摘要
目的探讨核受体亚家族1D组成员1(NR1D1)激动剂SR9009对白细胞介素-1β(IL-1β)诱导的小鼠软骨细胞退变的影响及其机制。方法2023年1月至2024年12月,酶消化法获取小鼠软骨细胞后,通过IL-1β(10 ng/ml)诱导构建体外软骨细胞退变模型,实验分组为:对照组(Control)、IL-1β组(IL-1β)、SR9009低剂量组(IL-1β+SR90095μmol/L)、SR9009高剂量组(IL-1β+SR900910μmol/L)。细胞计数试剂盒(CCK-8)检测细胞活力,流式细胞术检测细胞凋亡,酶联免疫吸附试验(ELISA)试剂盒检测炎症指标,活性氧(ROS)试剂盒检测氧化应激,实时荧光定量聚合酶链反应(RT-PCR)检测环氧合酶2(COX2),基质金属蛋白酶-3(MMP-3)、MMP-13和2型胶原(CollagenⅡ)mRNA表达,蛋白质印迹法(Western blot)检测NR1D1、COX2、MMP-3、MMP-13、CollagenⅡ、核因子-κB抑制因子α(IκBα),p-IκBα、核因子-κB-β(IKKβ)、p-IKKβ、核因子-κB p65(NF-κB p65)和p-p65的蛋白表达。两组间比较采用LSD-t检验,多组间比较采用方差分析(ANOVA)。结果SR9009低剂量组、高剂量组细胞活性高于IL-1β组(0.58±0.03、0.62±0.03比0.41±0.01,t=13.13、10.58,P<0.01),差异有统计学意义。SR9009低剂量组、高剂量组细胞早期凋亡率、晚期凋亡率低于IL-1β组[(13.77±1.44)%、(7.34±1.23)%比(19.05±2.41)%,(10.19±0.84)%、(4.47±0.93)%比(16.59±1.48)%,t=3.25、7.49、6.50、12.01,P<0.05],差异有统计学意义。SR9009高剂量组TNF-α、IL-6水平明显低于IL-1β组[(41.29±6.74)pg/ml比(123.0±11.14)pg/ml、(51.95±9.04)pg/ml比(152.59±7.45)pg/ml,t=14.88、10.88,P<0.01],差异有统计学意义。ROS检测结果显示,SR9009低剂量组、SR9009高剂量组ROS水平低于IL-1β组(2.89±0.23比4.71±0.70、2.02±0.29比4.71±0.70,t=4.23、6.19,P<0.05),差异有统计学意义。RT-PCR结果显示,SR9009高剂量组NR1D1、CollagenⅡ表达量高于IL-1β组(1.06±0.05比0.46±0.07、2.23±0.12比0.76±0.08,t=7.08、10.40,P<0.05),COX-2、MMP-3、MMP-13表达量低于IL-1β组(1.85±0.13比4.23±0.21、1.80±0.08比4.38±0.26、1.62±0.12比4.62±0.20,t=9.82、9.29、12.10,P<0.05),差异有统计学意义。Western blot结果显示,SR9009高剂量组NR1D1、CollagenⅡ表达量高于IL-1β组(2.15±0.15比0.86±0.12、2.03±0.16比1.02±0.11,t=6.88、6.75,P<0.05),COX-2、MMP-3、MMP-13表达量低于IL-1β组(1.01±0.07比3.43±0.42、1.32±0.12比3.57±0.34、1.21±0.08比4.14±0.34,t=8.89、8.95、10.97,P<0.05),差异有统计学意义。SR9009干预组在IL-1β刺激5 min时间点p-IκBα/IκBα、p-IKKβ/IKKβ和p-p65/p65表达量低于IL-1β组(0.65±0.04、0.56±0.05、0.73±0.06比1.32±0.11、1.44±0.07、1.97±0.11,t=6.624、6.723、6.726,P<0.05),差异有统计学意义。结论NR1D1激动剂SR9009抑制NF-κB通路降低炎症因子和基质金属蛋白酶的表达,减轻IL-1β诱导的软骨细胞退变,对关节软骨发挥保护作用。
Objective To investigate the effect of nuclear receptor subfamily 1 group D member 1(NR1D1)agonist SR9009 on interleukin-1β(IL-1β)-induced chondrocyte degeneration in mice and its underlying mechanism.Methods This research was conducted from January 2023 to December 2024.Mouse chondrocytes were isolated via enzymatic digestion,and an in vitro chondrocyte degeneration model was established using IL-1β(10 ng/ml)induction.The experimental groups included:control group,IL-1βgroup(IL-1β),SR9009 low-dose group(IL-1β+SR90095μmol/L),and SR9009 high-dose group(IL-1β+SR900910μmol/L).Cell viability was assessed by cell counting kit-8(CCK-8)assay,apoptosis was evaluated by flow cytometry,inflammatory markers were measured by enzyme linked immunosorbent assay(ELISA),oxidative stress was analyzed by reactive oxygen species(ROS)detection,and cyclooxygenase-2(COX-2),matrix metalloproteinase(MMP)-3,MMP-13,and collagenⅡmRNA expression levels were determined by RT-qPCR,while protein expression levels of NR1D1,COX-2,MMP-3,MMP-13,collagenⅡ,nuclear factor-κB inhibitorα(IκBα),phosphorylated IκBα(p-IκBα),IKKβ,phosphorylated IKKβ(p-IKKβ),nuclear factor-κB p65(NF-κB p65),and phosphorylated p65(p-p65)were analyzed by Western blotting.Statistical comparisons between two groups were performed using Student’s t-test,and comparisons among multiple groups were conducted using one-way analysis of variance(ANOVA).Results Compared with the IL-1βgroup(0.41±0.01),cell viability was significantly higher in both the SR9009 low-dose group[(0.58±0.03)]and the high-dose group[(0.62±0.03),t=13.13,10.58,P<0.01].Early and late apoptosis rates were lower in the SR9009 low-dose[(13.77±1.44)%,(10.19±0.84)%]and high-dose groups[(7.34±1.23)%,(4.47±0.93)%]than the IL-1βgroup[(19.05±2.41)%,(16.59±1.48)%,t=3.25,7.49,6.50,12.01,P<0.05].Levels of TNF-αand IL-6 were significantly reduced in the high-dose group[(41.29±6.74),(51.95±9.04)pg/ml]compared to the IL-1βgroup[(123.0±11.14),(152.59±7.45)pg/ml,t=7.15,10.24,14.88,10.88,P<0.01].ROS levels were also significantly lower in the SR9009 low-dose(2.89±0.23)and high-dose groups(2.02±0.29)than in the IL-1βgroup(4.71±0.70,t=4.23,6.19,P<0.05).RT-qPCR results demonstrated that NR1D1 and collagenⅡmRNA expression levels were significantly higher in the SR9009 intervention groups than in the IL-1βgroup(1.06±0.05 vs.0.46±0.07,2.23±0.12 vs.0.76±0.08,t=7.08,10.40,P<0.05),whereas COX-2,MMP-3,and MMP-13 mRNA expression levels were significantly lower(1.85±0.13 vs.4.23±0.21,1.80±0.08 vs.4.38±0.26,1.62±0.12 vs.4.62±0.20,t=9.82,9.29,12.10,P<0.05).Western blotting analysis confirmed that NR1D1 and collagenⅡprotein expression levels were significantly higher in the SR9009 high-dose groups than in the IL-1βgroup(2.15±0.15 vs.0.86±0.12,2.03±0.16 vs.1.02±0.11,t=6.88,6.75,P<0.05),while COX-2,MMP-3,and MMP-13 protein expression levels were significantly lower(1.01±0.07 vs.3.43±0.42,1.32±0.12 vs.3.57±0.34,1.21±0.08 vs.4.14±0.34,t=8.89,8.95,10.97,P<0.05).Furthermore,the expression levels of p-IκBα/IκBα,p-IKKβ/IKKβ,and p-p65/p65 at the 5-minute time point were significantly lower in the SR9009 intervention group than in the IL-1βgroup(0.65±0.04 vs.1.32±0.11,0.56±0.05 vs.1.44±0.07,0.73±0.06 vs.1.97±0.11,t=6.62,6.72,6.73,P<0.05).Conclusion The NR1D1 agonist SR9009 can suppress the expression of inflammatory factors and MMPs by inhibiting the NF-κB pathway,thereby alleviating IL-1β-induced chondrocyte degeneration and protecting articular cartilage.
作者
柳辉
沙米·艾合买提
迪丽胡玛尔·阿布力米提
阚云
陶圣祥
Liu Hui;Shami·AihemaitiDilihumaer·Abulimiti;Kan Yun;Tao Shengxiang(Department of Orthopaedic Trauma and Microsurgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)
出处
《中华实验外科杂志》
2025年第7期1217-1220,共4页
Chinese Journal of Experimental Surgery
基金
中央高校基本科研业务费专项基金(2042021kf0164)。
