胞外囊泡携带血吸虫多肽SJMHE1调节巨噬细胞自噬抑制关节炎的作用与机制研究

Pathogen molecules with high homology of mammalian host may have a stronger anti-inflammatory effect under the evolutionary pressure. Our previous studies showed that SJMHE1, a nationally patented Schistosoma japonicum peptide, could induce regulatory T cells in vivo and in vitro, inhibit the delayed hypersensitivity of mice and inflammation in arthritis mice, but its mechanism is unclear. In pre-experiment, we found that SJMHE1 inhibited autophagy of macrophages, activated PI3K/Akt/mTOR pathway, and reduced autophagic flux in RAW264.7 cells; Furthermore, SJMHE1 suppressed autophagy in CIA mice in vivo by LC3B-II immunohistochemical staining, while SJMHE1-treated macrophages inhibited the proliferation and activation of CD4+ T cells. Therefore, we hypothesize that SJMHE1 regulates the autophagy of macrophage and suppresses the proliferation and activation of pathogenic CD4+ T cells, thereby inhibiting joint inflammation in arthritis mice. In this study, mouse macrophages, arthritis model mice and the inflammatory cells from rheumatoid arthritis patients were used to observe the effects of SJMHE1 on macrophage autophagy and CD4+ T cells proliferation and activation, thus investigating the role and mechanism of SJMHE1 in inhibiting the arthritis inflammation. In order to improve the stability of SJMHE1 in vivo, the extracellular vesicles (EVs) as natural nanocarriers will be used to load the SJMHE1 peptide. We will evaluate the nano-drug characteristics, the way of drug release in vitro and in vivo, etc. The study will provide new technologies and targets for the treatment of autoimmune or inflammatory diseases.

在进化压力下与哺乳动物宿主同源的病原体分子可能具有更强的抗炎效应。我们前期发现，已获得国家发明专利的血吸虫多肽SJMHE1体内外能诱导调节性T细胞，抑制小鼠迟发性超敏反应及关节炎小鼠炎症，但其机制不清；预实验发现SJMHE1抑制巨噬细胞(MФs)自噬，活化PI3K/Akt/mTOR通路，减少MФs自噬流；抑制关节炎小鼠自噬；而SJMHE1处理的MФs抑制CD4+T细胞的增殖与活化，因此，我们提出SJMHE1调节MФs自噬抑制致病性CD4+T细胞的增殖与活化，抑制小鼠关节炎症的假说。本研究拟用小鼠MФs、关节炎模型小鼠及关节炎患者炎性细胞，观察SJMHE1对MФs自噬及CD4+T细胞增殖与活化的影响，抑制关节炎症反应的作用与机制，为了提高SJMHE1体内稳定性，本研究用天然纳米载体胞外囊泡负载多肽，并全面评价该纳米药物性能、体内外释药方式等，为自身免疫或炎症性疾病的治疗提供新的技术与靶点。

在进化压力下与哺乳动物宿主同源的病原体分子可能具有更强的抗炎效应。我们前期发现，已获得国家发明专利的血吸虫多肽SJMHE1体内外能诱导调节性T细胞，抑制小鼠迟发性超敏反应及关节炎小鼠炎症。本研究中，我们构建了SJMHE1的脂质体/胞外囊泡杂合载体，并检测了血吸虫多肽SJMHE1在哮喘、肠炎、周围神经损伤等多种炎症性疾病中的作用与机制；并拓展研究了人脐带间充质干细胞胞外囊泡(hUCMSC-EVs)干预周围神经损伤、哮喘等疾病的作用与机制；结果，我们成功构建了负载多肽的脂质体/胞外囊泡纳米载体，杂合囊泡的尺寸在100 nm左右。该纳米载体在PBST中释放最少，对小鼠脾细胞及脏器无损伤；血吸虫多肽SJMHE1可通过调节哮喘小鼠脾细胞、肺组织中细胞因子的表达、调节哮喘小鼠脾细胞中Th1/Th2/Th17/Treg细胞比例抑制哮喘小鼠的气道炎症。机制上，血吸虫多肽SJMHE1经miR-155调节哮喘小鼠的Th17/Treg细胞平衡。SJMHE1多肽调节结肠炎小鼠脾脏、肠系膜淋巴结免疫细胞亚群比例，调节结肠炎小鼠结肠组织、脾细胞中炎症相关细胞因子的表达，抑制急、慢性结肠炎小鼠的炎症反应；SJMHE1处理可促进坐骨神经离断大鼠功能恢复和髓鞘再形成。SJMHE1诱导巨噬细胞向M2极化，分泌抗炎因子IL-10和TGF-β；机制上，SJMHE1通过上调巨噬细胞中miR-106b-5p表达并靶向IRF1，从而诱导巨噬细胞向M2极化，抑制炎症反应，为周围神经的修复创建了适宜的微环境。hUCMSC-EVs聚集于大鼠神经缺损部位，下调神经损伤部位的IL-6、IL-1β，上调IL-10的表达，调节损伤部位的炎症反应，促进神经再生；缺氧环境下hUCMSC-EVs富含miR-146a-5p，减少人肺成纤维细胞(HLF-1)经TGF-β1刺激后p-smad2/3、α-SMA 和collagen-1的表达，减轻慢性哮喘小鼠气道炎症和气道重塑；基于SJMHE1多肽抑制哮喘、关节炎、肠炎等多种炎症反应，促进周围神经损伤的修复与再生；hUCMSC-EVs调节周围神经损伤、哮喘等炎症反应；SJMHE1多肽、hUCMSC-EVs将开辟多种复杂、难治性炎症性疾病新的临床治疗途径，具有较强的临床转化潜力。