磷酸酶Wip1介导中性粒细胞功能，调控重症脓毒性腹膜炎机制探讨

Emerging evidence highlights the importance of a new member of the PP2C family of phosphatase, Wild-type p53 induced phosphatase 1 (Wip1), in regulating DNA damage-induced and tumorigenesis-induced networks. Latest studies also showed that Wip1 played a critical role in regulating neural development and Hepatocyte regeneration. However, there is still no related reports on the exact role of Wip1 in the regulation of severe infection. In our clinical studies, we found that downrgulation of Wip1 occurs both in the neutrophils of non-severe septic peritonitis patients and severe septic peritonitis patients, while patients with non-severe septic peritonitis showed a sharpen reduction of Wip1 expression; with the use of animal model of severe septic peritonitis(CLP), we further found that Wip1 knock out mice showed resistent to severe septic peritonitis with more infiltration of neutrophils into abdominal cavity and less infiltration into lung tissues. With the help of cytometry, we discovered that the level of CXCR2 on neutrophils in Wip1 knock out mice after CLP was significantly higher than that in wild type mice. Taken together, these results suggested that Wip1 may influence the expression of CXCR2 on neutrophils to mediate the infiltration of neutrophils into abdominal cavity and further complete the function of eliminating pathogens. In the present study, we aimed to investigate the celluar and molecular mechanisms of Wip1 in regulating severe septic peritonitis. By employing transgenic mice such as Wip1-/- , CXCR2-/- and Wip1-/-CXCR2-/- mice, which would receive CLP and in vitro fuctional studies. Based on the experimental evidence, We hope to propose a new idea of the therapuetic strategy for severe septic peritonitis.

Wip1是磷酸酶PP2C家族的一员，调控着神经、肝细胞再生、细胞修复等多种生命活动，但在重症感染领域的研究尚属空白。我们的前期研究发现脓毒性腹膜炎患者与健康志愿者相比，外周血中性粒细胞内Wip1表达水平有所下调，但在重症患者，Wip1的下调程度不如轻症患者显著，且伴随有趋化能力减弱；通过构建小鼠重症脓毒性腹膜炎模型，我们发现Wip1-/-小鼠生存率显著高于Wip1+/+小鼠，且腹腔内菌落计数显著减少，进一步地运用流式细胞术，我们发现Wip1-/-小鼠外周血粒细胞CXCR2表达水平显著高于Wip1+/+小鼠，上述结果提示Wip1可能通过影响粒细胞表达CXCR2，介导其在感染灶的浸润，从而对重症脓毒性腹膜炎起到调控作用。因此，运用Wip1-/-CXCR2-/-、Wip1-/-、CXCR2-/-等多种基因敲除小鼠，深入探索Wip1调控重症脓毒性腹膜炎的机制，将为临床治疗提供新的思路。

中性粒细胞作为机体外周循环内数量最大的细胞群体，在抵御外源病原微生物入侵时发挥重要作用，然而其在脓毒症这一与感染密切相关的病理生理过程中，却存在趋化到原发感染灶及清除病原菌功能的降低，如何逆转这一现象、充分调动中性粒细胞的免疫功能是治疗脓毒症新的方向。我们的研究发现磷酸酶Wip1在调控中性粒细胞的发育及免疫应答方面扮演着重要角色。中性粒细胞内特异性缺失Wip1能显著增强中性粒细胞的趋化能力，促使其更多地浸润到原发感染灶，同时强化其杀伤菌群的能力，与Wip1缺失通过p38信号通路阻止了趋化因子受体CXCR2在脓毒症中的内吞有关；当用Wip1抑制剂预处理小鼠然后构建脓毒症模型后，抑制剂处理依然能够通过降低Wip1在中性粒细胞的表达，显著促进其在原发感染灶腹腔内的浸润，提高菌群清除能力，并改善脓毒症预后。在人类样本中，我们也发现类似的结果，脓毒症能降低Wip1在中性粒细胞内的表达，而伴有脓毒性休克的患者，其体内中性粒细胞Wip1下降程度显著高于不伴有脓毒性休克的患者，并且趋化能力及CXCR2的表达水平显著降低，提示Wip1的下降程度对于中性粒细胞功能的发挥呈现负调控作用；在体外实验中，当使用Wip1抑制剂处理中性粒细胞后，能显著逆转由于格兰阴性菌成分脂多糖刺激导致的中性粒细胞趋化能力的降低，进一步提示Wip1对于中性粒细胞趋化及杀菌功能的发挥的重要性，也为未来运用磷酸酶Wip1抑制剂治疗脓毒症提供理论依据。