β-伴大豆球蛋白通过p38/JNK MAPK信号通路引起IPEC-J2细胞损伤
|
摘要
采用细胞体外培养技术,研究不同浓度7S(β-伴大豆球蛋白)对IPEC-J2(猪小肠上皮细胞)的影响。实验分为6组,A(对照组)、B(1 mg·m L~(-1)7S)、C(5 mg·m L~(-1)7S)、D(10 mg·m L~(-1)7S)、E(5 mg·m L~(-1)7S+1μmol·L~(-1)SP600125)、F(5 mg·m L~(-1)7S+1μmol·L~(-1)SB202190),24 h后,用CCK-8法检测细胞存活率,收集细胞用ELISA法检测NO、5-HT、IL-6和IL~(-1)0含量,用Western blot法检测p-JNK、p-p38、Bcl-2蛋白表达量,用PCR法检测Bad、Bax、Bcl-2、Bcl-xL和Caspase-3 m RNA相对表达量。检测结果表明:C组和D组的IPEC-J2细胞活性极显著降低(P <0. 01),E组和F组的细胞活性显著高于C组(P <0. 05),说明7S促进炎性细胞因子NO、5-HT和IL-6的分泌,降低IL~(-1)0的分泌,添加抑制剂使细胞因子NO、5-HT和IL-6分泌减少,IL~(-1)0的分泌增多;同时7S促进p-JNK、p-p38蛋白表达,降低Bcl-2蛋白表达,添加抑制剂可抑制p-JNK、p-p38蛋白表达,使Bcl-2蛋白表达增高。Bcl-2/Bax m RNA比值随7S浓度增加而降低,Bax/Bcl-xl比值随7S浓度增加而升高,Caspase-3 m RNA相对表达量随7S浓度增加而升高。添加抑制剂后Bcl-2/Bax比值增高,Bax/Bcl-xl比值降低,Caspase-3 m RNA降低。结果表明,7S通过p38/JNK MAPK信号通路引起仔猪肠上皮细胞损伤。
In this study,in vitro culture techniques were used to study the effects of different concentrations of 7 S( β-conglycinin) on IPEC-J2( intestinal epithelial cells in piglets). The experiment was divided into six groups,A( control),B( 1 mg·m L~(-1)7 S),C( 5 mg·m L~(-1)7 S),D( 10 mg·m L~(-1)7 S),E( 5 mg·m L~(-1)7 S + 1μmol·L~(-1) SP600125),F( 5 mg·m L~(-1)7 S + 1 μmol·L~(-1) SB202190). After 24 hours,the viability of the cells was measured by CCK-8 assay. The contents of NO,5-HT,IL-6 and IL~(-1)0 were detected by ELISA,the expression levels of pJNK,p-p38 and Bcl-2 proteins were detected by western blot,the relative expression of Bad,Bax,Bcl-2 and Caspase-3 m RNA was detected by PCR. The results showed that the viability of IPEC-J2 cells was decreased in groups C and D( P < 0. 01). The viability of cells in groups E and F was significantly higher than that in group C( P < 0. 05). 7 S promoted the secretion of inflammatory cytokines NO,5-HT,and IL-6 and decreased the secretion of IL~(-1)0,the addition of inhibitors decreased the secretion of cytokines NO,5-HT,and IL-6,and increased the secretion of IL~(-1)0. 7 S promoted the expression of p-JNK and p-p38 proteins and decreased the expression of Bcl-2 protein. The addition of inhibitors inhibited the expression of p-JNK and p-p38 proteins and increased the expression of Bcl-2 protein. The ratio of Bcl-2/Bax m RNA decreased with the increase of 7 S concentration,and the ratio of Bax/Bcl-xlincreased with the increase of 7 S concentration. The relative expression of Caspase-3 m RNA increased with the increase of 7 S concentration. On contrary,often adding inhibitors,the ratio of Bcl-2/Bax increased,the ratio of Bax/Bcl-xldecreased,and the relative expression of Caspase-3 decreased. Our results indicated that 7 S caused damage to the intestinal epithelial cells of piglets through the p38/JNK MAPK signaling pathway.
In this study,in vitro culture techniques were used to study the effects of different concentrations of 7 S( β-conglycinin) on IPEC-J2( intestinal epithelial cells in piglets). The experiment was divided into six groups,A( control),B( 1 mg·m L~(-1)7 S),C( 5 mg·m L~(-1)7 S),D( 10 mg·m L~(-1)7 S),E( 5 mg·m L~(-1)7 S + 1μmol·L~(-1) SP600125),F( 5 mg·m L~(-1)7 S + 1 μmol·L~(-1) SB202190). After 24 hours,the viability of the cells was measured by CCK-8 assay. The contents of NO,5-HT,IL-6 and IL~(-1)0 were detected by ELISA,the expression levels of pJNK,p-p38 and Bcl-2 proteins were detected by western blot,the relative expression of Bad,Bax,Bcl-2 and Caspase-3 m RNA was detected by PCR. The results showed that the viability of IPEC-J2 cells was decreased in groups C and D( P < 0. 01). The viability of cells in groups E and F was significantly higher than that in group C( P < 0. 05). 7 S promoted the secretion of inflammatory cytokines NO,5-HT,and IL-6 and decreased the secretion of IL~(-1)0,the addition of inhibitors decreased the secretion of cytokines NO,5-HT,and IL-6,and increased the secretion of IL~(-1)0. 7 S promoted the expression of p-JNK and p-p38 proteins and decreased the expression of Bcl-2 protein. The addition of inhibitors inhibited the expression of p-JNK and p-p38 proteins and increased the expression of Bcl-2 protein. The ratio of Bcl-2/Bax m RNA decreased with the increase of 7 S concentration,and the ratio of Bax/Bcl-xlincreased with the increase of 7 S concentration. The relative expression of Caspase-3 m RNA increased with the increase of 7 S concentration. On contrary,often adding inhibitors,the ratio of Bcl-2/Bax increased,the ratio of Bax/Bcl-xldecreased,and the relative expression of Caspase-3 decreased. Our results indicated that 7 S caused damage to the intestinal epithelial cells of piglets through the p38/JNK MAPK signaling pathway.
引文
[1]MEINLSCHMIDT P,UEBERHAM E,LEHMANN J,et al.High pressure processing(HPP)assisted enzymatic hydrolysis:an innovative approach for the reduction of soybean allergy[C]//29th EFFoST International Conference,Athens,Greece,2015.
[2]游金明,王自蕊,瞿明仁,等.大豆抗原蛋白的生物学特性及其对仔猪的过敏反应[J].饲料工业,2007,28(21):53-56.YOU J M,WANG Z R,ZHAI M R,et al.Biological characteristics of soybean antigen protein and its allergic reaction to piglets[J].Feed Industry,2007,28(21):53-56.(in Chinese with English abstract)
[3]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].Bioscience Biotechnology and Biochemistry,2018,82(8):1408-1416.
[4]刘珊珊,武小霞,姜振峰,等.大豆7S球蛋白β-伴大豆球蛋白的研究现状[J].大豆科学,2007,26(3):417-422.LIU S S,WU X X,JIANG Z F,et al.Advances in soybean7S globulinβ-conglycinin[J].Soybean Science,2007,26(3):417-422.(in Chinese with English abstract)
[5]KRISHNAN H B,KIM W S,JANG S C,et al.All three subunits of soybean beta-conglycinin are potential food allergens[J].Journal of Agricultural and Food Chemistry,2009,57(3):938-943.
[6]王福慧,李颖丽,杨晓东,等.发酵豆粕对犊牛生长性能的影响[J].中国奶牛,2013(2):31-33.WANG F H,LI Y L,YANG X D,et al.Effects of fermented soybean meal on growth performance in calves[J].China Dairy Cattle,2013(2):31-33.(in Chinese with English abstract)
[7]邢秀苹,杨欢欢,韦庆勇,等.豆粕和膨化大豆粉对鲤鱼生长及其肌肉营养成分的影响[J].西北农林科技大学学报(自然科学版),2015,43(12):13-17.XING X P,YANG H H,WEI Q Y,et al.Effect of soybean meal and extruded soy flour on growth performance and nutritional composition of carp[J].Journal of Northwest A&F University(Natural Science Edition),2015,43(12):13-17.(in Chinese with English abstract)
[8]张瑜,王元红,汤雪冰,等.大豆抗原蛋白对仔猪的免疫作用[J].浙江农业学报,2018,30(4):560-567.ZHANG Y,WANG Y H,TANG X B,et al.Immune effect of soybean antigen protein in piglets[J].Acta Agriculturae Zhejiangensis,2018,30(4):560-567.(in Chinese with English abstract)
[9]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 with English abstract)
[10]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 F.Study on the distribution of gastro-intestinal tissue of piglets[J].Chinese Animal Husbandry and Veterinary Medicine,2014,41(5):176-179.(in Chinese with English abstract)
[11]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)
[12]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].Histochemistry and Cell Biology,2006,125(3):293-305.
[13]栾兆双,宋娟,胡彩虹.丝裂原活化蛋白激酶信号通路抑制剂对断奶仔猪小肠形态和肠通透性的影响[J].动物营养学报,2013,25(1):44-49.LUAN Z S,SONG J,HU C H.Inhibitors of mitogen activated protein kinase pathways:effects on intestinal morphology and permeability of weaner piglets[J].Acta Zoonutrimenta Sinica,2013,25(1):44-49.(in Chinese with English abstract)
[14]RIEDL S J,SHI Y.Molecular mechanisms of caspase regulation during apoptosis[J].Nature Reviews Molecular Cell Biology,2004,5(11):897-907.
[15]ADAMS J M,CORY S.The Bcl-2-regulated apoptosis switch:mechanism and therapeutic potential[J].Current Opinion in Immunology,2007,19(5):488-496.
[16]CHEN F,HAO Y,PIAO X S,et al.Soybean-derived betaconglycinin affects proteome expression in pig intestinal cells in vivo and in vitro[J].Journal of Animal Science,2011,89(3):743-753.
[17]ZHAO Y,QIN G,HAN R,et al.β-Conglycinin reduces the tight junction occludin and ZO-1 expression in IPEC-J2[J].International Journal of Molecular Sciences,2014,15(2):1915-1926.
[18]WU J J,ZHANG Y,DONG J H,et al.Allergens and intestinal damage induced by soybean antigen proteins in weaned piglets[J].Italian Journal of Animal Science,2016,15(3):536-544.
[19]WU J J,CAO C M,REN D D,et al.Effects of soybean antigen proteins on intestinal permeability,5-hydroxytryptamine levels and secretory Ig A distribution in the intestine of weaned piglets[J].Italian Journal of Animal Science,2016,15(1):174-180.
[20]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 andβ-conglycinin in Balb/c mice[D].Hangzhou:Zhejiang University,2008.(in Chinese with English abstract)
[21]MARKOU T,DOWLING A A,KELLY T,et al.Regulation of Bcl-2 phosphorylation in response to oxidative stress in cardiac myocytes[J].Free Radical Research,2009,43(9):809-816.
[22]NEUZIL J,WANG X F,DONG L F,et al.Molecular mechanism of‘mitocan’-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2family proteins[J].FEBS Letters,2006,580(22):5125-5129.
[23]ZONG W X,LINDSTEN T,ROSS A J,et al.BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak[J].Genes&Development,2001,15(12):1481-1486.
[24]MARTNEZ V R,AGUIRRE M V,TODARO J S,et al.Interaction of Zn with Losartan.Activation of Intrinsic apoptotic signaling pathway in lung cancer cells and effects on alkaline and acid phosphatases[J].Biological Trace Element Research,2018,186(2):413-429.
[2]游金明,王自蕊,瞿明仁,等.大豆抗原蛋白的生物学特性及其对仔猪的过敏反应[J].饲料工业,2007,28(21):53-56.YOU J M,WANG Z R,ZHAI M R,et al.Biological characteristics of soybean antigen protein and its allergic reaction to piglets[J].Feed Industry,2007,28(21):53-56.(in Chinese with English abstract)
[3]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].Bioscience Biotechnology and Biochemistry,2018,82(8):1408-1416.
[4]刘珊珊,武小霞,姜振峰,等.大豆7S球蛋白β-伴大豆球蛋白的研究现状[J].大豆科学,2007,26(3):417-422.LIU S S,WU X X,JIANG Z F,et al.Advances in soybean7S globulinβ-conglycinin[J].Soybean Science,2007,26(3):417-422.(in Chinese with English abstract)
[5]KRISHNAN H B,KIM W S,JANG S C,et al.All three subunits of soybean beta-conglycinin are potential food allergens[J].Journal of Agricultural and Food Chemistry,2009,57(3):938-943.
[6]王福慧,李颖丽,杨晓东,等.发酵豆粕对犊牛生长性能的影响[J].中国奶牛,2013(2):31-33.WANG F H,LI Y L,YANG X D,et al.Effects of fermented soybean meal on growth performance in calves[J].China Dairy Cattle,2013(2):31-33.(in Chinese with English abstract)
[7]邢秀苹,杨欢欢,韦庆勇,等.豆粕和膨化大豆粉对鲤鱼生长及其肌肉营养成分的影响[J].西北农林科技大学学报(自然科学版),2015,43(12):13-17.XING X P,YANG H H,WEI Q Y,et al.Effect of soybean meal and extruded soy flour on growth performance and nutritional composition of carp[J].Journal of Northwest A&F University(Natural Science Edition),2015,43(12):13-17.(in Chinese with English abstract)
[8]张瑜,王元红,汤雪冰,等.大豆抗原蛋白对仔猪的免疫作用[J].浙江农业学报,2018,30(4):560-567.ZHANG Y,WANG Y H,TANG X B,et al.Immune effect of soybean antigen protein in piglets[J].Acta Agriculturae Zhejiangensis,2018,30(4):560-567.(in Chinese with English abstract)
[9]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 with English abstract)
[10]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 F.Study on the distribution of gastro-intestinal tissue of piglets[J].Chinese Animal Husbandry and Veterinary Medicine,2014,41(5):176-179.(in Chinese with English abstract)
[11]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)
[12]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].Histochemistry and Cell Biology,2006,125(3):293-305.
[13]栾兆双,宋娟,胡彩虹.丝裂原活化蛋白激酶信号通路抑制剂对断奶仔猪小肠形态和肠通透性的影响[J].动物营养学报,2013,25(1):44-49.LUAN Z S,SONG J,HU C H.Inhibitors of mitogen activated protein kinase pathways:effects on intestinal morphology and permeability of weaner piglets[J].Acta Zoonutrimenta Sinica,2013,25(1):44-49.(in Chinese with English abstract)
[14]RIEDL S J,SHI Y.Molecular mechanisms of caspase regulation during apoptosis[J].Nature Reviews Molecular Cell Biology,2004,5(11):897-907.
[15]ADAMS J M,CORY S.The Bcl-2-regulated apoptosis switch:mechanism and therapeutic potential[J].Current Opinion in Immunology,2007,19(5):488-496.
[16]CHEN F,HAO Y,PIAO X S,et al.Soybean-derived betaconglycinin affects proteome expression in pig intestinal cells in vivo and in vitro[J].Journal of Animal Science,2011,89(3):743-753.
[17]ZHAO Y,QIN G,HAN R,et al.β-Conglycinin reduces the tight junction occludin and ZO-1 expression in IPEC-J2[J].International Journal of Molecular Sciences,2014,15(2):1915-1926.
[18]WU J J,ZHANG Y,DONG J H,et al.Allergens and intestinal damage induced by soybean antigen proteins in weaned piglets[J].Italian Journal of Animal Science,2016,15(3):536-544.
[19]WU J J,CAO C M,REN D D,et al.Effects of soybean antigen proteins on intestinal permeability,5-hydroxytryptamine levels and secretory Ig A distribution in the intestine of weaned piglets[J].Italian Journal of Animal Science,2016,15(1):174-180.
[20]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 andβ-conglycinin in Balb/c mice[D].Hangzhou:Zhejiang University,2008.(in Chinese with English abstract)
[21]MARKOU T,DOWLING A A,KELLY T,et al.Regulation of Bcl-2 phosphorylation in response to oxidative stress in cardiac myocytes[J].Free Radical Research,2009,43(9):809-816.
[22]NEUZIL J,WANG X F,DONG L F,et al.Molecular mechanism of‘mitocan’-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2family proteins[J].FEBS Letters,2006,580(22):5125-5129.
[23]ZONG W X,LINDSTEN T,ROSS A J,et al.BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak[J].Genes&Development,2001,15(12):1481-1486.
[24]MARTNEZ V R,AGUIRRE M V,TODARO J S,et al.Interaction of Zn with Losartan.Activation of Intrinsic apoptotic signaling pathway in lung cancer cells and effects on alkaline and acid phosphatases[J].Biological Trace Element Research,2018,186(2):413-429.