抑制溶血磷脂酸相关MAPK信号通路：四妙丸治疗类风湿关节炎的分子机制?

Rheumatoid arthritis is an autoimmune disease that arthritis is the main clinical manifestations, and its cause remain unclear. Recent researches have shown that dyslipidemia participate the whole process of RA. Lysophosphatidic acid (LPA)-mediated MAPK signaling pathways may be involved in the proliferation and activation of RA synovial cells. Previous evidence of our research group have shown that high fat diet may promote the progress of arthritis in CIA rats. Meanwhile, Simiao pill could regulate lipids and relieve arthritis symptoms. The mechanism could be associated with MAPK signaling pathway. TCM theory holds that lipids Blockaged in the joint and muscle may be the key risk factors of RA. According to point of view in TCM that is "The same syndrome for different disease". We hypothesize that the molecular mechanisms of Simiao pill to treat RA is related to the inhibition of LPA and the regulation of the MAPK signaling pathway. We will establish the arthritis model of mice and synovial cells of RA patients for the study, which using application of molecular biology, immunology and immunocytochemistry. The changes of LPA and its receptor, inflammatory cytokines, MAPK signaling, synovial cells will be observed in order to explain the effect of LPA in RA. We intervene the model with Simiao pill, so that we can prove academic hypothesis and explore its therapeutic mechanism of action and targets of RA. The successful completion of our proposal can provide scientific evidence for developing new treatments, improving the prognosis and guiding disease prevention.

类风湿关节炎(RA)是以关节炎为主要临床表现的自身免疫病，其病因尚不清楚。近期发现，血脂异常贯穿于RA发生发展全过程，溶血磷脂酸（LPA）介导的MAPK通路可能参与了RA滑膜细胞的增生与活化。课题组前期研究显示：高脂饲料促进关节炎模型的炎症反应，四妙丸既能调节血脂，还能缓解关节炎症状，其机制可能与MAPK通路相关。中医理论认为痹阻于关节肌肉中的“膏脂”是RA关键病因，基于"同证异病"的观点，提出假说：“抑制LPA及其介导的MAPK信号通路，是四妙丸治疗RA的分子机制”。将以关节炎小鼠模型和RA患者滑膜细胞为研究对象，应用分子生物学、免疫学及细胞免疫化学等技术，观察LPA及其受体、炎症因子、MAPK信号通路和滑膜细胞等的变化，明确LPA在RA中的作用，并用四妙丸加以干预，探讨其治疗RA的作用机制和靶点，以验证假说，为临床拓展RA治疗思路和指导预防提供科学依据。

研究背景：类风湿性关节炎是以关节炎症和滑膜增生为主要病理和临床表现的自身免疫病。越来越多的研究证据表明血脂异常贯穿于RA发生发展全过程，溶血磷脂酸（LPA）介导的MAPK通路可能参与了RA滑膜细胞的增生与活化。四妙丸是用来治疗类风湿性关节炎数百年的传统方剂，且此方其君药黄柏主要成分小檗碱均被发现有明显的调节血脂作用。研究方法：体内实验中通过CIA造模法，建立大鼠关节炎模型，并给与不同剂量的四妙丸，通过测定体重和关节炎评分评价四妙丸的临床改善作用，检测血清中TNF-α、IL-1β、CRP评价其炎症因子改善情况，踝关节micro-CT、HE染色、血清OPG检测评估关节破坏、滑膜增生及炎症浸润情况，检测血清及踝关节中ATX、LPA及LPA1评判四妙丸对LPA介导的MAPK上游蛋白影响，检测蛋白评价四妙丸对MAPK下游活性蛋白的影响；体外实验中，分子对接模拟小檗碱和靶向蛋白LPA1的作用方式，检测RA患者血浆LPA水平，提取原代FLS-RA细胞并模拟体内炎性环境，分为对照组、小檗碱组、小檗碱预处理+LPA处理、Ki16425预处理+小檗碱通过检测FLS-RA细胞活性、FLS-RA炎症因子分泌和MAPK通路活性蛋白探讨小檗碱的作用机制。研究结果：体内实验表明四妙丸可能通过降低LPA、ATX的分泌及LPA1受体的表达抑制滑膜增生和炎症、抑制滑膜及脾脏MAPK信号通路的活性；此外，四妙丸能改善骨质破坏；临床研究显示，RA患者血清中LPA明显高于正常对照，且滑膜明显增生伴有炎性浸润；体外实验表明四妙丸君药黄柏的主要化学成分小檗碱能可能通过结合LPA1抵抗LPA介导的MAPK蛋白的活化来抑制FLS-RA增殖及炎症因子的分泌，且提示同时可能有其他途径参与其中。科学意义：证实LPA参与RA的发展，发现四妙丸及其主要化学成分小檗碱不仅通过抑制LPA分泌，且拮抗其受体来抑制MAPK信号通路的活化，诠释了四妙丸治疗类风湿性关节炎的分子机制，为“祛痰化湿、活血化瘀”的RA治疗原则提供了科学依据，同时提示抑制LPA及其靶向受体LPA1可作为新的RA治疗模式候选之一。