The Effects Of GLP-2on The Tight Junction In Intestinal Epithelium Of Weaned Piglets And It’s Molecular Mechanism Research

The present experiment was conducted to study the effect of GLP-2(Glucagons-like petide-2) supplementation on the mRNA and protein expression of tight junction and protein distribution of tight junction in weaned piglets, and then explore the possible molecular mechanism of GLP-2regulate intestinal tight junction protein expression of weaned piglets in vitro.In order to measure the effect of different GLP-2concentrations on the key tight junction protein ZO-1, Occludin, Claudin-1mRNA and protein expression, the jejunal tissues were cultured in vitro72hours in the medium with GLP-2(1×10-8,1×10-7mol/L). After the dose of GLP-2which could promote the mRNA and protein expression of key tight junction protein was determined, added the pharmacological inhibition of ERK1/2activation U0126into medium. Furthermore, we sought to determine whether the pharmacological blockade of MAPK signaling pathway could influence the function of GLP-2on the tight junction gene, protein expression and distrbution. The RT-PCR and western bolting were used to measure the key tight junction gene and protein expression. Besides, the immunohistochemistry method was used to determine whether GLP-2could modulate the tight junction protein via ERK1/2signaling pathway.Compared with the control group, GLP-2supplementation significantly increased (P<0.05) the relative expression levels of ZO-1, Occludin and Claudin-1mRNA and protein. In1×10-8mol/L GLP-2concentration treatment, the relative expression of ZO-1, Occludin and Claudin-1mRNA respectively increased by29.0%,54.0%and120.0%(P<0.05) and the relative expression of ZO-1, Occludin and Claudin-1protein respectively increased by35.6%,30.4%and26.2%(P<0.05). In1～10-7mol/L GLP-2concentration treatment, the relative expression of ZO-1, Occludin and Claudin-1mRNA respectively increased by59.0%,142.0%and251%(P<0.05) and the relative expression of ZO-1, Occludin and Claudin-1protein increased by46.7%,56.7%and35.6%(P<0.05). At the same time, GLP-2also increased both extracellular signal-regulated kinase1/2(ERK1/2) and its’ downstream target p90RSK phosphorylation. In110"8mol/L GLP-2concentration treatment, the relative expression of phospho-ERKl/2-Thr202/Tyr204and phospho-p90RSK-Ser380respectively increased by34.7%and56.0%(P<0.05); In1×10-7mol/L GLP-2concentration treatment, the relative expression of phospho-ERK1/2-Thr202/Tyr204and phospho-p90RSK-Ser380protein respectively increased by69.5%and84.0%(P<0.05). When10μ mol/L U0126was used to block the MAPK signaling pathway, however, the gene and protein expression of ZO-1, Occludin and Claudin-1decreased significantly (P<0.05):compared with1×10-1mol/L GLP-2concentration treatment, the relative expression of ZO-1, Occludin, Claudin-1mRNA respectively decreased by74.0%,85.1%and87.7%(P<0.05); the relative expression of ZO-1, Occludin, Claudin-1protein respectively decreased by17.2%,24.6%and12.6%(P<0.05).Compared with the control group, adding GLP-2(1×10-7mol/L) influenced the distribution of ZO-1, Occludin and Claudin-1in the tissue by immunohistochemistry method. Adding GLP-2(1×10-7mol/L) could increase the optical density of ZO-1, Occludin and Claudin-1by18.6%,40.6%and27.6%respectively (P<0.05); while10μmol/L U0126were added into the medium, compared with the group (1×10-1mol/L GLP-2), the distribution of ZO-1, Occludin and Claudin-1weaked in the tissue and the optical density of ZO-1, Occludin and Claudin-1decreasd by9.80%,28.6%and16.2%(P<0.05).The results indicated that GLP-2could increase the expression of ZO-1, Occludin, Claudin-1and their distribution. The extra cellular signal related kinase ERKl/2pathway may be one of the most important signaling pathways that GLP-2modulates the expression of the weaned piglets’tight junction protein.