To Explore The Mechanism Of Guilu Erxian Gao Intervene In GIOP Rats By Regulating MiRNA Expression

Purpose:1.To investigate the differential expression of miRNAs in GIOP.2.To investigate the differential expression of miRNAs in rats with GIOP after the treatment of Guilu Erxian Gao.3.To investigate the possible mechanism and pathways in the pathogenesis of GIOP and the treatment of Guilu Erxian Gao using methods of GO enrichment analysis and KEGG enrichment analysis.Materials and Methods: 48 Wistar rats(aged 3 months)were divided randomly by weight into 6 groups,including normal group,model group,high-dose Guilu Erxian Gao group,medium-dose Guilu Erxian Gao group,low-dose Guilu Erxian Gao group,and alendronate sodium group.There were 8 animals in each group with half male.Except for the normal group,the other groups were injected intramuscularly with dexamethasone(2.5 mg/kg)twice a week to make models.The normal group and the model group were administered normal saline intragastrically,and other groups were administered corresponding drugs intragastrically.After receiving 9-week treatment,rats were anesthetized with 5% chloral hydrate by intraperitoneal injections and the femur and tibia of both hind limbs were taken.The bone mineral density(BMD)of femoral samples was measured by dual-energy X-ray absorptiometry(DXA).After HE staining,the morphology and structure of the femur bone were observed by the light microscope.The optimal concentration of Guilu Erxian was selected according to the results.The differential expression of miRNAs in the tibia bone tissue was screened from the normal group,the model group and the optimal concentration groups by high-throughput sequencing to predict the regulated target genes.Then the predicted genes were performed GO enrichment analysis and KEGG enrichment analysis to investigate the mechanism in the pathogenesis of GIOP and the treatment of Guilu Erxian Gao.Results:1.Compared with the normal group,the BMD of the model group decreased significantly(P< 0.05).Compared with the model group,the BMD of the high-dose,medium-dose and low-dose Guilu Erxian Gao group and the alendronate sodium group increased significantly(P < 0.05).The BMD of the medium-dose Guilu Erxian Gao group increased most significantly among the high-dose,medium-dose and low-dose Guilu Erxian Gao group.2.Under light microscope,the trabecular bones in the normal group were tightly arranged in a network-like structure,the microstructure of the bone in the model group was damaged with thinned and broken bone trabecula and the sparse bone trabecula,the sparse and fractured trabeculae were repaired in the high-dose,medium-dose and low-dose Guilu Erxian Gao group and the alendronate sodium group with increased total number of trabeculae in a gradual network-like structure.3.The sequencing results showed that compared with the normal group,the model group had11 differentially-expressed miRNA,with 10 out of them up-regulated and 1 down-regulated.Compared with the model group,the Guilu Erxian Gao group had 18 differentially-expressed miRNA,3 of which were up-regulated and 15 were down-regulated.Compared with the normal group,the Guilu Erxian Gao group had 6 differentially-expressed miRNA,all of which were up-regulated.4.The results of target gene prediction showed that 2468 target genes were predicted by the differentially-expressed miRNA in the model group compared with the normal group.Compared with the model group,4365 target genes were predicted by the differentially-expressed miRNA in the Guilu Erxian cream group.Compared with the normal group,2582 target genes were predicted by the differentially-expressed miRNA in the Guilu Erxian cream group.5.According to the results of bioinformatics analysis,GCs that binded to receptors through pathways like p53 signaling,affected the lifespan and differentiation of intraosseous cells,and promoted atherosclerotic vascular to change the blood supply in the bone and result in a decrease in bone mass might be the pathogenesis of GIOP.The main mechanism of Guilu Erxian Gao in the treatment of GIOP might be related to The Guilu Erxian Gao that could affect intracellular RNA polymerase II to regulate positively in transcription and protein phosphorylation through the MAPK signaling pathway,regulate cell proliferation,differentiation and lifespan might be the main mechanism of them in the treatment of GIOP.In addition,it may also affect cell differentiation and growth by regulating sphingolipid metabolism.Conclusion:1.The Guilu Erxian Gao could increase BMD of GIOP rats and improve the changes of bone microstructure,which is related to the differential expression of miRNA.2.The main pathogenesis of GIOP might be that GCs bind receptor proteins on the intraosseous cell membrane through pathways such as the p53 signaling pathway,MAPK signaling pathway and HIF-1 signaling pathway,affecting cell signal transduction,transcription regulation and redox process,changing the transferase activity and hydrolase activity in the cell,and then changing the internal structure of the cell such as the nucleus and cell composition,which could affect cell life and cell differentiation.In addition,it might be also related to GCs that affect the fluid shear stress of the intraosseous blood vessel,cause the atherosclerosis in the intraosseous vessles and change the blood supply in bones.3.The main mechanism for Guilu Erxian Gao in th treatment of GIOP might be the binding of metal ions in bone or receptor proteins in cell membrane and nucleus through MAPK signaling pathway,Wnt signaling pathway,m TOR signaling pathway and other signaling pathways,which positively regulates the transcription of RNA polymerase Ⅱ,affects the protein phosphorylation and protein homodimer activity.Thereby it could affect the combination of intracellular nucleotides and intracellular components and regulate cell proliferation,differentiation and lifespan.In addition,Guilu Erxian Gao could also affect the differentiation and growth of cells by the metabolism of sphingolipids in the cell membrane.