Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells

Figure 1 Effects of different doses of ghrelin on cell viability in human gastric epithelial cells induced by H/R. Cells were grouped as follows: normoxic culture for 6 h (N), 2 h hypoxia/4 h reoxygenation (H/R), alcohol vehicle postconditioning (DM) and ghrelin postconditioning at three doses (10^-9 mol/L, 10^-8 mol/L and 10^-7 mol/L). Cell viability was detected by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. mean ± SD. n = 6. ^aP < 0.05, ^bP < 0.01 vs H/R.

Figure 2 Effects of D-Lys3-GHRP-6, capsazepine and LY294002 in ghrelin postconditioning on cell viability in human gastric epithelial cells induced by hypoxia/reoxygenation. Cells were grouped as follows: normoxic culture for 6 h (N), 2 h hypoxia/4h reoxygenation (H/R), DMSO vehicle postconditioning (DM), ghrelin postconditioning (10^-7 mol/L) (GH), D-Lys3-GHRP-6 + ghrelin postconditioning (D + GH), capsazepine + ghrelin postconditioning (C + GH) and LY294002 + ghrelin postconditioning (L + GH). Cell viability was detected by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. mean ± SD. n = 6. ^bP < 0.01 vs H/R; ^cP < 0.05 vs GH.

Figure 3 Effects of D-Lys3-GHRP-6, capsazepine and LY294002 in ghrelin postconditioning on cell apoptosis in human gastric epithelial cells induced by H/R. Cells were grouped as follows: normoxic culture for 6 h (A), 2 h hypoxia/4 h reoxygenation (B), DMSO vehicle postconditioning (C), ghrelin postconditioning (10^-7 mol/L) (D), D-Lys3-GHRP-6 + ghrelin postconditioning (E), capsazepine + ghrelin postconditioning (F), and LY294002 + ghrelin postconditioning (G). mean ± SD. n = 6. ^bP < 0.01 vs B; ^dP < 0.01 vs D. Cells were then stained by Hoechst33258. The arrows indicate apoptotic cells.

Figure 4 Effects of D-Lys3-GHRP-6, capsazepine and LY294002 in ghrelin postconditioning on cell apoptosis in human gastric epithelial cells induced by hypoxia/reoxygenation. Cells were grouped as follows: normoxic culture for 6 h (A), 2 h hypoxia/4 h reoxygenation (B), DMSO vehicle postconditioning (C), ghrelin postconditioning (10^-7 mol/L) (D), D-Lys3-GHRP-6 + ghrelin postconditioning (E), capsazepine + ghrelin postconditioning (F), and LY294002 + ghrelin postconditioning (G). Subsequently, the cells were stained by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Apoptosis was detected by flow cytometry.

Figure 5 Effects of D-Lys3-GHRP-6, capsazepine and LY294002 in ghrelin postconditioning on the expression of Bcl-2, Bax, Akt and glycogen synthase kinase-3β in human gastric epithelial cells induced by hypoxia/reoxygenation. Cells were grouped as follows: normoxic culture for 6 h (N), 2 h hypoxia/4 h reoxygenation (H/R), DMSO vehicle postconditioning (DM), ghrelin postconditioning (10^-7 mol/L) (GH) D-Lys3-GHRP-6 + ghrelin postconditioning (D + GH), capsazepine + ghrelin postconditioning (C + GH) and LY294002 + ghrelin postconditioning (L + GH). The expression of β-actin was detected as an internal standard. Densitometry results are expressed as ratio of test over normal group. A, B: Bcl-2 expression; C, D: Bax expression; E: Akt expression; F: Glycogen synthase kinase (GSK)-3β expression. mean ± SD. n = 6. ^bP < 0.01 vs H/R, ^dP < 0.01 vs GH.

Figure 6 Effects of capsazepine in ghrelin postconditioning on the expression of vanilloid receptor subtype 1 in human gastric epithelial cells induced by hypoxia/reoxygenation. Cells were grouped as follows: normoxic culture for 6 h (A), 2 h hypoxia/4 h reoxygenation (B), DMSO vehicle postconditioning (C), ghrelin postconditioning (10^-7 mol/L) (D), and capsazepine + ghrelin postconditioning (E). The expression of vanilloid receptor subtype 1 (VR1) in each group was observed by immunocytochemistry. Cells were observed for photography under a phase contrast microscope (× 400). The arrows indicate the positive cells which express VR1.

Figure 7 Effects of LY294002 in ghrelin postconditioning on the expression of Akt in human gastric epithelial cells induced by hypoxia/reoxygenation. Cells were grouped as follows: normoxic culture for 6 h (A), 2 h hypoxia/4 h reoxygenation (B), DMSO vehicle postconditioning (C), ghrelin postconditioning (10^-7 mol/L) (D), and LY294002 + ghrelin postconditioning (E). The expression of Akt in each group was observed by immunocytochemistry. Cells were observed for photography under a phase contrast microscope (× 400). The arrows indicate the positive cells which express Akt.

Figure 8 Effects of LY294002 in ghrelin postconditioning on the expression of glycogen synthase kinase-3β in human gastric epithelial cells induced by hypoxia/reoxygenation. Cells were grouped as follows: normoxic culture for 6 h (A), 2 h hypoxia/4 h reoxygenation (B), DMSO vehicle postconditioning (C), ghrelin postconditioning (10^-7 mol/L) (D), and LY294002 + ghrelin postconditioning (E). Expression of glycogen synthase kinase (GSK)-3β in each group was observed by immunocytochemistry. Cells were observed for photography under a phase contrast microscope (× 400). The arrows indicate the positive cells which express GSK-3β.