miR-675靶向TBK1介导骨关节炎免疫微环境调控软骨细胞自噬及补肾活血中药干预研究

The incidence of osteoarthritis (OA) is closely related to the immune microenvironment and the level of chondrocyte autophagy. The effect of immune inflammation on the local microenvironment of the joint may be one of the main factors leading to OA. Chondrocyte autophagy is necessary for the maintenance of chondrocyte phenotype and repairing articular cartilage, so it is difficult for amaged articular cartilage to repair due to chondrocyte autophagy dysfunction, and then accelerate the progress of OA. MiR-675 can help repair cartilage injury by maintaining the chondrocyte phenotype via promoting the production of type 2 collagen.TBK1 is a essential phosphokinase for the functional activation of IRFs, which regulates the immune response, and autophagy may be a protective or balanced mechanism for inflammation caused by abnormal immune responses in normal cells. Therefore, we supposed that under the influence of inflammation, mechanical loading, aging and genetic factors, the expression of miR-675 in articular cartilage is down-regulated, which caused aberrant expression of TBK1, and then induced abnormal immune inflammatory reaction after activating IRFs, subsequently leaded to the inhibition of autophagy, thus accelerating articular cartilage injury. Traditional Chinese medicine has many potential and advantages in the prevention and control of OA with multiple targets and dual regulating functions. The subject is based on this hypothesis and the characteristics of traditional Chinese Medicine, explains the mechanism of chondrocyte autophagy dysfunction may be the essence of kidney deficiency and blood stasis of OA, and explores the mechanism of autophagy of OA cartilage and the molecular mechanism of protective effect on cartilage of traditional Chinese medicine, according to the perspective of immune microenvironment mediated by miR-675 targeting TBK1.

骨关节炎(OA)的发病与免疫微环境及软骨细胞自噬水平密切相关，机体免疫炎症对关节局部微环境产生的影响可能是导致OA的主要因素之一，而自噬失衡将不能维持软骨表型并导致损伤的关节软骨难以修复，加快OA的进展。miR-675可通过促进2型胶原的产生维持软骨表型，有助于软骨损伤后的修复，TBK1为IRFs活化所必需的磷酸激酶，可调节免疫反应，而自噬可能是正常细胞遭受异常免疫反应引起炎症时的一种保护或平衡机制。因此，课题组猜想在受炎症、力学负荷、增龄、遗传因素影响下，关节软骨中miR-675表达下调，引起TBK1异常表达，继而激活IRFs后诱导异常免疫炎性反应导致自噬被抑制，从而加速软骨损伤。本课题基于这一假说及中药的特性，阐述软骨细胞自噬功能失调机制可能是OA肾虚血瘀的本质；并以miR-675靶向TBK1介导的骨关节炎免疫微环境为视角，阐明OA软骨自噬的机制及中药靶向干预的分子机制。

骨关节炎（Osteoarthritis，OA）是多发于中老年群体的骨科临床常见慢性进展性疾病。然而OA发病机制尚不明确，给OA的临床预防和诊疗带来了巨大的挑战。中医药防治OA具有较大的潜力与优势，临床研究表明具有补肾活血功效的龙鳖胶囊在治疗OA上取得了满意的疗效，因此深入探索龙鳖胶囊保护关节软骨的分子机制将有利于补肾活血中药防治OA等相关疾病的推广应用。.结合前期的研究和预实验结果发现，在OA发病过程中关节软骨中的miR-675表达下调，导致靶分子TBK1异常表达，抑制细胞自噬，使MMPs降解受阻，从而加速了关节软骨损伤。因此，本研究首先在动物OA模型中观察miR-675和TBK1在关节软骨中的表达情况，接着利用RNA分子干扰技术和慢病毒载体干预细胞内miR-675和TBK1的表达，在明确miR-675靶向TBK1调控IRFs介导骨关节炎免疫微环境影响软骨生物学功能的前提下，进一步通过体内外实验验证龙鳖胶囊保护关节软骨的分子机制。相关结果显示OA大鼠的关节软骨中miR-675的表达减少，TBK1的表达增多，而且自噬水平也明显下降。干预细胞内miR-675和TBK1的表达后，结果显示过表达miR-675或抑制TBK1的表达后能增强软骨细胞自噬水平，促进软骨细胞增殖，抑制软骨细胞凋亡及软骨基质降解。也证实了TBK1是miR-675调控软骨细胞自噬、凋亡和免疫反应的一个重要靶点。细胞实验结果表明龙鳖胶囊含药血清具有增强软骨细胞活力、促进软骨细胞增殖及自噬、调节软骨基质代谢、抑制软骨细胞凋亡的作用，还可通过上调软骨细胞中miR-675表达或下调TBK1的表达达到抑制软骨细胞凋亡和促进软骨细胞增殖和自噬的作用。动物体内实验结果显示龙鳖胶囊能在一定程度上恢复关节软骨下骨的骨密度和骨质量；也能部分恢复关节软骨分泌软骨基质的能力，增强软骨的自噬水平，上调软骨中miR-675的表达，抑制TBK1的表达进而抑制关节软骨中免疫炎性反应的作用。.本项目的主要结果表明龙鳖胶囊可能是通过调控miR-675/TBK1信号轴抑制软骨细胞的免疫反应、抑制软骨细胞凋亡及基质降解、增强软骨细胞增殖活性和自噬水平从而起到保护关节软骨、延缓OA进展的作用。相关结果将为揭示OA的发病机制及补肾活血中药防治OA的分子机制提供新理论和实验依据。