11S通过NF-κB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),研究大豆球蛋白(11S)对猪小肠上皮细胞的影响。将IPEC-J2细胞分为6组[A(对照组)、B、C、D、E和F],各组中分别添加0、1、5、10、5、5 mg·mL~(-1)的11S,并且在E组和F组分别添加1μmol·L~(-1)吡咯烷二硫代氨基甲酸盐(PDTC)和1μmol·mL~(-1) Nω-硝基-L-精氨酸甲酯(L-NAME)。分别于8、16、24 h,用CCK-8法检测细胞存活率,用ELISA法检测细胞一氧化氮(NO)、二胺氧化酶(DAO)、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,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的相对表达量。结果表明:11S可以降低猪小肠上皮细胞存活率,添加PDTC和L-NAME可以提高小肠上皮细胞存活率;11S可促进NO、DAO、5-HT和IL-6的分泌,降低IL-10的分泌,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的相对表达量,添加PDTC和L-NAME可抑制11S的作用。综上,11S可引起猪小肠上皮细胞损伤,随着11S浓度的增大,损伤程度增加,PDTC和L-NAME可以降低11S对细胞的损伤。
In this study, porcine intestinal epithelial cells(IPEC-J2) were cultured in vitro to study the effect of different concentrations of glycinin(11 S) on IPEC-J2. IPEC-J2 cells were divided into six groups. A was control group, in groups B(1 mg·mL~(-1) 11 S), C(5 mg·mL~(-1) 11 S), D(10 mg·mL~(-1) 11 S), E(5 mg·mL~(-1) 11 S+1 μmol·L~(-1) PDTC) and F(5 mg·mL~(-1) 11 S+1 μmol·mL~(-1) L-NAME), 0, 1, 5, 10, 5, and 5 mg·mL~(-1) of 11 S were added respectively. In addition, 1 μmol·L~(-1) PDTC and 1 μmol·mL~(-1) L-NAME were added to groups E and F, respectively. After 8, 16 and 24 h, cell viability was measured by CCK-8 method, contents of NO, DAO, 5-HT, IL-6 and IL-10 were detected by ELISA, and p-NF-κB, p65, iNOS and COX-2 protein expression levels were detected by western blot, relative expression of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were determined by PCR. The results showed that 11 S reduced viability of porcine intestinal epithelial cells, promoted the secretion of NO, 5-HT and IL-6, decreased the secretion of IL-10, and increased expression of p-NF-κB p65, iNOS and COX-2 proteins, and expression of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were also increased. PDTC and L-NAME could inhibit the effects of 11 S. The results showed that 11 S caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME could reduce the damage of 11 S to cells.
In this study, porcine intestinal epithelial cells(IPEC-J2) were cultured in vitro to study the effect of different concentrations of glycinin(11 S) on IPEC-J2. IPEC-J2 cells were divided into six groups. A was control group, in groups B(1 mg·mL~(-1) 11 S), C(5 mg·mL~(-1) 11 S), D(10 mg·mL~(-1) 11 S), E(5 mg·mL~(-1) 11 S+1 μmol·L~(-1) PDTC) and F(5 mg·mL~(-1) 11 S+1 μmol·mL~(-1) L-NAME), 0, 1, 5, 10, 5, and 5 mg·mL~(-1) of 11 S were added respectively. In addition, 1 μmol·L~(-1) PDTC and 1 μmol·mL~(-1) L-NAME were added to groups E and F, respectively. After 8, 16 and 24 h, cell viability was measured by CCK-8 method, contents of NO, DAO, 5-HT, IL-6 and IL-10 were detected by ELISA, and p-NF-κB, p65, iNOS and COX-2 protein expression levels were detected by western blot, relative expression of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were determined by PCR. The results showed that 11 S reduced viability of porcine intestinal epithelial cells, promoted the secretion of NO, 5-HT and IL-6, decreased the secretion of IL-10, and increased expression of p-NF-κB p65, iNOS and COX-2 proteins, and expression of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were also increased. PDTC and L-NAME could inhibit the effects of 11 S. The results showed that 11 S caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME could reduce the damage of 11 S to cells.
引文
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[11] 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 with English abstract)
[12] 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. Preliminary analysis about expression and function of nitric oxide synthase in colon cancer [D]. Changchun: Jilin University, 2007. (in Chinese with English abstract)
[13] 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.
[14] SUN P, LI D, LI Z, 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.
[15] 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)
[16] 胡泉舟, 侯永清, 王猛. 血中二胺氧化酶活性与仔猪腹泻程度的相关性分析[J]. 猪业科学, 2007, 24(12):73-74. HU Q Z, HOU Y Q, WANG M. Correlation between blood diamine oxidase activity and diarrhea in piglets[J].Swine Industry Science, 2007, 24(12):73-74. (in Chinese)
[17] 黎君友, 吕艺, 付小兵, 等. 二胺氧化酶在创伤后肠道损伤中变化及意义[J]. 中华危重病急救医学, 2000, 12(8):482-484.LI J Y, LYU Y, FU X B, et al. The significance of changes in diamine oxidase activity in intestinal injury after trauma[J]. Chinese Critical Care Medicine, 2000, 12(8):482-484. (in Chinese with English abstract)
[18] 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 with English abstract)
[19] 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 with English abstract)
[20] 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 with English abstract)
[21] 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. Reaearch on mechanism of allergic reactions caused by soybean glycinin and β-conglycinin in Balb/c mice [D]. Hangzhou: Zhejiang University, 2008. (in Chinese with English abstract)
[22] 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.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. (in Chinese)
[2] 徐君. 大豆球蛋白和β-伴球蛋白对小鼠肠上皮细胞营养生理功能的影响与分子机理研究[D]. 雅安: 四川农业大学, 2009.XU J. The influence of the extracted soybean glycinin and β-conglycinin on the function and molecular mechanism of mice intestinal-epithelial cells in primary cultivation [D]. Ya'an: Sichuan Agricultural University, 2009. (in Chinese with English abstract)
[3] CHEN J, WANG J, SONG P, et al. Determination of glycinin in soybean and soybean products using a sandwich enzyme-linked immunosorbent assay[J]. Food Chemistry, 2014, 162(6):27-33.
[4] LAGEMAAT J, SILVáN J M, MORENO, F J, et al. In vitro glycation and antigenicity of soy proteins[J]. Food Research International, 2007, 40(1):153-160.
[5] WILSON S, BLASCHEK K, MEJIA E D. Allergenic proteins in soybean: processing and reduction of P34 allergenicity[J]. Nutrition Reviews, 2010, 63(2):47-58.
[6] 韩蕊. 大豆球蛋白对仔猪小肠上皮细胞机械屏障功能的影响[D]. 长春: 吉林农业大学, 2013.HAN R. Effects of glycinin on the mechanical barrier function of porcine intestinal epithelial cells [D]. Changchun: Jilin Agricultural University, 2013. (in Chinese with English abstract)
[7] 龚灵芝, 陈小连, 徐建雄. 大豆抗原蛋白的研究进展[J]. 饲料工业, 2008, 29(11):23-27.GONG L Z, CHEN X L, XU J X. Research progress on soybean antigen protein[J]. Feed Industry, 2008, 29(11):23-27. (in Chinese)
[8] 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)
[9] 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 Jiao Tong University, 2014. (in Chinese with English abstract)
[10] 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]. Journal of Animal Science, 2011, 89(3):743-753.
[11] 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 with English abstract)
[12] 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. Preliminary analysis about expression and function of nitric oxide synthase in colon cancer [D]. Changchun: Jilin University, 2007. (in Chinese with English abstract)
[13] 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.
[14] SUN P, LI D, LI Z, 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.
[15] 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)
[16] 胡泉舟, 侯永清, 王猛. 血中二胺氧化酶活性与仔猪腹泻程度的相关性分析[J]. 猪业科学, 2007, 24(12):73-74. HU Q Z, HOU Y Q, WANG M. Correlation between blood diamine oxidase activity and diarrhea in piglets[J].Swine Industry Science, 2007, 24(12):73-74. (in Chinese)
[17] 黎君友, 吕艺, 付小兵, 等. 二胺氧化酶在创伤后肠道损伤中变化及意义[J]. 中华危重病急救医学, 2000, 12(8):482-484.LI J Y, LYU Y, FU X B, et al. The significance of changes in diamine oxidase activity in intestinal injury after trauma[J]. Chinese Critical Care Medicine, 2000, 12(8):482-484. (in Chinese with English abstract)
[18] 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 with English abstract)
[19] 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 with English abstract)
[20] 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 with English abstract)
[21] 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. Reaearch on mechanism of allergic reactions caused by soybean glycinin and β-conglycinin in Balb/c mice [D]. Hangzhou: Zhejiang University, 2008. (in Chinese with English abstract)
[22] 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.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. (in Chinese)