Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Oct 14, 2012; 18(38): 5377-5388
Published online Oct 14, 2012. doi: 10.3748/wjg.v18.i38.5377
Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells
Zhang-Bo Liu, Su-Juan Fei, Sheng-Ping Zhu, Jin-Zhou Zhu, Hong-Xia Han, Qiu-Ju Dong, Jian-Fu Zhang
Zhang-Bo Liu, Su-Juan Fei, Sheng-Ping Zhu, Jin-Zhou Zhu, Hong-Xia Han, Qiu-Ju Dong, Department of Gastroenterology, The Affiliated Hospital of Xuzhou Medical College, 99 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China
Jian-Fu Zhang, Department of Physiology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China
Author contributions: Fei SJ and Zhang JF designed the research; Liu ZB, Han HX and Dong QJ performed the research; Zhu SP contributed to the analysis of new reagents; Zhu JZ drafted the paper.
Supported by National Natural Science Foundation of China, No. 30570671; the Educational Department Science Research Foundation of Jiangsu Province, No. 99KJB310005 and 05KJB310134
Correspondence to: Jian-Fu Zhang, Professor, Department of Physiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu Province, China. jfzhang@xzmc.edu.cn
Telephone: +86-516-83262105 Fax: +86-516-85582071
Received: December 26, 2011
Revised: January 16, 2012
Accepted: April 13, 2012
Published online: October 14, 2012
Abstract

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 dose-dependent 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-3β and 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.

Keywords: Human gastric epithelial cells; Ghrelin; Pharmacological postconditioning; Hypoxia/reoxygenation; Apoptosis