MFHAS1及其泛素化对脓毒症小鼠TLR2信号通路的作用机制研究

Sepsis is an important cause of mortality. However, to date, there have been no commercially available successful strategies for targeting the molecular pathways involved in sepsis. In our earlier studies, blood MFHAS1 (malignant fibrous histiocytoma amplified sequence 1) was found to be significantly elevated in septic patients, and MFHAS1 was more increased in mononuclear cells from septic patients. We also found that MFHAS1 had dual effects on TLR2 signaling pathway and inflammation, i.e., inhibitory effect at 6 hours, and then stimulatory effect after 24 hours. Moreover, according to our results of mass spectrometry analysis, praja2 (one of E3-ubiquitin ligases) was predicted to form a complex with MFHAS1, and MFHAS1 can be ubiquitylated. Based on these pre-experiments, we proposed the project and want to do: ① To specify the relationship of MFHAS1 and TLR2 in vivo, using septic mice. ② To clarify the effect of MFHAS1 on the development of sepsis and TLR2 signaling pathway, using MFHAS1-/- (MFHAS1 knockout) septic mice. ③ To confirm whether praja2 is the interaction protein of MFHAS1, and whether MFHAS1 can be ubiquitylated by praja2, using inflammatory cells stably expressing ubiquitylated MFHAS1, by immune co-precipitation, Q-PCR, laser confocal and so on. Implementation of this project will determine whether MFHAS1 could play an important role in the development of sepsis and whether the ubiquitination of MFHAS1 could affect the TLR2 signaling pathway, so as to determine a new target of sepsis treatment.

脓毒症长期以来是危重患者主要致死原因，目前仍没有成功的针对脓毒症的分子靶向治疗方案。课题组发现脓毒症患者血浆MFHAS1浓度较对照组显著升高；且MFHAS1对TLR2有特异性的双相调节作用：先抑制后刺激；通过质谱分析，我们还找到了可能与MFHAS1结合的E3泛素连接酶praja2。本项目拟开展：①建立脓毒症小鼠模型，观察MFHAS1和TLR2在脓毒症中的变化规律及其相互关系；②建立MFHAS1敲除脓毒症小鼠模型，明确MFHAS1对脓毒症发生发展及TLR2的影响；③建立MFHAS1泛素化炎症细胞模型，应用免疫共沉淀、蛋白共定位、泛素化实验等手段，确定MFHAS1能否被praja2泛素化从而影响其调控TLR2的功能。项目实施将确定MFHAS1对脓毒症的发生发展及体内TLR2的信号传导产生的影响，及其泛素化对TLR2信号通路调控的影响，明确MFHAS1能否作为脓毒症治疗的新靶点。

项目背景：脓毒症长期以来是危重患者主要致死原因，而脓毒症相关性脑病（SAE）是脓毒症的常见并发症，目前仍没有成功针对脓毒症和SAE的分子靶向治疗方案。 .主要研究内容与重要结果：本项目通过前期建立的脓毒症小鼠模型和MFHAS1（恶性纤维性组织细胞瘤扩增序列1）泛素化炎症细胞模型，利用免疫共沉淀、免疫荧光共定位、泛素化实验、fear conditioning、Barnes maze、siRNA脑室内注射和神经棘突高尔基染色等实验手段，首次发现：1）Pam3CSK4（TLR2配体）可诱导小鼠RAW264.7细胞和THP-1人单核细胞产生MFHAS1，且呈时间依赖性；MFHAS1对TLR2信号通路和炎症有双重作用；TLR2刺激后MFHAS1诱导JNK和p38磷酸化；2）E3泛素连接酶praja2可以直接与MFHAS1结合；3）praja2促进MFHAS1泛素化积累，但并不降解MFHAS1；4）praja2泛素化MFHAS1，对TLR2介导的JNK/p38通路有正向调节作用，促进巨噬细胞向M1极化、M2向M1转化和炎症反应；总结praja2泛素化MFHAS1对M1/M2巨噬细胞极化和炎症的影响：MFHAS1的泛素化对TLR2、JNK和p38通路下游的信号通路起正向调节作用，导致巨噬细胞从M2向M1转化，从而促进炎症反应；5）侧脑室注射MFHAS1-siRNA降低MFHAS1脑内表达，可改善脓毒症大鼠认知功能障碍，降低大鼠脑海马区TNF-α、IL-1β和Aβ表达，减少锥体神经元树突上棘突密度的下降，增加脓毒症大鼠脑部CA1区基底部树突分支。.科学意义：本项目结果提示MFHAS1的泛素化修饰可调节巨噬细胞M1/M2转化和极化，具有促炎和抑瘤作用。本项目揭示了praja2作为一种新的炎症相关蛋白，其与MFHAS1的共同表达能预测炎症反应，并且TLR2/JNK/P38/NF-κB与MFHAS1一起构成泛素－蛋白酶体系统（UPS）驱动的信号通路。本研究结果将有助于设计更为有效的靶向治疗策略，以治疗脓毒症患者的过度炎症反应。此外，本项目揭示脑内MFHAS1 knock-down可减轻SAE大鼠认知功能障碍、神经炎症和神经元棘突的减少，这些结果将有助于设计更为有效的靶向治疗策略来治疗SAE患者的认知功能障碍。.