神经营养因子MANF通过调控肝脏单核巨噬细胞功能发挥抗药物性肝损伤作用

Acetaminophen (APAP) overdose is the main cause of acute liver failure. Following liver injury, large numbers of Ly6C(hi) monocytes traffic to the liver. Ly6C(hi) monocytes aggravate inflammation and liver injury. Then Ly6C(hi) monocytes differentiate into Ly6C(low) monocyte-derived macrophages, which are involved in the resolution of liver injury. However, the precise mechanisms underlying the transition from inflammatory Ly6C(hi) monocytes into pro-resolution Ly6C(low) monocyte-derived macrophages are still unclear. Our previous studies have found that MANF deficiency in myeloid cells of mice impaired the recovery from APAP-induced liver injury(AILI). And the percentages and numbers of Ly6C(low) macrophages during resolution were reduced in MANF-deficient mice (myeloid-cell-specific deficiency). We hypothesize that MANF protects against AILI by regulating monocyte/macrophage function. To test our hypothesis, myeloid cell MANF deficient mice, hrMANF, and cell transfer experiments will be used to investigate the role of monocyte/macrophage-derived MANF in AILI. Then, the role of MANF in promoting the transition of inflammatory Ly6C(hi) monocytes into pro-resolution Ly6C(low) macrophages will be investigated. And the molecular mechanisms involved in the process will be also investigated by FACS analysis, RNA sequencing analysis, and immunofluorescence. Last, the above-mentioned findings will be confirmed in samples of patients with AILI. This proposal may clarify the mechanisms underlining regulating the function of monocytes/macrophages. This project also may provide some evidence to developing new therapeutic strategies in drug-induced liver injury through targeting MANF and monocytes/macrophages.

对乙酰氨基酚(APAP)摄入过量是引起急性肝衰竭的重要原因。肝细胞损伤后，单核细胞浸润到肝脏，参与炎症反应；炎性单核细胞随后转化为修复型巨噬细胞，促进损伤修复。目前调控单核巨噬细胞功能转化的机制仍不清楚。前期研究发现，神经营养因子MANF在小鼠髓系细胞中缺失，会延缓APAP诱导的肝脏损伤(AILI)修复，降低损伤恢复期修复型巨噬细胞的比例和数量。我们推测，MANF通过调控肝脏单核巨噬细胞功能来促进AILI修复。本课题拟使用髓系细胞特异性MANF敲除鼠、hrMANF和细胞转输实验，阐释单核巨噬细胞来源的MANF促进AILI修复；通过流式分析，RNA测序等揭示MANF调控单核巨噬由促炎型向修复型转化的分子机制；进一步利用APAP肝损伤病人标本对上述机制加以验证。单核巨噬细胞的调控机制的阐明，将为以MANF和单核巨噬细胞为靶点治疗药物性肝损伤提供理论和实验依据。

对乙酰氨基酚（APAP）服用过量是导致人类急性肝衰竭(ALF)的主要原因，可导致肝细胞坏死，继而激活天然免疫系统，进一步加重肝脏损伤。APAP过量后,具有表型可塑性的巨噬细胞在肝脏炎症、损伤、纤维化和消退中发挥着重要作用。了解巨噬细胞招募和功能的确切潜在机制将有助于确定新的治疗靶点和干预措施。我们研究发现APAP 腹腔注射野生型小鼠12 小时后，肝脏非实质细胞中神经营养因子（MANF）表达明显上调。在APAP 诱导的小鼠肝脏损伤模型中，髓系细胞特异性MANF 敲除鼠中肝脏损伤水平升高，修复型巨噬细胞比例和数量降低，并且IL10 表达水平下调。注射重组人源MANF蛋白(rhMANF)后，肝脏损伤水平降低，修复型巨噬细胞比例和数量升高，并且IL10 表达水平上调，巨噬细胞吞噬功能增强。本研究揭示MANF 通过p38 信号通路促进巨噬细胞分泌IL10，以及促进巨噬细胞吞噬功能来抑制DILI。这一研究将为以巨噬细胞和MANF 为靶点的新药开发提供理论依据。