摘要
目的 探讨SMAD泛素化调节因子1(SMURF1)泛素化调控μ型阿片受体(OPRM1)表达对白细胞介素-1β(IL-1β)诱导的人软骨细胞损伤的作用及机制.方法 IL-1β诱导构建体外细胞模型,分组为:对照组(Control)、IL-1β诱导组(IL-1β)、空载体组(IL-1 β+oe/siNC)、过表达组(IL-1 β+oe-OPRM1)、低表达组(IL-1β+siSMURF1)、回复实验组(IL-1 β+siSMURF1+siOPRM1).实时荧光定量聚合酶链反应和免疫印迹实验检测OPRM1与SMURF1的mRNA和蛋白表达;细胞计数试剂盒(CCK-8)检测细胞活力;流式细胞术检测凋亡;试剂盒检测水平;ELISA试剂盒检测氧化应激和炎症水平.免疫沉淀、免疫共沉淀和放线菌酮实验检测相互作用关系.结果 IL-1β+siSMURF1 组细胞活力高于IL-1 β+siNC 组[(69.33±5.13)％比(38.33±5.86)％,t=6.89,P＜0.05].IL-1 β+siSMURF1组凋亡率、氧化应激和炎症水平低于IL-1β+siNC组[(10.12±0.62)％比(15.28±1.16)％、(2.15±0.25)µm 比(4.24±0.12)μm、(2.63±0.11)μm 比(5.01±0.45)μm、(0.05±0.01)U/mg 比 0.12±0.01)U/mg、(88.68±5.13)pg/ml 比(135.33±10.21)pg/ml、(128.33±11.68)pg/ml 比(265.33±24.95)pg/ml,t=6.82、12.85、12.17、8.84、7.07、8.62,P＜0.05],免疫沉淀、免疫共沉淀结果显示,siSMURF1组OPRM1泛素化水平高于siNC组(196.32±8.15 比 217.68±10.05,t=3.10,P＜0.05).结论 SMURF1 通过泛素化调控 OPRM1的表达介导IL-1 β诱导的软骨细胞损伤以减轻骨关节炎.
Abstract
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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