7S β-伴大豆球蛋白通过NF-κB信号通路引起IPEC-J2细胞的炎性反应

7S β-conglycinin Triggered Inflammatory Response in IPEC-J2 Cells through NF-κB Signaling Pathway

采用细胞体外培养技术,研究不同浓度7Sβ-伴大豆球蛋白对猪小肠上皮细胞(IPEC-J2细胞)的影响。试验分为A、B、C、D、E、F 6组,其中A为对照组,其余各组中分别添加1、5、10、5、5 mg·mL^(-1)的β-伴大豆球蛋白,并且在E和F组分别添加1μmol·L^(-1) NF-κB(PDTC)和iNOS(L-NAME)抑制剂。用CCK-8法检测各组细胞存活率,用ELISA法检测细胞NO、DAO、5-HT、IL-6和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的相对转录量。结果显示:β-伴大豆球蛋白降低IPEC-J2细胞存活率,添加PDTC和L-NAME增高IPEC-J2细胞存活率,促进NO、DAO、5-HT和IL-6的分泌,并降低IL-10的分泌,同时增加p-NF-κB p65、iNOS、COX-2的蛋白表达量及NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对转录量,添加PDTC和L-NAME后抑制了β-伴大豆球蛋白的作用。结果表明,β-伴大豆球蛋白引起IPEC-J2细胞损伤,随浓度增大损伤增加,PDTC和L-NAME可以降低β-伴大豆球蛋白对细胞的作用。

In this study,IPEC-J2 cells(porcine intestinal epithelial cells)were cultured in vitro to study the effect of different concentrations of 7Sβ-conglycinin on IPEC-J2.The experiment was designed as six groups:A,B,C,D,E,and F,A was the control group,and the other groups were added 1,5,10,5 and 5 mg·mL^-1β-conglycinin,respectively,and 1μmol·L^-1 NF-κB(PDTC)and iNOS(L-NAME)inhibitors were added to groups E and F,respectively.Cell viability was measured by CCK-8 method,the contents of NO,DAO,5-HT,IL-6 and IL^-10 were detected by ELISA,p-NF-κB,p65,iNOS and COX-2 protein expression levels were detected by Western blot,the relative transcription of NF-κB p65,IKKβ,iNOS,IKKαand COX-2 mRNA were determined by qPCR.The results showed thatβ-conglycinin reduced the viability of IPEC-J2 cells,promoted the secretion of NO,5-HT and IL-6,decreased the secretion of IL^-10,and increased the expression of p-NF-κB p65,iNOS and COX-2 proteins,transcription of NF-κB p 65,IKKβ,i NOS,IKKαand COX-2 mRNA,PDTC and L-NAME can inhibit the effects ofβ-conglycinin.The results showed thatβ-conglycinin caused damage to IPEC-J2 and increased the damage with increasing concentration,PDTC and L-NAME can reduce the damage ofβ-conglycinin to cells.