CHOP调控M2型巨噬细胞促进血吸虫肝肉芽肿和纤维化的机制研究

Hepatic granulomas is due to the deposition of schistosome eggs in microvasculature of the liver. It precipitates the formation of liver fibrosis which obstructs blood flow, increases portal blood pressure, and ultimately attributes to much of the mortality of schistosomiasis. However, the mechanisms of granulomas formation and the development of liver fibrosis is still unclear. It was reported previously that the C/EBP homologous protein (CHOP), a pivotal pro-apoptotic regulatory factor, facilitated the formation of fibrosis in the heart, kidney and liver. The applicants have demonstrated that CHOP played an important role in the infiltration of macrophages, and then is tightly associated with renal and lung fibrosis. Based on the preliminary data, we speculate that CHOP should play a vital role in schistosome induced hepatic granulomas and liver fibrosis. This study is designed to reveal the regulatory function of CHOP in hepatic granulomas and liver fibrosis by detecting the morphological and pathological differences among human, CHOP deficiency and wild type C57BL/6 mice infected with schistosome. And then, to elaborate the cellular mechanism of CHOP in schistosomiasis by applying immunological techniques and in vitro cellular researches. Finally, to analyse the signal pathway of the CHOP regulation by a comprehensively analysis of the data from immunological studies, Chip sequence, immunoprecipitation and other molecular biology studies. The ultimate purpose of this study is to provide a treatment target for schistosomiasis at the early stage, and improve living quality of the patients of schistosomiasis.

血吸虫虫卵在肝脏沉积诱发肉芽肿及纤维化形成的病理机制十分复杂，目前仍是血吸虫病研究的核心科学问题，既往研究业已揭示M2型巨噬细胞在纤维化形成过程中发挥着重要作用。申报者前期研究发现C/EBP同源蛋白CHOP参与肺纤维化，通过对CHOP敲除小鼠的研究揭示CHOP参与巨噬细胞的浸润，且与肾和肺纤维化的形成有关， 但相关机制有待阐明。申报者依据上述结果假设：CHOP通过调节巨噬细胞M2极化作用促进日本血吸虫肝肉芽肿和纤维化。本课题拟在前期工作基础上，首先在血吸虫感染人群、CHOP敲除与野生型C57BL/6小鼠中，用形态学和病理学比对分析，确证CHOP在血吸虫所致肝肉芽肿及纤维化中的病理作用；继而解析CHOP介导血吸虫肝肉芽肿和纤维化的细胞学机制；最后拟阐明CHOP调控巨噬细胞朝M2型分化的分子机制。本研究将从内质网应激角度探究血吸虫性肝肉芽肿及纤维化的病理机制，为临床预防和早期治疗提供有效靶点。

血吸虫虫卵在肝脏沉积诱发肉芽肿及纤维化是血吸虫病研究的核心科学问题，肝脏纤维化程度与患者预后密切相关，预防、减轻、延缓甚至阻止血吸虫性肝脏纤维化仍是尚未解决的重大疾病防治问题。既往研究揭示M2型巨噬细胞在纤维化形成的过程中发挥重要作用。我们前期研究发现C/EBP同源蛋白CHOP参与肺纤维化，通过对CHOP敲除小鼠的研究揭示CHOP参与巨噬细胞的浸润，且与肾和肺纤维化的形成有关。CHOP及M2型巨噬细胞在血吸虫致肝纤维化中相关机制有待阐明。我们首先通过人群实验观察血吸虫病患者肝脏纤维化B超影像学变化、CHOP蛋白、肝纤维化四项（HA、LN、PCⅢ及Ⅳ-C）和相关炎症因子的表达，并分析其相关性。然后建立日本血吸虫尾蚴感染小鼠模型，检测感染小鼠在不同感染时期肝脏肉芽肿及纤维化的形态学改变、CHOP蛋白、巨噬细胞活化情况及相关通路STAT6、pSTAT6和KLF4表达。进一步在RAW264.7细胞水平，观察IL-4对CHOP、STAT6、pSTAT6、KLF4及IL-13Rα表达改变和细胞M2极化状态，而后使用STAT6磷酸化抑制剂及CHOP过表达质粒，观察细胞M2极化状态及STAT6、pSTAT6、KLF4表达，最后使用SEA对CHOP、STAT6、pSTAT6和KLF4表达改变。结果显示，日本血吸虫病致不同程度肝纤维化患者中，CHOP及炎症因子表达增加，M2巨噬细胞标志分子表达增加且与肝纤维化四项指标呈较强正相关。小鼠肝肉芽肿和纤维化随感染时间延长而加重，肝指数增加、肝脏胶原纤维沉积增加、M2巨噬细胞极化标志物表达增加，CHOP表达增加以及STAT6、pSTAT6及IL-13Rα表达增加，KLF4表达降低。进一步在RAW264.7细胞水平观察到，IL-4时间及剂量依赖性增加CHOP、STAT6、pSTAT6、KLF4及IL-13Rα表达，促进细胞向M2极化，而后使用STAT6磷酸化抑制剂后，抑制IL-4诱导的M2巨噬细胞活化及KLF4、CHOP和IL-13Rα的表达。使用CHOP过表达，发现M2型巨噬细胞增多，STAT6和KLF4增加。SEA刺激后p-STAT6、KLF4和CHOP蛋白表达增加，且SEA+IL-4组增加更明显。由此，我们提出，CHOP介导的M2型巨噬细胞极化参与了日本血吸虫致肝纤维化，其机制与IL-4/pSTAT6/KLF4信号通路介导有关。