Ghrelin Inhibits Apoptosis In Colonic Epithelial Cells And Its Signaling Pathways

ObjectiveOur study is to investigate if ghrelin inhibits apoptosis in colonic epithelial cells, observe the expression of some proteins relative to apoptosis and further explore if the effect of antiapoptosis of ghrelin in colonic epithelial cells is mediated by PI3K/Akt and ERK1/2 signaling pathways.Methods1.Detect if there is GHS-R1a expression in HT-29 cells via RT-PCR.2.Divide HT-29 cells into control and treated groups in wihch the cells are treated by 5-Fu(80μM) with or without ghrelin(10^-9M-10^-5 M) for 24h-48h.3.Assay the viability of different groups by MTT and conculate their survival rate.4.Stain the cells with DAPI and observe the changes of apoptotic nucleus in the different groups using fluorescence microscopy.5.Stain the cells with both Annexin V and PI,then assay and compare the apoptotic rate of the groups by flow cytometry.6.Observe the expression of some proteins relative to apoptosis such as caspase3,bcl-2,bax as well as several signal proteins Akt,P-Akt,ERK,P-ERK by western blot.Results1.We detected there was GHS-R1a expression in HT-29 cells by RT-PCR.2.MTT assay demonstrated ghrelin dose-dependently decreased growth inhibition of HT-29 cells induced by 5-Fu(P＜0.05).3.DAPI staining showed that the group treated with 5-Fu displayed chromatin condensation and nuclear fragmentation which are typical changes of apoptosis, while ghrelin reduced these changes.4.FCM detection after stainned with Annexin V and PI realved ghrelin dose-dependently decresed the apoptotic rate of HT-29 cells induced by 5-Fu(P＜0.05).5.Comparing with the group treated with only 5-Fu,the group with both 5-Fu and ghrelin had such differences:the activity of caspase3 weakened,bcl-2 expression improved while bax lowered,Akt and ERK were activated and the phosphorylation appeared stronger.conclusionsGhrelin can prevent colonic epithelial cells from apoptosis and the effect may be mediated by PI3K/Akt and ERK1/2 signaling pathways.