| The purpose of this research was to investigate the effect of glucagon-like peptide-2(GLP-2) on the tight junction expression and barrier function in lipopolysaccharide (LPS) stressed piglets jejunum epithelial IPEC-J2 cells and to examine the possible molecular mechanism that GLP-2 modulate the tight junctionand the barrier function (PI3K-Akt-mTOR signal transduction pathway) with deeply discussing the basic law and possible mechanism of GLP-2 effectting intestinal barrier function of piglet. Research included two experiments.Exp.1 Effect of GLP-2 on the TJ Expression and Barrier Function in LPS Stressed IPEC-J2 CellBased on the research of the effect of LPS and GLP-2 on tight junctions and barrier function of IPEC-J2 cells, this test was further investigating the impact of GLP-2 on the TJ expression and barrier function in LPS stressed IPEC-J2 cell.Firstly, single factor design was conduct to examine the effect of 100μg/ml LPS, 100nmol/L GLP-2 and 100μg/ml LPS with 100nmol/L GLP-2 treated with 24h on morphology, tight junctions’expression and barrier function in IPEC-J2 cells. Each treatment had 4 replicates. In order to investigate the protective effect of GLP-2 on LPS induced IPEC-J2 cells, the effect of GLP-2 on LPS stressed IPEC-J2 cell tight junctions and barrier function related indicators were been tested. The results showed that:1, Compared with the control group,100μg/ml LPS could significantly destroy cellular morphology and decrease tight junctions’ expression associated protein Occludin, Claudin-1 and ZO-1 mRNA in IPEC-J2 cell (P<0.01) respectively 46%,57% and 34% and can significantly decrease the IPEC-J2 cell tight junctionassociated protein Occludin, Claudin-1 and ZO-1 protein (P<0.01) respectively 76.5%,81.7% and 78.8%and can significantly decrease the IPEC-J2 cell’s transepithelial resistance (TER) (P<0.01) 92.4%.2, Compared with the control group, 100nmol/L GLP-2 could promote cellular morphology and significantly increase tight junctions’ expression associated protein Occludin, Claudin-1 and ZO-1 mRNA in IPEC-J2 cell (P<0.01) respectively 148%,261% and 54% and can significantly increase the IPEC-J2 cell tight junction associated protein Occludin, Claudin-1 and ZO-1 protein (P<0.01) respectively 35%,39% and 62% and can significantly increase the IPEC-J2 cell’s transepithelial resistance (TER) (P<0.01) 27.2%.3, Compared with the 100μg/ml LPS group, 100μg/ml LPS with 100nmol/L GLP-2 could improve cellular morphology and significantly increase tight junctions’ expression associated protein Occludin, Claudin-1 and ZO-1 mRNA in IPEC-J2 cell (P<0.01) respectively 46.3%,65.1% and 30.3% and can significantly increase the IPEC-J2 cells tight junction associated protein Occludin, Claudin-1 and ZO-1 protein (P<0.01) respectively 183%,300% and 231.8% and can significantly increase the IPEC-J2 cells’transepithelial resistance (TER) (P<0.01) 848.5%.The results revealed that GLP-2 can not only improve the expression of the tight junction associated protein Occludin, Claudin-1 and ZO-1 mRNA, protein and enhance the barrier function in normal IPEC-J2 cells but also can significantly inhibit the destroy of cellular morphology and decreasing of expression the tight junction associated protein Occludin, Claudin-1 and ZO-1 mRNA, protein caused by LPS stress and can maintain the barrier function of IPEC-J2 cells.Exp.2 The Research of the Molecular Mechanism that GLP-2 Modulate the Expression of Tight Junction and Barrier Function in IPEC-J2 CellsTrial 2 were carried out to study the effect of GLP-2 on TJ’s expression and barrier function in IPEC-J2 cells. Then Wortmannin (Wort) and LY294002 (LY), PI3K specific inhibitor, were added to investigate whether GLP-2 modulates TJ’s expression and barrier function in IPEC-J2 cells through PI3K-Akt-mTOR signal transduction pathway. Before the format test, the IPEC-J2 cells of each inhibitor group would be cultured in inhibitor medium without FBS for 1h. Four groups which were control group, 100nmol/L GLP-2 group,100nmol/L GLP-2+10nmol/L Wort treatment group,100nmol/LGLP-2+10μmol/L LY treatment group were conduct to test the expression of p-Akt and p-mTOR protein in the signal transduction of PI3K-Akt-mTOR and expression of IPEC-J2 cells tight junction associated protein Occludin, Claudin-1 and ZO-1 mRNA and protein, IPEC-J2 cells transepithelial resistance(TER). The results showed that:1, Compared with the control group, 100nmol/L GLP-2 could significantly increase Akt, mTOR, tight junctions expression associated protein Occludin, Claudin-1 and ZO-1 mRNA in IPEC-J2 cells (P<0.01) respectively 478%,590%,260%,370%and 159% and can significantly increase the IPEC-J2 cells p-Akt, p-mTOR, tight junction associated protein Occludin, Claudin-1 and ZO-1 protein (P<0.01) respectively 59.4%,67.3%,35.0%, 38.2%and 58.3% and can significantly increase the IPEC-J2 cells’transepithelial resistance (TER) (P<0.01) 23.5%.2, Compared with the 100nmol/L GLP-2 group, 100nmol/L GLP-2 with 10nmol/L Wort could significantly decrease Akt, mTOR, tight junctions expression associated protein Occludin, Claudin-1 and ZO-1 mRNA in IPEC-J2 cells (P<0.01) respectively 46.9%, 50.5%,38.1%,49.6% and 18.9% and can significantly decrease the IPEC-J2 cells p-Akt, p-mTOR, tight junction associated protein Occludin, Claudin-1 and ZO-1 protein (P<0.01) respectively 15.5%,15.4%,10.8%,14.2% and 27.6% and can significantly increase the IPEC-J2 cells’ transepithelial resistance (TER) (P<0.01) 5.4%.3, Compared with the 100nmol/L GLP-2 group, 100nmol/L GLP-2 with 10μmol/L LY could significantly decrease Akt, mTOR, tight junctions expression associated protein Occludin, Claudin-1 and ZO-1 mRNA in IPEC-J2 cells (P<0.01) respectively 67.2%, 70.8%,49.6%,60.9% and 25.8% and can significantly decrease the IPEC-J2 cells p-Akt, p-mTOR, tight junction associated protein Occludin, Claudin-1 and ZO-1 protein (P<0.01) respectively 24.2%,22.5%,13.7%,16.3% and 31.9% and can significantly increase the IPEC-J2 cells’ transepithelial resistance (TER) (P<0.01) 10.3%.The results revealed that PI3K specific inhibitor (Wort and LY) can not only decrease the expression of Akt and mTOR but also can significantly inhibit the increasing of expression the tight junction and barrier function in IPEC-J2 cells.In summary, the results showed that LPS stress significantly reduced expression of tight junction in IPEC-J2 cells and destroy the barrier function of IPEC-J2 cells. GLP-2 have protective effect on the LPS stressed IPEC-J2 cells.GLP-2 may modulate TJ’s expression and barrier function through PI3K-Akt-mTOR signal transduction pathway in IPEC-J2 cells. |
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