11S通过NF-κB信号通路引起猪小肠上皮细胞损伤

11S triggered porcine intestinal epithelial cells damage through NF-κB signaling pathway

通过体外培养猪小肠上皮细胞(IPEC-J2),研究大豆球蛋白(11S)对猪小肠上皮细胞的影响。将IPEC-J2细胞分为6组[A(对照组)、B、C、D、E和F],各组中分别添加0、1、5、10、5、5 mg·mL^(-1)的11S,并且在E组和F组分别添加1μmol·L^(-1)吡咯烷二硫代氨基甲酸盐(PDTC)和1μmol·mL^(-1) Nω-硝基-L-精氨酸甲酯(L-NAME)。分别于8、16、24 h,用CCK-8法检测细胞存活率,用ELISA法检测细胞一氧化氮(NO)、二胺氧化酶(DAO)、5-羟色胺(5-HT)、白细胞介素6(IL-6)和白细胞介素10(IL-10)含量,用western blot法检测细胞p-NF-κB p65、iNOS、COX-2的蛋白表达量,用qPCR法检测细胞NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对表达量。结果表明:11S可以降低猪小肠上皮细胞存活率,添加PDTC和L-NAME可以提高小肠上皮细胞存活率;11S可促进NO、DAO、5-HT和IL-6的分泌,降低IL-10的分泌,增加p-NF-κB p65、iNOS、COX-2的蛋白表达量及NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对表达量,添加PDTC和L-NAME可抑制11S的作用。综上,11S可引起猪小肠上皮细胞损伤,随着11S浓度的增大,损伤程度增加,PDTC和L-NAME可以降低11S对细胞的损伤。

In this study,porcine intestinal epithelial cells(IPEC-J2)were cultured in vitro to study the effect of different concentrations of glycinin(11S)on IPEC-J2.IPEC-J2 cells were divided into six groups.A was control group,in groups B(1 mg·mL^-1 11S),C(5 mg·mL^-1 11S),D(10 mg·mL^-1 11S),E(5 mg·mL^-1 11S+1μmol·L-1 PDTC)and F(5 mg·mL^-1 11S+1μmol·mL^-1 L-NAME),0,1,5,10,5,and 5 mg·mL^-1 of 11S were added respectively.In addition,1μmol·L-1 PDTC and 1μmol·mL^-1 L-NAME were added to groups E and F,respectively.After 8,16 and 24 h,cell viability was measured by CCK-8 method,contents of NO,DAO,5-HT,IL-6 and IL-10 were detected by ELISA,and p-NF-κB,p65,iNOS and COX-2 protein expression levels were detected by western blot,relative expression of NF-κB p65,IKKβ,iNOS,IKKαand COX-2 mRNA were determined by PCR.The results showed that 11S reduced viability of porcine intestinal epithelial cells,promoted the secretion of NO,5-HT and IL-6,decreased the secretion of IL-10,and increased expression of p-NF-κB p65,iNOS and COX-2 proteins,and expression of NF-κB p65,IKKβ,iNOS,IKKαand COX-2 mRNA were also increased.PDTC and L-NAME could inhibit the effects of 11S.The results showed that 11S caused damage to IPEC-J2 and increased the damage with increasing concentration,PDTC and L-NAME could reduce the damage of 11S to cells.