基于cPLA2/5-LO/LTs通路研究薯蓣皂苷调控LTs代谢合成及其受体功能治疗类风湿性关节炎的作用及机制

类风湿性关节炎（RA）是以关节滑膜异常增生为主要特征的自身免疫性疾病，其发病机制未明。白三烯(LTs：LTB4和CysLTs)由花生四烯酸代谢生成，其合成酶系（cPLA2、5-LO、LTA4H或LTC4S）和/或受体功能改变导致LTs异常增加及其受体功能障碍是启动RA发病的重要机制。最近我们首次发现薯蓣皂苷保护RA模型小鼠可能涉及其抑制LTB4生成。本项目拟增加原代培养RA病人滑膜成纤维细胞离体模型，系统性地研究RA期间：LTs异常增加与其合成酶系、受体亲和力及功能变化相关性；薯蓣皂苷抑制LTB4异常增加与其对LTB4合成酶系、BLT1/2受体亲和力及功能变化影响的相关性；薯蓣皂苷对CysLTs异常增加的作用与其对CysLTs合成酶系、CYSLT1/2受体亲和力及功能变化影响的相关性。阐释薯蓣皂苷调控LTs代谢合成及其受体功能治疗RA的作用及机制，为临床治疗此顽疾提供新靶点和实验理论依据。

Rheumatoid arthritis(RA) is a common autoimmune disease. Pathological changes are chronic synovial membrane inflammation or proliferation and joint erosion. At present, the exact pathogenesis of RA has not been described and its treatment is always difficult. .5-Lipoxygenase(5-LO) catalyzes leukotriene(LTs) biosynthesis. After cell stimulation 5-LO translocates to the nuclear membrane, where it colocalizes with FLAP and the cytosolic phospholipase A2 (cPLA2) which releases arachidonic acid (AA) from the membrane. AA is oxidized into 5-hydroperoxyeicosatetraenoic acid which is converted into the instable epoxide leukotriene A4(LTA4). LTA4 can be either metabolized to LTB4 by LTA4 hydrolase or by LTC4 synthase into the cysteinyl-leukotriene LTC4 which is then further degraded to LTD4 and LTE4. LTs synthesis enzymes and/or receptor dysfunction can lead to clinical signs and symptoms of RA. .Growing evidence indicates that LTs play a pivotal role in RA pathogenesis. As a potent chemotactic mediator, LTB4 can provoke most signs and symptoms in RA by initiating, coordinating, sustaining, and amplifying the inflammatory response, through recruitment of leukocytes. It is highly present in synovial fluid and serum of patients with active RA. LTB4 enhances the neutrophil-dependent increased microvascular permeability and exerts its activity by coupling to BLT1 and BLT2. CysLTs exhibit several biological activities through at least two specific CysLT1 and CysLT2. Koshihara et al demonstrated that CysLTs were detected in synovial fluid in RA patients. Xu et al discovered CysLTs signaling pathway play a vital role in the pathogenesis of collagen-induced arthritis (CIA) mouse. Despite the progresses, so far seldom systemic and integrated studies have been employed to explore why the abnormal increased LTs of AA metabolites presented in synovial fluid and serum of patients with active RA. LTs receptor dysfunction in RA pathogenesis is not fully clear..LTs are responsible for the progressive destruction of cartilage and bones, neither steroids, nor the non steroidal anti-inflammatory drugs, cannot slow down cartilage and bone destruction providing only symptomatic improvement. Long-term use of therapies for RA produces adverse effects; hence there is a need for safe and effective alternatives suitable for long term chronic use. Dioscin is a kind of important steroidal saponins which is the important raw material for synthesis of steroid hormone drugs. It has been reported for the treatment of CIA animal, however, the mechanism underlying its treating RA is far from fully elucidated. Recently, we firstly found that the protective effects of dioscornin tablets on CIA mouse may involve in its inhibiting LTB4 generation. Here, using several agonists and/or antagonists of these synthases/receptor in LTs pathway, we will conduct some experiments such as in vivo CIA mice and in vitro model of primary cultured synovial fibroblasts of RA patients to focus on elucidating the following topics: 1. explore systematically the correlations between the abnormal increased LTs and the alterations of cPLA2, 5-LO, LTA4H or LTC4S gene and protein expressions, distributions and activities together with the alteration of LTs receptor affinity and its pathways; 2.whether the effects of dioscin inhibiting LTB4 generation involved in its influences on the gene and protein expressions, distributions and activities of cPLA2, 5-LO, LTA4H, as well as on the alteration of BLT1/2 affinity and its pathway; 3. whether the dioscin actions on abnormal increased CysLTs involved in its influences on the gene and protein expressions, distributions and activities of cPLA2, 5-LO, LTC4S, as well as on the alteration of CYSLT1/2 affinity and its function. These works if completed will further clarify the pathogenesis of RA and the protective mechanism dioscin against RA. It will provide new ideas and targets for traditional Chinese medicine to clinically prevent and treat RA disease.