Study On The Effects And Mechanism Of Clopidogrel On Human Gastric Epithelial Cell Line Injury

Objective: To investigate the effect and probable mechanism of clopidogrel on proliferation of human gastric epithelial cells GES-1.Methods: GES-1 cells were cultured in vitro. After co-culture of clopidogrel solutions of 0.01, 0.1, 0.5 and 1mmol/L with GES-1 cells for 24h, 48h and 72h, the inhibitory rates of GES-1 cells in each group were examined using MTT assay. The dosage-response curve was drawed according to different concentrations of clopidogrel to GES-1 cells growth inhibition rates. The inhibitory concentration 50 and safe concentration ( more than 90% of survival rate) of clopidogrel were obtained based on the Bliss method. GES-1 cells morphology change after drug action was observed by inverted phase contrast microscope and transmission electron microscope. Flow cytometry was used to detect cells apoptosis after co-culture of clopidogrel solution with GES-1 cells for 24h. Immunocytochemistry was used to detect the expression of Occludin before and after clopidogrel was given.Results: After 24h, 48h and 72h,the inhibitory rates of GES-1 cells were 9.15%±3.37%, 21.96%±7.96%, 64.31%±8.59% and 81.46%±1.95%; 14.03%±3.67%, 20.11%±3.52%, 55.18%±10.50% and 81.60%±4.99%; 12.30%±2.28%, 24.98%±5.10%, 66.03%±7.17% and 85.83%±0.92% in groups with clopidogrel of 0.01, 0.1, 0.5 and 1mmol/L, respectively. GES-1 cells injury was induced by clopidogrel in a concentration-dependent manner, but in no obvious time-dependent manner. The inhibitory concentrations 50 and safe concentrations of clopidogrel after 24h, 48h and 72h were 0.36mmol/L(95%CI: 0.26-0.46), 0.51mmol/L(95%CI: 0.30-0.64)and 0.35mmol/L(95%CI: 0.24-0.44); 0.08mmol/L(95%CI: 0.03-0.13), 0.16mmol/L(95%CI: 0.03-0.28)and 0.08mmol/L(95%CI: 0.03-0.13)respectively. The number of adherent cells was reduced, with cells rounded and suspended, and part of the nucleus concentrated after drug action by microscopy. Cell damage was increased with drug concentration. Cell space became larger and the structure of tight junction was damaged by electron microscopy. Flow cytometric analysis displayed that apoptosis rates were 4.7%, 5.3%, 14.7%, 51.0% and 60.5% in groups with clopidogrel of 0, 0.01, 0.1, 0.5 and 1mmol/L, respectively. The apoptosis rate of GES-1 cells was increased with drug concentration. Immunocytochemistry showed that the expression of Occludin was significantly lower after clopidogrel was given.Conclusion: Clopidogrel could inhibit the proliferation of human gastric epithelial cells in a concentration-dependent manner. The damage was caused to GES-1 cells to reduce the expression of the tight junction protein of cells.