PKM2参与巨噬细胞活化促进关节炎症的分子机制

Macrophages play crucial roles on the initiation and development of inflammatory arthritides, especially in rheumatoid arthritis (RA). The activated macrophages during inflammation of joints may be regulated by some metabolic enzymes. Our preliminary data recently showed that pyruvate kinase muscle type 2 (PKM2) was feasibly involved in the development of pristane induced arthritis (PIA) in DA rats, which also participated in macrophage activation. So we propose the hypothesis that PKM2 regulates the activation and function of macrophages, contributing to the development of arthritis. We intend to prove our hypothesis by the following experiments. Firstly, the expression of PKM2 will be detected in spleen macrophages and synovial macrophages. The function of PKM2 in arthritis development will be investigated by RNAi in vivo in PIA rats. Secondly, the mechanism of PKM2 regulating macrophage activation will be studied from 2 aspects. The activated signal pathways will be screened and analyzed in PKM2 knocking-down macrophages by Western blotting to obtain the signal molecules and the downstream effect molecules which are associated with PKM2. The molecule which interacts with PKM2 could be captured by co-immunoprecipitation in PKM2 high-expressed macrophages. And then, we would like to use the computational biology method to mimic the domains of captured protein that PKM2 interact with. Thirdly, the regulatory molecules of PKM2 in activated macrophages are pinpointed in transcriptional and post-transcriptional levels. Finally, the function of PKM2 in arthritis development will be further confirmed by establishing the CIA animal model using macrophage conditional knockout mice and human RA samples. We insist that the results should offer strong evidence to support the hypothesis that the aerobic glycolysis enzyme PKM2 may participate in the initiation and development of inflammation of joints by regulating the activation and function of macrophages. Also, the findings will provide new insights into understanding the pathogenesis of RA and supply novel molecular targets for the prevention, diagnosis, therapy and new drug development of arthritis.

代谢分子影响巨噬细胞(Mφ)的活化，亦被发现参与关节炎症的发生；预实验提示丙酮酸激酶2（PKM2）可介导关节炎的进程，本研究拟证实PKM2调控Mφ活化和功能进而参与关节炎症的发展。首先在关节炎动物中确定高表达PKM2的细胞类型，分析PKM2与关节炎进程的关系；继而在PKM2敲低/过表达Mφ活化模型上检测信号通路活化情况，并用免疫共沉淀等手段确定其调控的效应分子，通过分子模拟、点突变等方法揭示PKM2与其相互作用分子之间的作用位点，阐明其对Mφ活化和功能调控机制；再利用免疫共沉淀、RNAi等方法从转录和转录后剪切过程解析调控PKM2高表达的机制；最后在Mφ条件性敲除小鼠关节炎模型和RA患者样本中检测PKM2及其相关分子的表达和活化情况，分析和关节炎发生发展的关系，确定PKM2调控Mφ介导关节炎中的作用。结果将丰富糖代谢调节酶调控Mφ参与关节炎发病机制的认识，为关节炎的诊疗提供理论和实验依据。

课题组前期在实验性关节炎的研究中发现，活化的巨噬细胞（Mφ）在关节炎的发病过程中起到了非常重要的作用，但Mφ的活化机制仍然未知。近年来，代谢分子调控免疫细胞功能逐渐受到人们的关注，因此，本研究从代谢分子PKM2入手，深入探究其在Mφ活化中的作用机制，提出“PKM2调控Mφ活化进而参与关节炎症的发生和发展”的假说。首先在构建的大鼠降植烷诱导的关节炎模型中，通过收集关节炎进程的大鼠脾脏组织，通过免疫印迹的方法，发现PKM2在诱导疾病后第6天即出现表达上调；进而通过免疫荧光的方法确定了高调的PKM2主要表达于ED1+的Mφ上。继而构建了Mφ经典活化模型，发现PKM2的表达也在经典活化的Mφ中显著增高；继而包装慢病毒感染巨噬细胞系RAW264.7，在PKM2敲低/过表达Mφ活化模型上通过检测信号通路的活化和免疫共沉淀等手段确定其下游活化通路和效应分子，发现PKM2可能作为信号通路中的接头子，与STAT和JAK结合，拉近其发生磷酸化修饰时二者的距离，进而调控JAK-STAT信号参与Mφ的活化过程。利用免疫印迹及RNAi的技术找到了在Mφ活化过程中调控pkm2 mRNA剪切的分子PTBP1，结果显示，PTBP1在Mφ活化后表达上调；干预PTBP1的表达，可显著抑制PKM2的表达，而促进PKM1的表达，且部分促炎因子的表达也随之下降；最后利用PKM2抑制剂Shikonin和RNAi的方法在体对PKM2进行了干预，发现降低PKM2的表达后，关节炎大鼠的临床计分、病情要显著低于NC组或者溶剂对照组；且关节组织中磷酸化的STAT1的表达也在干预后显著下调，促炎因子的表达也随之下降，说明大鼠的关节炎有了一定程度的改善和缓解。这些结果的揭示证明在发生关节炎症时，异常活化的代谢酶PKM2可能通过其非酶活性对Mφ的活化进行调节，而干预PKM2显著减轻关节炎症过程，则为日后选择治疗关节炎的靶标提供新的理论和实验依据。