PAD4抑制剂GSK484通过抑制H3Cit表达减轻小鼠脓毒症肺损伤后内皮功能障碍的发生  

GSK484,a PAD4 inhibitor,improves endothelial dysfunction in mice with sepsisinduced lung injury by inhibiting H3Cit expression

作　　者：苏晓飞 李霖 戴靖榕 肖宝 金子琦 刘斌[1] SU Xiaofei;LI Lin;DAI Jingrong;XIAO Bao;JIN Ziqi;LIU Bin(Department of Emergency Medicine,Central South University(Changsha First Hospital),Changsha 410005,China;Department of General Medicine,Affiliated Changsha Hospital of Xiangya School of Medicine,Central South University(Changsha First Hospital),Changsha 410005,China)

机构地区：[1]中南大学湘雅医学院附属长沙医院//长沙市第一医院急诊科,湖南长沙410005 [2]中南大学湘雅医学院附属长沙医院//长沙市第一医院全科医学科,湖南长沙410005

出　　处：《南方医科大学学报》2024年第12期2396-2403,共8页Journal of Southern Medical University

基　　金：长沙市自然科学基金(Kq2202010);睿意急诊医学研究专项基金(R2020009)。

摘　　要：目的探讨PAD4抑制剂(GSK484)对脓毒症后H3Cit表达的抑制作用,并探究其对脓毒症引起的内皮功能障碍的改善效果。方法C57BL/6小鼠18只通过盲肠结扎穿孔术(CLP)构建小鼠脓毒症模型,实验分为假手术组,模型组和GSK484治疗组,6只/组,治疗组在术后第2天腹腔注射给予GSK484(4 mg/kg)。ELISA检测小鼠血清VEGF、ESM-1、IL-6、IL-1β水平,HE染色观察肺组织病理学变化,免疫荧光和Western blotting检测各组小鼠肺组织中F-actin、VE-cadherin、ZO-1蛋白表达,Western blotting检测肺组织VE-Cadherin和H3Cit蛋白表达情况。肺组织原代分离培养肺微血管内皮细胞,使用脂多糖(LPS,10μg/mL)干预24 h构建脓毒症模型。检测细胞成管能力,CCK-8检测细胞增殖,流式检测细胞凋亡,ELISA检测细胞上清中VEGF、ESM-1、IL-6、IL-1β水平。结果与假手术组相比,脓毒症小鼠肺组织肺脏血管充血、肺泡断裂、肺泡腔及肺间质水肿、肺泡隔增厚、中性粒细胞浸润等,血清中炎症因子IL-6、IL-1β和内皮细胞因子VEGF、ESM-1含量均升高(P<0.05),肺组织中胞间连接蛋白F-actin、VE-cadherin、ZO-1表达降低,H3Cit蛋白表达升高(P<0.05),GSK484治疗可以有效改善这些变化。LPS诱导肺微血管内皮细胞上清中炎症因子表达量升高、内皮细胞因子VEGF、ESM-1含量均升高(P<0.05),细胞成管能力降低;2.5μmol/L浓度GSK484处理可降低炎症因子和内皮细胞因子表达(P<0.05)。结论GSK484的使用可有效抑制脓毒症后H3Cit表达,进一步改善脓毒症后内皮功能障碍的发生。Objective To investigate the inhibitory effect of GSK484,a PAD4 inhibitor,on H3Cit expression following sepsis and its effects for improving sepsis-induced endothelial dysfunction.Methods Eighteen C57BL/6 mice were randomized into sham-operated group,sepsis model group and GSK484 treatment group(n=6),and in the latter two groups,models of sepsis were established by cecal ligation and puncture(CLP).The mice in GSK484 treatment group were given an intraperitoneal injection of GSK484(4 mg/kg)on the second day following the surgery.Twenty-four hours after the injection,the mice were euthanized for measurement of serum levels of VEGF,ESM-1,IL-6 and IL-1βusing ELISA.Lung tissue pathology was observed with HE staining,and pulmonary expressions of F-actin,VE-cadherin,ZO-1 and H3Cit proteins were detected using immunofluorescence staining and Western blotting.In primary cultured of mouse lung microvascular endothelial cells,the effect of stimulation with LPS(10μg/mL)for 24 h on tube formation,proliferation,apoptosis and expressions of VEGF,ESM-1,IL-6 and IL-1βwere assessed using CCK-8 assay,flow cytometry and ELISA.Results Compared to the sham-operated mice,the septic mice exhibited significant lung tissue pathologies characterized by vascular congestion,alveolar rupture,edema,and neutrophil infiltration.Serum levels of IL-6,IL-1β,VEGF,and ESM-1 were elevated,pulmonary expressions of F-actin,VEcadherin,and ZO-1 were decreased,and H3Cit expression was increased significantly in the septic mice.GSK484 treatment effectively mitigated these changes in the septic mice.The LPS-stimulated endothelial cells showed increased productions of IL-6,IL-1β,VEGF and ESM-1,which were significantly reduced after treatment with 2.5μmol/L GSK484.Conclusion GSK484 treatment effectively suppresses H3Cit expression in septic mice to ameliorate sepsis-induced endothelial dysfunction.

关 键 词：脓毒症 肺损伤 PAD4抑制剂 小鼠肺微血管内皮细胞 内皮功能障碍 GSK484 

分 类 号：R459.7[医药卫生—急诊医学]