| Objective With the glucocorticoid(GC)widely used in the treatment of various autoimmune diseases and inflammatory diseases,excessive use of glucocorticoids can cause Glucoconicoid induced osteopomsis(GIOP),resulting in The loss of bone mass and the increase of fracture risk,the high incidence rate and the diseased part are mostly located in the central axis,which has a high disability rate and mortality rate.Currently,drugs for treating GIOP often cause various adverse side effects.Therefore,this experiment established a zebrafish juvenile osteoporosis model,using whole transcriptome sequencing analysis to explore the gene regulatory network involved in the pathogenesis of GIOP and to verify,to provide a new idea for further clarifying the pathogenesis of GIOP and targeted therapy.Methods1.Zebrafish were randomly divided into 6 groups.Alizarin red staining was used to observe the mineralization level of zebrafish at 8d,9d,and 10d after fertilization with 25 mM prednisolone.2.The zebrafish juveniles of the experimental one fertilized 10 dpf were sacrificed and analyzed by whole transcriptome sequencing.The total RNA was extracted and the cDNA library was constructed to prepare a transcriptome sequencing library.The differential gene expression of the two groups was calculated and analyzed.3.The resulting transcriptome sequencing library was subjected to gene GO function analysis and KEGG signal pathway enrichment analysis.4.?The expression levels of itgalO mRNA and itg?l1 mRNA in zebrafish GIOP model were detected by PCR to verify the reliability of sequencing results from the technical level.?The zebrafish itgalO and itg?l1 were constructed in vivo by using CRISPR/Cas9 gene knockout technology.Gene mutants,by comparing the mineralization area of zebrafish bone staining after intervention,to investigate the regulation of itg?10 and itg?l1 on bone metabolism of zebrafish juveniles;?to evaluate the inhibition of osteoporosis in zebrafish juveniles by evaluating exogenous itg?10 10 and itgA?l1 effect.5.Zebrafish were randomly divided into 4 groups(n=5)to observe the bone mineralization level of GIOP zebrafish by injecting exogenous itgalO and/or itg?l1 to the zebrafish.Results1.The fertilization level of 10 dpf was decreased under the intervention of zebrafish 25?M prednisolone(P<0.05),and the bone mineralization level of zebrafish juveniles was lower with the prolongation of action time(P<0.05).2.The transcriptomes of the two groups were successfully constructed by RNA-seq sequencing.Cluster analysis showed that there were differential expressions of zebrafish genes between the two groups.3.By analyzing the genome of the transcriptome for GO and KEGG,it is concluded that the extracellular matrix and collagen trimer may be involved in the whole biological process of glucocorticoid-induced osteoporosis,and the adhesion signaling pathway is the most significant difference in signal transduction,path.4.The expression of itgalO and itg?11 mRNA in GIOP zebrafish tissues was down-regulated compared with the wild control group.After knockout of itga10 and itg?l1,the stained area of bone mineralization was significantly lower after 10 days of zebrafish fertilization than in the model group(P<0.05).The modified itga10 and itg?l1 can increase the bone mineralization area of zebrafish to a certain extent.5.Under the intervention of exogenous itga10 and itg?l1 modified RNA,the staining area of bone mineralization was significantly higher than that of the model group 10 days after fertilization(P<0.05).Exogenous itg?10 and itg?l1 could alleviate zebrafish bone.Loose;in addition,after the injection of itg?10 and itg?l1 mRNA into the zebrafish single-cell fertilized egg,the stained area of bone mineralization increased significantly after 10 days of zebrafish fertilization(P<0.05),and itg?10 and itg?l1 were obtained.Modified RNA has a synergistic effect on the regulation of osteoporotic zebrafish.Conclusions1.25?M prednisolone induced osteoporosis-like phenotype in zebrafish juveniles,and the prolonged intervention time,the lower the bone mineralization level of zebrafish juveniles.2.From the analysis of gene function,it can be concluded that prednisolone may participate in the regulation of bone metabolism related signals by itga10 and itg?l1.3.Differential gene expression of GIOP is associated with enrichment of GO term and signaling pathways,with extracellular matrix and collagen trimers and adhesion signaling pathways being particularly important.4.itg?10 and itg?l1 genes can inhibit the down-regulation of prednisolone on bone mineralization of zebrafish.5.Exogenous itga10 and itg?l1 modified RNA can alleviate osteoporosis in zebrafish,and the two have synergistic effects. |
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