积雪草酸促进成骨细胞分化的机制

Mechanisms of asiatic acid promotes osteoblasts differentiation

目的研究积雪草酸在体外对成骨细胞分化的影响,并初步探讨其作用机制。方法使用小鼠胚胎成骨细胞前体细胞(MC3T3-E1)进行成骨分化相关实验,将积雪草酸作为成骨诱导条件下的附加刺激,用CCK8法检测药物对成骨细胞活力的影响;采用碱性磷酸酶(ALP)染色评价成骨细胞的早期分化能力;采用茜素红染色观察钙结节的形成数量,评估成骨细胞的矿化能力;用实时荧光定量PCR法检测成骨细胞标志物的水平;采用蛋白免疫印迹法检测MAPKAKT信号通路及凋亡相关蛋白的表达。结果1~40μmol·L^-1积雪草酸对细胞无毒性作用;随着积雪草酸刺激浓度的增加,ALP和茜素红的染色强度也随之增加。经积雪草酸刺激3d后,Ⅰ型胶原蛋白基因的表达水平出现明显变化;第7天时,ALP、骨形态发生蛋白2、转录因子、核心结核因子α1和骨钙素的表达水平显著增加。经积雪草酸刺激后,MC3T3-E1细胞内MAK-AKT信号通路激活;同时,抗凋亡蛋白的表达上调,促凋亡蛋白的表达下降。结论积雪草酸以浓度依赖的方式促进成骨细胞分化,抑制成骨细胞凋亡,该效应可能是通过激活MAPK-AKT信号介导。

OBJECTIVE To investigate the effects of asiatic acid on osteogenic differentiation of MC3T3 - El in vitro, and to explore the mechanism. METHODS MC3T3 - El was used for osteogenic differentiation experiments, with asiatic acid applied as an additional stimulus under osteogenic induction conditions, and the effect of drugs on osteoblast viability was detected by CCK8 method. Early differentiation ability of osteoblasts was evaluated by alkaline phosphatase ( ALP) staining. To evaluate the mineralization ability of osteoblasts, alizarin red staining was used to observe the number of calcium nodules formation. The level of osteoblast markers was detected by RT - qPCR, and the MAPK - AKT signaling pathway and apoptosis - related protein expression were detected by Western Blot. RESULTS Asiatic acid at concentrations of 1 -40μmol·L^-1 did not significantly affect cell growth after the treatment. The staining intensity of ALP and alizarin red were both increased with the concentration of asiatic acid. After 3 days of stimulation, the expression level of COL - 1 gene showed significant changes. At 7th day, the expression levels of ALP, BMP - 2, Osterix, Runx2 and OCN were significantly increased. After stimulation with asiatic acid, it activated the MAPK - AKT signaling pathway and related proteins, up-regulated anti 堦 apoptotic proteins, and down-regulated pro - apoptotic genes. CONCLUSION Asiatic acid promotes osteoblast differentiation and inhibits osteoblast apoptosis in a dose - dependent manner,possibly by activating MAPK - AKT signaling.