β-伴大豆球蛋白对IPEC-J2细胞损伤的作用机制研究  

Mechanism ofβ-conglycinin on IPEC-J2 cell injury

作　　者：孙智峰 王蕾 刘羽佳 丁红研[1] 王志[1] 李思婷 王承智 李玉[1] 王希春[1] 吴金节[1] SUN Zhifeng;WANG Lei;LIU Yujia;DING Hongyan;WANG Zhi;LI Siting;WANG Chengzhi;LI Yu;WANG Xichun;WU Jinjie(College of Animal Science and Technology,Anhui Agricultural University,Hefei,Anhui 230036,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《西北农林科技大学学报（自然科学版）》2022年第1期27-35,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31972750)。

摘　　要：【目的】研究β-伴大豆球蛋白通过核因子-κB(NF-κB)、诱导型一氧化氮合酶(iNOS)、c-Jun N端激酶(JNK)和p38丝裂原活化蛋白激酶(p38 MAPK)信号通路对猪肠上皮细胞(IPEC-J2)造成损伤的差异,为探索β-伴大豆球蛋白引发仔猪肠道黏膜损伤的分子机制提供理论依据。【方法】采用β-伴大豆球蛋白体外诱导IPEC-J2损伤,试验设对照组T0(猪肠上皮细胞不做处理),试验组T1(加入5 mg/mLβ-伴大豆球蛋白)、T2(加入5 mg/mLβ-伴大豆球蛋白+1μmol/L NF-κB抑制剂二硫氨基甲酸肽吡咯烷(PDTC))、T3(加入5 mg/mLβ-伴大豆球蛋白+1μmol/L iNOS抑制剂Nω-硝基-L-精氨酸甲酯(L-NAME))、T4(加入5 mg/mLβ-伴大豆球蛋白+1μmol/L JNK抑制剂SP600125)和T5(加入5 mg/mLβ-伴大豆球蛋白+1μmol/L p38抑制剂SB202190),处理24 h后观察细胞结构,测定各组细胞活性及细胞因子NO、TNF-α、IL-10、IFN-γ含量,细胞损伤相关基因NF-κB p65、iNOS、JNK、p38及其编码蛋白的表达水平。【结果】在IPEC-J2中加入5 mg/mLβ-伴大豆球蛋白24 h后,细胞数量减少,线粒体改变,染色质边集,细胞活性显著下降(P<0.05),NO、TNF-α、IFN-γ含量均极显著增加(P<0.01),而IL-10含量极显著减少(P<0.01),NF-κB p65、iNOS、JNK和p38 mRNA表达水平极显著升高(P<0.01),NF-κB、iNOS、JNK、p38蛋白表达量极显著上升(P<0.01);加入抑制剂后,细胞活性显著提高(P<0.05),细胞结构趋于完整和规则,IL-10含量极显著增加(P<0.01),而NO、TNF-α、IFN-γ含量极显著减少(P<0.01),NF-κB p65、iNOS、JNK和p38 mRNA及其编码蛋白表达受到抑制。其中T2组细胞活性显著高于T3、T4、T5组(P<0.05),细胞数量、形态和完整性最接近对照组细胞。【结论】β-伴大豆球蛋白主要通过NF-κB/iNOS信号通路对IPEC-J2造成损伤,PDTC对这种损伤作用的抑制效果最好。【Objective】This study investigated the effects ofβ-conglycinin on porcine intestinal epithelial cells(IPEC-J2)through nuclear factor-κB(NF-κB),inducible nitric oxide synthase(iNOS),c-Jun N-terminal kinase(JNK)and p38 mitogen-activated protein kinase(p38 MAPK)signaling pathways to provide basis for exploring molecular mechanism ofβ-conglycinin-induced intestinal mucosal injury in piglets.【Method】Porcine intestinal epithelial cells(IPEC-J2)were induced byβ-conglycinin in vitro.Treatments included control group T0(without treatment to porcine intestinal epithelial cells),experimental group T1(5 mg/mLβ-conglycinin),T2(5 mg/mLβ-conglycinin+1μmol/L NF-κB inhibitor PDTC),T3(5 mg/mLβ-conglycinin+1μmol/L iNOS inhibitor Nω-nitro-L-arginine methyl ester(L-NAME)),T4(5 mg/mLβ-conglycinin+1μmol/L JNK inhibitor SP600125)and T5(5 mg/mLβ-conglycinin+1μmol/L p38 inhibitor SB202190).After 24 h,cell structure was observed,and cell activity,contents of NO,TNF-α,IL-10 and IFN-γ,and expression levels of NF-κB p65,iNOS,JNK and p38 related to cell injury were determined.【Result】After adding 5 mg/mLβ-conglycinin into porcine intestinal epithelial cells(IPEC-J2)for 24 h,the number of cells decreased,mitochondria changed,chromatin margined,and cell activity significantly decreased(P<0.05).Contents of NO,TNF-αand IFN-γsignificantly increased(P<0.01),while content of IL-10 significantly decreased(P<0.01).The mRNA expression levels of NF-κB p65,iNOS,JNK and p38 significantly increased(P<0.01),and protein expression levels of NF-κB,iNOS,JNK and p38 significantly increased(P<0.01).After adding inhibitors,cell activity increased significantly(P<0.05),cell structure tended to be complete and regular,content of IL-10 increased significantly(P<0.01),and contents of NO,TNF-αand IFN-γdecreased significantly(P<0.01).NF-κB p65,iNOS,JNK and p38 mRNA and protein expression were inhibited.Among treatments,cell activity of T2 group was significantly(P<0.05)higher than that of T3,T4 and T5 groups.HE staining and electro

关 键 词：Β-伴大豆球蛋白 猪肠上皮细胞 细胞因子 免疫反应 

分 类 号：S856.5[农业科学—临床兽医学]