MAPK抑制剂对骨碎补总黄酮促进转基因成肌细胞成骨分化的影响

Influence of mitogen-activated protein kinase inhibitor in the process of assemble flavone of rhizome drynaria promoting the osteogentic differentiation of myoblasts

背景:前期研究中已证实低浓度骨碎补总黄酮含药血清对转睫状神经生长因子成肌细胞向成骨细胞分化具有促进作用,然而其潜在机制尚未明确。目的:观察MAPK抑制剂对骨碎补总黄酮促进转基因成肌细胞成骨分化的影响。方法:转睫状神经生长因子成肌细胞预处理接种后,在成骨诱导剂诱导下,检测空白血清、骨碎补总黄酮含药血清及加入p38 MAPK抑制剂SB203580各组碱性磷酸酶比活性变化,RT-PCR检测成骨相关基因核结合因子a1和碱性磷酸酶m RNA表达的差异,并采用Western-blotting技术定量分析加入p38MAPK抑制剂后成骨相关蛋白核结合因子a1和碱性磷酸酶蛋白表达情况的变化,进行统计学分析。结果与结论:1RT-PCR检测结果均显示骨碎补总黄酮血清组的成骨相关因子核结合因子a1 m RNA、碱性磷酸酶m RNA表达最高,加入p38抑制剂SB203580后核结合因子a1 m RNA、碱性磷酸酶m RNA表达明显下调;2Western-blot检测加入p38抑制剂SB203580后核结合因子a1 m RNA、碱性磷酸酶m RNA蛋白水平明显下降;3结果说明,p38 MAPK抑制剂可以下调骨碎补总黄酮促进转基因成肌细胞成骨相关基因表达及蛋白水平,骨碎补总黄酮可能通过激活p38 MAPK信号通路促进睫状神经生长因子成肌细胞向成骨细胞分化。

BACKGROUND：Previous studies have confirmed that drug-containing serum with low concentration of assemble flavone of rhizome drynaria （AFDR） promotes osteogentic differentiation of ciliary neurotrophic factor （CNTF）-modified myoblasts, but the underlying mechanism remains unclear. OBJECTIVE：To observe the influence of mitogen-activated protein kinase （MAPK） inhibitor in the process of AFDR promoting the osteogentic differentiation of myoblasts. METHODS：Transfected-CNTF myoblasts were preprocessed prior to osteogentic induction. Changes of alkaline phosphatase specific activities were detected in blank serum, AFDR drug serum and p38 pathway inhibitor SB203580 groups. mRNA and protein expression levels of core binding factor a1 and alkaline phosphatase were detected by real-time PCR and western blot, respectively. RESULTS AND CONCLUSION：Real-time PCR showed that the mRNA expression levels of core binding factor a1 and alkaline phosphatase in the AFDR group were the highest;while p38 inhibitor SB203580 significantly downregulated the above levels. Western blot findings showed that a significant reduction in the protein expression levels of core binding factor a1 and alkaline phosphatase after p38 inhibitor SB203580 addition. These results suggest that p38 pathway inhibitor can downregulate osteogenesis-related gene and protein expressions, and thus AFDR promoting the osteogentic differentiation of CNTF-modified myoblasts probably through activating p38 MAPK signaling pathway.