基于Keap1/Nrf2信号通路探索青蒿琥酯抗RA骨破坏的作用机制

Prevention the joint destruction is the new strategy for the treatmentment of RA. However, oxidative stress play an important in the bone destruction of inflamed joints of RA patients, and Keap1/Nrf2 axis is a central target that regulates osteoclast differentiation. It has been reported that anti-malarial drug artesunatea rtesunate have anti-inflammatory and immunosuppressive activities, and the anti-arthritic effect has been demonstrated by our preliminary study, but the mechanism is unknown. It demonstrated that the artesunate suppresses inflammation through Keap1/Nrf2 axis, thus, we speculate that artesunate can suppress oxidative stress and prevent bone destruction in arthritis by activating Keap1/Nrf2 axis. In our study, we explicit the anti-arthritic effects of artesunate by evaluating the clinical manifestation and bone destruction, explore the underlying mechanism by osteoclast differentiation in vitro and verify the mechanism and target by Nrf2 knock out mice. It is expected that our results will confirm the anti-arthritic effect and mechanism of artesunate, importantly, these findings will provide us a scientific experimental basis for the expansion of antimalarial drugs in the treatment of RA, and develop the mechanism of pharmacological effect of artesunate.

类风湿性关节炎（RA）最新治疗策略是自始至终防止和控制关节破坏。氧化应激在骨破坏中的角色近年备受关注，尤其Keap1/Nrf2轴成为调控破骨细胞分化的关键靶标。已知抗疟药物青蒿琥酯具有良好的抗炎免疫活性，我们前期的研究结果也发现其具有显著抑制RA破骨细胞生成作用，但机制未明。新近的研究报道青蒿琥酯可明显活化Keap1/Nrf2轴，籍此我们推测：青蒿琥酯能通过激活Keap1/Nrf2轴抑制氧化应激，从而发挥抗RA骨破坏作用。本项目拟以CIA大鼠为研究对象，通过病情评价及骨破坏检测，明确青蒿琥酯的抗RA作用特点；进而利用体外破骨细胞分化模型，以Keap1/Nrf2轴为切入点，明确其作用途径；最后通过Nrf2-/-小鼠反证，确证其分子靶点。预期研究结果将明确青蒿琥酯的抗风湿病情改善作用及机制，为扩大抗疟药物治疗RA的适应症提供实验依据，此外还发展了青蒿琥酯的药理作用机制。

类风湿关节炎（RA）最新治疗策略是自始至终防止和控制关节破坏。氧化应激在骨破坏中的角色近年备受关注，尤其p62/Keap1/Nrf2信号轴成为调控破骨细胞分化的关键靶标。已知抗疟药物青蒿琥酯具有良好的抗炎免疫活性，然而青蒿琥酯是否通过调控p62/Keap1/Nrf2信号轴抑制ROS从而抑制骨破坏尚不明确。本研究首先基于CIA大鼠模型明确了青蒿琥酯抑制大鼠关节肿胀度、发病率及临床积分且减轻大鼠关节软骨和骨破坏，以及破骨细胞形成。青蒿琥酯抑制CIA大鼠模型关节组织中CTSK、MMP-9和NFATc1表达的同时，也能明显抑制CIA大鼠模型关节组织中活性氧ROS的产生。进一步的机制探索证实，青蒿琥酯主要通过激活p62/Nrf2信号通路促进Nrf2核转位从而促进抗氧化酶的表达。与此同时，本研究采用体外RANKL诱导破骨细胞分化模型明确了青蒿琥酯明显抑制破骨细胞形成、分化和功能的同时，对体外破骨细胞分化体系中产生的ROS也具有明显的清除作用。且相关机制证实青蒿琥酯不仅能明显逆转破骨体系中HO-1和NQO1的显著降低，而且也明显激活p62的表达，促进Nrf2的表达以及核转位。与此同时，我们通过体外siRNA干扰技术进一步证实青蒿琥酯主要通过激活p62调控Nrf2及下游抗氧化酶抑制ROS的产生从而抑制破骨细胞分化以及骨吸收。本研究结果明确了青蒿琥酯的抗类风湿关节炎骨破坏作用机制是通过激活p62/Nrf2 信号通路抑制ROS，本研究结果为扩大抗疟药物治疗RA的适应症提供实验依据，此外还发展了青蒿琥酯的药理作用机制，并带动青蒿素类药物的研发。