内质网应激对膝骨关节炎大鼠关节软骨细胞的影响

Effect of endoplasmic reticulum stress on chondrocytes in a rat model of knee osteoarthritis

背景:研究认为,内质网应激与糖尿病、扩张性心肌病、神经退行性疾病等许多慢性疾病的发生相关,而它与骨关节炎的发生也密切相关。目的:分析内质网应激对大鼠关节软骨细胞生存状态的影响,以及内质网应激在体内对大鼠骨关节炎发生发展的影响。方法:在体外分离培养大鼠关节软骨细胞,应用10 mg/L衣霉素建立内质网应激模型。Western Blot检测内质网应激相关蛋白葡萄糖调节蛋白78和C/EBP同源蛋白的表达,并分别应用CCK-8和Annexin V-FITC(流式法)检测软骨细胞增殖活性及细胞凋亡情况。体内实验选取SD大鼠15只,行前交叉韧带切断、内侧半月板切除术制作膝关节骨关节炎模型,于关节腔分别注射内质网应激激动剂衣霉素、抑制剂牛磺熊去氧胆酸和空白对照PBS,4周后应用苏木精-伊红染色评估各组关节退变情况。结果与结论:(1)应用衣霉素刺激大鼠关节软骨细胞后,葡萄糖调节蛋白78和C/EBP同源蛋白的表达均显著升高;同时,软骨细胞的细胞增殖活性在衣霉素刺激后逐渐下降,而细胞凋亡率则明显升高;(2)在大鼠骨关节炎模型中,大体标本及苏木精-伊红染色结果均显示,关节腔注射衣霉素组骨关节炎进展加速,而关节腔注射牛磺熊去氧胆酸组骨关节炎进展减慢;(3)结论:内质网应激的过度激活导致体外培养的大鼠关节软骨细胞增殖活性降低及细胞凋亡增加,这可能是导致骨关节炎发生发展的重要机制之一;应用内质网应激抑制剂牛磺熊去氧胆酸可以有效地减少内质网应激导致的骨关节炎的进展。

BACKGROUND： Endoplasmic reticulum （ER） stress has been proved to be related to the occurrence of diabetes, dilated cardiomyopathy and neurodegenerative diseases. Indeed, it is closely associated with osteoarthritis. OBJECTIVE： To explore the effect of ER stress on the chondrocyte viability as well as the occurrence and development of osteoarthritis in rats. METHODS： Rat chondrocytes were isolated and cultured, and the ER stress in the rat chondrocytes was by 10 mg/L tunicamycin. The expression levels of ER stress markers C/EBP-homologous protein and 78 kDa glucose-regulated protein were detected by western blot assay, and the proliferation and apoptosis of chondrocytes were detected by cell counting kit-8 assay and AnnexinV-FITC flow cytometry, respectively. In the in vivo experiment, 15 Sprague-Dawley rats were selected and subjected to anterior cruciate ligament transection and medial meniscectomy to establish an animal model of osteoarthritis. Tunicamycin, tauroursodeoxycholic acid and PBS （blank control group） were respectively injected into the articular cavity, and then the progression of osteoarthritis was assessed by hematoxylin-eosin staining at 4 weeks after treatment. RESULTS AND CONCLUSION： After addition of tunicamycin, the expression levels of C/EBP-homologous protein and 78 kDa glucose-regulated protein were significantly upregulated, the viability of chondrocytes was decreased gradually, while the apoptotic rate was increased significantly. Results from gross observation and hematoxylin-eosin staining suggested that tunicamycin promoted the progression of osteoarthritis and tauroursodeoxycholic acid delayed the deterioration of cartilage in the rats. These findings indicate that ER stress results in the decreased chondrocyte viability and increased apoptosis, which may be an important pathogenesis of osteoarthritis. Additionally, tauroursodeoxycholic acid can effectively alleviate osteoarthritis induced by ER stress.