PEAR1的SUMO化修饰在小鼠肠缺血再灌注致肺损伤模型中的作用及其机制

ALI/ARDS is a common clinical disease with high mortality, but so far there is lack of effective treatment. The proliferation of pulmonary microvascular endothelial cells is identified to repair vascular barrier function, thus alleviating ALI/ARDS. Therefore, the proliferation of pulmonary microvascular endothelial cells has become a potential target for the treatment of ALI/ARDS. However, little is known about its molecular mechanism. The latest research indicated that platelet endothelial aggregation receptor-1 (PEAR1) suppressed the proliferation of endothelial cells. Our previous study found that knockout of PEAR1 could promote the proliferation of pulmonary microvascular endothelial cells and reduce lung injury; PEAR1 could be SUMOylated in the pulmonary of the mice, and PEAR1 SUMOylation decreased after intestinal ischemia/ reperfusion; PEAR1 SUMOylation may be associated with activation of PI3K(p110γ)/AKT pathway in the proliferation of pulmonary microvascular endothelial cells. In this paper we will establish the stable transfection cell lines of shPEAR1 human pulmonary microvascular endothelial cell and the model of intestinal ischemia/reperfusion inducing lung injury in mice, to further clarify the effect of PEAR1 SUMOylation on the proliferation of pulmonary microvascular endothelial cells and ALI/ARDS, and to explore its signal transduction mechanism. Our study will lay the foundation for the exploration of the mechanism and control methods of ALI/ARDS.

ALI/ARDS是病死率高但又缺乏有效治疗手段的临床常见疾病。肺微血管内皮细胞的增殖可以修复微血管屏障功能，减轻ALI/ARDS，是治疗ALI/ARDS备受关注的潜在靶点，但其相关分子机制尚不清楚。最新研究表明血小板内皮聚集受体-1（PEAR1）可以抑制内皮细胞的增殖。本课题的前期研究工作发现，敲除PEAR1可以促进肺微血管内皮细胞的增殖，减轻肺损伤；PEAR1可被SUMO化修饰，且在肠缺血再灌注后PEAR1的SUMO化减弱；PEAR1的SUMO化修饰可能通过激活PI3K(p110γ)/AKT通路来促进肺微血管内皮细胞增殖。本课题拟建立shPEAR1人肺微血管内皮细胞系和小鼠肠缺血再灌注致肺损伤模型，通过SUMO位点突变法，进一步明确PEAR1的SUMO化可以促进肺微血管内皮细胞增殖并在ALI/ARDS中起保护作用，并探讨其信号转导机制，为ALI/ARDS的治疗提供理论基础和潜在新靶点。

目的:课题组前期研究发现，敲除PEAR1可以促进肺微血管内皮细胞的增殖，减轻急性肺损伤的发生发展，然而具体的调控机制有待进一步明确。本研究旨在探讨PEAR1及其SUMO化修饰对肺微血管内皮细胞增殖的影响并在急性肺损伤中所起的作用，并阐明其信号转导机制。内容:利用PEAR1敲除小鼠和siPEAR1人肺微血管内皮细胞（HPMEC），建立急性肺损伤模型，分析PEAR1表达的变化，通过检测肺血管通透性、炎性因子和细胞增殖，研究PEAR1对肺微血管内皮细胞增殖作用的影响和对急性肺损伤发生发展的作用。使用PI3K抑制剂，通过CCK8计数和EdU细胞流式检测，观察PI3K／AKT途径在PEAR1调节来肺微血管内皮细胞增殖中所起的作用。构建PEAR1过表达质粒和其SUMO位点突变质粒，通过免疫共沉淀方法明确PEAR1的SUMO化修饰类型、位点和去SUMO化（deSUMO化）的SENP类型。在HPMEC中过表达PEAR1的SUMO位点突变质粒，使用EdU细胞流式检测和细胞划痕实验，观察肺微血管内皮细胞的增殖和迁移变化，同时利用PI3K抑制剂，观察PEAR1的SUMO化修饰是否通过PI3K／AKT通路调节肺微血管细胞的增殖。结果:急性肺损伤中肺微血管内皮细胞的PEAR1及其SUMO化修饰水平显著升高，并伴有细胞通透性增大、炎性因子增多等肺损伤加重的表现。PEAR1敲除小鼠和siPEAR1 HPMEC的细胞增殖水平显著高于WT小鼠和WT HPMEC，且肺损伤病理表现明显减轻。PEAR1的增高伴随着PI3K／AKT通路表达水平的下降。通过构建并过表达PEAR1及其SUMO化突变位点质粒，确定了PEAR1的SUMO化修饰条带、类型和位点，并发现PEAR1的SUMO位点突变组细胞的增殖和迁移能力显著强于SUMO化修饰组。使用PI3K抑制剂后，PEAR1 SUMO化修饰组和PEAR1 SUMO位点突变组细细胞的增殖和迁移能力都显著下降。结论与研究意义：本研究表明PEAR1及其SUMO化修饰通过调节PI3K／AKT途径来影响肺微血管内皮细胞的增殖和迁移，从而加重肺血管通透性和炎性因子的释放、加速急性肺损伤的发生发展。降低PEAR1的SUMO化修饰水平可以有效逆转或抑制肺微血管内皮细胞增殖受损的情况，为急性肺损伤提供潜在的治疗靶标。