胰高血糖素样肽-2对脂多糖应激的IPEC-J2细胞形态和紧密连接相关基因表达的影响

doi:10.11843/j.issn.0366-6964.2015.04.012

摘要/Abstract

摘要：

本研究旨在考察胰高血糖素样肽-2（Glucagons-like peptide-2，GLP-2）和脂多糖（Lipopolysaccharide，LPS）对仔猪空肠上皮细胞及其紧密连接(Tight Junctions，TJ)相关蛋白基因表达的影响，并探讨GLP-2调控仔猪肠道TJ蛋白基因表达可能的作用机理。试验一设对照、GLP-2、LPS、LPS+GLP-2 共4个组，考查各处理对IPEC-J2细胞形态和紧密连接关键蛋白Occludin、Claudin-1和ZO-1基因表达的影响。结果：添加100 nmol•L^-1 GLP-2能显著改善IPEC-J2细胞形态，显著提高Occludin、Claudin-1和ZO-1 mRNA表达（P<0.01）；100 μg•mL^-1 LPS处理能显著破坏细胞形态、降低IPEC-J2细胞紧密连接蛋白mRNA的表达（P<0.01）；在添加LPS基础上添加100 nmol•L^-1 GLP-2能有效维护细胞形态，显著增加IPEC-J2细胞TJ相关蛋白Occludin、Claudin-1和ZO-1 mRNA的表达（P<0.01），增加量分别为46.3%、65.1%和30.3%。试验二考察了添加PI3K特异性抑制剂Wortmannin（Wort）和LY294002（LY）阻断PI3K-Akt-mTOR信号转导途径后Occludin、Claudin-1和ZO-1 mRNA表达量的变化，试验共设对照、GLP-2、GLP-2+Wort、GLP-2+LY等4个组。结果：添加抑制剂Wort后可显著降低IPEC-J2细胞Akt、mTOR、Occludin、Claudin-1和ZO-1 mRNA的表达（P<0.01），降低量分别为46.9%、50.5%，38.1%、49.6%和18.9%；添加 LY后上述mRNA的表达量分别显著降低了67.2%、70.8%，49.6%、60.9%和25.8%（P<0.01）。以上结果表明：添加GLP-2能够有效抑制LPS应激对IPEC-J2细胞形态和TJ相关基因表达的损伤，PI3K-Akt-mTOR信号转导途径可能是GLP-2调控肠道紧密连接蛋白基因表达的重要信号通路之一。

Abstract:

The purpose of this research was to investigate the effect of glucagon-like peptide-2(GLP-2) and lipopolysaccharide (LPS) on the gene expression of tight junction in piglets jejunum epithelial IPEC-J2 cells and to deeply discuss the possible mechanism of GLP-2 regulating the expression of intestinal TJ gene of the piglet.Trial 1，single factor design was adopted and 4 treatments(control，GLP-2，LPS，LPS+GLP-2) were used to test the effect of GLP-2 and LPS on the mRNA expression of Occludin，Claudin-1 and ZO-1 in IPEC-J2 cells.The results showed that：100 nmol•L^-1 GLP-2 could improve cellular morphology and significantly increase the expression level of Occludin，Claudin-1 and ZO-1 mRNA in IPEC-J2 cell (P<0.01)；100 μg•mL^-1 LPS could significantly destroy cellular morphology and significantly reduce the mRNA expression of TJ in IPEC-J2 cells (P<0.01).LPS with 100 nmol•L^-1 GLP-2 could improve cellular morphology and significantly increase the expression of Occludin，Claudin-1 and ZO-1 mRNA in IPEC-J2 cell (P<0.01) for 46.3%，65.1% and 30.3%，respectively.Trial 2，PI3K specific inhibitor，Wortmannin (Wort) and LY294002 (LY)，were added to investigate whether GLP-2 modulates TJ’s mRNA expression in IPEC-J2 cells through PI3K-Akt-mTOR signal transduction pathway.Four treatments(control，GLP-2，GLP-2+Wort，GLP-2+LY) were designed.The results showed that：100 nmol•L^-1 GLP-2 with 10 nmol•L^-1 Wort could significantly decrease the expression of Akt，mTOR，Occludin，Claudin-1 and ZO-1 mRNA in IPEC-J2 cells (P<0.01) for 46.9%，50.5%，38.1%，49.6% and 18.9%，respectively；GLP-2 with LY could significantly decrease the expression of Akt，mTOR，Occludin，Claudin-1 and ZO-1 mRNA in IPEC-J2 cells (P<0.01) for 67.2%，70.8%，49.6%，60.9% and 25.8%，respectively.In summary，the results showed that GLP-2 can effectively inhibit the damnification of TJ mRNA expression by LPS.GLP-2 may modulate TJ’s mRNA expression through PI3K-Akt-mTOR signal transduction pathway in IPEC-J2 cells.

中图分类号:

S828
S815

余长松，贾刚，邓秋红，陈小玲，赵华，刘光芒，王康宁. 胰高血糖素样肽-2对脂多糖应激的IPEC-J2细胞形态和紧密连接相关基因表达的影响[J]. 畜牧兽医学报, 2015, 46(4): 592-599.

YU Chang-song，JIA Gang，DENG Qiu-hong，CHEN Xiao-ling，ZHAO Hua，LIU Guang-mang，WANG Kang-ning. The Effects of GLP-2 on Cell Morphology and the Gene Expression of Tight Junction in LPS Stressed IPEC-J2 Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(4): 592-599.

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参考文献

［1］NIEKAMP S R，SUTHERLAND M A，DAHL G E，et al.Immune responses of piglets to weaning stress：Impacts of photoperiod［J］.J Anim Sci，2007，85(1)：93-100.
［2］SMITH F，CLARK J E，OVERMAN B L，et al.Early weaning stress impairs development of mucosal barrier function in the porcine intestine［J］.Am J Physiol-Gastr L，2010，298(3)：G352-G363.
［3］ZHAO Y，QIN G，SUN Z，et al.Effects of soybean agglutinin on intestinal barrier permeability and tight junction protein expression in weaned piglets［J］.Int J Mol Sci，2011，12(12)：8502-8512.
［4］刘海萍，胡彩虹，徐勇.早期断奶对仔猪肠通透性和肠上皮紧密连接蛋白 Occludin mRNA 表达的影响［J］.动物营养学报，2008，20(4)：442-446.
LIU H P，HU C H，XU Y.Effects of early weaning on intestinal permeability and tight junction protein occludin mRNA expression levels of piglets［J］.Chinese Journal of Animal Nutrition，2008，20(4)：442-446.（in Chinese）
［5］SIGALET D L，DE HEUVEL E，WALLACE L，et al.Effects of chronic glucagon-like peptide-2 therapy during weaning in neonatal pigs［J］.Regul Peptides，2014，188：70-80.
［6］NAIMI R M，MADSEN K B，ASKOV-HANSEN C，et al.A dose-equivalent comparison of the effects of continuous subcutaneous glucagon-like peptide 2 (GLP-2) infusions versus meal related GLP-2 injections in the treatment of short bowel syndrome (SBS) patients［J］.Regul Peptides，2013，184：47-53.
［7］SHI X，WEN S，CHANG B，et al.GLP-2 receptor is required for glucose homeostasis and energy balance［J］.FASEB J，2013，27：1160.
［8］PETERSEN Y M，HARTMANN B，HOLST J J，et al.Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development［J］.J Nutr，2003，133(6)：1781-1786.
［9］BURRIN D G，PETERSEN Y，STOLL B，et al.Glucagon-like peptide 2：a nutrient-responsive gut growth factor［J］. J Nutr，2001，131(3)：709-712.
［10］BENJAMIN M A，MCKAY D M，YANG P C，et al.Glucagon-like peptide-2 enhances intestinal epithelial barrier function of both transcellular and paracellular pathways in the mouse［J］.Gut，2000，47(1)：112-119.
［11］CAMERON H L，PERDUE M H.Stress impairs murine intestinal barrier function：improvement by glucagon-like peptide-2［J］.J Pharmacol Exp Ther，2005，314(1)：214-220.
［12］DONG C X.Mechanisms of glucagon-like peptide-2-mediated effects on intestinal barrier function in health and irinotecan-induced enteritis［D］.Toronto，Ontario，Canada：University of Toronto，2013.
［13］GU M J，SONG S K，PARK S M，et al.Bacillus subtilis protects porcine intestinal barrier from deoxynivalenol via improved zonula occludens-1 expression［J］.Asian-Aust J Anim Sci，2014，27(4)：580-586.
［14］KOEHLER J A，HARPER W，BARNARD M，et al.Glucagon-like peptide-2 does not modify the growth or survival of murine or human intestinal tumor cells［J］.Cancer Res，2008，68(19)：7897-7904.
［15］JIANG Z Y，SUN L H，LIN Y C，et al.Effects of dietary glycyl-glutamine on growth performance，small intestinal integrity，and immune responses of weaning piglets challenged with lipopolysaccharide［J］.J Anim Sci，2009，87(12)：4050-4056.
［16］PASZTI-GERE E，MATIS G，FARKAS O，et al.The effects of intestinal LPS exposure on inflammatory responses in a porcine enterohepatic co-culture system［J］.Inflammation，2014，37(1)：247-260.
［17］RAZZUOLI E，VILLA R，AMADORI M.IPEC-J2 cells as reporter system of the anti-inflammatory control actions of interferon-alpha［J］.J Interf Cytok Res，2013，33(10)：597-605.
［18］HOU Y，WANG L，ZHANG W，et al.Protective effects of N-acetylcysteine on intestinal functions of piglets challenged with lipopolysaccharide［J］.Amino Acids，2012，43(3)：1233-1242.
［19］LIU Y，HUANG J，HOU Y，et al.Dietary arginine supplementation alleviates intestinal mucosal disruption induced by Escherichia coli lipopolysaccharide in weaned pigs［J］.Brit J Nutr，2008，100(3)：552-560.
［20］GEENS M M，NIEWOLD T A.Preliminary characterization of the transcriptional response of the porcine intestinal cell line IPEC-J2 to enterotoxigenic Escherichia coli，Escherichia coli，and E.coli lipopolysaccharide［J］. Comp Funct Genomics，2010，2010：469583.
［21］ARCE C，RAMIREZ-BOO M，LUCENA C，et al.Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium［J］.Comp Immunol Microb，2010，33(2)：161-174.
［22］DONG C X，ZHAO W，SOLOMON C，et al.The intestinal epithelial insulin-like growth factor-1 receptor links glucagon-like peptide-2 action to gut barrier function［J］.Endocrinology，2013，155(2)：370-379.
［23］MORAN G W，O′NEILL C，MCLAUGHLIN J T.GLP-2 enhances barrier formation and attenuates TNFα-induced changes in a Caco-2 cell model of the intestinal barrier［J］. Regul Peptides，2012，178(1)：95-101.
［24］CHEESEMAN C I.Upregulation of SGLT-1 transport activity in rat jejunum induced by GLP-2 infusion in vivo［J］.Am J Physiol-Reg I，1997，273(6)：R1965-R1971.
［25］YUSTA B，ESTALL J，DRUCKER D J.Glucagon-like peptide-2 receptor activation engages bad and glycogen synthase kinase-3 in a protein kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase［J］.J Biol Chem，2002，277(28)：24896-24906.
［26］SHI X，LI X，WANG Y，et al.Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway［J］.Amn J Physiol-Endoc M，2011，300(3)：E554-E563.
［27］KOEHLER J A，HARPER W，BARNARD M，et al.Glucagon-like peptide-2 does not modify the growth or survival of murine or human intestinal tumor cells［J］.Cancer Res，2008，68(19)：7897-7904.
［28］NAN L，JOSEF N.Glutamine deprivation alters intestinal tight junctions via a PI3-K/Akt mediated pathway in Caco-2 cells［J］.J Nutr，2009，139(4)：710-714.
［29］SHI X，ZHOU F，LI X，et al.Central GLP-2 enhances hepatic insulin sensitivity via activating PI3K signaling in POMC neurons［J］.Cell Metab，2013，18(1)：86-98.

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