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β-伴大豆球蛋白对IPEC-J2细胞损伤的作用机制研究
孙智峰1, 王 蕾1, 刘羽佳,等1
安徽农业大学 动物科技学院
摘要:
【目的】研究β-伴大豆球蛋白通过核因子-κB(NF-κB)、诱导型一氧化氮合酶(iNOS)、c-Jun N端激酶(JNK)和p38丝裂原活化蛋白激酶(p38 MAPK)信号通路对猪肠上皮细胞(IPEC-J2)造成损伤的差异,为探索β-伴大豆球蛋白引发仔猪肠道黏膜损伤的分子机制提供理论依据。【方法】采用β-伴大豆球蛋白体外诱导IPEC-J2损伤,试验设对照组T0(猪肠上皮细胞不做处理),试验组T1(加入5 mg/mL β-伴大豆球蛋白)、T2(加入5 mg/mL β-伴大豆球蛋白+1 μmol/L NF-κB抑制剂二硫氨基甲酸肽吡咯烷(PDTC))、T3(加入5 mg/mL β-伴大豆球蛋白+1 μmol/L iNOS抑制剂Nω-硝基-L-精氨酸甲酯(L-NAME))、T4(加入5 mg/mL β-伴大豆球蛋白+1 μmol/L JNK抑制剂SP600125)和T5(加入5 mg/mL β-伴大豆球蛋白+1 μmol/L p38抑制剂SB202190),处理24 h后观察细胞结构,测定各组细胞活性及细胞因子NO、TNF-α、IL-10、IFN-γ含量,细胞损伤相关基因NF-κB p65、iNOS、JNK、p38及其编码蛋白的表达水平。【结果】在IPEC-J2中加入5 mg/mL β-伴大豆球蛋白24 h后,细胞数量减少,线粒体改变,染色质边集,细胞活性显著下降(P<0.05),NO、TNF-α、IFN-γ含量均极显著增加(P<0.01),而IL-10含量极显著减少(P<0.01),NF-κB p65、iNOS、JNKp38-mRNA表达水平极显著升高(P<0.01),NF-κB、iNOS、JNK、p38蛋白表达量极显著上升(P<0.01);加入抑制剂后,细胞活性显著提高(P<0.05),细胞结构趋于完整和规则,IL-10含量极显著增加(P<0.01),而NO、TNF-α、IFN-γ含量极显著减少(P<0.01),NF-κB p65、iNOS、JNKp38-mRNA及其编码蛋白表达受到抑制。其中T2组细胞活性显著高于T3、T4、T5组(P<0.05),细胞数量、形态和完整性最接近对照组细胞。【结论】β-伴大豆球蛋白主要通过NF-κB/iNOS信号通路对IPEC-J2造成损伤,PDTC对这种损伤作用的抑制效果最好。
关键词:  β-伴大豆球蛋白  猪肠上皮细胞  细胞因子  免疫反应
DOI:
分类号:
基金项目:国家自然科学基金项目(31972750)
Mechanism of β-conglycinin on IPEC-J2 cell injury
SUN Zhifeng,WANG Lei,LIU Yujia,et al
Abstract:
【Objective】This study investigated the effects of β-conglycinin on porcine intestinal epithelial cells (IPEC-J2) through nuclear factor-κB (NF-κB),inducible nitric oxide synthase (iNOS),c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK) signaling pathways to provide basis for exploring molecular mechanism of β-conglycinin induced intestinal mucosal injury in piglets.【Method】Porcine intestinal epithelial cells (IPEC-J2) were induced by β-conglycinin in vitro.Treatments included control group T0 (without treatment to porcine intestinal epithelial cells),experimental group T1 (5 mg/mL β-conglycinin),T2 (5 mg/mL β-conglycinin+1 μmol/L NF-κB inhibitor PDTC),T3 (5 mg/mL β-conglycinin+1 μmol/L iNOS inhibitor Nω-nitro L-arginine methyl ester (L-NAME)),T4 (5 mg/mL β-conglycinin+1 μmol/L JNK inhibitor SP600125) and T5 (5 mg/mL β-conglycinin+1 μmol/L p38 inhibitor SB202190).After 24 h,cell structure was observed,and cell activity,contents of NO,TNF-α,IL-10 and IFN-γ,and expression levels of NF-κB p65,iNOS,JNK and p38 related to cell injury were determined.【Result】After adding 5 mg/mL β-conglycinin into porcine intestinal epithelial cells (IPEC-J2) for 24 h,the number of cells decreased,mitochondria changed,chromatin margined,and cell activity significantly decreased (P<0.05).Contents of NO,TNF-α and IFN-γ significantly increased (P<0.01),while content of IL-10 significantly decreased (P<0.01).The mRNA expression levels of NF-κB p65,iNOS,JNK and p38 significantly increased (P<0.01),and protein expression levels of NF-κB,iNOS,JNK and p38 significantly increased (P<0.01).After adding inhibitors,cell activity increased significantly (P<0.05),cell structure tended to be complete and regular,content of IL-10 increased significantly (P<0.01),and contents of NO,TNF-α and IFN-γ decreased significantly (P<0.01).NF-κB p65,iNOS,JNK and p38 mRNA and protein expression were inhibited.Among treatments,cell activity of T2 group was significantly (P<0.05) higher than that of T3,T4 and T5 groups.HE staining and electron microscopy showed that number,morphology and integrity of cells in T2 group were closest to those in the control group.【Conclusion】β-coglycinin damages IPEC J2 cells mainly through NF-κB/iNOS signal pathway and PDTC has the best inhibitory effects.
Key words:  β-conglycinin  IPEC-J2 cell  cell factor  immune response