HIF-1α信号通路对脂多糖诱导鸡巨噬细胞炎症的调节作用研究  

Study on the Regulatory Role of the HIF-1αSignaling Pathway on Lipopolysaccharide-induced Inflammation in Chicken Macrophages

作　　者：冯晓梦 高超 陶新磊 李小方 吕晓萍[1] 高雪丽[1] 赵一 李亚楠 刘超男[1] FENG Xiaomeng;GAO Chao;TAO Xinlei;LI Xiaofang;LYU Xiaoping;GAO Xueli;ZHAO Yi;LI Yanan;LIU Chaonan(Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine,College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]东北农业大学动物医学院,黑龙江省实验动物与比较医学重点实验室,哈尔滨150030 [2]中国农业大学动物医学院,北京100193

出　　处：《中国畜牧兽医》2024年第5期2071-2080,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31472169);黑龙江省教育厅科学技术研究项目基金(12511029)。

摘　　要：【目的】探究鸡巨噬细胞(HD11)在脂多糖(LPS)诱导条件下,缺氧诱导因子-1α(HIF-1α)信号通路和线粒体功能对细胞炎症反应的影响。【方法】试验以HD11细胞为研究对象,分别用不同浓度LPS作用于细胞,培养24 h后检测细胞活力,筛选LPS最佳作用浓度;同时在最佳LPS作用浓度下,筛选LPS最佳作用时间。将HD11细胞分为对照组和模型组,模型组加入最佳作用浓度LPS培养,对照组加等量的完全培养液,按照LPS最佳作用时间培养后,提取细胞总RNA进行转录组测序,并对差异表达基因进行GO功能注释和KEGG通路富集分析。利用实时荧光定量PCR和Western blotting分别检测HIF-1α信号通路相关因子mRNA和蛋白表达量;通过流式细胞术检测线粒体膜电位和活性氧(ROS)水平变化。【结果】LPS诱导的HD11细胞炎症模型中最适条件为1μg/mL LPS培养12 h,以此条件成功建立了细胞炎症模型。与对照组相比,模型组共检测到2063个差异表达基因,其中1319个上调,744个下调。GO功能注释结果显示,差异表达基因显著富集到免疫系统应答、对外部刺激的反应和细胞因子受体结合等过程。KEGG通路富集分析结果显示,差异表达基因显著富集在50条信号通路,主要涉及免疫细胞介导的炎症反应和Toll样受体、核转录因子-κB(NF-κB)、HIF-1α等信号通路。其中有13个显著上调表达的基因集中在HIF-1α相关信号通路,包括Toll样受体4(TLR4)、NF-κB、HIF-1α、血管内皮生长因子(VEGF)基因等。实时荧光定量PCR和Western blotting结果显示,NF-κB p65、HIF-1α、VEGF等mRNA和蛋白表达水平均显著上调(P<0.05)。流式细胞术检测结果显示,与对照组相比,模型组线粒体膜电位极显著下降(P<0.01),ROS水平显著升高(P<0.05)。【结论】成功建立LPS诱导鸡巨噬细胞炎症模型,HIF-1α与NF-κB信号通路相互串扰共同参与LPS诱导的细胞炎症过程。同时,细胞线粒体功能下降,导致ROS�【Objective】This study was aimed to investigate the impact of the hypoxia inducible factor-1α(HIF-1α)signaling pathway and mitochondrial function on inflammatory responses in chicken macrophages(HD11)under lipopolysaccharide(LPS)-induced condition.【Method】HD11 cells were treated with LPS at different concentrations and cultured for 24 h to detect cell viability and screen the optimal concentration.At the same time,the optimal action time of LPS was screened under the optimal concentration.HD11 cells were divided into control and model groups.The HD11 cells in model group were cultured with LPS at the optimal concentration,while the cells in control group were received an equivalent amount of complete culture medium.After culture according to the optimal time of LPS action,total RNA was extracted for transcriptome sequencing,and GO function annotation and KEGG pathway enrichment analysis were performed for differentially expressed genes.mRNA and protein expression of HIF-1αsignaling pathway related factors were detected by Real-time quantitative PCR and Western blotting.Mitochondrial membrane potential and reactive oxygen species(ROS)were detected by flow cytometry.【Result】The optimal condition of LPS-induced HD11 cell inflammation model was 1μg/mL LPS culture for 12 h,and the cell inflammation model was successfully established under this condition.Compared with control group,a total of 2063 differentially expressed genes were identified in model group,with 1319 genes upregulated and 744 genes downregulated.GO functional annotation analysis showed that differentially expressed genes were significantly enriched in processes such as immune system response,response to external stimuli and cytokine receptor binding.KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in 50 signaling pathways,mainly involved in immune cell-mediated inflammatory response and Toll-like receptor,nuclear transcription factor-κB(NF-κB),HIF-1αand other signaling pathways.A

关 键 词：鸡巨噬细胞 脂多糖(LPS) HIF-1Α 炎症反应 线粒体功能 

分 类 号：S831.7[农业科学—畜牧学]