非产肠毒素型脆弱拟杆菌通过下调NF-κB通路抑制TNF-α诱导的结肠上皮细胞炎症反应  

Non-enterotoxin-producing Bacteroides fragilis inhibits TNF-α-induced inflammatory response in colonic epithelial cells by down-regulating the NF-κB signaling pathway

作　　者：何秋月 黄秋玲[1] 卯建[1] 赵永师 陈莹萱 张艳[1] 杜艳[1] He Qiuyue;Huang Qiuling;Mao Jian;Zhao Yongshi;Chen Yingxuan;Zhang Yan;Du Yan(Department of Clinical Laboratory,the First Affiliated Hospital of Kunming Medical University,Yunnan Key Laboratory of Laboratory Medicine,Yunnan Province Clinical Research Center for Laboratory Medicine,Kunming 650032,China)

机构地区：[1]昆明医科大学第一附属医院医学检验科,云南省检验医学重点实验室,云南省医学检验临床医学研究中心,昆明650032

出　　处：《中华微生物学和免疫学杂志》2024年第10期829-837,共9页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(82260415);国家级重大科技成果培育项目(2023zdpy05)。

摘　　要：目的探讨非产肠毒素型脆弱拟杆菌(NTBF)抑制TNF-α诱导的人正常结肠上皮细胞hcoEPIC炎症反应的机制,为结肠炎的预防和治疗探索新的益生菌疗法。方法建立NTBF与hcoEPIC细胞共培养体系,分别检测NTBF的黏附和侵袭能力;NTBF与hcoEPIC细胞共培养4 h后加入TNF-α诱导细胞炎症,24 h后CCK-8试验和AnnexinⅤ-FITC/PI法检测细胞存活率及凋亡情况;Western blot和RT-qPCR检测不同处理组中hcoEPIC细胞内NF-κB信号通路关键蛋白质的变化;ELISA法检测该通路下游细胞因子IL-1β、TNF-α、IL-10的表达;体内动物实验评估NTBF干预对葡聚糖硫酸钠(DSS)诱导的结肠炎小鼠的影响。结果NTBF能够黏附于hcoEPIC细胞,对细胞无毒害作用。与对照组相比,NTBF单独处理并不影响hcoEPIC细胞增殖(P>0.05),但显著降低由TNF-α诱导的细胞损伤和凋亡(P<0.05);与TNF-α单独处理组相比,NTBF+TNF-α组中磷酸化NF-κB p65和磷酸化IκBα蛋白表达水平与NF-κB和IκBαmRNA水平显著降低(P<0.05);细胞上清液中IL-1β和TNF-α减少,IL-10增加(P<0.05)。动物实验表明NTBF能有效缓解DSS诱导的溃疡性结肠炎模型小鼠症状,主要表现为抑制结肠炎小鼠体重减轻、降低疾病活动指数评分、改善结肠缩短、减轻结肠病理损伤。结论NTBF可能通过下调NF-κB通路抑制TNF-α诱导的结肠上皮细胞炎症反应。ObjectiveTo investigate the mechanism by which non-enterotoxin-producing Bacteroides fragilis(NTBF)inhibits TNF-α-induced inflammatory responses in human normal colonic epithelial cells hcoEPIC,and explore new probiotic therapies for the prevention and treatment of colitis.MethodsThe co-culture system of NTBF and hcoEPIC cells was established,and the adhesion and invasion ability of NTBF were detected,respectively.TNF-αwas added to induce cellular inflammation after 4 h of co-culture of NTBF and hcoEPIC cells,and cell survival and apoptosis were detected by the CCK-8 assay and the AnnexinⅤ-FITC/PI assay respectively after 24 h.Key proteins of the NF-κB signalling pathway in hcoEPIC cells in different treatment groups were detected by Western blot and RT-qPCR,and the expression of downstream cytokines of this pathway incluing IL-1β,TNF-αand IL-10 were detected by ELISA.The effect of NTBF intervention on dextran sodium sulfate(DSS)-induced colitis mice was assessed by in vivo animal experiments.ResultsNTBF adhered to hcoEPIC cells,and was non-toxic to the cells.Compared with control group,NTBF treatment alone did not affect cell survival and apoptosis of hcoEPIC cells(P>0.05),but significantly reduced cell damage and apoptosis induced by TNF-α(P<0.05);Compared with the TNF-αtreatment alone group,the expression levels of p-NF-κB p65 and p-IκBαprotein as well as NF-κB and IκBαmRNA were significantly reduced(P<0.05);the production of IL-1βand TNF-αin the cell supernatant was reduced and the release of IL-10 was increased(P<0.05).Animal experiments demonstrated that NTBF was indeed effective in alleviating DSS-induced colitis in ulcerative colitis model mice,which was mainly manifested by inhibiting weight loss,lowering DAI scores,improving colonic shortening,and attenuating colonic pathological damage in colitis-induced mice.ConclusionsNTBF may inhibit TNF-α-induced inflammatory responses in colonic epithelial cells by down-regulating the NF-κB pathway.

关 键 词：非产肠毒素型脆弱拟杆菌 结肠炎 NF-κB信号通路 炎症反应 

分 类 号：R574.62[医药卫生—消化系统]