Experimental Study On Bushen Jianpi Huoxue Recipe Regulating Osteoblast Proliferation And Mineralization By MiR-210

Objective1.To observe the effect of Bushen Jianpi Huoxue Fang on the proliferation and mineralization of osteoblasts;2.To observe the effect of miR-210 transfection on the proliferation and mineralization of osteoblasts;3.Analysis of target genes,signal pathways and molecular mechanisms of miR-210 in regulating osteoporosis through bioinformatics.Methods1.Bushen Jianpi Huoxue Fang interferes with primary osteoblasts and miR-210 transfected primary osteoblasts:①Different concentrations of Bushen Jianpi Huoxue Fang were prepared,and then the primary osteoblasts were intervened,and the osteoblast activity was measured by CCK-8.The optimal concentration of Bushen Jianpi Huoxue Fang interferes with primary osteoblasts,and BCIP-NBT is used to detect ALP expression of osteoblasts;②Primary osteoblasts were transfected with miR-210 at different concentrations,and then osteoblast activity was measured by CCK-8.The optimal concentration of miR-210 was used to transfect primary osteoblasts and BCIP-NBT was used to detect ALP expression of osteoblasts;RT-qPCR was used to detect Runx2,OPG,OPN,β-catenin mRNA expression;2.Explore the mechanism of miR-210 in regulating osteoporosis based on bioinformatics:This study analyzed the expression of miR-210 in the microarray chips GSE74209 and GSE93883.The three databases of TargetScan,miRTarBase and miRDB predicted the miR-210 target genes,and the three databases of Gene,GenaCards and OMIM were used to screen for osteoporosis targets.The study sought to find the target genes of miR-210 for osteoporosis by mapping,and completed the GO and KEGG enrichment analysis of target genes to explain its molecular mechanism.Results1.Bushen Jianpi Huoxue Fang interferes with primary osteoblasts and miR-210 transfected osteoblasts:①Bushen Jianpi Huoxue Recipe at a concentration of 100-500ug/ml can promote the proliferation of osteoblasts.Bushen Jianpi Huoxue Recipe at a concentration of 500ug/ml interferes with osteoblasts on the third day,and its proliferation effect is the most obvious.②After the intervention of primary kidney osteoblasts by Bushen Jianpi Huoxue Fang,the positive rate of ALP staining was higher than that of the control group.③When miR-210 was transfected at a concentration of 50 nmol/L and the time was 2 days,osteoblast proliferation activity was the strongest.Intervention of miR-210 in primary osteoblasts can promote cell proliferation,promote osteogenic mineralization,and promote the expression of Runx2,OPG,OPN.2.Explore the mechanism of miR-210 in regulating osteoporosis based on bioinformatics:①miR-210 is differentially expressed in bone and blood of osteoporosis.②The core target genes of miR-210 for osteoporosis include STAT1,MAPK1,CSF1,HIF1A,TFRC,BTK,HDAC4,PAX5,FOXP3,P4HB,SMAD4,APOA1,SYK,ARRB1,BDNF,CD38,CTTN,GBA,CIITA,TWIST1,RUNX3.③miR-210 participates in the regulation of osteoporosis mainly through the MAPK signal pathway,cAMP signal pathway,cGMP-PKG signal pathway,PI3K-Akt signal pathway,TGF-β signal pathway,and NF-K B signal pathway.Conclusion1.Bushen Jianpi Huoxue Fang intervention in primary osteoblasts can increase cell viability and positive rate of ALP staining,and promote osteogenic mineralization.Transfection of miR-210 into osteoblasts can increase the positive rate of ALP staining,promote the expression of Runx2,OPG,OPN,and promote osteogenic mineralization.2.MiR-210 is differentially expressed in osteoporosis.Its core target genes include STAT1,MAPK1,CSF1,HIF1A,TFRC,BTK,HDAC4,PAX5,FOXP3,P4HB,SMAD4,APOA1,SYK,ARRB1,BDNF,CD38,CTTN,GBA,CIITA,TWIST1,RUNX3.It mainly participates in regulating osteogenic mineralization through MAPK signal pathway,cAMP signal pathway,and cGMP-PKG signal pathway.