Increased endoplasmic reticulum stress response is involved in clopidogrel-induced apoptosis of gastric epithelial cells

Abstract

Background: The widespread use of clopidogrel alone or in combination with aspirin may result in gastrointestinal mucosal injury, clinically represented as recurrent ulceration and bleeding complications. Our recent work suggested that clopidogrel significantly induced human gastric epithelial cell (GES-1) apoptosis and disrupted gastric mucosal barrier, and that a p38 MAPK inhibitor could attenuate such injury. However, their exact mechanisms are largely unknown.

Methods: The GES-1 cells were used as a model system, the effects of clopidogrel on the whole gene expression profile were evaluated by human gene expression microarray and gene ontology analysis, changes of the mRNA and protein expression were determined by real-time PCR and Western blot analysis, and cell viability and apoptosis were measured by MTT assay and flow cytometry analysis, respectively.

Results: Gene microarray analysis identified 79 genes that were differentially expressed (P<0.05 and fold-change >3) when cells were treated with or without clopidogrel. Gene ontology analysis revealed that response to stress and cell apoptosis dysfunction were ranked in the top 10 cellular events being affected, and that the major components of endoplasmic reticulum stress-mediated apoptosis pathway - CHOP and TRIB3- were up-regulated in a concentration- and time-dependent manner when cells were treated with clopidogrel. Pathway analysis demonstrated that multiple MAPK kinases were phosphorylated in clopidogrel-treated GES-1 cells, but that only SB-203580 (a p38-specific MAPK inhibitor) attenuated cell apoptosis and CHOP over-expression, both of which were induced by clopidogrel.

Conclusions: Increased endoplasmic reticulum stress response is involved in clopidogrel-induced gastric mucosal injury, acting through p38 MAPK activation.

Figure 1. Hierarchical cluster analysis of 79 differentially expressed genes in all 6 samples.

Hierarchical cluster analysis was performed as described in the Materials and Methods section. Each column represents one sample, and each gene is depicted by one row, where red denotes an increase in gene expression and green denotes a decrease in gene expression as compared with the other group. The brighter the color, the higher the gene expression level.

Figure 2. The GO category for the up-regulated genes in clopidogrel group.

A P value <0.05 was used as a cut-off threshold to select significant GO categories. The higher the enrichment, the more significant the biological processes.

Figure 3. The GO category for the down-regulated genes in clopidogrel group.

A P value <0.05 was used as a cut-off threshold to select significant GO categories. The higher the enrichment, the more significant the biological processes.

Figure 4. Effects of clopidogrel on CHOP and TRIB3 mRNA expression in the GES-1 cells.

The mRNA expression levels of both CHOP and TRIB3 were up-regulated in the GES-1 cells in a concentration- and time-dependent manner when treated with clopidogrel, as measured by real-time PCR. Data are a representative of three independent experiments. **P<0.05 vs vehicle control; *P<0.05 vs clopidogrel (0.5 mM); #P<0.05 vs clopidogrel (1.5 mM); ⁺⁺P<0.05 vs clopidogrel (1.5 mM for 12 h); ⁺⁺⁺P<0.05 vs clopidogrel (1.5 mM for 24 h).

Figure 5. Effects of clopidogrel on CHOP and TRIB3 protein expression in the GES-1 cells.

The protein expression levels of both CHOP and TRIB3 were up-regulated in the GES-1 cells in a concentration- and time-dependent manner, consistent with their mRNA expression profiles as measured by real-time PCR. Data are a representative of three independent experiments. **P<0.05 vs vehicle control; #P<0.05 vs clopidogrel (1.5 mM); ⁺⁺P<0.05 vs clopidogrel (1.5 mM for 12 h); ⁺⁺⁺P<0.05 vs clopidogrel (1.5 mM for 24 h).

Figure 6. Effects of ER stress on ATF expression in the GES-1 cells.

GES-1 cells were treated with clopidogrel 1.5 mM for 24 h. Data are expressed as mean ± SD, representative of three independent experiments. *P<0.05 vs control.

Figure 7. Suppression of clopidogrel-induced CHOP up-regulation by the p38 MAPK inhibitor.

As expected, only the p38 MAPK inhibitor SB-203580 significantly attenuated CHOP up-regulation. Data are expressed as mean ± SD, representative of three independent experiments. *P<0.05 vs control; #P<0.05 vs clopidogrel alone (1.5 mM).

Figure 8. The p38 MAPK inhibitor suppressed clopidogrel-induced cell proliferation inhibition and apoptosis.

After pretreatment of a p38 MAPK inhibitor SB-203580, clopidogrel-induced cell proliferation inhibition and apoptosis were significantly attenuated. Data are expressed as mean ± SD, representative of three independent experiments. *P<0.05 vs control; #P<0.05 vs clopidogrel alone.