跨膜蛋白208可影响人软骨细胞的自噬和线粒体功能

Transmembrane protein 208 affects autophagy and mitochondrial function in chondrocytes

背景:软骨细胞自噬水平下降是骨关节炎发病的重要机制之一。自噬抑制基因跨膜蛋白208在晚期膝骨关节炎患者软骨组织中表达升高,该基因可能通过抑制软骨细胞自噬促进骨关节炎发展。目的:通过体外骨关节炎模型,观察软骨细胞跨膜蛋白208基因表达对自噬和线粒体功能的影响。方法:软骨组织来源于膝关节表面置换患者术中废弃软骨,非负重面轻度病变软骨(OuterbridgeⅠ-Ⅱ级)作为早期组,负重面重度病变软骨(OuterbridgeⅢ-Ⅳ级)作为晚期组,检测不同软骨组织跨膜蛋白208含量。实验方案符合北京大学第一医院对研究的相关伦理要求,软骨组织供者及其家属在充分了解治疗方案的前提下签署了“知情同意书”。取第4代人正常关节软骨细胞,通过白细胞介素1β刺激人正常软骨细胞诱导骨关节炎体外模型;另外加入自噬激活剂雷帕霉素或自噬抑制剂3-MA预处理软骨细胞后,再加入白细胞介素1β刺激12,24,48h,观察软骨细胞状态。Real Time PCR检测不同病变软骨组织跨膜蛋白208基因的表达;Western Blot检测自噬相关蛋白轻链蛋白3B、P62表达评价自噬水平;AnnexinV-FITC法评估软骨细胞的凋亡情况;MitoSOX Red dye和JC-1染料荧光显色评价线粒体损伤情况。结果与结论:①骨关节炎患者重度退变软骨组织跨膜蛋白208表达升高;②用白细胞介素1β刺激软骨细胞12,24和48h后,软骨细胞跨膜蛋白208表达水平先降后升,自噬水平先升后降,凋亡水平逐渐升高,线粒体损伤逐渐加重;③雷帕霉素能明显降低跨膜蛋白208的表达量,提高自噬水平,降低白细胞介素1β刺激后软骨细胞的凋亡和线粒体损伤;④结论:跨膜蛋白208可抑制软骨细胞自噬,其表达增高可能导致软骨细胞应激状态下的损伤增加。

BACKGROUND:Decreased level of autophagy in chondrocytes is an important mechanism of osteoarthritis.The autophagy inhibitory gene transmembrane protein 208 is elevated in late-stage osteoarthritis cartilage,and this gene may promote the development of osteoarthritis by inhibiting autophagy of chondrocytes.OBJECTIVE:To investigate the effects of chondrocyte transmembrane protein 208 gene on autophagy and mitochondrial function in osteoarthritis model in vitro.METHODS:The cartilage tissue was from the discarded cartilage of patients undergoing knee arthroplasty.The mild lesion cartilage on non-weight-bearing face(Outerbridge Ⅰ-Ⅱ grade)was as early-term group,and the severe lesion cartilage on heavy-weight surface(Outerbridge Ⅲ-Ⅳ grade)was as the late-term group.The content of transmembrane protein 208 in different cartilage tissues was detected.The study was in accordance with the ethical criteria of Peking University First Hospital,and the cartilage donors and their family members signed an informed consent form.Passage 4 normal human chondrocytes were obtained and then stimulated by interleukin-1β to establish the in vitro osteoarthritis.The chondrocytes were pretreated by autophagy activator rapamycin or autophagy inhibitor 3MA,followed by interleukin-1β added for 12,24 and 48 hours to observe the morphology of chondrocytes.The content of transmembrane protein 208 gene was detected by real-time PCR.Western blot assay was used to detect autophagy-associated protein light chain proteins 3B and P62 to evaluate autophagy levels.The apoptosis of chondrocytes was detected by Annexin V-FITC method.Mitochondrial damage was assessed by fluorescent detection with MitoSOX Red dye and JC-1 dye.RESULTS AND CONCLUSION:(1)The expression of transmembrane protein 208 was significantly increased in severe osteoarthritis cartilage.(2)After stimulation with interleukin-1β for 12,24 and 48 hours,the expression level of transmembrane protein 208 in chondrocytes decreased initially,and then increased.The level of autophagy rose initially and then fell.The apoptotic level gradually increased,and mitochondrial damage gradually became severe.(3)Rapamycin could significantly reduce the expression of transmembrane protein 208,increase the level of autophagy,and reduce the apoptosis and mitochondrial damage in chondrocytes after interleukin-1β stimulation.(4)To conclude,transmembrane protein 208 inhibits autophagy in chondrocytes,and increased expression of this gene may lead to increased damage of chondrocytes under stress.