腺相关病毒过表达IL22可有效改善葡聚糖硫酸钠诱导的小鼠结肠炎  

Overexpression of IL22 by adeno-associated virus effectively ameliorates dextran sulfate sodium-induced colitis in mice

作　　者：吴天宇 宋娅岚 吴静峰 郑植元 张宁[2] 周子默 唐红[2] 王锋超[2] 陈文生 WU Tianyu;SONG Yalan;WU Jingfeng;ZHENG Zhiyuan;ZHANG Ning;ZHOU Zimo;TANG Hong;WANG Fengchao;CHEN Wensheng(Department of Gastroenterology,First Affiliated Hospital,Institute of Combined Injury,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;State Key laboratory of Trauma,Burns and Combined Injury,Institute of Combined Injury,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院消化内科,重庆400038 [2]陆军军医大学(第三军医大学)军事预防医学系复合伤研究所,重庆400038

出　　处：《陆军军医大学学报》2023年第19期1995-2006,共12页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81872556);陆军军医大学创新能力提升项目(2021XJS03);重庆市院士专项(基础研究与前沿探索类)(cstc2018jcyj-yszxX0004)。

摘　　要：目的探讨白介素22(IL22)在小鼠葡聚糖硫酸钠(dextran sulfate sodium salt,DSS)诱导的急性结肠炎中的作用及其机制。方法选取8周龄雄性同窝IL22敲除小鼠(IL22-/-)及对照基因型小鼠(IL22+/+)各3只,给予2组小鼠2.5%DSS自由饮水6 d、正常饮水2 d构建小鼠急性结肠炎模型,记录小鼠体质量变化及疾病活动指数(disease activity index,DAI),干预8 d后行小鼠肠道内窥镜检查,测量2组小鼠结肠长度以及主要免疫器官脏器指数,采用Western blot检测白介素1α(IL1α)、白介素1β(IL1β)、白介素6(IL6)、肿瘤坏死因子α(TNFα)等促炎细胞因子蛋白水平表达;选取8周龄雄性C57BL/6小鼠建立腺相关病毒(adeno-associated virus,AAV)转染模型,给予DSS诱导结肠炎模型,设置PBS组(n=5)、AAV-mock+DSS组(n=5)、AAV-IL22+DSS组(n=5),采用免疫荧光检测各组结肠闭合小环蛋白1(ZO-1)、钙粘附蛋白(E-claudin)表达,采用Western blot检测IL22、闭合蛋白(Occludin)、信号传导及转录激活蛋白3(Stat3)、p-Stat3蛋白表达水平。结果与IL22+/+组比较,IL22-/-组小鼠在DSS诱导后第8天体质量显著降低(P<0.05),DAI评分(P<0.01)和肠镜评分显著升高(P<0.05),组织损伤加重,病理评分显著升高(P<0.01),促炎细胞因子IL1α、IL1β、cleaved-IL1β、IL6、TNFα表达上调(P<0.05)。与AAV-mock+DSS组相比,AAV-IL22+DSS组可逆转DSS诱导结肠炎,结肠组织炎症水平显著降低(P<0.05),免疫荧光结果示E-claudin表达显著升高(P<0.05),Western blot结果显示Occludin、p-Stat3表达水平显著增高(P<0.01)。结论IL22缺乏加重小鼠DSS诱导结肠炎,而通过AAV过表达IL22能增强结肠紧密连接蛋白表达,减少炎症浸润,缓解DSS肠炎,其机制可能与Jak/Stat3通路激活有关。Objective To investigate the effects of interleukin22(IL22)in acute colitis induced by dextran sulfate sodium salt(DSS)in mice and its underlying mechanism.Methods Three IL22 knockout mice(IL22^(-/-))and 3 control mice(IL22^(+/+))of 8-week-old males were subjected from the same litter,and then had free-choice drinking of 2.5%DSS water for 6 d followed by normal water for 2 d to construct a mouse model of acute colitis.The changes in body mass weight and disease activity index(DAI)were measured and calculated.After the intervention of 8 d,mouse enteroscopy was performed,and the length of colon and the organ index of major immune organs were measured in both groups.Protein levels of pro-inflammatory cytokines,such as IL1α,IL1β,IL6 and TNFαwere detected by Western blotting.Adeno-associated virus(AAV)transfection model was established in 8-week-old male C57BL/6 mice,which were then treated by 2.5%DSS drinking.The mice were randomly divided into PBS group(n=5),AAV-mock+DSS group(n=5)and AAV-IL22+DSS group(n=5).The expression levels of colonic closed small loop protein 1(ZO-1)and calcium adhesion protein(E-claudin)in different groups were detected by immunofluorescence assay.The protein levels of IL22,Occludin,Stat3 and p-Stat3 were detected by Western blotting.Results Compared with the IL22^(+/+)group,the mice in the IL22^(-/-)group had significantly decreased body weight on day 8(P<0.05),obviously increased DAI score(P0.01),endoscopy score(P<0.05)and pathological score(P0.01)after DSS induction,and notable up-regulation of pro-inflammatory cytokines including IL1α,IL1β,cleaved-IL1β,IL6,and TNFαin the colon tissues(P<0.05).While AAV-IL22 transfection reversed DSS-induced colitis.Significantly reduced inflammation in colonic tissues(P<0.05),enhanced E-claudin expression(P<0.05)and up-regulation of p-Stat3 and Occludin(P0.01)were observed in the AAV-IL22+DSS group than the AAV-mock+DSS group.Conclusion IL22 deficiency aggravates DSS-induced colitis in mice,and its overexpression by AAV-IL22 alleviates DSS enterit

关 键 词：白细胞介素22 腺相关病毒 炎症性肠病 葡聚糖硫酸钠 Jak/Stat3通路 

分 类 号：R-332[医药卫生] R574.62R976