| 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、JNK和p38-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、JNK和p38-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： |
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| 基金项目:国家自然科学基金项目（31972750） |
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| Mechanism of β-conglycinin on IPEC-J2 cell injury |
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SUN Zhifeng,WANG Lei,LIU Yujia,et al
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| 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 |
