基于Nrf2/NQO1/GPX4 信号通路探讨益气通腑泻热方改善小鼠急性肺损伤的作用机制

Exploration on the Mechanism of Yiqi Tongfu Xiere Prescription in Ameliorating Acute Lung Injury in Mice Based on the Nrf2/NQO1/GPX4 Signaling Pathway

摘要:

目的：基于核因子E2相关因子2（Nrf2） /NAD（P） H醌氧化还原酶1（NQO1） /谷胱甘肽过氧化 物酶4（GPX4） 信号通路探究益气通腑泻热方改善小鼠急性肺损伤（ALI） 的作用机制。方法：取SPF级雄性 ICR小鼠50只，其中8只设为对照组（气管滴注生理盐水），其余42只采用气管滴注脂多糖（LPS） 建立ALI模 型。从造模小鼠中随机取6只作为模型验证组进行模型验证，确认造模成功后，将剩余36只造模小鼠随机分为 模型组、溶剂组、益气通腑泻热方低剂量组（低剂量组）、益气通腑泻热方高剂量组（高剂量组），每组8只（剩 余4只作为备损耗）。对照组正常小鼠每天给予等体积生理盐水灌胃；模型组仅抓取固定，不予灌胃；溶剂组 给予等体积生理盐水灌胃；低、高剂量组分别给予5 g/kg、10 g/kg益气通腑泻热方灌胃，每天1次，连续3天。 比较对照组及模型验证组小鼠肺湿干重比，苏木素-伊红（HE） 染色肺组织病理变化。比较各组小鼠肺组织碾 磨液炎症因子水平及Nrf2/NQO1/GPX4信号通路相关分子表达水平。结果：与对照组比较，模型验证组小鼠肺 湿干重比、病理学损伤评分升高（P＜0.05）。与对照组比较，模型组和溶剂组小鼠白细胞介素（IL） -1β、IL-6、 肿瘤坏死因子-α （TNF-α） 水平均升高（P＜0.05）， Nrf2、NQO1、GPX4 的mRNA 和蛋白表达水平均降 低（P＜0.05）；模型组与溶剂组间各指标比较，差异均无统计学意义（P＞0.05）。与溶剂组比较，低、高剂量 组小鼠IL-1β、IL-6、TNF-α 水平均降低（P＜0.05），Nrf2、NQO1、GPX4 的mRNA 和蛋白表达水平均升 高（P＜0.05）；与低剂量组比较，高剂量组小鼠IL-1β、IL-6、TNF-α水平均降低（P＜0.05），Nrf2、NQO1、 GPX4的mRNA和蛋白表达水平均升高（P＜0.05）。结论：益气通腑泻热方可改善ALI小鼠的炎症反应程度， 可能与其激活Nrf2/NQO1/GPX4信号通路相关。

Abstract:

Abstract： Objective： To investigate the mechanism of Yiqi Tongfu Xiere Prescription in ameliorating acute lung injury （ALI） in mice based on the nuclear factor erythroid 2-related factor 2 （Nrf2）/NAD（P）H： quinone oxidoreductase 1 （NQO1）/glutathione peroxidase 4 （GPX4） signaling pathway. Methods：Fifty SPF-grade male ICR mice were used. Among them，8 mice were assigned to the control group （intratracheal instillation of normal saline）， and the remaining 42 mice received intratracheal instillation of lipopolysaccharide （LPS） to establish ALI models. Six mice were randomly selected from the model-induced mice for model validation. After successful modeling，the model mice were randomly divided into model group， vehicle group， low-dose Yiqi Tongfu Xiere Prescription group （lowdose group）， and high-dose Yiqi Tongfu Xiere Prescription group （high-dose group）， with 8 mice in each group （the remaining 4 mice serving as reserves）. Mice in the control group received an equal volume of normal saline by gavage daily. Mice in the model group were only subjected to grasping and fixation without gavage. Mice in the vehicle group received an equal volume of normal saline by gavage. Mice in the low-dose and high-dose groups received Yiqi Tongfu Xiere Prescription at doses of 5 g/kg and 10 g/kg by gavage，respectively，once daily for three consecutive days. The lung wet-to-dry weight ratio and hematoxylin-eosin （HE） staining of lung tissue pathological changes were compared between the control group and the model validation group. The levels of inflammatory factors in lung tissue homogenate and the expression levels of molecules related to the Nrf2/NQO1/GPX4 signaling pathway were compared among all groups. Results：Compared with the control group，the lung wet-to-dry weight ratio and pathological injury score in the model validation group were increased （P＜0.05）. Compared with the control group， the levels of interleukin （IL）-1β，IL-6，and tumor necrosis factor-alpha （TNF-α） in the model group and the vehicle group were increased （P＜0.05）， while the mRNA and protein expression levels of Nrf2， NQO1， and GPX4 were significantly decreased （P＜0.05）. No statistically significant differences were observed between the model group and the vehicle group for any of these parameters （P＞0.05）. Compared with the vehicle group， the low-dose and high-dose groups showed significantly decreased levels of IL-1β，IL-6，and TNF-α（ P＜0.05），and significantly increased mRNA and protein expression levels of Nrf2，NQO1，and GPX4 （P＜0.05）. Compared with the low-dose group，the high-dose group exhibited significantly decreased levels of IL-1β， IL-6， and TNF-α （P＜0.05）， and significantly increased mRNA and protein expression levels of Nrf2， NQO1， and GPX4 （P＜0.05）. Conclusion： Yiqi Tongfu Xiere Prescription can ameliorate the inflammatory response in ALI mice，and this effect may be associated with the activation of the Nrf2/NQO1/GPX4 signaling pathway.