南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (8): 1142-1149.doi: 10.12122/j.issn.1673-4254.2021.08.03

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大鼠膝骨关节炎模型中高迁移率族蛋白1的高表达机制

梅 伟，洪博文，黄桂成   

  1. 南京中医药大学附属医院骨伤科，江苏 南京 210000
  • 出版日期:2021-08-20 发布日期:2021-09-07

Mechanism of high expression of high mobility group protein 1 in a rat model of knee osteoarthritis

MEI Wei, HONG Bowen, HUANG Guicheng   

  1. Department of Orthopedics and Traumatology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210000, China
  • Online:2021-08-20 Published:2021-09-07

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摘要： 目的 研究膝骨关节炎病理进程中滑膜成纤维细胞（FLSs）焦亡与其释放高迁移率族蛋白1（HMGB1）的分子机制。方法在动物实验中，将SD大鼠利用随机数字表法分为2 组，6只/组，空白对照组（无任何处理），膝骨关节炎组（膝骨关节炎模型），使用前交叉韧带切断法（ACLT）建立大鼠膝骨关节炎模型，通过HE染色及Mankin'评分观察大鼠膝关节软骨的破坏情况，通过蛋白免疫印迹法检测膝关节滑膜组织中细胞焦亡相关的蛋白和HMGB1蛋白的表达情况。在细胞实验中，将细胞随机分为3组，空白对照组（等量的PBS处理），细胞焦亡组（LPS+ATP处理），小干扰RNA转染组（LPS+ATP+siRNA处理）。使用脂多糖+三磷酸腺苷（LPS+ATP）诱导滑膜成纤维细胞发生焦亡，分别使用不同的小干扰RNA抑制滑膜成纤维细胞发生焦亡，使用RT-qPCR验证不同的小干扰RNA抑制NLRP1、NLRP3 、ASC和caspase-1的转染效果，通过Western blot检测滑膜成纤维细胞中细胞焦亡相关的蛋白和HMGB1蛋白的表达情况；使用ELISA检测细胞上清中HMGB1的蛋白水平。结果 动物实验中，与空白对照组相比，膝骨关节炎组滑膜组织中焦亡相关的蛋白及HMGB1蛋白表达明显升高（P<0.05），软骨组织Mankin'评分明显升高（P<0.05）；细胞实验中，小干扰RNA能够明显抑制NLRP1、NLRP3、ASC和caspase-1的mRNA表达（P<0.05），细胞焦亡组中细胞焦亡相关的蛋白及HMGB1蛋白表达明显高于空白对照组和小干扰RNA转染组（P<0.05），各细胞焦亡组细胞上清中HMGB1的蛋白水平明显高于空白对照组和小干扰RNA转染组（P<0.05）。结论 在膝骨关节炎病理进程中，NLRPs炎性小体介导的滑膜成纤维细胞焦亡可促进HMGB1的大量释放，这与细胞焦亡下游因子caspase-1蛋白的活化有关。

关键词: 膝骨关节炎；滑膜成纤维细胞；焦亡；高迁移率族蛋白1

Abstract: Objective To investigate the molecular mechanism triggering pyroptosis of synovial fibroblast-like synoviocytes (FLSs) and the release of high mobility group protein 1 (HMGB1) in a rat model of knee osteoarthritis (KOA). Methods Twelve SD rats were randomized equally into blank control group without any treatment and KOA group with anterior cruciate ligament amputation (ACLT) to induce KOA. HE staining and Mankin score were used to evaluate the damage of knee cartilage. Western blotting was used to detect the expression of pyroptosis-related proteins and HMGB1 in the synovial tissue. In the cell experiment, rat FLSs were treated with PBS (control group), LPS +ATP (to induce cell pyroptosis), or LPS+ATP+siRNAs (to inhibit pyroptosis of the FLSs), and the cellular expressions of apoptosis-related proteins and HMGB1 were detected using Western blotting; the level of HMGB1 in the culture supernatant was detected with ELISA. Results In the rat models of KOA, the expressions of pyroptosis- related proteins and HMGB1 in the synovial tissue and Mankin score were significantly increased as compared with those in the control group (P<0.05). In cultured rat FLSs, the expressions of apoptosis related proteins and HMGB1 were significantly higher in the pyroptosis group than in the control group and in cells transfected with the siRNAs targeting NLRP1, NLRP3, ASC and caspase-1 (P<0.05). The protein level of HMGB1 in the culture supernatant was significantly higher in pyroptosis group than in the control and siRNA groups (P<0.05). Conclusion In the pathological process of KOA, NLRPs inflammasome- mediated FLS pyroptosis causes massive release of HMGB1, which is associated with the activation of the downstream molecule caspase-1.

Key words: knee osteoarthritis; fibroblast-like synoviocytes; pyroptosis; high mobility group protein 1