E3蛋白泛素连接酶WWP1调控TLR4介导的炎症性肠病炎性聚集中的作用  

The role of E3 protein ubiquitin ligase WWP1 in regulation of TLR4-mediated inflammatory aggregation in inflammatory bowel disease

作　　者：徐舒佳 杨剑 李燕飞 XU Shu-jia;YANG Jian;LI Yan-Fei(Department of Gastroenterology,Shekou Free Trade Zone Hospital of Shenzhen Qianhai,Shenzhen 518000,China)

机构地区：[1]深圳市前海蛇口自贸区医院消化内科,广东深圳518000

出　　处：《解剖学研究》2023年第1期51-56,共6页Anatomy Research

基　　金：深圳市南山区卫生科技计划项目(深南科[2021]NS046)。

摘　　要：目的 探讨E3蛋白泛素连接酶WWP1调控TLR4介导的炎症性肠病炎性聚集中的作用及机制。方法 构建髓系细胞中特异性敲除E3蛋白泛素连接酶WWP1基因的小鼠,检测WPP1基因是否被敲除,随后进行分组,分为WWP敲除组(WWP1^(+/+))和WWP未敲除组(WWP1-/-),两组小鼠再分别喂养菊聚糖硫酸钠(DSS)建立炎症性肠病模型(DSS组)和喂养生理盐水(无菌水组),每组小鼠共10只,喂养7 d,每天检测小鼠结肠炎的临床症状,结束后处死小鼠,测量结肠长度,评估小鼠结肠炎损伤情况,Elisa检测小鼠结肠黏膜组织炎性因子IL-6、IL-10、TNF-α水平,采用RT-qPCR和Western blot检测TLR4表达水平。同时使用基因沉默方法敲除RAW 264.7细胞的WWP1基因,使用LPS刺激构建结肠炎症情况下的巨噬细胞环境,检测WWP1+组与WWP1-组细胞中IL-6、IL-10、TNF-α和TLR4、WWP1表达水平。结果 WWP1^(+/+)组和RAW264.7细胞沉默组中WWP1-mRNA表达均明显下调,提示成功敲除WWP1基因。DSS组WWP1-/-小鼠体质量减轻程度明显大于DSS组WWP1^(+/+)小鼠,DSS组WWP1-/-小鼠的DAI值明显大于DSS组WWP1^(+/+)小鼠,DSS组WWP1^(+/+)小鼠结肠长度明显长于DSS组WWP1-/-小鼠;与WWP1-/-相比,WWP1^(+/+)的小鼠组织病理损伤程度更轻,结肠炎的症状更轻,同时小鼠IL-6、TNF-α表达水平明显更低,IL-10表达水平明显更高,炎症损伤程度更轻,RT-qPCR和Western blot检测结肠组织和细胞中TLR4的表达也显示,与WWP1-/-相比,WWP1^(+/+)的小鼠TLR4表达水平明显更低,在细胞株中,WWP1+组TLR4表达水平明显更低(P<0.05)。结论 E3蛋白泛素连接酶WWP1可以对炎症性肠病中炎性聚集起到保护作用,这种保护作用的发挥可能是通过TLR4通路实现的。Objective To investigate the role and mechanism of E3 protein ubiquitin ligase WWP1 in the regulation of TLR4-mediated inflammatory aggregation in inflammatory bowel disease.Methods Mice with specific knockout of E3 protein ubiquitin ligase WWP1 gene in myeloid cells were constructed,and WPP1 gene was detected.Then the mice were divided into WWP knockout group(WWP1^(+/+) )and WWP non-knockout group(WWP1^(-/-) ).Mice in the two groups were fed inulan sodium sulfate(DSS)to establish inflammatory bowel disease model(DSS group)and normal saline(sterile water group)for 7 days,and the clinical symptoms of colitis were detected every day.After the mice were sacrificed,colonic length was measured,and colitis injury was evaluated.The levels of inflammatory factors IL-6,IL-10 and TNF-A were detected by Elisa,and the expression of TLR4 was detected by RT-qPCR.At the same time,WWP1 gene of RAW 264.7 cells was knocked down by gene silencing method,and the macrophage environment under colonic inflammation was constructed by LPS stimulation.The expression levels of IL-6,IL-10,TNF-A,TLR4 and WWP1 in WWP1+and WWP1-groups were detected.Results WWP1-mRNA expression was significantly down-regulated in both WWP1^(+/+) group and RAW 264.7 cell silencing group,suggesting that WWP1 gene was successfully knocked out.The weight loss degree of WWP1^(-/-) mice in DSS group was significantly greater than WWP1^(+/+) mice in DSS group,the DAI value of WWP1^(-/-) mice in DSS group was significantly greater than WWP1^(+/+) mice in DSS group,and the colon length of WWP1^(+/+) mice in DSS group was significantly longer than WWP1^(-/-) mice in DSS group.Compared with WWP1^(-/-) ,mice with WWP1^(+/+) have less pathological damage and less symptoms of colitis.Meanwhile,mice with WWP1^(+/+) have significantly lower expression levels of IL-6 and TNF-α,significantly higher expression levels of IL-10,and less inflammatory damage.RT-qPCR and Western blot detection of TLR4 expression in colon tissues and cells also showed that compared with WWP1^(-/-) ,th

关 键 词：炎症性肠病 泛素化修饰 含WW结构域的E3泛素连接酶1(WWP1) 作用机制 

分 类 号：R574[医药卫生—消化系统]