摘要
目的:探讨牛磺酸对白介素诱导的椎间盘退变大鼠模型髓核细胞凋亡、炎症和氧化应激的作用及其作用机制。方法:采用Ⅱ型胶原酶分离培养原代大鼠髓核细胞,采用番红染色和甲苯胺蓝染色对第2代细胞进行鉴定;将第3代髓核细胞随机分为5组(1个正常组和4个模型组):Ctrl组、IL-1β组、IL-1β+Taurine(100μg)组、IL-1β+Taurine(200μg)组和IL-1β+Taurine(400μg)组,并用Hochest33342染色检测各组细胞凋亡率,蛋白印迹实验检测各组髓核细胞Caspase-3、-9蛋白表达;试剂盒检测细胞氧化应激因子SOD、MDA、GSH和LDH的含量;ELISA检测细胞炎症因子IL-6、iNOS和IL-10的表达水平;同时蛋白印迹实验检测NF-κB p65磷酸化和TNF-α表达水平,免疫荧光检测p65入核情况。结果:采用Ⅱ型胶原酶成功分离得到较纯的大鼠髓核细胞;牛磺酸能负向调节IL-1β对凋亡相关蛋白Caspase-3、-9(P<0.01)和髓核细胞凋亡率(P<0.01)的诱导作用;能剂量依赖性的抑制IL-1β对氧化应激因子SOD、GSH含量的下调作用(P<0.01)和对MDA、LDH含量的上调作用(P<0.01);另外,随着牛磺酸浓度的增大,IL-1β对IL-6、iNOS表达的促进作用逐渐减弱(P<0.01),同时其对IL-10的抑制作用得到解除(P<0.01);IL-1β对髓核细胞NF-κB p65磷酸化、TNF-α表达和p65入核的促进作用均被牛磺酸扭转(P<0.01),且这种效应随牛磺酸浓度增大而明显化。结论:牛磺酸能抑制白介素诱导的椎间盘退变大鼠模型髓核细胞发生凋亡、炎症和氧化应激的发生。
Objective:To investigate the effects and mechanisms of taurine on apoptosis,inflammation and oxidative stress in intervertebral disc degeneration model of rat nucleus pulposus cells.Methods:Primary rat nucleus pulposus cells were isolated and cultured with type Ⅱ collagenase.The second generation cells were identified by safranin O staining and toluidine blue staining.The third generation cells were randomly divided into five groups(1 control + 4 model groups):Ctrl group,IL-1β group,IL-1β+Taurine(100 μg),IL-1β+Taurine(200 μg) group and IL-1β+Taurine(400 μg) group.The apoptotic rate of cells was detected by Hochest33342 staining,the expression of Caspase-3 and 9 in cells were detected by Western blot,and the content of SOD,MDA,GSH and LDH in cells were detected by kit,the expression levels of inflammatory factors IL-6,iNOS and IL-10 were detected by ELISA,and the expression of NF-κB p65 and TNF-α were detected by Western blot,P65 was detected by immunofluorescence.Results:Rat nucleus pulposus cells were successfully isolated by type Ⅱ collagenase;taurine could negatively regulate the induction of apoptosis-related protein Caspase-3,9(P<0.01) and apoptotic rate of nucleus pulposus cells(P<0.01);dose-dependent inhibit the down-regulation of oxidative stress factors SOD and GSH(P<0.01),and up-regulation the contents of MDA and LDH(P<0.01).In addition,while increasing the concentration,taurine decreased the promotion of IL-1β on the expression of IL-6 and iNOS(P<0.01),and relieved the inhibition of IL-10(P<0.01);IL-1β promoted the phosphorylation of NF-κB p65,the expression of TNF-α and the entry of P65 into nucleus of nucleus pulposus cells(P<0.01).They were all reversed by taurine(P<0.01),and this effect became apparent with the increase of taurine concentration.Conclusion:Taurine can inhibit apoptosis,inflammation and oxidative stress induced by interleukin in intervertebral disc degeneration model of rat nucleus pulposus cells.
作者
史锐
朱振军
杨永波
SHI Rui;ZHU Zhen-Jun;YANG Yong-Bo(Department of Orthopedics,Xinxiang Central Hospital,Xinxiang 453000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第9期1097-1102,共6页
Chinese Journal of Immunology
关键词
牛磺酸
白介素
椎间盘退变
髓核细胞
凋亡
炎症
氧化应激
Taurine
Interleukin
Intervertebral disc degeneration
Nucleus pulposus cells
Apoptosis
Inflammation
Oxidative stress
