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1μmol/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、p 38 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p 38 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p 38 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�
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 1μmol/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 p 38 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-γand 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-γand 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
作者
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
WANG Lei;SUN Zhifeng;PENG Chenglu;DING Hongyan;WANG Zhi;LI Siting;WAGN Chengzhi;LI Yu;WANG Xichun;WU Jinjie(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230061,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2021年第3期1663-1674,共12页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金(31972750)。
