Expression Mechanism Of JAK2-STAT3 Signaling Pathway In Steroid-induced Osteonecrosis Of The Femoral Head

Background:Steroid-induced Osteonecrosis of Femoral Head(SONFH)is a metabolic disease caused by the use of Glucocorticoids(GCs)drugs.Since GCs has been widely used in the treatment of various clinical diseases,the number and incidence of SONFH have been increasing year by year.At present,long-term or large-scale application of GCs has become the primary pathogenesis of Osteonecrosis of Femoral Head(ONFH).SONFH mainly affects young and middle-aged patients,and most of them have bilateral necrosis with a wide range of necrosis.The early conservative treatment is not effective.With the progress of the disease,the collapse of the femoral head eventually leads to hip joint dysfunction,and eventually most patients need hip arthroplasty.At present,the pathogenesis of SONFH has not been fully elucidated.Most scholars generally believe that the accumulation of hormones in the human body to a certain extent can cause hypercoagulable state of blood,dyslipidemia,ischemia caused by embolism of the blood supply vessels of the femoral head,reduced osteogenic ability,and finally lead to the occurrence of osteonecrosis.SONFH brings great psychological pressure and economic burden to patients,their families and society due to the difficulty in repairing osteonecrosis,high disability rate,and the need for long-term use of GCs after being diagnosed as ONFH.Therefore,it is of great significance to study the pathogenesis of SONFH and find new treatment methods for the prevention,diagnosis and treatment of SONFH.Objective:1.The appropriate SONFH gene chips were selected from the GEO database for bioinformatics analysis,in order to find the important differential genes and key signaling pathways involved in the development of SONFH,and then to further explore its related pathogenesis.2.To explore the expression mechanism of JAK2-STAT3 signaling pathway in SONFH by constructing SONFH rat model in vivo and in vitro.Methods:1.The SONFH gene expression profile dataset GSE123568 was obtained from the GEO database,including a total of 40 serum samples.Among them,the control group was serum samples from 10 patients who did not develop ONFH after the use of GCs,and the SONFH group was serum samples from 30 patients who developed ONFH after the use of GCs.The obtained gene expression data sets were preprocessed and the Differential Expressed Genes(DEGs)of the two groups of serum samples were analyzed.The selected DEGs were subjected to Gene ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(Kyoto Encyclopedia of Genes and Genomes,Kyoto Encyclopedia of Genes and Genomes,China).KEGG)pathway enrichment analysis was used to further explore the key signal pathways in steroid-induced osteonecrosis of the femoral head.2.The mechanism of JAK2-STAT3 signaling pathway in SONFH was further explored by establishing a rat model of steroid-induced osteonecrosis of the femoral head.The experimental rats were randomly divided into three groups:(1)blank control group;(2)SONFH model group;(3)SONFH+AG490 group.HE staining was used to evaluate and analyze the occurrence of empty lacunae and SONFH in the femoral head of the three groups of rats.TUNEL staining was used to detect the apoptotic osteoblasts in the femoral head of the three groups of rats to verify the SONFH model and detect the effect of JAK2-STAT3 signaling pathway specific inhibitor AG490 on SONFH rats.The growth curve of rat osteoblasts was measured.The primary osteoblasts were divided into three groups:(1)blank control group;(2)Dex group;(3)Dex+AG490 group.TUNEL staining was used to detect the apoptosis level of primary osteoblasts in the three groups.The expression levels of JAK2-STAT3 signaling pathway related proteins(p-JAK2,p-STAT3)and apoptosis proteins in osteoblasts were detected by Western-Blot.Alkaline phosphatase(ALP)activity was measured to evaluate the effect of AG490 on osteogenic activity and apoptosis of osteoblasts.Results:1.In this study,a total of 719 DEGs were obtained after screening the gene expression dataset GSE123568,of which 429 genes were up-regulated and 290 genes were down-regulated in SONFH samples.Further enrichment analysis of DEGs showed that DEGs were significantly enriched in inflammatory response,immune response,signal transduction,cytoplasmic membrane,cytosol,cell exosomes,signal receptor activity,protein homodimerization activity,etc.KEGG pathway enrichment analysis showed that DEGs were mainly enriched in osteoclast differentiation,chemokine signaling pathway,B cell receptor signaling pathway,FoxO signaling pathway,mTOR signaling pathway,JAK-STAT signaling pathway,etc.2.After the SONFH rat model was established,HE staining showed that the rate of empty lacunae in the femoral head of the model group was significantly higher than that of the blank control group and AG490 group.The results of TUNEL staining showed that the apoptosis of osteoblasts in the femoral head of the model group was significantly higher than that of the blank control group and the AG490 group.HE staining and TUNEL staining confirmed that the rat femoral head necrosis model was successfully established.It suggests that AG490 may effectively reduce the rate of empty lacunae in the femoral head of SONFH rats and the apoptosis of osteoblasts induced by GCs by inhibiting JAK2-STAT3 signaling pathway.3.The results of Western-Blot showed that the JAK2-STAT3 signaling pathway-related proteins p-JAK2 and p-STAT3 in the SONFH model group were significantly higher than those in the control group and the treatment group(P < 0.05).Compared with the model group,AG490 increased the expression of Bcl-2 in osteoblasts.The expression of Bax pro-apoptotic protein was down-regulated.Compared with the model group,AG490 increased the ALP activity of osteoblasts.AG490 may reduce the apoptosis of osteoblasts by inhibiting JAK2-STAT3 signaling pathway,suggesting that GCs may regulate apoptosis-related proteins by activating JAK2-STAT3 pathway and participate in the development of steroid-induced SONFH in rats.4.The results of TUNEL staining of rat primary osteoblasts showed that the apoptosis of primary osteoblasts induced by Dex was consistent with that of rat femoral head osteoblasts,and the intervention of AG490 reduced the apoptosis induced by Dex.It is further demonstrated that GCs may be involved in the development of SONFH by regulating osteoblast apoptosis and activity through JAK2-STAT3 signaling pathway.Conclusion:1.A total of 719 DEGs were identified in this study,of which 429 genes were up-regulated and290 genes were down-regulated in SONFH samples.Subsequent enrichment analysis showed the main functions and signaling pathways of DEGs,suggesting that JAK2-STAT3 pathway may be involved in the development of SONFH.2.GCs may regulate the apoptosis and osteogenic activity of osteoblasts by activating JAK2-STAT3 signaling pathway,and participate in the occurrence and development of SONFH in rats.3.AG490 can effectively improve the occurrence and development of steroid-induced osteonecrosis of the femoral head in rats,which is expected to become a new way to prevent and treat SONFH.