IL-18在脓毒症肺损伤发生发展过程中的作用及机制研究

Role of IL-18 on occurrence and development of sepsis induced lung injury and its mechanism

目的:研究IL-18在脓毒症肺损伤发展过程中的作用及调控机制。方法:成年雄性野生型C57BL/6(WT)和IL-18基因敲除(IL-18-/-)小鼠分为野生型小鼠对照组(WT组)、脂多糖(LPS)处理的野生型小鼠组(WT LPS组)、IL-18基因敲除的小鼠对照组(IL-18-/-组)、LPS处理的IL-18基因敲除小鼠组(IL-18-/-LPS组)。腹腔注射LPS(15 mg/kg)建立小鼠脓毒症模型,对照组注射等量生理盐水。观察各组小鼠72 h生存率并处死小鼠,HE染色观察肺部病理组织变化,RT-PCR及Western blot检测各组小鼠肺组织IL-18 mRNA及蛋白表达,免疫荧光检测肺组织IL-18表达及定位,TUNEL染色检测肺组织细胞凋亡,流式细胞术检测肺组织Treg/Th17比例,ELISA检测炎症因子TNF-α、IL-17A、TGF-1β、IL-10表达。Western blot检测各组小鼠肺组织RORγt、FoxP3蛋白表达及STAT3磷酸化水平。结果:LPS诱导后,小鼠肺组织高表达IL-18。与WT LPS组相比,IL-18-/-LPS组小鼠生存率显著提高,肺组织病理损伤减轻,凋亡细胞显著减少,Treg/Th17比例提高,抑炎因子TGF-1β、IL-10表达增加,而促炎因子TNF-α、IL-17A表达明显减少,肺组织RORγt蛋白表达增加,而STAT3磷酸化水平降低。结论:IL-18可通过上调STAT3磷酸化水平,促进Treg/Th17免疫失衡及炎症因子分泌,从而加剧脓毒症急性肺损伤。

Objective:To study role of IL-18 on development of sepsis lung injury and its regulatory mechanism.Methods:Adult male wild-type C57BL/6(WT)and IL-18 gene knockout(IL-18-/-)mice were divided into wild-type mice control group(WT group),lipopolysaccharide(LPS)treated wild-type mice group(WT LPS group),IL-18 gene knockout mice control group(IL-18-/-)and LPS treated IL-18 gene knockout mice group(IL-18-/-LPS group).Sepsis model was established by intraperitoneal injection of LPS(15 mg/kg)in mice,and control group was injected with equivalent normal saline.Survival rate of mice in each group was observed for 72 h and were killed,pathological changes of lung tissues were observed by HE staining.Expressions of mRNA and protein of IL-18 in lung tissues of mice in each group were detected by RT-PCR and Western blot.Expression and localization of IL-18 in lung tissue were detected by immunofluorescence;apoptosis in lung tissue was detected by TUNEL staining;proportions of Treg/Th17in lung tissue were detected by flow cytometry;expressions of TNF-α,IL-17A,TGF-1βand IL-10 were detected by ELISA.Western blot was used to detect expressions of RORγt,FoxP3 and phosphorylation of STAT3.Results:After LPS induction,IL-18 was highly expressed in lung tissues of mice.Compared with WT LPS group,survival rate of mice in IL-18-/-LPS group was significantly increased,lung tissue damage was relieved,apoptotic cells in lung tissue were reduced,proportions of Treg/Th17 were increased,expressions of anti-inflammatory factors TGF-1β,IL-10 were increased,while expressions of pro-inflammatory factors TNF-αand IL-17A were decreased,expression of RORγt protein was increased,and phosphorylation level of STAT3 were decreased.Conclusion:IL-18 can increase STAT3 phosphorylation level,promote immune imbalance of Treg/Th17 and secretion of pro-inflammatory factors,so as to aggravate acute lung injury in sepsis.