动物营养学报 >

2021 , Vol. 33 >Issue 3: 1663 - 1674

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.03.047

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

11S球蛋白通过核因子-κB、诱导型一氧化氮合酶、c-Jun N端激酶、p38丝裂原活化蛋白激酶信号通路诱导猪小肠上皮细胞损伤的研究

  • 王蕾 ,
  • 孙智峰 ,
  • 彭成璐 ,
  • 丁红研 ,
  • 王志 ,
  • 李思婷 ,
  • 王承智 ,
  • 李玉 ,
  • 王希春 ,
  • 吴金节
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  • 安徽农业大学动物科技学院, 合肥 230061
王蕾(1986-),女,河南桐柏人,硕士研究生,从事畜禽营养代谢病研究。E-mail:350939708@qq.com

收稿日期: 2020-08-09

  网络出版日期: 2021-03-18

基金资助

国家自然科学基金（31972750）

收起

Research on IPEC-J2 Cell Injury Induced by 11S Globulin via Signal Pathway of Nuclear Factor-κB, Inducible Nitric Oxide Synthase, c-Jun N-Terminal Kinase and p38 Mitogen Activated Protein Kinase

  • WANG Lei ,
  • SUN Zhifeng ,
  • PENG Chenglu ,
  • DING Hongyan ,
  • WANG Zhi ,
  • LI Siting ,
  • WAGN Chengzhi ,
  • LI Yu ,
  • WANG Xichun ,
  • WU Jinjie
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  • College of Animal Science and Technology, Anhui Agricultural University, Hefei 230061, China

Received date: 2020-08-09

  Online published: 2021-03-18

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摘要

本研究旨在利用细胞体外培养技术，分析11S球蛋白通过核因子-кB（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）信号通路诱导猪小肠上皮细胞（IPEC-J2细胞）损伤的作用差异。试验随机分为6组：A组（对照组）无添加；B组添加5 mg/mL的11S球蛋白；C、D、E和F组分别添加1 μmol/L的NF-κB抑制剂二硫氨基甲酸肽吡咯烷（PDTC）、iNOS抑制剂Nω-硝基-L-精氨酸甲酯（L-NAME）、JNK抑制剂SP600125和p38 MAPK抑制剂SB202190预处理后，分别添加5 mg/mL的11S球蛋白。培养24 h后，CCK-8检测细胞活性，酶联免疫吸附测定（ELISA）法检测一氧化氮（NO）、肿瘤坏死因子-α（TNF-α）、干扰素-γ（INF-γ）和白细胞介素-10（IL-10）含量，苏木精-伊红（HE）染色法观察细胞及细胞核形态，用透射电子显微镜观察细胞超微结构，实时荧光定量PCR检测NF-κB、iNOS、JNK、p38 MAPK mRNA相对表达量，Western blot检测NF-κB、iNOS、JNK、p38 MAPK蛋白表达水平。结果显示：1）与A组相比，B组细胞活性极显著降低（P<0.01）；与B组相比，C、D、E和F组细胞活性极显著升高（P<0.01），且C组细胞活性显著高于D、E和F组（P<0.05）。2）与A组相比，B组TNF-α、INF-γ和NO含量极显著升高（P<0.01），IL-10含量极显著降低（P<0.01）；与B组相比，C、D、E和F组TNF-α、INF-γ和NO含量极显著降低（P<0.01），IL-10含量极显著升高（P<0.01）。3）与A组相比，B组NF-κB、iNOS、JNK和p38 MAPK蛋白表达水平和mRNA相对表达量极显著升高（P<0.01）。4）与B组相比，C和F组NF-κB、iNOS、JNK和p38 MAPK mRNA相对表达量显著或极显著降低（P<0.05或P<0.01），D组NF-κB、iNOS和JNK mRNA相对表达量极显著降低（P<0.01），E组NF-κB、JNK和p38 MAPK mRNA相对表达量显著或极显著降低（P<0.05或P<0.01）。5）与B组相比，C、E和F组NF-κB、iNOS、JNK和p38 MAPK蛋白表达水平极显著降低（P<0.01），D组NF-κB、iNOS和JNK蛋白表达水平极显著降低（P<0.01）。6） HE染色及透射电镜观察可见，B组细胞损伤、胞质空泡化、核染色质聚集，C、D、E和F组细胞损伤受到抑制，且C组细胞结构形态的完整性优于D、E和F组。由此可见，11S球蛋白通过JNK/p38 MAPK/NF-κB/iNOS信号通路诱导IPEC-J2细胞损伤，且NF-κB信号通路在诱导细胞损伤的过程中发挥关键作用。

关键词： 大豆球蛋白; IPEC-J2细胞; 信号通路; 细胞损伤

本文引用格式

王蕾 , 孙智峰 , 彭成璐 , 丁红研 , 王志 , 李思婷 , 王承智 , 李玉 , 王希春 , 吴金节 . 11S球蛋白通过核因子-κB、诱导型一氧化氮合酶、c-Jun N端激酶、p38丝裂原活化蛋白激酶信号通路诱导猪小肠上皮细胞损伤的研究[J]. 动物营养学报, 2021 , 33(3) : 1663 -1674 . DOI: 10.3969/j.issn.1006-267x.2021.03.047

Abstract

The present study aimed to analyze the discrepancies in 11S glycinin induced porcine intestinal epithelial cell (IPEC-J2 cell) injury via signal pathways of nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS), c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK) using cell culture technology in vitro. The experiment was randomly divided into 6 groups: group A (control group) was no added; group B was added 5 mg/mL 11S glycinin; groups C, D, E and F were pretreated with 1 μmol/L NF-κB inhibitor of pyrrolidine dithiocarbamte (PDTC), iNOS inhibitor of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), JNK inhibitor of SP600125) and p38 MAPK inhibitor of SB202190, respectively, and then were added 5 mg/mL 11S glycinin. After 24 hours culture, cell viability was detected by CCK-8. The contents of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ) and interleukin-10 (IL-10) were measured by enzyme-linked immunosorbent assay (ELISA) method. The morphology of cells and nuclei were observed by hematoxylin-eosin (HE)staining. The ultrastructure of cells was observed by transmission electron microscope. The mRNA relative expression levels of NF-κB, iNOS, JNK and p38 MAPK were determined by real-time quantitative PCR. The protein expression levels of NF-κB, iNOS, JNK and p38 MAPK were detected by Western blot. The results showed as follows: 1) compared with group A, the cell viability of group B was significantly decreased (P<0.01); compared with group B, the cell viability of groups C, D, E and F were significantly increased (P<0.01), and the cell viability of group C was significantly higher than that of groups D, E and F (P<0.05). 2) Compared with group A, the contents of TNF-α, INF-γ and NO of group B were significantly increased (P<0.01), but the IL-10 content was significantly decreased (P<0.01); compared with group B, the contents of TNF-α, INF-γ and NO of groups C, D, E and F were significantly decreased (P<0.01), but the IL-10 content was significantly increased (P<0.01). 3) Compared with group A, the protein expression levels and the mRNA relative expression levels of NF-κB, iNOS, JNK and p38 MAPK of group B were significantly increased (P<0.01). 4) Compared with group B, the mRNA relative expression levels of NF-κB, iNOS, JNK and p38 MAPK of group C and F were significantly decreased (P<0.05 or P<0.01), the mRNA relative expression levels of NF-κB, iNOS and JNK of group D were significantly decreased (P<0.01), the mRNA relative expression levels of NF-κB, JNK and p38 MAPK of group E were significantly decreased (P<0.05 or P<0.01). 5) Compared with group B, the protein expression levels of NF-κB, iNOS, JNK and p38 MAPK of group C ,E and F were significantly decreased (P<0.01), the protein expression levels of NF-κB, iNOS and JNK of group D were significantly decreased (P<0.01). 6) Observation of the HE staining and transmission electron microscope showed cells damage, cytoplasmic vacuolation and aggregation of nuclear chromatin in group B, the injury of cells were inhibited in groups C, D, E and F, and the integrity of cell structure and morphology in group C was better than that in groups D, E and F. In conclusion, the 11S glycinin can induce IPEC-J2 cell damage via JNK/p38 MAPK/NF-κB/iNOS signaling pathways, and the NF-κB signaling pathway plays a critical role in the process of inducing cell damage.

Key words： glycinin; IPEC-J2 cells; signal pathway; cell damage

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