E3蛋白泛素连接酶WWP1...炎症性肠病炎性聚集中的作用_徐舒佳.pdf

解剖学研究2023年第45卷第1期Anat Res,2023,Vol.45,No.1E3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徐舒佳，杨剑，李燕飞深圳市前海蛇口自贸区医院消化内科，广东深圳518000论著【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IL6、IL10、TNF水平，采用RTqPCR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 IL6、IL10、TNF 和 TLR4、WWP1 表达水平。结果WWP1+/+组和 RAW264.7 细胞沉默组中WWP1mRNA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 IL6、TNF 表达水平明显更低，IL10 表达水平明显更高，炎症损伤程度更轻，RTqPCR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（P0.05）。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。【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）；作用机制基金项目：深圳市南山区卫生科技计划项目（深南科 2021 NS046）DOI：10.20021/ki.16710770.2023.01.09The role of E3 protein ubiquitin ligase WWP1 in regulation of TLR4mediated inflammatory aggregation ininflammatory bowel diseaseXU Shujia，YANG Jian，LI YanFeiDepartment of Gastroenterology，Shekou Free Trade Zone Hospital of Shenzhen Qianhai，Shenzhen 518000，China【Abstract】ObjectiveTo investigate the role and mechanism of E3 protein ubiquitin ligase WWP1 in theregulation of TLR4mediated inflammatory aggregation in inflammatory bowel disease.MethodsMice with specificknockout 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 nonknockout group（WWP1-/-）.Mice in the two groups were fed inulan sodium sulfate（DSS）to establish inflammatory bowel disease model（DSSgroup）and normal saline（sterile water group）for 7 days，and the clinical symptoms of colitis were detected everyday.After the mice were sacrificed，colonic length was measured，and colitis injury was evaluated.The levels of inflammatory factors IL6，IL10 and TNFA were detected by Elisa，and the expression of TLR4 was detected by RTqPCR.At the same time，WWP1 gene of RAW 264.7 cells was knocked down by gene silencing method，and themacrophage environment under colonic inflammation was constructed by LPS stimulation.The expression levels of IL6，IL10，TNFA，TLR4 and WWP1 in WWP1+and WWP1-groups were detected.ResultsWWP1mRNA expression was significantly downregulated in both WWP1+/+group and RAW 264.7 cell silencing group，suggesting thatWWP1 gene was successfully knocked out.The weight loss degree of WWP1-/-mice in DSS group was significantly51解剖学研究2023年第45卷第1期Anat Res,2023,Vol.45,No.1greater than WWP1+/+mice in DSS group，the DAI value of WWP1-/-mice in DSS group was significantly greaterthan WWP1+/+mice in DSS group，and the colon length of WWP1+/+mice in DSS group was significantly longer thanWWP1-/-mice in DSS group.Compared with WWP1-/-，mice with WWP1+/+have less pathological damage and lesssymptoms of colitis.Meanwhile，mice with WWP1+/+have significantly lower expression levels of IL6 and TNF，significantly higher expression levels of IL10，and less inflammatory damage.RTqPCR and Western blot detectionof TLR4 expression in colon tissues and cells also showed that compared with WWP1-/-，the expression level ofTLR4 in WWP1+/+mice was significantly lower，and in cell lines，the expression level of TLR4 in WWP1+group wassignificantly lower（P0.05）.ConclusionE3 ubiquitin ligase WWP1 may play a protective role in inflammatory aggregation in inflammatory bowel disease，which may be realized through TLR4 pathway.【Key words】Inflammatory bowel disease；Ubiquitination modification；WW domaincontaining E3 ubiquitin protein ligase 1（WWP1）；Mechanism of action炎症性肠病（Inflammatory bowel disease，IBD）是一类慢性反复发作的肠道疾病，以胃肠道炎症及不受控的上皮损伤为特征1-2，目前炎症性肠病的病因及发病机制尚不完全明确，多项研究提示IBD 的发生与多种调控机制异常引起的肠道黏膜屏障、炎症、免疫失调有关3-5。泛素化修饰是一种蛋白质翻译后修饰方式，对蛋白质的降解，蛋白质之间相互作用，蛋白质功能等进行调节6。泛素化过程的实现与过E1泛素激活酶、E2泛素结合酶和 E3 泛素连接酶等相关7，研究表明，E3 泛素连接酶可以通过多种信号调节通路参与炎症及免疫反应，并且有大量研究表明E3泛素连接酶的活化异常与 IBD 的炎症及免疫损伤发生机制相关8-10。E3 及其相应的靶蛋白控网络对炎症及免疫反应调控呈现双向调控，可诱导也可以抑制，具体作用的发挥取决于 E3 及其靶蛋白的具体类型11-12。目前关于含 WW 结构域的 E3 泛素连接酶 1（WW domaincontaining E3 ubiquitin protein ligase 1，WWP1）在IBD中的作用及作用机制尚不明确，因此，本研究探讨 E3 蛋白泛素连接酶 WWP1负性调控 TLR4 介导的炎症性肠病炎性聚集中的作用，现报道如下。1材料与方法1.1材料1.1.1实验动物本研究中使用小鼠为 C57BL/6雄性小鼠40只，基因缺陷小鼠为WWP1基因敲除的 C57BL/6 雄性小鼠，小鼠购于江苏艾菱菲生物科技有限公司。所有小鼠均为 68 周龄，体质量为 1825 g 的 SPF 级小鼠。所有的小鼠均在送风隔离独立笼具中进行饲养，饲养笼具保持恒温（223），湿度为 30%70%，光照时间昼夜各半，给予小鼠常规灭菌饲料及饮用水自由喂养。1.1.2实验细胞选取一株RAW264.7细胞株，购自武汉普诺赛生命科技有限公司。1.2方法1.2.1DSS 小鼠急性结肠损伤模型构建所有小鼠 分 为 WWP1+/+DSS 组WWP1 敲 除 小 鼠 喂 养2.5%葡聚糖硫酸钠（DSS）构建程炎症性肠病模型、WWP1+/+无菌水组（WWP1敲除小鼠喂养生理盐水作为对照）、WWP1-/-DSS 组（正常小鼠喂养DSS构建程炎症性肠病模型）和WWP1-/-无菌水组（正常小鼠喂养生理盐水），每组 10 只小鼠，所有小鼠正常自由喂养，共喂养7 d。1.2.2各组小鼠处理饲养 7 d 期间每日对小鼠所表现出的结肠损伤症状进行监测，监测的症状包括体质量、大便性状、小鼠行为状态。饲养 7 d后处死小鼠，剖腹取小鼠结肠，所取结肠段为全结肠取自回肠末段及回盲部交点至肛门口，使用直尺测量肛门至回盲部距离作为结肠长度。取远端结肠组织，沿纵轴切开，并沿中线切成两半卷成瑞士卷状进行苏木精-伊红（HE）染色制片，采用免疫组化、RTqPCR 和 We