| BackgroundOsteoarthritis(OA)is a very common degenerative disease characterized by progressive chondrocyte apoptosis and cartilage degeneration.The activation of Wnt/β-catenin signaling pathway can cause chondrocyte apoptosis and cartilage degradation,which plays a key role in the occurrence and development of osteoarthritis and is considered as one of the effective targets for osteoarthritis.However,the current conservative treatment of osteoarthritis has several problems,such as inexact therapeutic effect and lots of serious side effects.Chinese herbal medicine is of great interest to researchers because of its good therapeutic effect,little toxicity and side effects,sustainable use but low cost.Many studies have shown that some active ingredients extracted from traditional Chinese medicine play an anti-osteoarthritis role by down-regulating inflammatory mediators,inhibiting inflammatory signal pathways,promoting chondrocyte proliferation,preventing chondrocyte apoptosis,maintaining the homeostasis of chondroextracellular matrix and anti-oxidation mechanisms.The aim of this study is to explore the exactly mechanism of the active ingredients of traditional Chinese medicine that can effectively inhibit chondrocyte apoptosis and cartilage degeneration,and to lay the experimental foundation for the development of new drugs for osteoarthritis.PART Ⅰ USP49 inhibits IL-1β-induced degeneration of cartilageObjective To investigate the effect of USP49 on chondrocyte apoptosis and expression of proteins related to cartilage metabolism induced by IL-1β.Method The expression of USP49 in OA patients was determined based on the GSE57218 database.Primary Rat articular chondrocytes were cultured and identified by immunofluorescence of Collagen II and SOX9.After pretreated with 0,5,10,and20 ng/m L of IL-1β respectively,Chondrocytes proliferation was measured with CCK-8 Kit(Cell Counting kit-8),USP49 gene expression was detected by RT-PCR,and USP49 protein level was determined by Western blot.Appropriate IL-1βconcentration was selected to simulate osteoarthritis.Primary chondrocytes were transfected with overexpressed USP49 plasmid to constructe overexpressed USP49 chondrocytes.The transfection was verified by RT-PCR and Western blot.After incubation with 10 ng/m L IL-1β,the apoptosis of USP49 overexpressed chondrocyte was detected by flow cytometry,and the expression of β-catenin,anti-apoptotic protein Survivin,proteins related to cartilage degradation(MMP-1,MMP-13)were detected by Western blot.Result Analysis based on the GSE57218 database showed that the expression of USP49 was significantly decreased in patients with osteoarthritis compared with normal samples.CCK-8、RT-PCR and Western blot showed that IL-1β significantly inhibited chondrocytes proliferation and USP49 expression in a concentration-dependent manner.10 ng/ml IL-1β has had a significant effect and was selected for further experiments.Pretransfected chondrocytes with the USP49 overexpression vector,the expression of USP49 was significantly up-regulated.Flow cytometry and Western blot showed that the overexpression of USP49 significantly attenuated the apoptosis of chondrocytes induced by IL-1β,up-regulated the expression of USP49 and anti-apoptotic protein Survivin,inhibited the signaling pathway of Wnt/β-catenin and the expression of proteins related to cartilage degradation(MMP-1,MMP-13).Summary USP49 inhibits chondrocyte apoptosis and alleviates cartilage degradation induced by IL-1β through the Wnt/β-catenin signaling pathway.PART Ⅱ 6-gingerol upregulates USP49 and inhibits IL-1β-induced cartilage degenerationObjective To explore the effect and mechanism of 6-gingerol on chondrocyte apoptosis and cartilage degradation in osteoarthritis.Method The active ingredient of traditional Chinese medicine with the most obvious upregulated expression of USP49 was screened from Baicalin,Paeoniflorin,Curcumin,6-gingerol,Echinacoside and Astragaloside IV.Subsequently,chondrocytes induced by IL-1β were treated with different concentrations of6-gingerol(0,5,10,and 20 μmol/L).Chondrocytes apoptosis was detected by flow cytometry,and expression of USP49,β-catenin,Survivin,chondrocytes metabolism-related proteins(MMP-1,MMP-13)were determined by Western blot.After transfection with si RNA,the apoptosis of USP49 knockdown chondrocytes was detected by flow cytometry.Western blot was used to detect expression of β-catenin,Survivin and chondrometabolic-related proteins(MMP-1,MMP-13)in USP49 knockdown chondrocytes.After respective treatment with 10 μmol/L XAV939(Wnt/β-catenin inhibitor)and 6-gingerol in USP49 knockdown chondrocytes,apoptosis was detected by flow cytometry,and expression of β-catenin,Survivin,MMP-1 and MMP-13 was detected by Western blot.Result It was found that 6-gingerol could significantly counteract the effect of IL-1β and significantly promote the expression of USP49.After treatment of IL-1β-induced chondrocytes with 6-gingerol at different concentrations,flow cytometry and Western blot showed that 6-gingerol significantly inhibited IL-1β-induced apoptosis and Wnt/β-catenin signaling pathway,down-regulated expression of proteins related to cartilage metabolism(MMP-1,MMP-13).After successful construction of USP49 knockdown chondrocytes,It showed that the knockdown of USP49 significantly increase the apoptosis of chondrocytes,downregulate the expression of Survivin,promote the production of β-catenin,MMP-1 and MMP-13.Further studies showed that 6-gingerol and XAV939 could strongly counteract the effect of USP49 knockdown,significantly inhibit chondrocytes apoptosis,downregulate the expression of β-catenin,MMP-1,MMP-13.Summary 6-gingerol regulates the activation of Wnt/β-catenin signaling pathway by USP49,inhibits chondrocyte apoptosis and the expression of proteins related to cartilage degradation.PART Ⅲ USP49 inhibits the Wnt/β-catenin signaling pathway by deubiquitination of AxinObjective To investigate the mechanism of USP49 inhibiting Wnt/β-catenin signaling pathway.Method Gene transcription and protein expression of Axin in USP49 knockdown and overexpression chondrocytes were detected by RT-PCR and Western blot respectively.The interaction between USP49 and Axin was studied by immunoprecipitation.After IL-1β intervention,the difference of Axin ubiquitination between USP49 overexpression chondrocytes and normal chondrocytes was detected by Western blot.The impact of proteasome inhibitor MG132 on IL-1β-treated USP49 knockdown chondrocytes was determined by Western blot.Results Overexpression or knockdown USP49 had significant effect on the level of Axin protein,but did not interfere Axin m RNA expression.Immunoprecipitation revealed a clear interaction between USP49 and Axin,and Western blot showed the overexpression of USP49 significantly inhibited the ubiquitination of Axin.Further experiments showed that MG132 could effectively counteract the effect of USP49 downregulation and significantly up-regulate the level of Axin protein.Summary USP49 inhibited Wnt/β-catenin signal pathway by deubiquitination of Axin.Conclusion6-gingerol inhibits Wnt/β-catenin signaling pathway to down-regulating IL-1β-induced chondrocyte apoptosis and extracellular matrix degradation by deubiquitination Axin with USP49.Therefore,6-gingerol has a therapeutic effect on cartilage degeneration. |
PDF Full Text Request