Study On The Mechanism Of Icariin Regulating Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By MiRNA

Objective: Based on the theory of traditional Chinese medicine,"kidney dominates bone and generates marrow",the mechanism of icariin regulating the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)through mi RNA was preliminarily discussed,providing new ideas and experimental basis for the clinical treatment of delayed fracture healing and nonunion.Methods: Gene chips related to fracture healing in rats were screened from the Gene Expression Omnibus(GEO)database.The original data of the data set were downloaded and standardized by robust multi-array average(RMA)to extract and analyze the m RNA expression level of dual-specificity phosphatase 1(DUSP1).Mi RDB,Diana-micro T and Target Scan databases were used to predict mi RNAs which may bound to DUSP1 m RNA specifically,and the hypothesis of regulatory mechanism of mi RNA was established by bioinformatics methods.The optimal concentration of icariin was screened by CCK-8 cell proliferation experiment,and the osteogenic effect of icariin on BMSCs in rats was analyzed by alkaline phosphatase(ALP)activity detection.Western blotting and RT-q PCR were used to analyze the expression of Osterix(OSX)and osteocalcin(OCN),target genes DUSP1 and mi RNA during the osteogenic differentiation of rat BMSCs promoted by icariin,and the m RNA and protein expression of target gene DUSP1 in rat BMSCs with overexpression of mi RNA.The targeted relationship betweenmi RNA and DUSP1 m RNA was analyzed by double luciferase report assay.Results: 1.By mining the GSE594 data set,it was found that the expression levels of DUSP1 m RNA in the bone samples of rats in the juvenile group,the youth group and the middle and old group were significantly different at different dates after fracture.In addition,with the progress of fracture healing in rats,there was a trend of fluctuation from rise to fall.2.Combining the prediction results of three mi RNA databases,six mi RNAs targeting DUSP1 m RNA were obtained,including rno-mi R-101a-3p,rno-mi R-101b-3p,rno-mi R-137-3p,rno-mi R-429,rno-mi R-200c-3p and rno-mi R-200b-3p.3.Icariin(concentration of 10μg/m L)could significantly improve the proliferation activity,ALP activity and expression of osteogenic marker genes OCN and OSX in BMSCs in rats.4.Icariin could inhibit the expression of rno-mi R-200c-3p in BMSCs of rats and promote the expression of DUSP1 protein.5.The dual-luciferase reporting assay verified that rno-mi R-200c-3p could specifically bind to DUSP1 m RNA.6.The expression levels of DUSP1 m RNA and protein decreased in BMSCs of rats with overexpression of rno-mi R-200c-3p.Conclusion: Icariin can increase the expression of DUSP1 by inhibiting the expression of rno-mi R-200c-3p in rat BMSCs,and promote the proliferation and osteogenic differentiation of rat BMSCs,which may play a role in promoting fracture healing.