7S β-伴大豆球蛋白通过NF-κB信号通路引起IPEC-J2细胞的炎性反应
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摘要
采用细胞体外培养技术,研究不同浓度7Sβ-伴大豆球蛋白对猪小肠上皮细胞(IPEC-J2细胞)的影响。试验分为A、B、C、D、E、F 6组,其中A为对照组,其余各组中分别添加1、5、10、5、5 mg·mL~(-1)的β-伴大豆球蛋白,并且在E和F组分别添加1μmol·L~(-1) NF-κB(PDTC)和iNOS(L-NAME)抑制剂。用CCK-8法检测各组细胞存活率,用ELISA法检测细胞NO、DAO、5-HT、IL-6和IL-10含量,用Western blot法检测细胞p-NF-κB p65、iNOS、COX-2的蛋白表达量,用qPCR法检测细胞NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对转录量。结果显示:β-伴大豆球蛋白降低IPEC-J2细胞存活率,添加PDTC和L-NAME增高IPEC-J2细胞存活率,促进NO、DAO、5-HT和IL-6的分泌,并降低IL-10的分泌,同时增加p-NF-κB p65、iNOS、COX-2的蛋白表达量及NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对转录量,添加PDTC和L-NAME后抑制了β-伴大豆球蛋白的作用。结果表明,β-伴大豆球蛋白引起IPEC-J2细胞损伤,随浓度增大损伤增加,PDTC和L-NAME可以降低β-伴大豆球蛋白对细胞的作用。
In this study, IPEC-J2 cells(porcine intestinal epithelial cells) were cultured in vitro to study the effect of different concentrations of 7 S β-conglycinin on IPEC-J2. The experiment was designed as six groups: A, B, C, D, E, and F, A was the control group, and the other groups were added 1, 5, 10, 5 and 5 mg·mL~(-1)β-conglycinin, respectively, and 1 μmol·L~(-1) NF-κB(PDTC) and iNOS(L-NAME) inhibitors were added to groups E and F, respectively. Cell viability was measured by CCK-8 method, the contents of NO, DAO, 5-HT, IL-6 and IL-10 were detected by ELISA, p-NF-κB, p65, iNOS and COX-2 protein expression levels were detected by Western blot, the relative transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were determined by qPCR. The results showed that β-conglycinin reduced the viability of IPEC-J2 cells, promoted the secretion of NO, 5-HT and IL-6, decreased the secretion of IL-10, and increased the expression of p-NF-κB p65, iNOS and COX-2 proteins, transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA, PDTC and L-NAME can inhibit the effects of β-conglycinin. The results showed that β-conglycinin caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME can reduce the damage of β-conglycinin to cells.
In this study, IPEC-J2 cells(porcine intestinal epithelial cells) were cultured in vitro to study the effect of different concentrations of 7 S β-conglycinin on IPEC-J2. The experiment was designed as six groups: A, B, C, D, E, and F, A was the control group, and the other groups were added 1, 5, 10, 5 and 5 mg·mL~(-1)β-conglycinin, respectively, and 1 μmol·L~(-1) NF-κB(PDTC) and iNOS(L-NAME) inhibitors were added to groups E and F, respectively. Cell viability was measured by CCK-8 method, the contents of NO, DAO, 5-HT, IL-6 and IL-10 were detected by ELISA, p-NF-κB, p65, iNOS and COX-2 protein expression levels were detected by Western blot, the relative transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were determined by qPCR. The results showed that β-conglycinin reduced the viability of IPEC-J2 cells, promoted the secretion of NO, 5-HT and IL-6, decreased the secretion of IL-10, and increased the expression of p-NF-κB p65, iNOS and COX-2 proteins, transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA, PDTC and L-NAME can inhibit the effects of β-conglycinin. The results showed that β-conglycinin caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME can reduce the damage of β-conglycinin to cells.
引文
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[19] ZHAO Y,QIN G X,HAN R,et al.β-conglycinin reduces the tight junction occludin and ZO-1 expression in IPEC-J2[J].Int J Mol Sci,2014,15(2):1915-1926.
[20] 王俊.β-伴大豆球蛋白及其酶解肽对仔猪肠上皮细胞完整性的影响[D].长春:吉林农业大学,2013.WANG J.β-conglycinin and its enzymatic peptide on the integrity of procine intestinal epithelial cells[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[21] 曹程鸣,李宝,张瑜,等.日粮中添加大豆抗原蛋白对断奶仔猪血清中IgG和IgE水平以及肠道ZO-1表达的影响[J].中国兽医科学,2017,47(3):389-396.CAO C M,LI B,ZHANG Y,et al.Effects of adding soybean antigen proteins on serum IgG and IgE levels and expression of intestinal ZO-1 in weaning piglets[J].Chinese Veterinary Science,2017,47(3):389-396.(in Chinese)
[22] WU J J,ZHANG Y,DONG J H,et al.Allergens and intestinal damage induced by soybean antigen proteins in weaned piglets[J].Ital J Anim Sci,2016,15(3):437-445.
[23] WU J J,CAO C M,REN D D,et al.Effects of soybean antigen proteins on intestinal permeability,5-hydroxytryptamine levels and secretory IgA distribution in the intestine of weaned piglets[J].Ital J Anim Sci,2016,15(1):174-180.
[24] 刘欣.大豆球蛋白glycinin和β-conglycinin引发Balb/c小鼠过敏反应及其机理的研究[D].杭州:浙江大学,2008.LIU X.Research on mechanism of allergic reactions caused by soybean glycinin and β-conglycinin in Balb/c mice[D].Hangzhou:Zhejiang University,2008.(in Chinese)
[25] 张建中,卢建军.大豆抗原蛋白对断奶仔猪生长性能及肠道免疫因子的影响[J].浙江农业学报,2010,22(6):843-847.ZHANG J Z,LU J J.Effects of soybean antigen protein on the growth performance of weaning piglets[J].Acta Agriculturae Zhejiangensis,2010,22(6):843-847.(in Chinese)
[26] LEE Y B,YUNE T Y,BAIK S Y,et al.Role of tumor necrosis factor-α in neuronal and glial apoptosis after spinal cord injury[J].Exp Neurol,2000,166(1):190-195.
[27] NéMETH Z H,DEITCH E A,SZABó C,et al.Hyperosmotic stress induces nuclear factor-κB activation and interleukin-8 production in human intestinal epithelial cells[J].Am J Pathol,2002,161(3):987-996.
[28] 王新,闻勤生,黄裕新.L-NAME对肝硬化大鼠胃肠道中一氧化氮合酶亚型表达的影响[J].第四军医大学学报,2001,22(9):817-820.WANG X,WEN Q S,HUANG Y X.L-NAME and expression of NOS isoforms in cirrhotic rat intestines[J].Journal of the Fourth Military Medical University,2001,22(9):817-820.(in Chinese)
[2] MURAKAMI H,OGAWA T,TAKAFUTA A,et al.Identification of the 7S and 11S globulins as percutaneously sensitizing soybean allergens as demonstrated through epidermal application of crude soybean extract[J].Biosci,Biotechnol,Biochem,2018,82(8):1408-1416.
[3] MUJOO R,TRINH D T,NG P K W.Characterization of storage proteins in different soybean varieties and their relationship to tofu yield and texture[J].Food Chem,2003,82(2):265-273.
[4] 孙泽威,秦贵信,娄玉杰.大豆抗原及其对仔猪和犊牛的影响[J].动物营养学报,2005,17(1):20-24.SUN Z W,QIN G X,LOU Y J.Soybean antigens and its influence on piglets and calves[J].Acta Zoonutrimenta Sinica,2005,17(1):20-24.(in Chinese)
[5] 李进杰,宋志甫.β-伴大豆球蛋白在仔猪胃肠组织中分布规律的研究[J].中国畜牧兽医,2014,41(5):176-179.LI J J,SONG Z P.Study on the distribution of β-conglycinin in gastro-intestinal tissue of piglets[J].China Animal Husbandry & Veterinary Medicine,2014,41(5):176-179.(in Chinese)
[6] 乔维洲.免疫性病变与肠道菌群相关性研究进展[J].国际检验医学杂志,2013,34(23):3199-3201.QIAO W Z.Research progress on the correlation between immune diseases and intestinal flora[J].International Journal of Laboratory Medicine,2013,34(23):3199-3201.(in Chinese)
[7] 朱丽慧.早期断奶仔猪肠细胞凋亡分子机理及营养调控研究[D].上海:上海交通大学,2014.ZHU L H.Molecular mechanism of apoptosis in weaned piglets and nutrition regulation[D].Shanghai:Shanghai Jiaotong University,2014.(in Chinese)
[8] SCHIERACK P,NORDHOFF M,POLLMANN M,et al.Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine[J].Histochem Cell Biol,2006,125(3):293-305.
[9] SALVEMINI D,ISCHIROPOULOS H,CUZZOCREA S.Roles of nitric oxide and superoxide in inflammation[M]//WINYARD P G,WILLOUGHBY D A.Inflammation Protocols.Totowa:Humana Press,2003,225:291-303.
[10] 范精华,刘康,刘保林.NO在炎症及免疫应答中的调节作用[J].中外医疗,2009,28(25):163,166.FAN J H,LIU K,LIU B L.Regulation of NO in inflammation and immune response [J].China & Foreign Medical Treatment,2009,28(25):163,166.(in Chinese)
[11] TORRES N,TORRE-VILLALVAZO I,TOVAR A R.Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders[J].J Nutr Biochem,2006,17(6):365-373.
[12] CHEN F,HAO Y,PIAO X S,et al.Soybean-derived β-conglycinin affects proteome expression in pig intestinal cells in vivo and in vitro[J].J Anim Sci,2011,89(3):743-753.
[13] HAYDEN M S,GHOSH S.Signaling to NF-κB[J].Genes Dev,2004,18(18):2195-2224.
[14] WILSMANN-THEIS D,KOCH S,MINDNICH C,et al.Generation and functional analysis of human TNF-α/iNOS-producing dendritic cells (Tip-DC)[J].Allergy,2013,68(7):890-898.
[15] CARNOVALE C E,RONCO M T.Role of nitric oxide in liver regeneration[J].Ann Hepatol,2012,11(5):636-647.
[16] 周艳梅.NF-κB特异性抑制剂PDTC对鼠黑素瘤B16细胞作用的实验研究[D].合肥:安徽医科大学,2012.ZHOU Y M.Experimental research of NF-κB specific inhibitor PDTC on mouse melanoma cell line B16 in vitro[D].Hefei:Anhui Medical University,2012.(in Chinese)
[17] 宋红梅.一氧化氮合酶抑制剂L-NAME对慢性脑缺血大鼠行为学及海马iNOS表达的影响[D].长春:吉林大学,2007.SONG H M.Effects of nitric oxide synthase inhibitor L-NAME on cognitive behavior and the expression of iNOS in hippocampus of chronic cerebral ischemia rats[D].Changchun:Jilin University,2007.(in Chinese)
[18] CHEN C C,LEE T T,HSU C B,et al.Associations of allergenic soybean proteins with piglet skin allergic reaction and application of polyclonal antibodies[J].Anim Prod Sci,2011,51(11):1008-1014.
[19] ZHAO Y,QIN G X,HAN R,et al.β-conglycinin reduces the tight junction occludin and ZO-1 expression in IPEC-J2[J].Int J Mol Sci,2014,15(2):1915-1926.
[20] 王俊.β-伴大豆球蛋白及其酶解肽对仔猪肠上皮细胞完整性的影响[D].长春:吉林农业大学,2013.WANG J.β-conglycinin and its enzymatic peptide on the integrity of procine intestinal epithelial cells[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[21] 曹程鸣,李宝,张瑜,等.日粮中添加大豆抗原蛋白对断奶仔猪血清中IgG和IgE水平以及肠道ZO-1表达的影响[J].中国兽医科学,2017,47(3):389-396.CAO C M,LI B,ZHANG Y,et al.Effects of adding soybean antigen proteins on serum IgG and IgE levels and expression of intestinal ZO-1 in weaning piglets[J].Chinese Veterinary Science,2017,47(3):389-396.(in Chinese)
[22] WU J J,ZHANG Y,DONG J H,et al.Allergens and intestinal damage induced by soybean antigen proteins in weaned piglets[J].Ital J Anim Sci,2016,15(3):437-445.
[23] WU J J,CAO C M,REN D D,et al.Effects of soybean antigen proteins on intestinal permeability,5-hydroxytryptamine levels and secretory IgA distribution in the intestine of weaned piglets[J].Ital J Anim Sci,2016,15(1):174-180.
[24] 刘欣.大豆球蛋白glycinin和β-conglycinin引发Balb/c小鼠过敏反应及其机理的研究[D].杭州:浙江大学,2008.LIU X.Research on mechanism of allergic reactions caused by soybean glycinin and β-conglycinin in Balb/c mice[D].Hangzhou:Zhejiang University,2008.(in Chinese)
[25] 张建中,卢建军.大豆抗原蛋白对断奶仔猪生长性能及肠道免疫因子的影响[J].浙江农业学报,2010,22(6):843-847.ZHANG J Z,LU J J.Effects of soybean antigen protein on the growth performance of weaning piglets[J].Acta Agriculturae Zhejiangensis,2010,22(6):843-847.(in Chinese)
[26] LEE Y B,YUNE T Y,BAIK S Y,et al.Role of tumor necrosis factor-α in neuronal and glial apoptosis after spinal cord injury[J].Exp Neurol,2000,166(1):190-195.
[27] NéMETH Z H,DEITCH E A,SZABó C,et al.Hyperosmotic stress induces nuclear factor-κB activation and interleukin-8 production in human intestinal epithelial cells[J].Am J Pathol,2002,161(3):987-996.
[28] 王新,闻勤生,黄裕新.L-NAME对肝硬化大鼠胃肠道中一氧化氮合酶亚型表达的影响[J].第四军医大学学报,2001,22(9):817-820.WANG X,WEN Q S,HUANG Y X.L-NAME and expression of NOS isoforms in cirrhotic rat intestines[J].Journal of the Fourth Military Medical University,2001,22(9):817-820.(in Chinese)