异佛司可林抑制伯氏疏螺旋体膜蛋白BmpA诱发的促炎细胞因子风暴的机制研究

Lyme disease is an important vector-borne disease caused by Borrelia burgdorferi, Lyme arthritis is most common damage in patients with Lyme disease. The pathogenesis of Lyme arthritis is not fully elucidated. Based on the early series of studies, we found that Lyme Borrelia burgdorferi outer membrane protein gene bmpA is closely associated with Lyme arthritis; gene product rBmpA of bmpA can cause arthritis in mice, and strongly stimulate monocyte-macrophage to produce pro-inflammatory cytokines in vitro, but the specific mechanism is unknown. Medical compound isoforskolin from Yunnan endemic medicinal herb has good anti inflammation effects, we found that it significantly inhibited the production of proinflammatory cytokines induced by BmpA, but its mechanism of action is unclear.Based on our previous studies, we hypothesize that Borrelia membrane protein BmpA may induce specific proinflammatory cytokine production by TLRs, thereby initiating the process of arthritis. To confirm this hypothesis, we plan to do: ( 1 ) through TLR blocking experiments and immune co-precipitation techniques, determine specific rBmpA-binding TLR; (2) Selecting key molecules and cytokines in TLR signaling pathway as a research targets, explore specific signaling pathways of BmpA-TLR interactions and key pro-inflammatory cytokines; ( 3 ) Identifying key molecules targeted by isoforskolin in TLR signaling pathway, based on our findings that isoforskolin significantly inhibited BmpA-induced proinflammatory cytokine production. Through these studies, exposing the pathogenesis of BmpA-induced arthritis and molecular targets of isoforskolin’s anti-inflammatory effects, providing scientific basis for using yunnan endemic herbs to prevention and treatment of Lyme arthritis.

莱姆关节炎是伯氏疏螺旋体（Bb）感染引起的关节炎症。我们通过前期研究，找到了与莱姆关节炎密切相关的Bb外膜蛋白基因bmpA及其产物rBmpA，但作用机制未完全清楚；云南特产药物异佛司可林具有良好抗炎作用，我们发现它明显抑制BmpA诱导的细胞因子产生，具体机制不明。我们推测，膜蛋白BmpA可能通过TLRs诱导促炎细胞因子风暴，启动关节炎进程，异佛司可林通过抑制TLRs信号通路发挥作用。为此本项目拟（1）确定与rBmpA特异结合的TLR；（2）选择TLR信号传导通路的关键分子和相关细胞因子作为研究靶点，探索BmpA刺激特定TLR诱导促炎细胞因子风暴的信号传导通路及关键分子；（3）研究云南特产药物异佛司可林显著抑制BmpA诱生促炎细胞因子风暴的信号通路靶点。通过上述研究，揭示BmpA诱发关节炎的发病机理和异佛司可林的抗炎作用靶点，为云南特产药异佛司可林开发利用于莱姆关节炎的防治提供科学依据。

莱姆关节炎是伯氏疏螺旋体引起的重要虫媒病，发生率高，危害最大，但病机理未充分阐明。我们通过功能基因组学、生物信息学和动物实验研究，找到了与莱姆关节炎密切相关的伯氏疏螺旋体外膜蛋白基因bmpA；发现注射bmpA基因产物rBmpA能引起小鼠关节炎，在体外能强力刺激促炎细胞因子产生，但具体致病机理不明。基于前期研究，我们认为，膜蛋白BmpA可能通过Toll样受体（TLRs）诱导特定促炎细胞因子产生，从而启动关节炎进程。为此，依托该项目，我们开展了下列工作：（1）通过通过TLR受体封闭实验，确定了与rBmpA特异结合的TLR；（2）选择TLR信号传导通路的关键分子和相关细胞因子，应用免疫荧光技术、免疫组化技术、实时定量PCR技术和ELISA技术等，探索了BmpA刺激特定TLR诱导促炎细胞因子的信号传导通路及关键分子；（3）研究了云南特产药物异佛司可林(isoforskolin)显著抑制BmpA诱生促炎细胞因子的信号通路靶点。通过上述研究，揭示了BmpA诱发关节炎的发病机理和异佛司可林的抗炎作用靶点，为莱姆关节炎防治提供了科学依据。本项目按照任务书要求，完成了相关科研研究内容、人才培养任务和科研成果如下：① 找到疏螺旋体表面膜蛋白BmpA 的TLRs 受体类别是TLR和TLR2，确定BmpA 通过TLR1/TLR2引发促炎细胞因子产生的信号传导通路，进一步证明BmpA 是伯氏疏螺旋体的主要致关节炎毒力因子之一，为进一步深入探索莱姆关节炎的的发病机理提供有力支撑。② 通过异佛司可林干预实验，确定异佛司可林通过调控TLR信号传导通路的分子靶点，为难治性莱姆关节炎和相关关节炎治疗提供了新思路，为进一步深入探索莱姆关节炎的新手段打下基础。