摘要
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(11 S) on IPEC-J2. IPEC-J2 cells were divided into six groups. A was control group, in groups B(1 mg·mL~(-1) 11 S), C(5 mg·mL~(-1) 11 S), D(10 mg·mL~(-1) 11 S), E(5 mg·mL~(-1) 11 S+1 μmol·L~(-1) PDTC) and F(5 mg·mL~(-1) 11 S+1 μmol·mL~(-1) L-NAME), 0, 1, 5, 10, 5, and 5 mg·mL~(-1) of 11 S were added respectively. In addition, 1 μmol·L~(-1) PDTC and 1 μmol·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, IKKα and COX-2 mRNA were determined by PCR. The results showed that 11 S 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, IKKα and COX-2 mRNA were also increased. PDTC and L-NAME could inhibit the effects of 11 S. The results showed that 11 S caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME could reduce the damage of 11 S to cells.
引文
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