三基序蛋白59通过调控巨噬细胞HIF-1α介导的乳酸分泌和IL-10表达发挥抑制炎症和肺损伤的作用

Tripartite motif-containing protein 59 inhibits inflammation and lung injury by regulating HIF-1α-mediated lactic acid secretion and IL-10 expression in macrophages

目的探究三基序蛋白59(tripartite motif-containing protein 59, TRIM59)对巨噬细胞糖代谢的影响及其在炎症状态下调控巨噬细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)/IL-10轴的作用。方法对高表达TRIM59的巨噬细胞及转染TRIM59空载质粒的巨噬细胞(对照组)进行差异表达基因GO、KEGG聚类分析。RT-qPCR和Western blot检测TRIM59高表达的RAW264.7巨噬细胞经脂多糖(lipopolysaccharide, LPS)刺激不同时间后HIF-1α的表达水平。取TRIM59-cKO和TRIM59flox/flox小鼠骨髓诱导成骨髓来源巨噬细胞(bone marrow-derived macrophages, BMDMs), 收集LPS刺激后3、6、12、24 h的上清液, ELISA检测IL-10水平。此外, 建立小鼠盲肠结扎手术(cecum ligation and puncture, CLP)模型, 收集相同时间点的肺泡灌洗液, ELISA检测IL-10水平, 并采集肺组织进行HE染色观察组织病理变化。采用Student′st检验进行统计学分析。结果 TRIM59高表达的巨噬细胞中糖代谢相关基因变化显著, 乳酸含量明显升高。LPS刺激后, TRIM59-cKO小鼠BMDMs中HIF-1α基因表达下调(P<0.05), IL-10水平在3 h和24 h升高, 在6 h和12 h与对照组相比差异无统计学意义。TRIM59基因缺失后, CLP模型鼠肺泡灌洗液中IL-10含量随着时间的推移出现升高现象, 在24 h出现下降趋势, 但均高于对照组(P<0.05或P<0.01)。结论 TRIM59可抑制巨噬细胞HIF-1α介导的乳酸分泌以及IL-10表达, 进而使其发挥抑制炎症及减轻肺损伤的作用。本研究可为基于TRIM59开发新型抑制脓毒血症药物提供参考。

Objective To investigate the effect of tripartite motif-containing protein 59(TRIM59)on glucose metabolism in macrophages and its role in regulating hypoxia-inducible factor-1α(HIF-1α)/IL-10 axis in macrophages under inflammatory conditions.Methods The differentially expressed genes between macrophages with high expression of TRIM59 and control cells transfected with empty TRIM59 plasmid were analyzed by GO and KEGG.The expression of HIF-1αby RAW264.7 macrophages with high expression of TRIM59 was detected at different time points after lipopolysaccharide(LPS)stimulation by RT-qPCR and Western blot.Bone marrow was isolated from TRIM59-cKO and TRIM59flox/flox mice and induced to differentiate into bone marrow-derived macrophages(BMDMs).These BMDMs were stimulated with LPS and the supernatants of cell culture were collected at 3,6,12 and 24 h after stimulation to detect IL-10 level by ELISA.In addition,mouse models of cecal ligation and puncture(CLP)were established,and bronchoalveolar lavage fluid(BALF)samples were collected at the same time points to detect IL-10 level by ELISA.Histopathological changes in lung tissues were observed after HE staining.Results There was a significant change in glucose metabolism-related genes in macrophages with high expression of TRIM59,and the content of lactic acid increased significantly.Compared with the control group,the expression of HIF-1αat mRNA level in BMDMs from TRIM59-cKO mice decreased after LPS stimulation(P<0.05);the level of IL-10 increased at 3 h and 24 h in the TRIM59-cKO group,but there was no significant difference in IL-10 level at 6 h or 12 h between the two groups.In the TRIM59-cKO mouse model of CLP,the levels of IL-10 in the BALF samples increased with time,but decreased at 24 h.The level of IL-10 was higher in the TRIM59-cKO mouse model group than that in the control group at each time point(P<0.05 or P<0.01).Conclusions TRIM59 can inhibit inflammation and lung injury by decreasing HIF-1α-mediated lactate secretion and IL-10 expression in macrophages.This study provides a new idea for developing novel anti-sepsis drugs based on TRIM59.