The research on the mechanism of SMURF1 regulating OPRM1 expression through ubiquitination to inhibit chondrocyte damage and alleviate osteoarthritis
Objective Investigate the function and underlying mechanism of SMAD ubiquitination regulatory factor 1(SMURF1)in modulating the expression of µ-opioid receptor(OPRM1)via ubiquitina-tion,as well as its impact on chondrocyte damage in humans caused by interleukin-1 β(IL-1β).Methods The cells were divided into the following groups:Control group,IL-1[3-induced group(IL-1 β),empty vec-tor group(IL-1 β+oe/siNC),overexpression group(IL-1[3+oe-OPRM1),low expression group(IL-1 β+siSMURF1),and rescue experiment group(IL-1β+siSMURF1+siOPRM1).Real-time fluorescent quanti-tative PCR and immunoblotting experiments were conducted to detect the mRNA and protein expression lev-els of OPRM1 and SMURF1.MTT assays were used to assess cell viability.Flow cytometry was employed to detect apoptosis.The levels of oxidative stress and inflammation were detected by kits.Immunoprecipitati-on,co-immunoprecipitation,and cycloheximide chase experiments were performed to investigate the inter-action between OPRM1 and SMURF1.Result The cell viability in IL-1 β+siSMURF1 group was higher than that in IL-1 β+siNC group[(69.33±5.13)％vs.(38.33±5.86)％,t=6.89,P＜0.05].The ap-optosis rate,oxidative stress and inflammation levels in IL-1 β+siSMURF1 group were lower than those in IL-1β+siNC group[(10.12±0.62)％vs.(15.28±1.16)％,(2.15±0.25)μm vs.(4.24±0.12)µm,(2.63±0.11)μm vs.(5.01±0.45)µm,(0.39+0.09)U/mg vs.(0.18+0.04)U/mg,(0.05±0.01)U/mg vs.(0.12±0.01)U/mg,(88.68±5.13)pg/ml vs.(135.33±10.21)pg/ml,(128.33±11.68)pg/ml vs.(265.33±24.95)pg/ml,t=6.82,12.85,12.17,8.84,7.07,8.62,P＜0.05],The results of immunoprecipitation and co-immunoprecipitation showed that the level of OPRM1 ubiquitination in siSMURF1 group was higher than that in siNC group(196.32±8.15 vs.217.68±10.05,t=3.10,P＜0.05).The results of cycloheximide experiment showed that the degradation rate of OPRM1 protein in siSMURF1 group was lower than that in siNC group at 6 h and 12 h(0.72±0.07 vs.0.47±0.10,0.64±0.06vs.0.25±0.035,t=3.71,9.85,P＜0.05).Conclusion SMURF1 medi-ates IL-1 β-induced chondrocyte damage to alleviate osteoarthritis by regulating the expression of OPRM1 through ubiquitination.
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