胰岛素通过激活Nrf2/GPX4通路抑制铁死亡改善脂多糖诱导的急性肺损伤

Insulin inhibits ferroptosis via activation of Nrf2/GPX4 pathway to ameliorate lipopolysaccharide induced acute lung injury

【摘要】目的 探究胰岛素对脂多糖(LPS)诱导的急性肺损伤(ALI)的作用及具体调控机制。方法 采用6~8周龄的C57BL/6雄性小鼠,按照随机数表法分为对照组(WT组)、LPS组、LPS+胰岛素组(LPS+INS组)和LPS+胰岛素+ML385(Nrf2蛋白抑制剂)组(LPS+INS+ML385组),每组6只。通过气管内滴注LPS建立LPS诱导的ALI模型,造模12h后处死,收集小鼠肺组织并进行检测。采用苏木精-伊红染色法检测肺损伤严重程度,采用吉姆萨染色检测支气管肺泡灌洗液(BALF)中炎症细胞水平,应用酶联免疫吸附试验测定BALF中促炎因子水平及肺组织中铁死亡相关指标,采用免疫印迹法测定铁死亡相关蛋白的表达水平。采用SPSS 19.0和GraphPad Prism 8软件进行数据分析。两组间比较采用t检验,多组间差异比较采用单因素方差分析。结果 与WT组比较,LPS组小鼠肺组织病理损伤加重,BALF中炎症细胞及促炎因子显著增加(均P＜0.05)。与LPS组相比,LPS+INS组小鼠肺组织中丙二醛显著下降,谷胱甘肽水平增加；LPS+INS组小鼠肺组织中核因子红细胞2相关因子2(Nrf2)、谷胱甘肽过氧化物酶4(GPX4)蛋白水平显著升高,与LPS+INS组相比,LPS+INS+ML385组Nrf2及GPX4蛋白水平显著下降(均P＜0.05)。结论 在ALI中,胰岛素通过激活Nrf2/GPX4通路减轻铁死亡,为临床干预ALI提供新靶点。

基金项目:国家自然科学基金青年项目(82200091)；国家自然科学基金面上项目(82370083)

【Abstract】Objective To investigate the role of insulin (INS) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the underlying mechanism. Methods Male C57BL/6 mice (6-8 weeks) were randomly divided into four groups (6 mice per group):control (WT), LPS, LPS+INS, and LPS+INS+ML385 (Nrf2 inhibitor) groups. Mouse model of ALI was established with intratracheal LPS instillation, and the mice were sacrificed to collect lung tissues in 12 h after modelling. HE staining was used to observe the severity of lung injury, Giemsa staining was employed to detect the inflammatory cells in bronchoalveolar lavage fluid (BALF), enzyme-linked immunosorbent assay was ultilized to measue the contents of pro-inflammatory cytokines and ferroptosis-related indicators in lung tissues, and Western blotting was applied to determine the expression of ferroptosis-related proteins. SPSS statistics 19.0 and GraphPad Prism 8 were used for statistical analysis. Student′s t test was conducted for comparison between two groups, and one-way analysis of variance was performed for comparison among multiple groups. Results Compared to the WT group, the LPS group exhibited significantly severer lung injury, more BALF inflammatory, and pro-inflammatory cytokines in lung tissues (all P<0.05). INS treatment resulted in decreased malondialdehyde and increased glutathione contents, and elevated protein levels of Nrf2 and GPX4 in lung tissues when compared with the LPS group (P<0.05). Compared with the LPS+INS group, the protein levels of Nrf2 and GPX4 in the LPS+INS+ML385 group decreased significantly (P<0.05). Conclusion Insulin alleviates ferroptosis in ALI by activating the Nrf2/GPX4 pathway, suggesting a promising therapeutic target for ALI treatment.

This work was supported by the National Natural Science Foundation for Young Scholars of China (82200091) and the General Program ofNational Natural Science Foundation of China (82370083).