卫矛醇通过调节Toll样受体4/髓样分化因子88/核因子-κB信号通路缓解脂多糖诱导的猪肠道上皮细胞损伤  

Dulcitol Alleviates Lipopolysaccharide Induced Porcine Small Intestinal Epithelial Cells Injury by Modulating Toll-Like Receptor 4/Myeloid Differentiation Factor 88/Nuclear Factor-κB Signaling Pathway

作　　者：王晗[1,2] 刘正群 朱龙博 李宁 郑梓[2] 闫峻[2] 穆淑琴[2] 孙超[1] WANG Han;LIU Zhengqun;ZHU Longbo;LI Ning;ZHENG Zi;YAN Jun;MU Shuqin;SUN Chao(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;Tianjin Engineering Research Center of Animal Healthy Farming,Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology,Institute of Animal Science and Veterinary,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]天津市农业科学院畜牧兽医研究所,天津市畜禽分子育种与生物技术重点实验室,天津市畜禽健康养殖工程技术中心,天津300381

出　　处：《动物营养学报》2024年第6期3952-3962,共11页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：天津市中央引导地方科技发展专项(22ZYCGSN00130);天津市生猪产业技术体系创新团队(ITTPRS2021007);天津市农业科学院财政种业创新研究项目(2023ZYCX011)。

摘　　要：本试验旨在通过构建猪肠道上皮细胞(IPEC-J2细胞)经脂多糖(LPS)刺激后细胞损伤模型,揭示卫矛醇(DUL)对LPS处理后的IPEC-J2细胞屏障受损和炎症损伤的缓解作用及其可能的分子机制。采用细胞计数试剂盒-8(CCK-8)检测IPEC-J2细胞活力,以确定LPS造模浓度(150μg/mL)和最佳DUL处理浓度(200μmol/L)。试验设3个组:对照(CON)组、LPS组和DUL组。IPEC-J2细胞贴壁后,CON组先用完全培养基处理24 h,然后用DMEM培养基处理24 h;LPS组先用完全培养基处理24 h,然后用含有150μg/mL LPS的DMEM培养基处理24 h;DUL组先用含有200μmol/L DUL的完全培养基处理24 h,然后用含有150μg/mL LPS的DMEM培养基处理24 h。观察各组IPEC-J2细胞生长状态,划痕试验评价细胞的迁移和增殖能力,实时荧光定量PCR(RT-qPCR)法和蛋白质免疫印迹法(Western blot)检测屏障和炎症相关基因的mRNA和蛋白相对表达水平。结果表明:1)在IPEC-J2细胞上,确定200μmol/L的DUL为最佳处理浓度,150μg/mL的LPS为有效致炎浓度。2)相较于LPS组,DUL预处理缓解了细胞密度降低、细胞空泡增多、细胞边界不清晰的情况。3)相较于LPS组,DUL预处理显著增加了细胞迁移距离和细胞迁移面积(P<0.05)。4)相较于LPS组,DUL预处理显著升高了屏障相关基因闭锁小带蛋白-1(ZO-1)、闭锁蛋白(OCLN)、封闭蛋白1(CLDN1)的mRNA和蛋白相对表达水平及黏蛋白2(MUC2)、大肿瘤抑制激酶1(LATS1)、YES相关蛋白1(YAP1)的mRNA相对表达水平(P<0.05)。5)相较于LPS组,DUL预处理显著降低了通路相关基因髓样分化因子88(Myd88)、核因子-κB抑制蛋白-α(IκB-α)、核因子-κB(NF-κB)、白细胞介素-1β(IL-1β)的mRNA和蛋白相对表达水平和Toll样受体4(TLR4)的蛋白相对表达水平以及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、白细胞介素-18(IL-18)的mRNA表达相对表达水平(P<0.05)。由此可见,DUL通过下调TLR4/Myd88/NF-κB信号通路相关蛋白表达,调节炎�This experiment was conducted to reveal the mitigating effect of dulcitol(DUL)on lipopolysaccharide(LPS)induced cellular barrier and inflammatory injury of porcine small intestinal epithelial cells(IPEC-J2 cells)and its possible molecular mechanism by establishing a IPEC-J2 cells damage model after stimulated with LPS.The IPEC-J2 cell viability was detected by the cell count kit-8(CCK-8),to determine the c LPS modeling concentration(150μg/mL)and the appropriate DUL treatment concentration(200μmol/L).Three groups were setup in the experimental group:control(CON)group,LPS group and DUL group.After IPEC-J2 cells adhesion,the CON group was firstly treated with complete culture medium for 24 hours,then treated with DMEM medium for 24 hours;the LPS group firstly treated with complete culture medium for 24 hours,then treated with DMEM medium which contained 150μg/mL LPS for 24 hours;the DUL group firstly treated with complete culture medium which contained 200μmol/L DUL for 24 hours,then treated with DMEM medium which contained 150μg/mL LPS for 24 hours.The IPEC-J2 cells growth state in each group was observed,the migration and proliferation ability of the cells was detected by wound healing assay,and the mRNA and protein relative expression levels of the barrier and inflammatory related genes was detected by quantitative real-time PCR(RT-qPCR)and Western blot.The results showed as follows:1)in IPEC-J2 cells,the appropriate treatment concentration of DUL was 200μmol/L,and the effective inflammatory concentration of LPS was 150μg/mL.2)Compared with the LPS group,the DUL pretreatment alleviated the decreased cell density,increased cell vacuoles and poorly defined cell edges.3)Compared with the LPS group,the DUL pretreatment significantly increased the cell migration distance and cell migration area(P<0.05).4)Compared with the LPS group,the DUL pretreatment significantly increased the mRNA and protein relative expression levels of the barrier-associated genes zonula occludens-1(ZO-1),occludin(OCLN)and claudin-1(CLDN

关 键 词：卫矛醇 IPEC-J2细胞 TLR4/Myd88/NF-κB信号通路 炎性损伤 肠道屏障 

分 类 号：S828[农业科学—畜牧学]