AhR对急性胰腺炎大鼠肠道微环境及屏障功能的影响_李娟.pdf

doi:10.3969/j.issn.1006-5709.2023.01.003急性胰腺炎论著第一作者简介:李娟,初级技师,研究方向:医学检验。E-mail:lilijuan286 AhR 对急性胰腺炎大鼠肠道微环境及屏障功能的影响李娟,马肖,王建成首都医科大学附属北京友谊医院检验科,北京 100050【摘要】目的探讨芳香烃受体(aryl hydrocarbon receptor,AhR)对急性胰腺炎(acute pancreatitis,AP)大鼠肠道微环境、屏障功能及核因子 E2 相关因子 2(nuclear factor E2-related factor 2,Nrf-2)通路的影响。方法通过胆胰管注射 5%牛磺胆酸钠的方法建立AP 大鼠模型,将 50 只大鼠随机分组:AP 组、NC 组(尾部注射 5 l 慢病毒空白载体)、AhR 过表达组(尾部注射 5 l 过表达 AhR 慢病毒载体)、AhR 抑制剂组(腹腔注射 10 g 2-甲基-2H-吡唑-3-羧酸)、AhR 过表达+ML385 组(在 AhR 过表达组基础上,腹腔注射20 mg/kg ML385),另取 10 只大鼠为假手术组。心脏采血,检测血清中脂肪酶、淀粉酶水平以及 SOD、MDA、CRP 水平;分离胰腺组织,观察胰腺组织损伤程度及 AhR、Nrf-2、HO-1 蛋白表达水平;分离回肠组织,观察肠黏膜损伤情况并进行评分。结果与假手术组相比,模型组大鼠回肠、胰腺病理评分、CRP、MDA 含量、脂肪酶、淀粉酶水平均升高,SOD 含量、Nrf-2、HO-1、AhR 表达显著降低(P0.05);与模型组、NC 组相比,AhR 过表达组大鼠回肠、胰腺病理评分、CRP、MDA 含量、脂肪酶、淀粉酶水平均降低,SOD 含量、Nrf-2、HO-1、AhR 蛋白表达水平显著增加(P0.05);与模型组相比,AhR 抑制剂组大鼠回肠、胰腺病理评分、CRP、MDA 含量、脂肪酶、淀粉酶水平均升高,SOD 含量、Nrf-2、HO-1、AhR 蛋白表达水平显著降低(P0.05);ML385 逆转了 AhR 过表达对 AP 大鼠的保护作用(P0.05)。结论增强 AhR 表达可以改善 AP 大鼠肠道微环境,恢复肠道功能,缓解大鼠胰腺和肠黏膜损伤,可能与激活Nrf-2 通路有关。【关键词】AhR;急性胰腺炎大鼠;屏障功能;Nrf-2 通路;肠道微环境中图分类号:R576文献标识码:A文章编号:1006-5709(2023)01-0009-06收稿日期:2022-07-04Impacts of AhR on intestinal microenvironment and barrier function in rats with acute pancreatitisLI Juan,MA Xiao,WANG JianchengDepartment of Laboratory,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China【Abstract】ObjectiveTo investigate the impacts of aryl hydrocarbon receptor(AhR)on intestinal microenvironment,barrier function and nuclear factor E2-related factor 2(Nrf-2)pathway in acute pancreatitis(AP)rats.MethodsThe AP rat model was established by injecting 5%sodium taurocholate into the bile-pancreatic duct.Fifty rats were randomly di-vided into 5 groups:AP group,NC group(injected with 5 l lentiviral blank vector in the tail),AhR overexpression group(injected with 5 l of the overexpressed AhR lentiviral vector in the tail),AhR inhibitor group(intraperitoneal injection of 10 g 2-methyl-2H-pyrazole-3-carboxylic acid),and AhR overexpression+ML385 group(intraperitoneal injection of 20 mg/kg ML385 on the basis of AhR overexpression group),and another 10 rats were taken as the sham operation group.Blood was collected from the heart to detect the levels of lipase,amylase,SOD,MDA and CRP in ser-um;the pancreatic tissue was isolated,and the degree of pancreatic tissue damage and the protein expression levels of AhR,Nrf-2 and HO-1 were observed;the ileum tissue was isolated,and the intestinal mucosal injury was observed and scored.ResultsCompared with the sham operation group,the pathological scores of ileum and pancreas,the contents of CRP and MDA,the levels of lipase and amylase in the model group were increased,while the content of SOD and the expressions of Nrf-2,HO-1 and AhR were significantly decreased(P0.05).Compared with the model group and the NC group,the pathological scores of ileum and pancreas,the contents of CRP and MDA,the levels of lipase and amyl-ase in the AhR overexpression group were decreased,while the content of SOD and the protein expressions of Nrf-2,HO-1 and AhR were significantly increased(P0.05).Compared with the model group,the pathological scores of ile-um and pancreas,the contents of CRP and MDA,the levels of lipase and amylase in the AhR inhibitor group were in-creased,while the content of SOD and the protein expressions of Nrf-2,HO-1 and AhR were significantly decreased(P0.05);ML385 reversed the protective effect of AhR overexpression on AP rats(P0.05).ConclusionEnhan-cing the expression of AhR can improve the intestinal microenvironment of AP rats,restore intestinal function,and re-9胃肠病学和肝病学杂志2023 年 1 月第 32 卷第 1 期Chin J Gastroenterol Hepatol,Jan 2023,Vol.32,No.1lieve the damage of rat pancreas and intestinal mucosa,which may be related to the activation of Nrf-2 pathway.【Key words】Aryl hydrocarbon receptor;Acute pancreatitis rats;Barrier function;Nrf-2 pathway;Intestinal micro-environment急性胰腺炎(acute pancreatitis,AP)是一种自身消化性胰腺疾病,病因复杂,死亡率高,且 AP 发病率在全世界约为 4/10 000,且每年都在大幅度增加1。研究表明,细胞氧化损伤、炎性反应、凋亡与钙超载等与 AP 的发生和发展息息相关2。核因子 E2 相关因子 2(nuclear factor E2-related factor 2,Nrf-2)通过调节抗氧化反应元件(antoxidant response element,ARE)信号通路,活化下游血红素氧合酶 1(heme oxgenase-1,HO-1)等表达,降低炎性因子的分泌,在内源性抗氧化通路中发挥重要作用3。芳香烃受体(aryl hydrocarbon receptor,AhR)是一种核受体/转录因子,通过控制炎症信号(包括细胞因子和趋化因子信号)调节各种免疫相关疾病4。研究发现,在免疫系统中,AhR 已被明确确定为可以将环境、饮食或微生物信号整合到先天性及适应性免疫反应中的关键调节因素5,但 AhR 在 AP 疾病的作用及 Nrf-2 通路之间的关系尚不清楚。本研究旨在探讨 AhR 对 AP 大鼠肠道微环境、屏障功能及Nrf-2 通路的影响。1材料与方法1.1材料1.1.1实验动物:北京维通利华实验动物技术有限公司提供健康、SPF 级、雄性 SD 大鼠 60 只,8 周龄,体质量 200230 g,许可证号:SYXK(京)2021-0044。将所有大鼠饲养在 24 的动物室单笼中,光/暗循环为12 h/12 h,并用标准的实验室啮齿动物食物和水喂养1 周。所有动物实验均按照伦理委员会批准的相关伦理标准和规定进行(伦理批号:20220810)。1.1.2实验试剂与仪器:美国 Sigma 公司提供 AhR拮抗剂 2-甲基-2H-吡唑-3-羧酸(2-methyl-2H-pyrazole-3-carboxylic acid)(C8124-5MG)、牛磺胆酸钠;上海吉玛制药技术有限公司提供慢病毒空白载体以及过表达 AhR 慢病毒载体(5-CAGGAACACCTACATCTAGAAGG-3);Med-chem Express 公司提供 Nrf-2 通路抑制剂 ML385(HY-100523);上海酶联生物科技有限公司提供超氧化歧化酶(superoxide dismutase,SOD)(m1094766)、丙二醛(malondialdehyde,MDA)(m1094963)、C 反应蛋白(C-reactive protein,CRP)试剂盒(m1092609);珠海贝索生物技 术 有 限 公 司 提 供 HE 染 色 试 剂 盒(BA4025);Abcam 公司提