β-伴大豆球蛋白通过p38/JNK MAPK信号通路引起IPEC-J2细胞损伤

采用细胞体外培养技术,研究不同浓度7S(β-伴大豆球蛋白)对IPEC-J2(猪小肠上皮细胞)的影响。实验分为6组,A(对照组)、B(1 mg·mL^-1 7S)、C(5 mg·mL^-1 7S)、D(10 mg·mL^-1 7S)、E(5 mg·mL^-1 7S+1 μmol·L^-1 SP600125)、F(5 mg·mL^-1 7S+1 μmol·L^-1 SB202190),24 h后,用CCK-8法检测细胞存活率,收集细胞用ELISA法检测NO、5-HT、IL-6和IL-10含量,用Western blot法检测p-JNK、p-p38、Bcl-2蛋白表达量,用PCR法检测Bad、Bax、Bcl-2、Bcl-x_L和Caspase-3 mRNA相对表达量。检测结果表明:C组和D组的IPEC-J2细胞活性极显著降低(P<0.01),E组和F组的细胞活性显著高于C组(P<0.05),说明7S促进炎性细胞因子NO、5-HT和IL-6的分泌,降低IL-10的分泌,添加抑制剂使细胞因子NO、5-HT和IL-6分泌减少,IL-10的分泌增多;同时7S促进p-JNK、p-p38蛋白表达,降低Bcl-2蛋白表达,添加抑制剂可抑制p-JNK、p-p38蛋白表达,使Bcl-2蛋白表达增高。Bcl-2/Bax mRNA比值随7S浓度增加而降低,Bax/Bcl-x_l比值随7S浓度增加而升高,Caspase-3 mRNA相对表达量随7S浓度增加而升高。添加抑制剂后Bcl-2/Bax比值增高,Bax/Bcl-x_l比值降低,Caspase-3 mRNA降低。结果表明,7S通过p38/JNK MAPK信号通路引起仔猪肠上皮细胞损伤。

β-Conglycinin triggered IPEC-J2 cell damage through p38/JNK MAPK signaling pathway

In this study, in vitro culture techniques were used to study the effects of different concentrations of 7S (β-conglycinin) on IPEC-J2 (intestinal epithelial cells in piglets). The experiment was divided into six groups, A (control), B (1 mg·mL^-1 7S), C (5 mg·mL^-1 7S), D (10 mg·mL^-1 7S), E (5 mg·mL^-1 7S+1μmol·L^-1 SP600125), F (5 mg·mL^-1 7S+1 μmol·L^-1 SB202190). After 24 hours, the viability of the cells was measured by CCK-8 assay. The contents of NO, 5-HT, IL-6 and IL-10 were detected by ELISA, the expression levels of p-JNK, p-p38 and Bcl-2 proteins were detected by western blot, the relative expression of Bad, Bax, Bcl-2 and Caspase-3 mRNA was detected by PCR. The results showed that the viability of IPEC-J2 cells was decreased in groups C and D (P<0.01). The viability of cells in groups E and F was significantly higher than that in group C (P<0.05). 7S promoted the secretion of inflammatory cytokines NO, 5-HT, and IL-6 and decreased the secretion of IL-10, the addition of inhibitors decreased the secretion of cytokines NO, 5-HT, and IL-6, and increased the secretion of IL-10. 7S promoted the expression of p-JNK and p-p38 proteins and decreased the expression of Bcl-2 protein. The addition of inhibitors inhibited the expression of p-JNK and p-p38 proteins and increased the expression of Bcl-2 protein. The ratio of Bcl-2/Bax mRNA decreased with the increase of 7S concentration, and the ratio of Bax/Bcl-x_l increased with the increase of 7S concentration. The relative expression of Caspase-3 mRNA increased with the increase of 7S concentration. On contrary, often adding inhibitors, the ratio of Bcl-2/Bax increased, the ratio of Bax/Bcl-x_l decreased, and the relative expression of Caspase-3 decreased. Our results indicated that 7S caused damage to the intestinal epithelial cells of piglets through the p38/JNK MAPK signaling pathway.