ABSTRACT
Background:With the development of capsule endoscopy,small intestinal injury induced by non-steroidal anti-inflammatory drugs( NSAIDs)has become an issue of growing concern. Although there are a variety of drugs used for NSAIDs-induced gastric mucosal injury,small intestinal injury caused by NSAIDs is lack of effective prevention and treatment modalities. Aims:To investigate the protective effect and possible mechanism of rebamipide on human colon cancer cell line Caco-2 injury induced by aspirin. Methods:In aspirin group,Caco-2 cells were treated with aspirin 10 mmol/L;in rebamipide group,Caco-2 cells were treated with aspirin and different concentrations of rebamipide(0. 1, 0. 5,1. 0 mmol/L),and a negative control group was established. Cell proliferation inhibition was measured by MTT assay. Cell apoptosis was determined by flow cytometry. Morphological changes of cells were observed under inverted phase contrast microscope. Permeability of cells was assessed by Transwell assay. Expressions of tight junction proteins occludin and zonula occluden-1(ZO-1),as well as mitogen activated protein kinase(MAPK)signaling pathway-associated proteins including extracellular signal-regulated kinase(ERK)1/2,phosphorylated ERK1/2(p-ERK1/2),p38,p-p38,c-Jun N-terminal kinase( JNK),and p-JNK,were determined by Western blotting. Results:Proliferation inhibition rate,apoptosis rate and permeability of Caco-2 cells in rebamipide 0. 1,0. 5,1. 0 mmol/L groups were significantly lower than those in aspirin group in a dose-dependent manner(P<0. 05). Injuries of Caco-2 cells were seen in aspirin group by inverted phase contrast microscope and rebamipide could reduce these injuries. Expressions of occludin,ZO-1 and p-JNK were significantly higher and expressions of p-p38 and p-ERK1/2 were significantly lower in rebamipide 0. 1,0. 5,1. 0 mmol/L groups than those in aspirin group in a dose-dependent manner(P<0. 05). Conclusions:Rebamipide have a protective effect against aspirin-induced Caco-2 cell injury,probably through regulating MAPK signaling pathway( inhibiting p38 and ERK1/2 phosphorylation,stimulating JNK phosphorylation),and subsequently up-regulating the expressions of tight junction proteins and decreasing the permeability of cells.
ABSTRACT
Objective To investigate the protective effects and its mechanism of rebamipide on aspirin-induced injury in human gastric mucosal epithelium cells (GES-1).Methods GES-1 cells monolayer culture model was established in vitro.Then the cells were divided into negative control group,aspirin injured group and combination of rebamipide at different concentration (0.2,0.5,1.0 mrnol/L) and aspirin groups.The cell proliferation,the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) of each group were detected.The ultrastructural changes of each group were observed by transmission electron microscopy (TEM).The expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) at protein level in the cells of each group were detected by Western blot.Nrf2 interfering suppression test was performed and then the influence of Nrf2 small interfering RNA (siRNA) on the expression of HO-1 protein was observed.One-way analysis of variance was performed for comparison among multi-groups and t-test was used for comparison between the two groups.Results The cell viability of aspirin injured group and combination of rebamipide at different concentration (0.2,0.5,1.0 mmol/L) and aspirin groups were (49.56±3.88)％,(59.34±4.36) ％,(70.79 ± 5.96) ％ and (86.07 ± 5.20) ％,respectively,and the difference was statistically significant (F=30.634,P＜ 0.01).Compared with aspirin injured group,the content of MDA significantly lowered in combination of rebamipide at different concentration (0.2,0.5,1.0 mmol/L) and aspirin groups ((2.26±0.25) nrnol/rng vs (1.85±0.13) nmol/mg vs (1.62±0.11) nmol/mg vs (1.13±0.15) nmol/mg),and the difference was statistically significant (F=23.821,P＜0.05).Compared with aspirin injured group,the activity of SOD significantly increased in combination of rebamipide at 0.5 and 1.0 mmol/L and aspirin groups ((8.49±0.89) U/rng vs (11.50±1.03) U/mg vs (13.74±0.76) U/mg),the difference was statistically significant (F=25.666,P＜0.05).Under TEM,the cell ultrastrucmral was obviously inured in aspirin treated,while rebamipide could relieve the injury.The differences of relative expression quantity of Nrf2 and HO-1 at protein level among combination of rebamipide at 0.2,0.5 and 1.0 mmol/L and aspirin groups and aspirin injured group were statistically significant (0.35±0.04 vs 0.46± 0.05 vs 0.84±0.08 vs 0.15±0.02,0.72±0.09 vs 0.93±0.11 vs 1.29±0.14 vs 0.39±0.07,F=92.550and 38.235,both P＜0.05).After transfected with Nrf2 siRNA,the expression of HO-1 was 0.38±0.04 in aspirin injured group and 0.62±0.08 in combination of rebamipide and aspirin group,which was lower than that before transfection (0.61 ± 0.05,1.33± 0.09),respectively.The differences were statistically significant (t =6.276 and 10.444,both P＜0.05).Conclusion Rebamipide may activate Nrf2/HO-1 pathway and relieve aspiriwinduced oxidative stress in GF1 ceils.