基于Hsp90-NLRP3途径研究雷公藤红素治疗类风湿性关节炎的新机制

Rheumatoid arthritis (RA) is a common chronic inflammatory and systemic autoimmune disease, which is characterized by cytokine mediated inflammation of the synovium and destruction of cartilage and bone. Nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome complex had been associated with the development of RA and played an important role in the pathogenesis of RA. Celastrol, a bioactive component of the traditional Chinese medicine Celastrus aculeatus Merr, had been proven effective in the treatment of RA, probably with its good anti-inflammatory and antioxidant properties. Our previous study has shown that, celastrol exert therapeutic effects on CIA mice, with the progression and development of CIA blocked in a dose-dependent manner. Consistent with the clinical scoring, measurements of paw swelling in CIA mice also showed celastrol to be highly effective. Especially, our results shown that the new anti-inflammation mechanism of celastrol through inhibiting of the NLRP3 inflammasome. May be, celastrol prevent the NLRP3 inflammasome activity through Hsp90, and this hypothesis needs other experiments to prove.. According to the role of inflammation in RA and our previous research about celastrol, we will find out the underlying mechanism of therapeutic effect of celastrol. Firstly, studying the binding and inhibition function of celastrol to Hsp90 with molecular and cellular biological methods. Also, we will discusses how celastrol inhibit the activation of NLRP3 inflammasome through Hsp90. And then, study the anti-arthritic effects of celastrol CIA in mice in vivo, by obtaining clinical scores, radiographic evaluations, histopathological assessments. Also, explore its underlying mechanism with immunofluorescence, Western blot, RT-PCR and siRNA technology, through the signaling pathways of Hsp90-NLRP3, TLR4-NF-κB/MAPK and PI3K/AKT/mTOR. All the above studies can provide the foundation for the development and application of celastrol, and offer a new strategy for the treatment of RA.

类风湿性关节炎（RA）是多种细胞因子介导的慢性自身免疫性疾病，研究表明，炎症小体NLRP3在RA发病机制中发挥重要作用。雷公藤红素具有良好的抗炎与抗氧化作用，在治疗RA方面具有多层次、多靶点的独特优势。前期研究发现，抑制NLRP3炎症小体的激活是雷公藤红素新的抗炎机制，雷公藤红素如何通过调控NLRP3通路治疗RA仍需要进一步明确。首先运用分子及细胞生物学方法对雷公藤红素与Hsp90的结合作用以及抑制NLRP3炎症小体激活的分子机制进行深入研究。通过关节炎评分、X光摄片、组织病理学等探索雷公藤红素对RA的治疗特点；利用免疫共沉淀、免疫荧光、Western blot、RT-PCR和siRNA等技术，通过Hsp90-NLRP3、TLR4-NF-κB/MAPK、PI3K/AKT/mTOR等通路，阐释雷公藤红素阻断炎症反应、抑制滑膜增生的机制，为雷公藤红素的研究提供依据，为RA的治疗提供新策略。

类风湿性关节炎（RA）为慢性炎性自身免疫性疾病，其中炎症小体NLRP3在RA的发病机制中发挥重要作用。雷公藤红素又称为南蛇藤素，属于醌甲基三萜，来源于雷公藤的根皮，是治疗类风湿病雷公藤多甙片的有效成分之一，在治疗RA方面具有多层次、多靶点的独特优势。前期研究发现，抑制NLRP3炎症小体激活是雷公藤红素新的抗炎机制，雷公藤红素是否能够通过调控NLRP3通路治疗RA，及其深入的机制仍需要进一步明确。首先建立佐剂型关节炎大鼠模型和胶原诱导性关节炎（CIA）小鼠模型，细胞水平以人单核巨噬细胞PMA-THP-1和人成纤维样滑膜细胞（FLS）为研究对象，在动物水平与细胞水平通过关节炎评分、X光摄片、组织病理学等探索雷公藤红素对RA的治疗特点；利用免疫共沉淀、免疫荧光、Western blot、RT-PCR和siRNA等技术，评价雷公藤红素对Hsp90-NLRP3、TLR4-NF-κB等通路的影响，探索雷公藤红素治疗RA可能的机制。进而运用分子及细胞生物学方法对雷公藤红素与Hsp90的结合作用以及抑制NLRP3炎症小体激活的分子机制进行深入研究。动物实验结果显示雷公藤红素对佐剂型关节炎大鼠、胶原性关节炎小鼠的的关节炎症状有一定改善作用，且不良反应低，其机制与抑制炎症因子分泌，抑制NF-κB-NLRP3信号通路活化密切相关。进一步研究发现雷公藤红素能通过ROS-NF-κB-NLRP3等通路抑制细胞水平的炎症反应；同时能够抑制TNF-α诱导滑膜细胞增殖，抑制细胞因子分泌，这与调控Hsp90-NLRP3通路密切相关。体外分子水平实验结果显示雷公红素能够直接与Hsp90结合，进而影响其客户蛋白NLRP3的稳定性。该项目在动物水平完成了雷公藤红素治疗RA的药效学特点评价，并通过细胞实验进一步阐释了雷公藤红素阻断炎症反应、抑制滑膜增生、治疗RA可能的机制，而且在分子水平阐释了雷公藤红素通过Hsp90调控NLRP3炎症小体的深入机制。本项目为进一步开发雷公藤红素的临床应用奠定实验基础，也为类风湿性关节炎的治疗提供新思路与策略。