SGK1 Regulates Chondrocyte Anabolism And Catabolism In Osteoarthritis And Its Underlying Molecular Mechanism

Background Osteoarthritis(OA)is a common chronic joint disease,which is based on progressive degeneration of articular cartilage.It can cause synovitis,subchondral osteosclerosis,formation of bone cysts and osteophytes,structural changes of articular capsules and their internal and external soft tissues,resulting in affected joint pain,deformity and dysfunction,and seriously affecting the daily life and working status of patients,as well as imposing a heavy burden on society and the economy.In recent years,with the prolongation of life expectancy,the incidence of OA has increased significantly.At present,the clinical treatment of OA is only to control pain,because there is no effective disease-modifying treatments.Therefore,the exploration of the mechanism of OA articular cartilage degeneration and new clinical treatment has been one of the hotspots in orthopedic research today.Traditionally,articular cartilage degeneration is caused by mechanical tear and wear,however current studies have demonstrated that inflammation,genetic susceptibility,aging,gender,obesity,trauma are associated with OA cartilage degeneration.Although the specific pathogenesis of OA cartilage degeneration is still unclear,it is generally accepted that chondrocyte anabolism and catabolism imbalance is the fundamental factor leading to cartilage degeneration.Therefore,it is an effective way to explore new treatment of OA cartilage degeneration by focusing on the specific molecules regulating chondrocyte anabolism and catabolism and their underlying molecular mechanism.ObjectiveIn view of the above research background and ideas,the present study investigated the role of serum and glucocorticoid-inducible kinase 1(SGK1)in regulating chondrocyte anabolism and catabolism,as well as exploring the possibility of using SGK1 as a therapeutic target to treat OA,providing new clues to the drug development and new therapeutic strategies for OAMethods1.We retrieved Gene Expression Omnibus(GEO)public database,using R-studiosoftware to analyze differentially expressed genes of GSE113825,which is a transcriptome datasets containing 5 normal cartilage samples and 5 OA cartilage samples;moreover,we verified the differential expression of SGK1 between normal cartilage and OA cartilage specimens by q RT-PCR,Western blot and immunohistochemical staining.2.Human primary chondrocytes were isolated and cultured,then SGK1 was knockeddown by small interfering RNA for SGK1(small interfering RNA for SGK1,si-SGK1),followed by treated with pro-inflammatory cytokine IL-1? to simulate the microenvironment of OA in vitro.Differences in expression of anabolic markers(Collagen II,Aggrecan)and catabolic markers(ADAMTS-5,MMP-13)of chondrocytes were compared by western blot,as well as differences in glycosaminoglycans(GAGs)deposition by cell safranin O staining.3.Knockdown of SGK1 in chondrocytes and the cells were cultured in OAmicroenvironment in vitro,the expression of autophagy-related proteins(Beclin-1,LC3,P62)were detected by western blot.Additionally,MDC staining were employed to observe the difference in the number of autophagosomes.4.Co-IP was used to detect the direct binding of SGK1 to Forkhead box protein O1(FoxO1)in human primary chondrocytes;Knockdown of SGK1 in chondrocytes and the cells were cultured in OA microenvironment in vitro,the protein level ofphosphorylation-Fox O1(p-Fox O1)and Fox O1 were detected by western blot;moreover,changes in subcellular localization of Fox O1 was examined by western blot analysis and immunofluorescence staining.5.Function rescue experiments were carried out by using chloroquine to inhibitautophagy and AS1842856 to inhibit Fox O1,respectively.Results1.SGK1 m RNA and protein expression levels were significantly elevated in OAcartilage tissues.2.Knockdown of SGK1 in chondrocytes and the cells were cultured in OAmicroenvironment in vitro for 24 hours,the expression of Collagen II and Aggrecan were significantly up-regulated while the expression of ADAMTS-5 and MMP-13 were significantly down-regulated;after 7 days of culture,the deposition of GAGs increased.3.Knockdown of SGK1 in chondrocytes and the cells were cultured in OAmicroenvironment in vitro for 24 hours,the expression of Beclin-1 was significantly up-regulated,the ratio of LC3 II /LC3 I was significantly increased and the expression of P62 was significantly down-regulated,while the number of autophagosomes increased.4.SGK1 and Fox O1 directly bind in human primary chondrocytes;knockdown of SGK1in chondrocytes and the cells were cultured in OA microenvironment in vitro,the ratio of p-Fox O1/Fox O1 was significantly decreased,and nuclear Fox O1 protein level was increased.5.SGK1 regulates the anabolic and catabolic metabolism of human primarychondrocytes in OA microenvironment via autophagy,and regulates the level ofutophagy through Fox O1.Conclusion1.The expression of SGK1 is aberrantly elevated in OA cartilage tissues.2.Knockdown of SGK1 promotes the metabolic balance of chondrocytes in OAmicroenvironment in vitro,and this effect is achieved by inhibiting FoxO1 phosphorylation,increasing nuclear Fox O1 protein level,and activating the level of autophagy.3.Inhibition of aberrantly up-regulated expression of SGK1 in OA cartilage may be anew treatment of OA articular cartilage degeneration,which deserves further study.