大豆球蛋白通过p38丝裂原活化蛋白激酶/c-Jun氨基端激酶信号通路引起猪小肠上皮细胞损伤
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摘要
通过体外培养猪小肠上皮细胞(IPEC-J2),研究不同浓度大豆球蛋白(11S)对猪小肠上皮细胞的影响。试验分为6组:A、B、C、D、E、F组,其中A组为对照组,其余各组分别在培养液中添加1、5、10、5、5 mg/mL的11S,并且在E、F组分别添加1μmol/L的c-Jun氨基端激酶(JNK)和p38抑制剂。培养24 h后,用CCK-8法检测细胞存活率;用酶联免疫吸附测定(ELISA)法检测细胞一氧化氮(NO)、5-羟色胺(5-HT)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)含量;Western blot检测细胞磷酸化JNK(p-JNK)、磷酸化p38(p-p38)和B淋巴细胞瘤-2(Bcl-2)蛋白表达量;实时荧光定量PCR(qRT-PCR)检测细胞Bcl-2/Bcl-xL相关凋亡蛋白(Bad)、Bcl-2相关X蛋白(Bax)、Bcl-2和半胱氨酸天冬氨酸蛋白酶-3(caspase-3) mRNA相对表达量。结果表明:1)与A组相比,C、D组细胞存活率极显著降低(P<0.01); E、F组细胞存活率显著或极显著高于C组(P<0.05或P<0.01)。2)与A组相比,C、D组NO、5-HT、IL-6、IL-10含量均极显著增加(P<0.01);与C组相比,E、F组NO、5-HT、IL-6、IL-10含量显著或极显著降低(P <0.05或P <0.01)。3)与A组相比,C、D组p-JNK、p-p38蛋白表达量显著或极显著增加(P <0. 05或P <0. 05),C、D组Bcl-2蛋白表达量极显著降低(P<0.01);与C组相比,E、F组p-JNK、p-p38蛋白表达量显著或极显著降低(P<0.05或P<0.01),E、F组Bcl-2蛋白表达量显著或极显著增加(P<0.05或P<0.01)。4)与A组相比,B、C和D组Bcl-2/Bax极显著降低(P<0.01),C、D组Bax/Bcl-xL、Bad和caspase-3 mRNA相对表达量显著或极显著增加(P<0.05或P<0.01);与C组相比,E、F组Bcl-2/Bax极显著增加(P<0.01),E、F组Bax/Bcl-xL和caspase-3 mRNA相对表达量极显著降低(P <0.01),F组Bad mRNA相对表达量极显著降低(P<0.01)。结果说明,11S通过p38 MAPK/JNK信号通路引起猪小肠上皮细胞损伤。
This experiment was aimed to study the effects of different concentrations of glycinin( 11 S) on porcine intestinal epithelial cells( IPEC-J2) cultured in vitro. The experiment was divided into six groups: groups A,B,C,D,E and F,group A was the control group,and the other groups were added 1,5,10,5 and 5 mg/mL11 S,respectively,and 1 μmol/L JNK and p38 inhibitors were added to groups E and F,respectively. After 24 h culture,cell viability was measured by CCK-8 method; the contents of nitric oxide( NO),5-hydroxytryptamine( 5-HT),interleukin-6( IL-6),and interleukin-10( IL-10) were detected by enzyme linked immunosorbent assay( ELISA) method; phospho-c-Jun N-terminal kinase( p-JNK),phospho-p38( p-p38) and B lymphocytoma-2( Bcl-2) protein expression levels were detected by Western blot; the relative expression levels of Bcl-2/Bcl-xL related apoptotic protein( Bad),Bcl-2 related X protein( Bax),Bcl-2 and caspase-3 mRNA were determined by qRT-PCR. The results showed as follows: 1) compared with group A,cell viability in groups C and D significantly decreased( P<0.01); compared with group C,cell viability in groups E and F significantly increased( P<0.05 or P < 0.01). 2) Compared with group A,the contents of NO,5-HT,IL-6 and IL-10 significantly increased( P<0.01); compared with group C,the contents of NO,5-HT,IL-6 and IL-10 significantly decreased( P<0.05 or P<0.01). 3) Compared with group A,the expression levels of p-JNK and p-p38 protein in groups C and D significantly increased( P<0.05 or P<0.01),and the expression level of Bcl-2 protein in groups C and D significantly decreased( P<0.01); compared with group C,the expression levels of p-JNK and p-p38 protein in groups E and F significantly decreased( P<0.05 or P<0.01),and the expression level of Bcl-2 protein in groups E and F significantly increased( P<0.05 or P < 0.01). 4) Compared with group A,Bcl-2/Bax in groups B,C and D significantly increased( P<0.01),and Bax/Bcl-xL and the relative expression levels of Bad and caspase-3 mRNA in groups C and D significantly increased( P<0.05 or P<0.01); compared with group C,Bcl-2/Bax in groups E and F significantly increased( P<0.01),Bax/Bcl-xL and the relative expression level of caspase-3 mRNA in groups E and F significantly decreased( P<0.01),and the relative expression level of Bad mRNA significantly decreased( P<0.01). The results indicate that 11 S can induce IPEC-J2 damage through the p38 MAPK/JNK signaling pathway.
This experiment was aimed to study the effects of different concentrations of glycinin( 11 S) on porcine intestinal epithelial cells( IPEC-J2) cultured in vitro. The experiment was divided into six groups: groups A,B,C,D,E and F,group A was the control group,and the other groups were added 1,5,10,5 and 5 mg/mL11 S,respectively,and 1 μmol/L JNK and p38 inhibitors were added to groups E and F,respectively. After 24 h culture,cell viability was measured by CCK-8 method; the contents of nitric oxide( NO),5-hydroxytryptamine( 5-HT),interleukin-6( IL-6),and interleukin-10( IL-10) were detected by enzyme linked immunosorbent assay( ELISA) method; phospho-c-Jun N-terminal kinase( p-JNK),phospho-p38( p-p38) and B lymphocytoma-2( Bcl-2) protein expression levels were detected by Western blot; the relative expression levels of Bcl-2/Bcl-xL related apoptotic protein( Bad),Bcl-2 related X protein( Bax),Bcl-2 and caspase-3 mRNA were determined by qRT-PCR. The results showed as follows: 1) compared with group A,cell viability in groups C and D significantly decreased( P<0.01); compared with group C,cell viability in groups E and F significantly increased( P<0.05 or P < 0.01). 2) Compared with group A,the contents of NO,5-HT,IL-6 and IL-10 significantly increased( P<0.01); compared with group C,the contents of NO,5-HT,IL-6 and IL-10 significantly decreased( P<0.05 or P<0.01). 3) Compared with group A,the expression levels of p-JNK and p-p38 protein in groups C and D significantly increased( P<0.05 or P<0.01),and the expression level of Bcl-2 protein in groups C and D significantly decreased( P<0.01); compared with group C,the expression levels of p-JNK and p-p38 protein in groups E and F significantly decreased( P<0.05 or P<0.01),and the expression level of Bcl-2 protein in groups E and F significantly increased( P<0.05 or P < 0.01). 4) Compared with group A,Bcl-2/Bax in groups B,C and D significantly increased( P<0.01),and Bax/Bcl-xL and the relative expression levels of Bad and caspase-3 mRNA in groups C and D significantly increased( P<0.05 or P<0.01); compared with group C,Bcl-2/Bax in groups E and F significantly increased( P<0.01),Bax/Bcl-xL and the relative expression level of caspase-3 mRNA in groups E and F significantly decreased( P<0.01),and the relative expression level of Bad mRNA significantly decreased( P<0.01). The results indicate that 11 S can induce IPEC-J2 damage through the p38 MAPK/JNK signaling pathway.
引文
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[20]陈海华,陈海华,周贤龙,等.p38 MAPK和JNK信号通道在TGF-β1诱导的肺泡上皮细胞上皮-间质转化中的意义[J].国际呼吸杂志,2014,34(20):1544-1550.
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[22]JEONG J W,LEE H H,CHOI E O,et al.Schisandrae fructus inhibits IL-1β-induced matrix metalloproteinases and inflammatory mediators production in SW1353 human chondrocytes by suppressing NF-κB and MAPKactivation[J].Drug Development Research,2015,76(8):474-483.
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[24]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.
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[2]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.
[3]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):13-17,28.
[4]HOLZHAUSER T,WACKERMANN O,BALLMER-WEBER B K,et al.Soybean(Glycine max)allergy in Europe:Gly m 5(β-conglycinin)and Gly m 6(glycinin)are potential diagnostic markers for severe allergic reactions to soy[J].Journal of Allergy and Clinical Immunology,2009,123(2):452-458.e4.
[5]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.
[6]LUO D H,ZHAO Q Z,ZHAO M M,et al.Effects of limited proteolysis and high-pressure homogenisation on structural and functional characteristics of glycinin[J].Food Chemistry,2010,122(1):25-30.
[7]USUI M,TAMURA H,NAKAMURA K,et al.Enhanced bactericidal action and masking of allergen structure of soy protein by attachment of chitosan through Maillardtype protein-polysaccharide conjugation[J].Die Nahrung,2004,48(1):69-72.
[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.
[9]王红云,李同洲,赵杰.膨化全脂大豆在断奶仔猪上的应用研究进展[J].饲料博览,2002(6):12-13.
[10]刘辉,姚咏明.细胞内炎症信号通路交汇作用研究进展[J].感染.炎症.修复,2003,4(4):241-245.
[11]REUSTLE A,TORZEWSKI M.Role of p38 MAPK in atherosclerosis and aortic valve sclerosis[J].International Journal of Molecular Sciences,2018,19(12):3761.
[12]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.
[13]YEE Y H,CHONG S J F,PERVAIZ S.The anti-oxidant and pro-oxidant dichotomy of Bcl-2[J].Biological Chemistry,2016,397(7):585-593.
[14]韩蕊,赵元,潘丽,等.大豆球蛋白对仔猪小肠上皮细胞Occludin mRNA表达的影响[J].畜牧兽医学报,2013,44(8):1258-1262.
[15]SUN P,LI D F,LI Z J,et al.Effects of glycinin on IgE-mediated increase of mast cell numbers and histamine release in the small intestine[J].Journal of Nutritional Biochemistry,2008,19(9):627-633.
[16]刘欣.大豆球蛋白glycinin和β-conglycinin引发Bal b/c小鼠过敏反应及其机理的研究[D].博士学位论文.杭州:浙江大学,2008.
[17]张频捷,朱立新,耿小平.p38 MAPK信号传导通路及其抑制剂的研究现状[J].安徽医药,2010,14(5):596-598.
[18]ALMEIDA A,BOLAOS J P.A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons[J].Journal of Neurochemistry,2010,77(2):676-690.
[19]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.
[20]陈海华,陈海华,周贤龙,等.p38 MAPK和JNK信号通道在TGF-β1诱导的肺泡上皮细胞上皮-间质转化中的意义[J].国际呼吸杂志,2014,34(20):1544-1550.
[21]SMITH W E,KANE A V,CAMPBELL S T,et al.Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells[J].Infection and Immunity,2003,71(3):1497-1504.
[22]JEONG J W,LEE H H,CHOI E O,et al.Schisandrae fructus inhibits IL-1β-induced matrix metalloproteinases and inflammatory mediators production in SW1353 human chondrocytes by suppressing NF-κB and MAPKactivation[J].Drug Development Research,2015,76(8):474-483.
[23]KESHET Y,SEGER R.The MAP kinase signaling cascades:a system of hundreds of components regulates a diverse array of physiological functions[J].Methods in Molecular Biology,2010,661:3-38.
[24]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.
[25]ADAMS J M,CORY S.Bcl-2-regulated apoptosis:mechanism and therapeutic potential[J].Current Opinion in Immunology,2007,19(5):488-496.