首页|Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells
Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells
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AIM:To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells.METHODS:The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells.Cells were divided into seven groups:normal control group (N); H/R postconditioning group;DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 +ghrelin postconditioning group (L + GH).3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability.Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1 cells.Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH).Protein expression of Bcl-2,Bax,Akt,and glycogen synthase kinase (GSK)-3β was determined by western blotting.Expression of vanilloid receptor subtype 1 (VR1),Akt and GSK-3β was observed by immunocytochemistry.RESULTS:Compared with the H/R group,cell viability of the GH group was significantly increased in a dosedependent manner (55.9％ ± 10.0％ vs 69.6％ ± 9.6％,71.9％ ± 17.4％,and 76.3％ ± 13.3％).Compared with the H/R group,the percentage of apoptotic cells in the GH group significantly decreased (12.38％ ± 1.51％ vs 6.88％ ± 0.87％).Compared with the GH group,the percentage of apoptotic cells in the D + GH group,C + GH group and L + GH groups significantly increased (11.70％ ± 0.88％,11.93％ ± 0.96％,10.20％ ± 1.05％vs 6.88％ ± 0.87％).There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38％ ± 1.51％ vs13.00％± 1.13％).There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ±105.29 U/L).There was a significant increase in LDH release in the D + GH,C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L,870.95 ± 64.06 U/L,838.62 ± 118.45 U/L vs 561.58± 64.01 U/L).There were no significant differences in LDH release between the H/R and DM groups (1062.45± 105.29 U/L vs 1017.65 ± 68.90 U/L).Compared with the H/R group,expression of Bcl-2 and Akt increased in the GH group,whereas expression of Bax and GSK-3β decreased.Compared with the GH group,expression of Bcl-2 decreased and Bax increased in the D + GH,C + GH and L + GH groups,and Akt decreased and GSK-3β increased in the L + GH group.The H/R group also upregulated expression of VR1 and GSK-3βand downregulated Akt.The number of VR1-positive and Akt-positive cells in the GH group significantly increased,whereas the number of GSK-3β-positive cells significantly decreased.These effects of ghrelin were reversed by capsazepine and LY294002.CONCLUSION:Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells,which indicated that this protection might be associated with GHS-R,VR1 and the PI3K/Akt signaling pathway.
Human gastric epithelial cellsGhrelinPharmacological postconditioningHypoxia/reoxygenationApoptosis
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