摘要
目的:探究布托啡诺调节HMGB1-RAGE信号通路对IL-1β诱导的人关节软骨细胞凋亡的影响。方法:体外培养人关节软骨细胞,CCK-8法测定0、1、2、4、6、8μmol/L布托啡诺处理的10 ng/ml IL-1β诱导前后人关节软骨细胞存活率,选出布托啡诺最佳作用浓度。体外培养人关节软骨细胞并随机分为对照组、IL-1β组、IL-1β+布托啡诺组、IL-1β+空载组、IL-1β+布托啡诺+HMGB1过表达组,除对照组外,其余各组以10 ng/ml IL-1β诱导建立骨关节炎(OA)体外细胞模型,布托啡诺和质粒分组处理各组细胞后CCK-8、EdU染色检测各组细胞增殖;流式细胞实验检测各组细胞凋亡;2',7'-二氯荧光素二乙酸酯(DCFH-DA)荧光探针检测各组人关节软骨细胞ROS含量;ELISA检测各组细胞炎症相关因子IL-6、TNF-α、IL-18产生水平;试剂盒检测各组细胞抗氧化酶过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性水平;免疫印迹检测各组细胞凋亡蛋白(Bax、Cleaved Caspase-3)与HMGB1-RAGE通路相关蛋白表达。结果:与对照组相比,IL-1β组人关节软骨细胞存活率、增殖率、CAT、SOD、GSH-Px水平显著降低(P<0.05),凋亡率、ROS相对含量、IL-6、TNF-α与IL-18产生水平、Bax、Cleaved Caspase-3、HMGB1、RAGE蛋白表达显著升高(P<0.05)。与IL-1β组相比,IL-1β+布托啡诺组人关节软骨细胞存活率、增殖率、CAT、SOD、GSH-Px水平升高(P<0.05),凋亡率、ROS相对含量、IL-6、TNF-α与IL-18产生水平、Bax、Cleaved Caspase-3、HMGB1、RAGE蛋白表达降低(P<0.05);IL-1β+空载组各指标无明显变化(P>0.05)。与IL-1β+布托啡诺组相比,IL-1β+布托啡诺+HMGB1过表达组人关节软骨细胞存活率、增殖率、CAT、SOD、GSH-Px水平降低(P<0.05),凋亡率、ROS相对含量、IL-6、TNF-α与IL-18产生水平、Bax、Cleaved Caspase-3、HMGB1、RAGE蛋白表达升高(P<0.05)。结论:布托啡诺可通过降低HMGB1-RAGE信号活性减轻IL-1β诱导的人关节软骨细胞炎症,增强其抗氧化活性,从而抑制软骨细胞凋亡。
Objective:To investigate effect of butorphanol on apoptosis of human articular chondrocytes induced by IL-1βby regulating HMGB1-RAGE signal pathway.Methods:Human articular chondrocytes were cultured in vitro,CCK-8 method was applied to determine survival rate of human articular chondrocytes before and after induction of 10 ng/ml IL-1βtreated with 0,1,2,4,6 and 8μmol/L butorphanol,optimal concentration of butorphanol was selected.Human articular chondrocytes were cultured in vitro and randomly separated into control group,IL-1βgroup,IL-1β+butorphanol group,IL-1β+empty group and IL-1β+butorphanol+HMGB1 overexpression group,except control group,other groups were induced with 10 ng/ml IL-1βto establish an cell model of osteoarthritis(OA)in vitro,after grouping and treatment of butorphanol and plasmid,CCK-8 and EdU staining were applied to detect cell proliferation in each group;flow cytometry was applied to detect cell apoptosis in each group;2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe was applied to detect ROS content of human articular chondrocytes in each group;ELISA was applied to detect levels of inflammatory related factors IL-6,TNF-αand IL-18 in each group;kits were applied to detect activity levels of cell antioxidant enzymes catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)in each group;Western blot was applied to detect expressions of apoptotic proteins(Bax,Cleaved Caspase-3)and HMGB1-RAGE pathway related proteins in each group.Results:Compared with control group,survival rate,proliferation rate,CAT,SOD,GSH-Px levels of human articular chondrocytes in IL-1βgroup were obviously reduced(P<0.05),apoptosis rate,relative ROS content,IL-6,TNF-αand IL-18 produc-tion levels,Bax,Cleaved Caspase-3,HMGB1 and RAGE protein expressions were obviously increased(P<0.05).Compared with IL-1βgroup,survival rate,proliferation rate,CAT,SOD,GSH-Px levels of human articular chondrocytes in IL-1β+butorphanol group were obviously increased(P<0.05),apoptosis rate,relative ROS content,IL-6,TNF-αand IL-18 production levels,Bax,Cleaved Caspase-3,HMGB1 and RAGE protein expressions were obviously reduced(P<0.05);there was no obvious change in all indicators in IL-1β+empty group(P>0.05).Compared with IL-1β+butorphanol group,survival rate,proliferation rate,CAT and SOD,GSH-Px levels of human articular chondrocytes in IL-1β+butorphanol+HMGB1 overexpression group were obviously reduced(P<0.05),apoptosis rate,relative ROS content,IL-6,TNF-αand IL-18 production levels,Bax,Cleaved Caspase-3,HMGB1 and RAGE protein expressions were obviously increased(P<0.05).Conclusion:Butorphanol can alleviate IL-1βinduced inflammation of human articular chondrocytes,enhance its antioxidant activity,thereby inhibit chondrocyte apoptosis by reducing HMGB1-RAGE signaling activity.
作者
刘凯
张田田
韩洋
LIU Kai;ZHANG Tiantian;HAN Yang(Department of Anesthesiology,Tengzhou Central People's Hospital,Zaozhuang 277500,China)
出处
《中国免疫学杂志》
北大核心
2025年第8期1933-1939,共7页
Chinese Journal of Immunology
