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