MANF通过调控巨噬细胞PRDX6表达及活性抑制急性肺损伤的作用及机制研究

MANF is an endoplasmic reticulum stress response protein with anti-inflammatory effect, but its function in acute lung injury (ALI) has not been reported in home and abroad. It is well known that the up-regulated expression of PRDX6 and the increasing of its phospholipase activity are closely related with ALI. Results from our pre- experiment showed the up-regulated expression of MANF in LPS-induced lung tissues, and its co-localization with PRDX6 in macrophages. We also found that the expression of MANF and PRDX6 was negatively correlated in LPS-induced lung tissue. The administration of exogenous recombinant human MANF can alleviate lung injury. We assume that MANF alleviate ALI through inhibition of PRDX6 and its phospholipase activity in macrophages. To demonstrate the hypothesis, we will firstly detected the expression of MANF in lung and demonstrate the anti-inflammatory role of MANF in LPS induced lung injury, using wild type mice and macrophage MANF gene knockout mice, combined with administration of recombinant human MANF; secondly, we will demonstrate that the anti-inflammatory role of MANF depends on PRDX6, using PRDX6 gene knockout mice; thirdly, we will demonstrate that MANF plays an anti-inflammatory role in ALI by inhibiting the expression and activity of PRDX6. This study will elucidate the inhibiting role of MANF in ALI and the mechanism, and provide new ideas for MANF as a target drug in ALI treatment.

MANF是内质网应激反应蛋白，具有抗炎作用，其在急性肺损伤（ALI）中的作用国内外未见报道。PRDX6表达上调及其磷脂酶（iPLA2）活性增加与ALI密切相关。我们前期发现:MANF在ALI小鼠肺组织中表达升高，在巨噬细胞中与PRDX6有共定位，在体外与PRDX6的表达负相关；重组人MANF能减少ALI小鼠肺组织炎症细胞浸润。推测MANF可能通过下调巨噬细胞PRDX6表达及抑制其iPLA2活性减轻ALI。为此，我们将以野生型及巨噬细胞MANF特异性敲除鼠为研究对象，用LPS诱导ALI模型，观察MANF表达，给予重组人MANF，确认MANF抑制ALI的作用；用PRDX6基因敲除鼠制备ALI模型，证明MANF抑制ALI的作用依赖于PRDX6；证明MANF通过抑制PRDX6的表达及其活性发挥抗ALI作用。该研究将阐明MANF抑制ALI的作用及机制，为MANF作为治疗ALI的靶点药物提供新思路。

MANF能够抑制多种炎症，但其在急性肺损伤（ALI）中的作用未见报道。PRDX6表达上调及其磷脂酶（iPLA2）活性增加与ALI密切相关。激活的巨噬细胞是iPLA2的主要来源。我们前期发现: MANF在ALI小鼠肺组织中表达明显升高，在巨噬细胞中与PRDX6有共定位，但与PRDX6的表达量负相关；重组人MANF能够减少ALI小鼠肺组织炎症细胞浸润。推测: MANF通过下调巨噬细胞PRDX6表达及其iPLA2活性抑制ALI。我们发现，MANF在人炎症性肺组织及LPS诱导ALI小鼠肺组织及肺泡巨噬细胞中的表达增高，而给予外源性的MANF蛋白可以抑制小鼠ALI。单核巨噬细胞MANF敲除会引起肺部炎症，并加重LPS诱导的肺部损伤。此外，我们还发现ALI大鼠肺组织中的PRDX6表达增加，而与PRDX6磷脂酶活性密切相关的ROS产生也增加；给予外源性的MANF会减少ALI大鼠PRDX6的蛋白表达及肺组织ROS产生；而PRDX6基因敲除后，针对ALI，MANF不再具有显著的抑制作用。本项目的研究结果表明，MANF对ALI具有保护性作用，可以抑制巨噬细胞的过度激活。在进一步的分子机制研究中，我们发现MANF能够抑制ALI大鼠PRDX6的表达。而PRDX6主要来源于激活的巨噬细胞，其表达和磷脂酶活性与组织损伤介质ROS的产生密切相关。本项目揭示了MANF对ALI的保护性作用及细胞和分子机制，为ALI药物治疗提供新思路。