ERK通路在脂多糖诱导HD11细胞炎性反应中的作用机制

doi:10.16431/j.cnki.1671-7236.2025.08.038

摘要/Abstract

摘要： 【目的】阐明ERK信号通路在家禽巨噬细胞炎性反应过程中的交叉调控机制。【方法】将鸡巨噬细胞(HD11)分为4组：对照组(CON)、脂多糖组(LPS)、抑制剂＋脂多糖组(AST-Ⅳ＋LPS)、抑制剂组(AST-Ⅳ)。LPS组HD11细胞中加入1 μg/mL脂多糖，AST-Ⅳ组HD11细胞中加入5 μg/mL ERK抑制剂黄芪甲苷(AST-Ⅳ)，AST-Ⅳ＋LPS组HD11细胞用5 μg/mL ERK抑制剂处理12 h后再加入1 μg/mL脂多糖处理6 h，对照组加等量DMEM培养基，每组设置3个生物学重复。利用实时荧光定量PCR和Western blotting检测经典Ras-MEK-ERK信号通路相关因子及炎症因子基因和蛋白的表达，同时检测转录因子缺氧诱导因子-1α(HIF-1α)、核因子κB(NF-κB)的蛋白表达；采用荧光探针法检测细胞内活性氧(ROS)的变化，并用流式细胞术检测线粒体膜电位。【结果】与对照组相比，当脂多糖诱导细胞炎性反应时，经典ERK通路关键因子MAP3K1、MEK、ERK与下游转录因子p-c-Jun、p-c-Fos及炎症因子白细胞介素-6(IL-6)、IL-8和肿瘤坏死因子-α(TNF-α)的表达量显著升高(P＜0.05)；线粒体膜电位显著降低，ROS生成显著增多(P＜0.05)，转录因子HIF-1α、NF-κB的蛋白表达显著增多(P＜0.05)。通过预先加入ERK抑制剂，可显著降低HD11细胞产生的IL-6、IL-8和TNF-α及ROS、HIF-1α、NF-κB的表达量(P＜0.05)，线粒体膜电位显著升高(P＜0.05)。【结论】巨噬细胞发生炎性损伤时，可通过经典ERK通路上调炎症因子的表达，损伤线粒体内膜完整性并促进ROS的生成，经ROS-HIF-1α/NF-κB进一步促进炎症因子表达，恶性循环并加重细胞炎性反应。本研究为揭示ERK通路与其他炎症通路间串扰现象及其在炎症发生发展过程中的作用机制奠定基础。

关键词: 脂多糖(LPS); ERK信号通路; 炎性反应

Abstract: 【Objective】 The purpose of the experiment was to elucidate the cross-regulatory mechanism of ERK signaling pathway in the inflammatory response process of avian macrophages. 【Method】 The chicken macrophage cells (HD11) were divided into four groups:Control group (CON),lipopolysaccharide group (LPS),inhibitor＋lipopolysaccharide group (AST-Ⅳ＋LPS),and inhibitor group (AST-Ⅳ).HD11 cells in LPS group were treated with 1 μg/mL lipopolysaccharide,HD11 cells in AST-Ⅳ group waere treated with 5 μg/mL ERK inhibitor astragaloside Ⅳ (AST-Ⅳ),HD11 cells in AST-Ⅳ＋LPS group were treated with 5 μg/mL ERK inhibitor for 12 h and then treated with 1 μg/mL LPS for 6 h,and HD11 cells in control group were treated with an equal volume of DMEM culture medium.Each group was set up with three biological replicates.Real-time quantitative PCR and Western blotting were used to detect the expression of classical Ras-MEK-ERK signaling pathway-related factors and inflammatory factor genes and proteins,while hypoxia inducible factor-1α (HIF-1α) and nuclear factor kappa-B (NF-κB) protein expression were also detected.The changes of intracellular reactive oxygen species (ROS) were detected by fluorescence probe method,and the mitochondrial membrane potential was detected by flow cytometry. 【Result】 Compared with control group,when LPS induced an inflammatory response in cells,the expression of key classical ERK pathway factors MAP3K1,MEK and ERK,as well as downstream transcription factors p-c-Jun,p-c-Fos,and inflammatory factors interleukin-6 (IL-6),IL-8,and tumor necrosis factor-α (TNF-α) were significantly increased (P＜0.05).Mitochondrial membrane potential was decreased,ROS generation was increased (P＜0.05),and the expression of transcription factor HIF-1α and NF-κB protein were also increased significantly (P＜0.05).However,the expression of IL-6,IL-8,TNF-α, ROS,HIF-1α and NF-κB produced by HD11 cells could be significantly reduced by pre-addition of ERK inhibitors (P＜0.05),and mitochondrial membrane potential was increased (P＜0.05). 【Conclusion】 When macrophages underwent inflammatory damage,they could upregulate inflammatory factor expression through the classical ERK pathway,damaging the integrity of the mitochondrial inner membrane and promoting the generation of ROS.ROS-HIF-1α/NF-κB further promoted inflammatory factor expression in a vicious cycle,exacerbating the cellular inflammatory response.This study laid a foundation for elucidating the crosstalk between the ERK pathway and other inflammation pathways and the mechanisms of inflammation development.

Key words: lipopolysaccharide (LPS); ERK pathway; inflammation response

中图分类号:

S852.4^＋2

李小方, 高超, 赵亮, 刘颖, 高雪丽, 吕晓萍, 郑世民, 刘超男. ERK通路在脂多糖诱导HD11细胞炎性反应中的作用机制[J]. 中国畜牧兽医, 2025, 52(8): 3896-3906.

LI Xiaofang, GAO Chao, ZHAO Liang, LIU Ying, GAO Xueli, LYU Xiaoping, ZHENG Shimin, LIU Chaonan. Mechanism of ERK Pathway in the Inflammatory Response of HD11 Cells Induced by Lipopolysaccharide[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3896-3906.

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