miR-146a调控骨关节炎固有免疫分子机制及参威骨痹片干预作用研究

The morbidity and disability rate of osteoarthritis(OA) is high ,and become one of the main causes of the global burden of disease.Over-activation of innate immune TLR signaling pathway is closely related to inflammation and autoimmune diseases. MiR-146a negatively regulates innate immunity and plays an important role in the development of OA.The Shenweigubi Soup is confirmed effective after clinical trials.Shenweigubi tablets were developed from its soup dosage,and could down-regulate the expression of Tumor Necrosis Factor-α(TNF-α) in the OA model rabbit joint fluid.We find that Shenweigubi Soup treats OA can down-regulate the level of P50 and P65 in TLR4 pathway in recent trials.Inherent immunity plays a key role in the pathogenesis of OA,liver and kidney are origin of inherence,and liver and kidney deficiency is the root cause of OA according to TCM theory.Based on the consistency in the concept between east and west and pre-studies,we made a hypothesis that Shenweigubi tablets maybe intervene the OA through regulating the innate immune,nevertheless,the mechanism is not yet exactly.The target of Shenweigubi tablets treat OA is about miR-146a,molecule level of signal or levels of transcription factor?Those are what we plan to be studied.Therefore,this study is hope to bring the mechanism of how miR-146a regulates the innate immune in OA and the real mechanism of Shenweigubi tablets treat OA to light,and then find out the mechanism and targets of innate immune,improve the manufacturing technique and provide the basis for clinical application ultimately.

骨关节炎(OA)患病率及致残率高，是造成全球疾病负担的主要因素之一。固有免疫TLR信号通路过度激活与炎症和自身免疫性疾病密切相关，miRNA-146a负性调控其强度，在OA发病中起重要作用。参威骨痹汤临床有效，由其研制的参威骨痹片能下调OA模型兔关节液中TNF-α。新近发现参威骨痹汤能下调OA患者TLR4通路中TLR4、P50、P65。固有免疫是一种先天性免疫，在OA发病中起关键作用；中医认为肝肾为人之先天，肝肾亏虚是OA发病之本。基于中西方理念一致及前期研究，推测参威骨痹片可能通过调节固有免疫应答干预OA。然miRNA-146a调控OA固有免疫分子机制尚不完全清楚，参威骨痹片干预OA的靶点是miR-146a?还是TLR4水平？信号分子水平？转录因子水平？有待深入研究。本项目拟探讨miR-146a调控OA固有免疫应答机制和参威骨痹片的作用，有望揭示其机制及靶标，为参威骨痹片研发奠定基础。

骨关节炎是由多种因素引起的关节退行性疾病，病理特点包括关节软骨变性破坏、滑膜炎、软骨下骨硬化或囊性变、关节边缘骨质增生、骨赘形成等症状，是导致残疾的最大原因。本课题对服用参威骨痹片后的KOA模型鼠取其关节软骨、滑膜、关节液，并对其中炎症因子、TLR4/MyD88/NF-κB通路因子的表达进行检测；并完善细胞实验，对软骨细胞中的miR-146a 表达、炎症因子以及通路相关因子的mRNA以及蛋白表达变化进行检测，并检测细胞凋亡率以及细胞存活率变化；为了进一步验证LncRNA MINCR在OA组织和IL-1β诱导的软骨细胞中的表达情况，对诱导后的软骨细胞增殖、凋亡和ECM变性进行检测；验证MINCR对miR-146a-5p/ BMPR2的调控作用。通过大量实验，我们发现参威骨痹片后KOA模型鼠关节软骨、滑膜以及关节液中的炎症因子的浓度均明显下降，通路相关因子的mRNA、蛋白表达以及p-P65/P65均明显下降，且呈剂量依赖趋势；参威骨痹片刺激后，软骨细胞中的miR-146a 表达均明显上升；软骨细胞提取成功；参威骨痹片刺激后，软骨细胞中的炎症因子以及通路相关因子的mRNA以及蛋白表达明显下降，细胞凋亡率均明显下降，而细胞存活率明显上升，且呈剂量依赖趋势；抑制miR-146a表达后，软骨细胞中的炎症因子以及通路相关因子的mRNA以及蛋白表达明显上升，细胞凋亡率均明显上升，而细胞存活率明显下降，且呈剂量依赖趋势，当进一步抑制TLR4表达时，上述结果被逆转。