关节挛缩是临床常见病之一,关节囊纤维化被认为是关节挛缩最重要的病理改变之一。然而,关节囊纤维化的潜在机制仍存在争议。本研究旨在建立大鼠膝关节伸展性关节挛缩的动物模型,并利用该动物模型研究缺氧介导的焦亡在关节挛缩进展中的作用。选择36只雄性SD大鼠,其中6个未固定,作为对照组,将30只大鼠分为I-1组(自由运动7周后固定1周),I-2组(自由运动6周后固定2周),I-4组(4周自由运动后固定4周),I-6组(固定6周,2周后自由运动)和I-8组(固定8周)根据不同的固定时间。通过测量膝关节活动范围来评估关节挛缩的进展,用Masson染色检查关节囊中的胶原沉积,HIF-1α的蛋白表达水平,NLRP3,Caspase-1,GSDMD-N,使用蛋白质印迹法评估关节囊中的TGF-β1,α-SMA和p-Smad3,透射电镜观察成纤维细胞的形态变化。总挛缩和关节源性挛缩的程度从第一周开始进展,并持续到固定后的前八周。固定后的前四周,总挛缩和关节源性挛缩的程度迅速发展,然后缓慢发展。Masson染色表明,固定后的前8周内,关节囊中的胶原蛋白沉积逐渐增加。Westernblotting分析显示HIF-1α的蛋白水平在固定的前8周持续增加,固定后的前4周,焦亡相关蛋白NLRP3,Caspase-1,GSDMD-N的蛋白水平持续升高,然后下降。纤维化相关蛋白TGF-β1,p-Smad3和α-SMA的蛋白质水平在固定后的前8周内持续增加。透射电镜显示4周的固定化诱导细胞膜破裂和细胞内容物溢出,这进一步表明焦亡的激活。外固定矫形器可建立大鼠膝关节扩张关节挛缩动物模型,缺氧介导的焦亡的激活可能在关节囊纤维化和关节挛缩的过程中起刺激作用。
Joint
contracture is one of the common diseases clinically, and joint capsule fibrosis is considered to be one of the most important pathological changes of joint
contracture. However, the underlying mechanism of joint capsule fibrosis is still controversial. The present study aims to establish an animal model of knee extending joint
contracture in rats, and to investigate the role of hypoxia-mediated pyroptosis in the progression of joint
contracture using this animal model. 36 male SD rats were selected, 6 of which were not immobilized and were used as control group, while 30 rats were divided into I-1 group (immobilized for 1 week following 7 weeks of free movement), I-2 group (immobilized for 2 weeks following 6 weeks of free movement), I-4 group (immobilized for 4 weeks following 4 weeks of free movement), I-6 group (immobilized for 6 weeks following 2 weeks of free movement) and I-8 group (immobilized for 8 weeks) according to different immobilizing time. The progression of joint contracture was assessed by the measurement of knee joint range of motion, collagen deposition in joint capsule was examined with Masson staining, protein expression levels of HIF-1α, NLRP3, Caspase-1, GSDMD-N, TGF-β1, α-SMA and p-Smad3 in joint capsule were assessed using western blotting, and the morphological changes of fibroblasts were observed by transmission electron microscopy. The degree of total and arthrogenic contracture progressed from the first week and lasted until the first eight weeks after immobilization. The degree of total and arthrogenic contracture progressed rapidly in the first four weeks after immobilization and then progressed slowly. Masson staining indicated that collagen deposition in joint capsule gradually increased in the first 8 weeks following immobilization. Western blotting analysis showed that the protein levels of HIF-1α continued to increase during the first 8 weeks of immobilization, and the protein levels of pyroptosis-related proteins NLRP3, Caspase-1, GSDMD-N continued to increase in the first 4 weeks after immobilization and then decreased. The protein levels of fibrosis-related proteins TGF-β1, p-Smad3 and α-SMA continued to increase in the first 8 weeks after immobilization. Transmission electron microscopy showed that 4 weeks of immobilization induced cell membrane rupture and cell contents overflow, which further indicated the activation of pyroptosis. Knee extending joint
contracture animal model can be established by external immobilization orthosis in rats, and the activation of hypoxia-mediated pyroptosis may play a stimulating role in the process of joint capsule fibrosis and joint contracture.