从LncRNA调控软骨细胞及其基质代谢角度探讨独活寄生汤治疗膝骨关节炎的作用机制

Cartilage degeneration is the most important pathological changes in osteoarthritis (OA), therefore, effectively protecting articular cartilage is the crux in preventing and treating osteoarthritis. Recently research has found that Long non-coding RNA (LncRNA) plays an important role in the development of OA, which will cause cartilage degeneration when LncRNA is abnormally activated. Therefore, blocking LncRNA GAS5 and LncRNA CIR’s abnormally activated is a new way in the treatment of OA. Previously, we found that Duhuo Jisheng Decoction can effectively delay cartilage degeneration by promoting chondrocyte proliferation and inhibiting chondrocyte apoptosis, indicating that effectively regulating LncRNA GAS 5 and LncRNA CIR may be a potential target of the protection of Duhuo Jisheng Decoction on articular cartilage. Therefore, in order to further approach the mechanism in the cure on OA by Duhuo Jisheng Decoction from epigenetic aspect, and reveal the scientific connotation of the clinical efficacy of Duhuo Jisheng Decoction, we observe the effect of Duhuo Jisheng Decoction on LncRNA GAS5, LncRNA CIR, and chondrocyte function and extracellular matrix metabolism regulated by LncRNA GAS5 and LncRNA CIR by using small animal nuclear magnetic resonance, Real-time PCR, Western blot, RNAi silence, and so on, based on research platforms with knee OA animal model on SD rats and degeneration of cartilage cells, in vivo and vitro.

软骨退变是骨关节炎(OA)最主要病理变化，围绕保护关节软骨以防治OA成为治疗的关键。研究发现长链非编码RNA(LncRNA)在OA发生发展过程中起重要作用，LncRNA GAS5和CIR的异常激活将引起软骨退变，阻断其异常激活是OA药物治疗新思路。课题组前期研究发现独活寄生汤能有效促进软骨细胞增殖、抑制其凋亡，延缓软骨退变，提示有效调节LncRNA GAS5和CIR可能是独活寄生汤保护关节软骨的作用机制之一。本项目通过体内外实验，以SD大鼠膝OA动物模型和退变软骨细胞模型为研究平台，运用小动物核磁成像、Real-time PCR、Western blot和RNAi沉默等技术方法，观察独活寄生汤对LncRNA GAS5、LncRNA CIR及其调控的软骨细胞功能和细胞外基质代谢的影响，从表观遗传学角度，正反两方面探讨独活寄生汤治疗膝OA的作用机制，为揭示其临床疗效的科学内涵提供实验基础。

独活寄生汤是治疗骨关节炎的经方，但其作用机制尚未完全阐明。软骨退变是骨关节炎主要病理变化，长链非编码RNA(LncRNA)在骨关节炎发生发展中起重要作用，LncRNA GAS5和LncRNA H19的异常激活将引起软骨退变。因此，本研究以骨关节炎的动物模型和细胞模型为研究对象，采用小动物核磁成像、Real-time PCR、Western blot和慢病毒过表达、沉默等方法，观察独活寄生汤对LncRNA GAS5、LncRNA H19及其调控的软骨细胞功能和细胞外基质代谢的影响。结果显示：(1)独活寄生汤可改善膝骨关节炎大鼠软骨形态，延缓软骨退变；通过调控LncRNA GAS5/miR-21，下调MMP-3、MMP-9、MMP-13、ADAMTS-5表达，减轻软骨细胞外基质降解，抑制软骨细胞凋亡；通过调控LncRNA H19/miR-675，上调Aggrecan、Collagen II表达，增强软骨细胞外基质合成，促进软骨细胞增殖。(2)独活寄生汤含药血清可通过调控LncRNA GAS5/miR-21，下调MMP-3、MMP-9、MMP-13、ADAMTS-5表达，抑制软骨细胞凋亡；通过调控LncRNA H19/miR-675，上调Aggrecan、Collagen II表达，促进软骨细胞增殖。(3)慢病毒过表达LncRNA GAS5实验证实独活寄生汤可通过LncRNA GAS5调控软骨细胞外基质降解；慢病毒沉默LncRNA H19实验进一步证实独活寄生汤可通过LncRNA H19调控软骨细胞外基质合成。以上研究结果表明独活寄生汤可通过调控LncRNA GAS5与LncRNA H19，调节软骨细胞功能和细胞外基质降解与合成，延缓骨关节炎的病理进程，为独活寄生汤临床治疗膝骨关节炎提供实验依据。 .本研究发表学术论文6篇，其中SCI源期刊收录论文2篇。