低聚壳聚糖硒抗玉米赤霉烯酮对IPEC-J2细胞紧密连接蛋白表达的影响

Effects of Selenium-oligochitosan Against Zearalenone on the Expression of Tight Junction Protein in IPEC-J2 Cells

试验旨在探究低聚壳聚糖硒抗玉米赤霉烯酮(zearalenon,ZEN)对猪小肠上皮细胞(IPEC-J2)紧密连接蛋白表达的影响。试验分为对照组(C)、ZEN(30μg/mL ZEN)、ZEN+0.5 Se(30μg/mL ZEN+0.5μmol/L低聚壳聚糖硒,以Se计)、ZEN+1.5 Se和ZEN+3 Se组。待细胞长至60%~70%汇合时,ZEN+0.5 Se、ZEN+1.5 Se和ZEN+3 Se组培养液更换为含对应Se浓度的培养液,C和ZEN组更换为正常培养液,12 h后向含ZEN组加入30μg/mL ZEN,继续培养24 h。收集各组细胞培养液,检测碱性磷酸酶(AKP)活性;Western blotting法检测闭锁连接蛋白(zonula occludens-1,ZO-1)、闭合蛋白(Occludin)的表达,免疫荧光法检测ZO-1和Occludin蛋白的表达和定位情况。AKP活性检测结果表明,与对照组相比,ZEN、ZEN+0.5 Se、ZEN+1.5 Se组AKP活性均显著升高(P<0.05),ZEN+3 Se组AKP活性差异不显著(P>0.05);与ZEN组相比,ZEN+1.5 Se和ZEN+3 Se组AKP活性均显著降低(P<0.05)。Western blotting检测结果表明,与对照组比,ZEN组ZO-1和Occludin蛋白表达水平均显著降低(P<0.05);与ZEN组比,ZEN+0.5 Se、ZEN+1.5 Se和ZEN+3 Se组ZO-1表达水平均差异不显著(P>0.05),但随着Se浓度的增加,ZO-1表达水平逐渐升高,ZEN+0.5 Se、ZEN+1.5 Se、ZEN+3 Se组Occludin表达水平均显著升高(P<0.05)。免疫荧光结果表明,低聚壳聚糖硒可缓解ZEN引起的ZO-1和Occludin蛋白荧光信号减弱现象,并恢复ZO-1和Occludin蛋白的定位分布。由此说明,低聚壳聚糖硒可降低ZEN引起的IPEC-J2细胞AKP活性升高,上调ZEN引起的ZO-1、Occludin蛋白表达水平下降,并调节其定位分布。

The aim of this study was to investigate the effects of selenium-oligochitosan against zearalenon(ZEN)on the expression of tight junction protein of IPEC-J2 cells.The experiment was divided into control group(C),ZEN(30μg/mL),ZEN+0.5 Se(30μg/mL ZEN+0.5μmol/L selenium-oligochitosan,in terms of Se),ZEN+1.5 Se and ZEN+3 Se groups.When the confluence of cells reached 60%-70%,the culture medium of ZEN+0.5 Se,ZEN+1.5 Se and ZEN+3 Se groups was replaced with the culture medium containing the corresponding Se concentration,C and ZEN groups were replaced with normal culture medium.After 12 h,30μg/mL ZEN was added to the ZEN-containing groups,and the culture was continued for 24 h.The cell culture medium of each group was collected to detect the activity of alkaline phosphatase(AKP),the expressions of zonula occludens-1(ZO-1)and Occludin were detected by Western blotting,the expression and localization of ZO-1 and Occludin proteins were detected by immunofluorescence.The results of AKP activity showed that compared with control group,the AKP activity was significantly increased in ZEN,ZEN+0.5 Se and ZEN+1.5 Se groups(P<0.05),but there was no significant difference in ZEN+3 Se group(P>0.05).Compared with ZEN group,the AKP activity of ZEN+1.5 Se and ZEN+3 Se groups was significantly reduced(P<0.05).Western blotting results showed that compared with control group,the protein expression levels of ZO-1 and Occludin in ZEN group were significantly decreased(P<0.05).When compared with ZEN group,there were no significant differences in ZO-1 expression levels in ZEN+0.5 Se,ZEN+1.5 Se and ZEN+3 Se groups(P>0.05),but with the increase of Se concentration,the protein expression level of ZO-1 was gradually increased,the protein expression level of Occludin was significantly increased in ZEN+0.5 Se,ZEN+1.5 Se and ZEN+3 Se groups(P<0.05).The immunofluorescence results showed that selenium-oligochitosan could relieve the decreased fluorescence signal of ZO-1 and Occludin caused by ZEN,and restore the location distribution of the proteins.In conclusion,selenium-oligochitosan could reduce the increase of AKP activity in IPEC-J2 cells caused by ZEN,up-regulate the decreased expression levels of ZO-1 and Occludin in IPEC-J2 cells caused by ZEN,and adjust its location distribution.