丙酸钠对IPEC-J2细胞紧密连接及炎症细胞因子的影响
|
摘要
[目的]本文以猪肠道上皮IPEC-J2细胞为模型,探究不同水平丙酸钠对细胞紧密连接和炎症细胞因子的影响。[方法]分别用1和10 mmol·L~(-1)丙酸钠处理IPEC-J2细胞,以不加丙酸钠处理IPEC-J2细胞为对照组,分别处理12 h和24 h后测定各组细胞活力、跨膜电阻(TEER)值、紧密连接相关蛋白及炎症细胞因子基因表达和细胞上清液中炎症因子浓度。[结果]丙酸钠处理24 h后,处理组细胞活力显著下降(P<0.05),细胞的TEER值则显著上升(P<0.05)。RT-qPCR结果显示,紧密连接相关蛋白Claudin中,不同浓度丙酸钠处理12和24 h后,显著下调Claudin-1 mRNA的表达(P<0.05),但显著上调Claudin-2、Claudin-3和Claudin-4 mRNA的表达(P<0.05)。紧密连接相关蛋白ZO中,处理12和24 h时,1和10 mmol·L~(-1)丙酸钠分别显著上调ZO-2 mRNA和ZO-1 mRNA的表达(P<0.05),但10 mmol·L~(-1)丙酸钠则显著下调ZO-2 mRNA的表达(P<0.05)。不同浓度丙酸钠处理12和24 h均显著上调炎症细胞因子IL-8、IL-18和TNF-αmRNA的表达(P<0.05),显著下调IL-6 mRNA的表达(P<0.05)。ELISA测定结果表明,丙酸钠显著刺激IL-8和TNF-α的分泌(P<0.05),对IL-18和IL-6的分泌则无显著影响(P>0.05)。[结论]丙酸钠选择性上调紧密连接蛋白家族相关基因的表达,一定程度上维护小肠上皮细胞屏障功能的完整性,同时调节细胞炎症反应,揭示丙酸在调节小肠上皮细胞屏障功能以及炎症反应过程中发挥重要作用。
[Objectives]This study aims to investigate the effects of sodium propionate on the expression of tight junction and inflammatory cytokines in IPEC-J2 cells. [Methods]IPEC-J2 cells were treated by different doses of sodium propionate( 1 and 10 mmol·L~(-1),0 mmol·L~(-1)as control group),and the tight junction permeability and expression of tight junction complex and inflammatory cytokines were measured by trans-epithelial electrical resistance( TEER),RT-qPCR and enzyme linked immunosorbent assay( ELISA),respectively. [Results]Compared with the control group,sodium propionate dose-dependently increased the TEER of IPEC-J2 cells after being incubated for 12 h and 24 h( P<0.05). The expression of tight junction complex Claudin-2,Claudin-3 and Claudin-4 were significantly up-regulated by different levels of sodium propionate( P< 0.05),whereas the expression of Claudin-1 was down-regulated( P<0.05). In addition,1 mmol·L~(-1)and 10 mmol·L~(-1)sodium propionate up-regulated the expression of ZO-2 and ZO-1,respectively( P<0.05). However,ZO-2 expression decreased by 10 mmol·L~(-1)sodium propionate( P < 0. 05). The mRNA expression results of inflammatory cytokines showed that the sodium propionate administration significantly up-regulated the expression of pro-inflammatory cytokines except IL-6( P<0.05). Moreover,ELISA results showed that sodium propionate significantly stimulated the release of IL-8 and TNF-α in IPEC-J2 cells( P<0.05),but had no effects on the concentration of IL-6 and IL-18( P>0.05). [Conclusions]Taken together,this study indicates that sodium propionate selectively up-regulates the mRNA expression of tight junction related protein,partly maintains the intestinal epithelial barrier function,and regulates inflammation process,reveals the role of propionate in the regulation of small intestinal epithelial barrier function and inflammatory reaction.
[Objectives]This study aims to investigate the effects of sodium propionate on the expression of tight junction and inflammatory cytokines in IPEC-J2 cells. [Methods]IPEC-J2 cells were treated by different doses of sodium propionate( 1 and 10 mmol·L~(-1),0 mmol·L~(-1)as control group),and the tight junction permeability and expression of tight junction complex and inflammatory cytokines were measured by trans-epithelial electrical resistance( TEER),RT-qPCR and enzyme linked immunosorbent assay( ELISA),respectively. [Results]Compared with the control group,sodium propionate dose-dependently increased the TEER of IPEC-J2 cells after being incubated for 12 h and 24 h( P<0.05). The expression of tight junction complex Claudin-2,Claudin-3 and Claudin-4 were significantly up-regulated by different levels of sodium propionate( P< 0.05),whereas the expression of Claudin-1 was down-regulated( P<0.05). In addition,1 mmol·L~(-1)and 10 mmol·L~(-1)sodium propionate up-regulated the expression of ZO-2 and ZO-1,respectively( P<0.05). However,ZO-2 expression decreased by 10 mmol·L~(-1)sodium propionate( P < 0. 05). The mRNA expression results of inflammatory cytokines showed that the sodium propionate administration significantly up-regulated the expression of pro-inflammatory cytokines except IL-6( P<0.05). Moreover,ELISA results showed that sodium propionate significantly stimulated the release of IL-8 and TNF-α in IPEC-J2 cells( P<0.05),but had no effects on the concentration of IL-6 and IL-18( P>0.05). [Conclusions]Taken together,this study indicates that sodium propionate selectively up-regulates the mRNA expression of tight junction related protein,partly maintains the intestinal epithelial barrier function,and regulates inflammation process,reveals the role of propionate in the regulation of small intestinal epithelial barrier function and inflammatory reaction.
引文
[1]张亚南,余凯凡,朱伟云.肠道微生物调控宿主食欲的研究进展[J].微生物学报,2017,57(7):951-960.Zhang Y N,Yu K F,Zhu W Y. Recent progress on the role of the gut microbiota in host appetite control[J]. Acta Microbiologica Sinica,2017,57(7):951-960(in Chinese with English abstract).
[2] Ashida H,Ogawa M,Kim M,et al. Bacteria and host interactions in the gut epithelial barrier[J]. Nature Chemical Biology,2011,8(1):36-45.
[3] Guilloteau P,Martin L,Eeckhaut V,et al. From the gut to the peripheral tissues:the multiple effects of butyrate[J]. Nutrition Research Reviews,2010,23(2):366-384.
[4] Feng W,Wu Y,Chen G,et al. Sodium butyrate attenuates diarrhea in weaned piglets and promotes tight junction protein expression in colon in a GPR109A-dependent manner[J]. Cellular Physiology and Biochemistry,2018,47(4):1617-1629.
[5] Yan H,Ajuwon K M. Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway[J]. PLoS One,2017,12(6):e0179586.
[6] Xia Z,Han Y,Wang K,et al. Oral administration of propionic acid during lactation enhances the colonic barrier function[J]. Lipids in Health and Disease,2017,16(1):62.
[7] Tong L C,Wang Y,Wang Z B,et al. Propionate ameliorates dextran sodium sulfate-induced colitis by improving intestinal barrier function and reducing inflammation and oxidative stress[J]. Frontiers in Pharmacology,2016,7:e970936.
[8] Wang L N,Chen X L,Li X G,et al. Evaluation of adrenocorticotropin regulated glucocorticoid synthesis pathway in adrenal of different breeds of pigs[J]. Livestock Science,2014,169:185-191.
[9] Liu W H,Mi S M,Ruan Z,et al. Dietary tryptophan enhanced the expression of tight junction protein ZO-1 in intestine[J]. Journal of Food Science,2017,82(2):562-567.
[10] Zhou X L,Kong X F,Lian G Q,et al. Dietary supplementation with soybean oligosaccharides increases short-chain fatty acids but decreases protein-derived catabolites in the intestinal luminal content of weaned Huanjiang mini-piglets[J]. Nutrition Research,2014,34(9):780-788.
[11] Alizadeh A,Braber S,Akbari P,et al. Deoxynivalenol impairs weight gain and affects markers of gut health after low-dose,short-term exposure of growing pigs[J]. Toxins,2015,7(6):2071-2095.
[12] Feng Z M,Li T J,Wu C L,et al. Monosodium L-glutamate and dietary fat exert opposite effects on the proximal and distal intestinal health in growing pigs[J]. Applied Physiology Nutrition and Metabolism,2015,40(4):353-363.
[13] Pie S,Lalles J P,Blazy F,et al. Weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets[J].Journal of Nutrition,2004,134(3):641-647.
[14] Schmitz H,Fromm M,Bentzel C J,et al. Tumor necrosis factor-alpha(TNFalpha)regulates the epithelial barrier in the human intestinal cell line HT-29/B6[J]. Journal of Cell Science,1999,112(Pt1):137-146.
[15] Suzuki T,Yoshida S,Hara H. Physiological concentrations of short-chain fatty acids immediately suppress colonic epithelial permeability[J].The British Journal of Nutrition,2008,100(2):297-305.
[16] Hammer A M,Morris N L,Earley Z M,et al. The first line of defense:the effects of alcohol on post-burn intestinal barrier,immune cells,and microbiome[J]. Alcohol Research,2015,37(2):209-222.
[17] Suzuki T. Regulation of intestinal epithelial permeability by tight junctions[J]. Cellular and Molecular Life Sciences,2013,70(4):631-659.
[18] Forster C. Tight junctions and the modulation of barrier function in disease[J]. Histochemistry and Cell Biology,2008,130(1):55-70.
[19] Piche T,Barbara G,Aubert P,et al. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome:involvement of soluble mediators[J]. Gut,2009,58(2):196-201.
[20] Al-Sadi R,Khatib K,Guo S H,et al. Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier[J]. American Journal of Physiology-Gastrointestinal and Liver Physiology,2011,300(6):1054-1064.
[21] Hamer H M,Jonkers D,Venema K,et al. Review article:the role of butyrate on colonic function[J]. Alimentary Pharmacology and Therapeutics,2008,27(2):104-119.
[22] Tedelind S,Westberg F,Kjerrulf M,et al. Anti-inflammatory properties of the short-chain fatty acids acetate and propionate:a study with relevance to inflammatory bowel disease[J]. World Journal of Gastroenterology,2007,13(20):2826-2832.
[23] Vinolo M A,Rodrigues H G,Hatanaka E,et al. Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils[J]. The Journal of Nutritional Biochemistry,2011,22(9):849-855.
[24] Fusunyan R D,Quinn J J,Ohno Y,et al. Butyrate enhances interleukin(IL)-8 secretion by intestinal epithelial cells in response to IL-1 beta and lipopolysaccharide[J]. Pediatric Research,1998,43(1):84-90.
[25] Mirmonsef P,Zariffard M R,Gilbert D,et al. Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands[J]. American Journal of Reproductive Immunology,2012,67(5):391-400.
[26] Kim M H,Kang S G,Park J H,et al. Short-chain fatty acids activate GPR41 and GPR43 on intestinal epithelial cells to promote inflammatory responses in mice[J]. Gastroenterology,2013,145(2):396-406.
[2] Ashida H,Ogawa M,Kim M,et al. Bacteria and host interactions in the gut epithelial barrier[J]. Nature Chemical Biology,2011,8(1):36-45.
[3] Guilloteau P,Martin L,Eeckhaut V,et al. From the gut to the peripheral tissues:the multiple effects of butyrate[J]. Nutrition Research Reviews,2010,23(2):366-384.
[4] Feng W,Wu Y,Chen G,et al. Sodium butyrate attenuates diarrhea in weaned piglets and promotes tight junction protein expression in colon in a GPR109A-dependent manner[J]. Cellular Physiology and Biochemistry,2018,47(4):1617-1629.
[5] Yan H,Ajuwon K M. Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway[J]. PLoS One,2017,12(6):e0179586.
[6] Xia Z,Han Y,Wang K,et al. Oral administration of propionic acid during lactation enhances the colonic barrier function[J]. Lipids in Health and Disease,2017,16(1):62.
[7] Tong L C,Wang Y,Wang Z B,et al. Propionate ameliorates dextran sodium sulfate-induced colitis by improving intestinal barrier function and reducing inflammation and oxidative stress[J]. Frontiers in Pharmacology,2016,7:e970936.
[8] Wang L N,Chen X L,Li X G,et al. Evaluation of adrenocorticotropin regulated glucocorticoid synthesis pathway in adrenal of different breeds of pigs[J]. Livestock Science,2014,169:185-191.
[9] Liu W H,Mi S M,Ruan Z,et al. Dietary tryptophan enhanced the expression of tight junction protein ZO-1 in intestine[J]. Journal of Food Science,2017,82(2):562-567.
[10] Zhou X L,Kong X F,Lian G Q,et al. Dietary supplementation with soybean oligosaccharides increases short-chain fatty acids but decreases protein-derived catabolites in the intestinal luminal content of weaned Huanjiang mini-piglets[J]. Nutrition Research,2014,34(9):780-788.
[11] Alizadeh A,Braber S,Akbari P,et al. Deoxynivalenol impairs weight gain and affects markers of gut health after low-dose,short-term exposure of growing pigs[J]. Toxins,2015,7(6):2071-2095.
[12] Feng Z M,Li T J,Wu C L,et al. Monosodium L-glutamate and dietary fat exert opposite effects on the proximal and distal intestinal health in growing pigs[J]. Applied Physiology Nutrition and Metabolism,2015,40(4):353-363.
[13] Pie S,Lalles J P,Blazy F,et al. Weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets[J].Journal of Nutrition,2004,134(3):641-647.
[14] Schmitz H,Fromm M,Bentzel C J,et al. Tumor necrosis factor-alpha(TNFalpha)regulates the epithelial barrier in the human intestinal cell line HT-29/B6[J]. Journal of Cell Science,1999,112(Pt1):137-146.
[15] Suzuki T,Yoshida S,Hara H. Physiological concentrations of short-chain fatty acids immediately suppress colonic epithelial permeability[J].The British Journal of Nutrition,2008,100(2):297-305.
[16] Hammer A M,Morris N L,Earley Z M,et al. The first line of defense:the effects of alcohol on post-burn intestinal barrier,immune cells,and microbiome[J]. Alcohol Research,2015,37(2):209-222.
[17] Suzuki T. Regulation of intestinal epithelial permeability by tight junctions[J]. Cellular and Molecular Life Sciences,2013,70(4):631-659.
[18] Forster C. Tight junctions and the modulation of barrier function in disease[J]. Histochemistry and Cell Biology,2008,130(1):55-70.
[19] Piche T,Barbara G,Aubert P,et al. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome:involvement of soluble mediators[J]. Gut,2009,58(2):196-201.
[20] Al-Sadi R,Khatib K,Guo S H,et al. Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier[J]. American Journal of Physiology-Gastrointestinal and Liver Physiology,2011,300(6):1054-1064.
[21] Hamer H M,Jonkers D,Venema K,et al. Review article:the role of butyrate on colonic function[J]. Alimentary Pharmacology and Therapeutics,2008,27(2):104-119.
[22] Tedelind S,Westberg F,Kjerrulf M,et al. Anti-inflammatory properties of the short-chain fatty acids acetate and propionate:a study with relevance to inflammatory bowel disease[J]. World Journal of Gastroenterology,2007,13(20):2826-2832.
[23] Vinolo M A,Rodrigues H G,Hatanaka E,et al. Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils[J]. The Journal of Nutritional Biochemistry,2011,22(9):849-855.
[24] Fusunyan R D,Quinn J J,Ohno Y,et al. Butyrate enhances interleukin(IL)-8 secretion by intestinal epithelial cells in response to IL-1 beta and lipopolysaccharide[J]. Pediatric Research,1998,43(1):84-90.
[25] Mirmonsef P,Zariffard M R,Gilbert D,et al. Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands[J]. American Journal of Reproductive Immunology,2012,67(5):391-400.
[26] Kim M H,Kang S G,Park J H,et al. Short-chain fatty acids activate GPR41 and GPR43 on intestinal epithelial cells to promote inflammatory responses in mice[J]. Gastroenterology,2013,145(2):396-406.