MC3T3-E1 cells, immunofluorescence technique (IF) was used to detect the expression levels of Runx2 and COL1A1. Results Compared with the control group, after 1d of intervention, it was found that the mRNA expression levels of COL1A1 (P < 0.001), Runx2 (P < 0.05) and OSX (P < 0.05) in BAI group increased during osteogenic differentiation, after 3d of intervention, compared with the control group, the mRNA expressions of ALP (P < 0.05) and Runx2 (P < 0.001) in BAI group increased, after 7d of intervention, the mRNA expression level of COL1A1 (P < 0.05) and the protein expression levels of BMP-2 and p-smad1 / Smad1 in BAI group were higher than the control group (P < 0.05). The expression of osteogenic marker proteins Runx2 and COL1A1 increased in IF (P < 0.05). Conclusion BAI can promote osteogenic differentiation of MC3T3-E1 by activating BMP-2 / Smad pathway.