分子营养 Molecular Nutrition

苏氨酸对大鼠小肠上皮细胞系IEC-6细胞活性和紧密连接蛋白表达的影响

展开
  • 安徽科技学院动物科学学院, 凤阳 233100
任曼(1986-),女,安徽淮北人,副教授,博士,从事仔猪肠道健康和氨基酸营养相关研究。E-mail:renman@yeah.net

收稿日期: 2018-03-16

  网络出版日期: 2018-10-20

基金资助

国家自然科学基金(31501968);安徽省自然科学基金(1608085QC72);安徽省高校自然科学研究项目(KJ2015A296)

Effects of Threonine on Activity and Tight Junction Protein Expression of Rat Intestinal Epithelia Cell Line IEC-6 Cell

Expand
  • College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China

Received date: 2018-03-16

  Online published: 2018-10-20

摘要

本研究旨在探讨苏氨酸对大鼠小肠上皮细胞活性和增殖的影响,并研究苏氨酸是否对大鼠小肠上皮细胞紧密连接蛋白表达具有调控作用。试验采用不同浓度的苏氨酸(0、0.1、0.5、1.0、5.0、10.0 mmol/L)处理大鼠小肠上皮细胞系IEC-6细胞24 h,用噻唑兰(MTT)法测定IEC-6细胞活性,苏木精-伊红(HE)染色观察IEC-6细胞增殖情况,Western-blot法检测IEC-6细胞紧密连接蛋白表达情况。结果表明:各浓度的苏氨酸均能不同程度地提高IEC-6细胞的活性,0.5 mmol/L苏氨酸处理24 h时IEC-6细胞的活性最高,且与未经苏氨酸处理的对照组差异显著(P<0.05)。不同浓度的苏氨酸均能促进IEC-6细胞的增殖,且在苏氨酸浓度为0.5 mmol/L时促增殖作用最明显。与未经苏氨酸处理的对照组相比,0.1 mmol/L的苏氨酸对紧密连接蛋白claudin-3的表达未产生显著影响(P>0.05),但显著促进了occludin的表达(P<0.05);苏氨酸浓度为0.5、1.0、5.0、10.0 mmol/L时均显著促进上述2种紧密连接蛋白的表达(P<0.05),使claudin-3的相对表达量分别增加了1.84、7.08、8.71、3.83倍,occludin的相对表达量分别增加了2.65、2.71、2.82、3.88倍。综上,适宜浓度的苏氨酸对大鼠小肠上皮细胞系IEC-6细胞的活性和增殖具有促进作用,且在浓度为0.5 mmol/L时作用最显著,同时促进了紧密连接蛋白claudin-3和occludin的表达。

本文引用格式

任曼, 黄金歌, 靳二辉, 车传燕, 胡倩倩, 周金星, 李升和 . 苏氨酸对大鼠小肠上皮细胞系IEC-6细胞活性和紧密连接蛋白表达的影响[J]. 动物营养学报, 2018 , 30(10) : 4081 -4086 . DOI: 10.3969/j.issn.1006-267x.2018.10.033

Abstract

This experiment was to investigate the effects of threonine on the activity and proliferation of rat intestinal epithelial cells, and to explore whether threonine had regulation effect on tight junction protein expression of rat intestinal epithelial cells. Rat intestinal epithelial cell line IEC-6 cells were treated with different concentrations (0, 0.1, 0.5, 1.0, 5.0 and 10.0 mmol/L) of threonine for 24 h. The IEC-6 cell activity was determined using MTT method, the IEC-6 cell proliferation was observed through hematoxylin and eosin (HE) staining, and the expression of IEC-6 cell tight junction proteins claudin-3 and occludin were tested by Western-blot method. The results showed that different concentrations of threonine could increase the IEC-6 cell activity in varying degree, and the IEC-6 cell activity was the highest after explored to 0.5 mmol/L threonine for 24 h, and it significantly higher that the control group which not treated by threonine (P<0.05). IEC-6 cell proliferation also was enhanced by different concentrations of threonine, and the 0.5 mmol/L threonine group had best effect. Compared with the control group which not treated by threonine, threonine with the concentration of 0.1 mmol/L had no effect on the tight junction protein claudin-3 expression (P>0.05), but significantly increased the occludin expression (P<0.05). Compared with the control group which not treated by threonine, threonine with the concentrations of 0.5, 1.0, 5.0 and 10.0 mmol/L significantly increased the occludin and claudin-3 expression, the relative expression level of claudin-3 increased by 1.84, 7.08, 8.71 and 3.83 times, respectively, and the relative expression level of occluding increased by 2.65, 2.71, 2.82 and 3.88 times, respectively. In conclusion, threonine with properly concentration can improve the activity and proliferation of IEC-6, and 0.5 mmol/L threonine has the most significant effect. Meanwhile, the 0.5 mmol/L threonine up-regulates the expression of tight junction proteins claudin-3 and occludin.

参考文献

[1] SCHAART M W,SCHIERBEEK H,VAN DER SCHOOR R D,et al.Threonine utilization is high in the intestine of piglets[J].The Journal of Nutrition,2005,135(4):765-770.  

[2] LI P,YIN Y L,LI D F,et al.Amino acids and immune function[J].British Journal of Nutrition,2007,98(2):237-252.  

[3] MAO X,ZENG X,QIAO S,et al.Specific roles of threonine in intestinal mucosal integrity and barrier function[J].Frontiers in Bioscience,2011,3:1192-1200.

[4] FAURE M,MOËNNOZ D,MONTIGON F,et al.Dietary threonine restriction specifically reduces intestinal mucin synthesis in rats[J].The Journal of Nutrition,2005,135(3):486-491.  

[5] MUNASINGHE L L,ROBINSON J L,HARDING S V,et al.Protein synthesis in mucin-producing tissues is conserved when dietary threonine is limiting in piglets[J].The Journal of Nutrition,2017,147(2):202-210.  

[6] AZZAM M M M,ZOU X T,DONG X Y,et al.Effect of supplemental L-threonine on mucin 2 gene expression and intestine mucosal immune and digestive enzymes activities of laying hens in environments with high temperature and humidity[J].Poultry Science,2011,90(10):2251-2256.  

[7] TREVISI P,CORRENT E,MAZZONI M,et al.Effect of added dietary threonine on growth performance,health,immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to Escherichia coli infection and challenged with E.coli K88ac[J].Journal of Animal Physiology and Animal Nutrition,2015,99(3):511-520.  

[8] GÖKE M N,SCHNEIDER M,BEIL W,et al.Differential glucocorticoid effects on repair mechanisms and NF-κB activity in the intestinal epithelium[J].Regulatory Peptides,2002,105(3):203-214.  

[9] 韩国全,余冰,陈代文,等.苏氨酸对体外培养感染伪狂犬病毒猪空肠上皮细胞免疫相关基因表达的影响[J].动物营养学报,2012,24(3):487-496.

[10] WANG X,QIAO S Y,YIN Y L,et al.A deficiency or excess of dietary threonine reduces protein synthesis in jejunum and skeletal muscle of young pigs[J].The Journal of Nutrition,2007,137(6):1442-1446.  

[11] REN M,LIU X T,WANG X,et al.Increased levels of standardized ileal digestible threonine attenuate intestinal damage and immune responses in Escherichia coli K88+ challenged weaned piglets[J].Animal Feed Science and Technology,2014,195:67-75.

[12] CUMMINS A G,THOMPSON F M.Effect of breast milk and weaning on epithelial growth of the small intestine in human[J].Gut,2002,51(5):748-754.  

[13] 李艳云,张艳英,史秋梅,等.紫锥菊多糖对LPS损伤后IEC-6细胞的增殖作用[J].安徽农业科学,2015,43(14):19-21,73.

[14] CHIBA H,OSANAI M,MURATA M,et al.Transmembrane proteins of tight junctions[J].Biochimcaet Biophysica Acta:Biomembranes,2008,1778(3):588-600.  

[15] MITIC L,VAN TIALLIE C M,ANDERSON J M.Molecular physiology and pathophysiology of tight junctions Ⅰ.Tight junction structure and function:lessons from mutant animals and proteins[J].American Journal of Physiology:Gastrointestinal and Liver Physiology,2000,279(2):G250-G254.

[16] FURUSE M,HATE M,FURUSE K,et al.Claudin-based tight junctions are crucial for the mammalian epidermal barrier:a lesson from claudin-1-deficient mice[J].The Journal of Cell Biology,2002,156(6):1099-1111.  

[17] ZHANG S H,REN M,ZENG X F,et al.Leucine stimulates ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line (IPEC-J2) through PI3K/Akt/mTOR and ERK signaling pathways[J].Amino Acids,2014,46(12):2633-2642.  

[18] REN M,ZHANG S H,LIU X T,et al.Different lipopolysaccharide branched-chain amino acids modulate porcine intestinal endogenous β-defensin expression through the Sirt1/ERK/90RSK pathway[J].Journal of Agricultural and Food Chemistry,2016,64(17):3371-3379.  

[19] XIAO H,ZENG L M,SHAO F Y,et al.The role of nitric oxide pathway in arginine transport and growth of IPEC-1 cells[J].Oncotarget,2017,8(18):29976-29983.
文章导航

/