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

doi:10.16431/j.cnki.1671-7236.2024.05.028

摘要/Abstract

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1 μg/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2 063个差异表达基因,其中1 319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生成增多,进而促进HIF-1α的表达,共同加重了炎性反应和代谢紊乱。

关键词: 鸡巨噬细胞; 脂多糖(LPS); HIF-1α; 炎症反应; 线粒体功能

Abstract: 【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-1α signaling 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 1 μg/mL LPS culture for 12 h,and the cell inflammation model was successfully established under this condition.Compared with control group,a total of 2 063 differentially expressed genes were identified in model group,with 1 319 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-1α and other signaling pathways.Among them,13 significantly upregulated genes were concentrated in HIF-1α-related signaling pathways,including Toll-like receptor 4 (TLR4),NF-κB,HIF-1α,and vascular endothelial growth factor (VEGF) genes. Flow cytometry determination results showed that compared with control group,mitochondrial membrane potential was extremely significantly decreased in model group (P＜0.01),and ROS level was significantly increased (P＜0.05).【Conclusion】 The LPS-induced inflammation model of chicken macrophages was established successfully.The crosstalk of HIF-1α and NF-κB signaling pathway was involved in the LPS-induced inflammation process.At the same time,the mitochondrial function of cells decreased,resulting in the increase of ROS production,and then promoted the expression of HIF-1α,and jointly aggravated the inflammatory response and metabolic disorders.

Key words: chicken macrophages; lipopolysaccharide (LPS); HIF-1α; inflammation; mitochondrial function

中图分类号:

S831.7

冯晓梦, 高超, 陶新磊, 李小方, 吕晓萍, 高雪丽, 赵一, 李亚楠, 刘超男. 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[J]. 中国畜牧兽医, 2024, 51(5): 2071-2080.

FENG Xiaomeng, GAO Chao, TAO Xinlei, LI Xiaofang, LYU Xiaoping, GAO Xueli, ZHAO Yi, LI Yanan, LIU Chaonan. Study on the Regulatory Role of the HIF-1α Signaling Pathway on Lipopolysaccharide-induced Inflammation in Chicken Macrophages[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2071-2080.

参考文献

[1] THFNER I,CHRISTENSEN J P.Bacterial Diseases in Poultry[M].London:Academic Press is an Imprint of Elsevier,2021.
[2] MINGYANG S,CHAN A T.Environmental factors,gut microbiota,and colorectal cancer prevention[J]. Clinical Gastroenterology & Hepatology,2018,17(2):275-289.
[3] 董平祥.饲料污染对动物生产的影响[J].饲料博览,2018,7:12-15. DONG P X.The impact of feed pollution on animal production[J]. Feed Review,2018,7:12-15.(in Chinese)
[4] 刘颖,田旭,冯晓梦,等.黄芪多糖对肠炎雏鸡小肠黏膜损伤的保护作用[J].中国畜牧兽医,2023,50(1):76-85. LIU Y,TIAN X,FENG X M,et al.Protective effect of Astragalus polysaccharides on intestinal mucosal injury in chicks with enteritis[J].China Animal Husbandary & Veterinary Medicine,2023,50(1):76-85.(in Chinese)
[5] 王潇娅,徐庆宝,何家全.DUSP8通过JNK/p38 MAPK通路来改善LPS诱导的巨噬细胞炎症反应[J].中国生物化学与分子生物学报,2021,37(2):229-235. WANG X Y,XU Q B,HE J Q.DUSP8 overexpression alleviates LPS-induced macrophage inflammatory responses through JNK/p38 MAPK pathways[J]. Chinese Journal of Biochemistry and Molecular Biology,2021,37(2):229-235.(in Chinese)
[6] 韩孝先,刘泽宇,周庆彪,等.线粒体在炎症调控中的作用研究进展[J].癌变.畸变.突变,2017,29(6):467-475. HAN X X,LIU Z Y,ZHOU Q B,et al.Research progress on the role of mitochondria in inflammatory regulation[J].Carcinogenesis,Teratogenesis & Mutagenesis,2017,29(6):467-475.(in Chinese)
[7] 袁清,刘江华,曾召林.线粒体功能障碍在代谢性炎症中的作用[J].江苏大学学报:医学版,2021,31(5):448-454. YUAN Q,LIU J H,ZENG Z L.The role of mitochondrial dysfunction in metabolic inflammation[J].Journal of Jiangsu Universuty (Medicine Edition),2021,31(5):448-454.(in Chinese)
[8] 张焱,赵丹.BML-111通过调节NLRP3炎症激活ROS产生来介导慢性阻塞性肺疾病的抗炎作用[J].国际呼吸杂志,2020,40(20):1543-1548. ZHANG Y,ZHAO D.BML-111 mediates the anti-inflammatory effect of chronic obstructive pulmonary disease by regulating the production of ROS activated by NLRP3 inflammation[J].International Journal of Respiration,2020,40(20):1543-1548.(in Chinese)
[9] HUANG B,DING J,GUO H R.SIRT3 regulates the ROS-FPR1/HIF-1α axis under hypoxic conditions to influence lung cancer progression[J].Cell Biochemistry and Biophysics,2023,81(4):813-821.
[10] TORRES-AGUILAR H,SOSA-LUIS S A,AGUILAR-RUIZ S R.Infections as triggers of flares in systemic autoimmune diseases:Novel innate immunity mechanisms[J]. Current Opinion in Rheumatology,2019,31(5):1.
[11] 吴晓玲.TFF2调控脂多糖诱导的巨噬细胞活化并拮抗炎症的功能与机制[D].福州:厦门大学,2017. WU X L.TFF2 regulates LPS-induced macrophages polarization and inhibits inflammation[D].Fuzhou:Xiamen University,2017.(in Chinese)
[12] 谢兵兵,杨亚东,丁楠,等.整合分析多组学数据筛选疾病靶点的精准医学策略[J].遗传,2015,37(7):655-663. XIE B B,YANG Y D,DING N,et al.Identification of disease targets for precision medicine by integrative analysis of multi-omics data[J].Hereditas,2015,37(7):655-663.(in Chinese)
[13] 汪颀浩.基于转录组学研究附子治疗急性心衰大鼠的作用机制[D].成都:成都中医药大学,2019. WANG X H.The studies of the mechanism of Aconiti Lateralis Radix in the treatment of rats with acute heart failure based on the transriptional analysis[D].Chengdu:Chengdu University of Traditional Chinese Medicine,2019.(in Chinese)
[14] 戴超辉,冯海悦,吴圣龙,等.猪源Toll样受体家族(TLRs)及其在抗病育种中的应用[J].浙江农业学报,2018,30(3):507-520. DAI C H,FENG H Y,WU S L,et al. Advances in porcine Toll-like receptor family (TLRs) and its application in resistance breeding[J].Acta Agriculturae Zhejiangensis,2018,30(3):507-520.(in Chinese)
[15] FENG Y,CUI Y,GAO J L,et al.Resveratrol attenuates neuronal autophagy and inflammatory injury by inhibiting the TLR4/NF-κB signaling pathway in experimental traumatic brain injury[J].International Journal of Molecular Medicine,2016,37(4):921-930.
[16] 杨柳,廖巧静,郑燕芳,等.芪药鸡金粥含药血清通过TLR4/NF-κB通路对Caco-2炎症细胞模型的影响[J].世界中西医结合杂志,2021,16(4):653-657. YANG L,LIAO Q J,ZHENG Y F,et al.Effect of Qi Yao Ji Jin Zhou medicated serum on the expressions of inflammatory model of Caco-2 cell through TLR4/NF-κB pathway[J].World Journal of Integrated Traditional and Western Medicine,2021,16(4):653-657.(in Chinese)
[17] CHOU H T C.Magnolol suppresses hypoxia-induced angiogenesis via inhibition of HIF-1α/VEGF signaling pathway in human bladder cancer cells[J].Biochemical Pharmacology,2013,85(9):1278-1287.
[18] 崔卫娜.NF-κB p65、HIF-1α及VEGF在变应性鼻炎小鼠鼻黏膜中的表达及其相关性研究[D].石家庄:河北医科大学,2012. CUI W N.Expression and correlation studies of nuclear factor-κB p65,HIF-1α and VEGF in the nasal mucosa of allergic rhinitis mice[D].Shijiazhuang:Hebei Medical University,2012.(in Chinese)
[19] 钟伦坤,周兴玮,朱佳丽,等.基于ROS/HIF-1通路探讨白花蛇舌草多糖对鼻咽癌裸小鼠肿瘤的抑制作用及机制[J].现代肿瘤医学,2022,30(15):2698-2706. ZHONG L K,ZHOU X W,ZHU J L,et al.Exploring the tumor suppressive effect and mechanism of polysaccharides extracts from Hedyotis diffusa on nasopharyngeal carcinoma nude mice based on ROS/HIF-1 pathway[J]. Journal of Modern Oncology, 2022,30(15):2698-2706.(in Chinese)
[20] DONG S,LIANG S,CHENG Z,et al.ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer[J].Journal of Experimental & Clinical Cancer Research,2022,41(1):15.
[21] 李文茜,王学红,朱思雨,等.胃炎及胃癌患者血清HIF-1α和ROS表达水平与Hp感染的关系及临床意义[J].检验医学与临床,2023,20(11):1532-1535. LI W Q,WANG X H,ZHU S Y,et al.Relationship between the expression levels of serum HIF-1α and ROS and Hp infection in patientsn with gastritis and gastric cancer and their clinical significance[J].Laboratory Medicine and Clinic,2023,20(11):1532-1535.(in Chinese)