Cigarette Smoke Extract-induced SEPCR And MEPCR Expression In Human Umbilical Vein Endothelial Cells And Simvastatin Intervention Role

BackgroudA lot of research surveys have shown that cigarette smoking could damage the vascular endothelial cells and lead to vascular endothelial cells dysfunction, increasing the risk of thrombosis. In recent years, the relationgship beween endothelial cell protein C receptor and thrombosis aroused great attention from academic circle. When a variety of factors lead to endothelial cell damage, membrane associated EPCR by endothelial cell falls off, the soluble EPCR concentration increases, make thrombosis risk increase significantly. Therefore, the determination sEPCR become one of the sensitive indicators reflect thrombosis. Simvastatin in addition to its lipid role besides, also has the the endothelial protection, anticoagulation, inhibiting platelet aggregation and anti-inflammatory functions. Simvastatin whether can improve the CSE mediated endothelial cells blood coagulation disorders, has not been reported.ObjectiveTo investigate the effects of simvastatin on cigarette smoke extract-induced expression levels of soluble endothelial cell protein C receptor(sEPCR) and membrane associated endothelial cell protein C receptor(mEPCR) in human umbilical vein endothelial cells(HUVECs).Methods(1) HUVECs were cultured in24-well tissue-culture plates and randomly divided into the control group and CSE treated group.①Concentration-dependent effect of cigarette smoke extract on the expression of sEPCR:HUVECs were incubated with0%、2.5%、5%and10%CSE for8hours respectively.②Time-dependent effect of cigarette smoke extract on the expression of sEPCR:The cells were exposured to5%CSE for0,4,8,12,24hours respectively. The cells of control groups were exposured to the same volume of PBS instead of CSE at each time points. The supematants were collected respectively. The expression of sEPCR were determined by the enzyme linked immunosorbent assay (ELISA).(2) Simvastatin intervention trials:HUVECs were cultured in25cm×25cm culture flasks and randomly divided into control group,5%CSE group, simvastatin groups and simvastatin+CSE groups, In simvastatin groups, HUVECs were incubated with simvastatin at the concentrations of50、100and200μmol/L for24h. In simvastatin+CSE groups, the cells were treated with simvastatin at the concentrations of50、100and200μmol/L for2h, and then exposed to CSE for24h. The protein level of sEPCR in the culture supernatants was measured by ELISA. The cells were collected for determining the mRNA expression of mEPCR by real-time PCR. Results(1) Expression levels of sEPCR protein of5%、10%CSE treated group were increased significantly (P<0.05).but beween the two group have no statistically significant. While the2.5%CSE group sEPCR content is not statistically significant. The stimulated with5%CSE for0,4,8,12,24hours, the levels of sEPCR protein increased over time and reached the peak at24hours, which were significantly higher than those of control groups (P<0.05), and showed time-dependent.(2) Simvastatin intervention trials:Compared with control group,the protein level of sEPCR was significantly increased, and the mRNA expression of mEPCR was significantly decreased (all P<0.05)in5%CSE group. The protein levels of sEPCR were significantly increased, and the mRNA expression of mEPCR was significantly decreased in100μ mol/Land200μ mol/L simvastatin groups. However, the protein levels of sEPCR were lower, and the mRNA expression of mEPCR was significantly higher in100μ mol/Land200μ mol/L simvastatin groups than those in5%CSE group.Compared with5%CSE group, the protein levels of sEPCR in simvastatin+CSE groups were significantly decreased, but higher than in control group and simvastatin group with corresponding concentration; On the contrary, The mRNA expression of mEPCR in simvastatin+CSE groups was significantly increased, but lower than that in control group and simvastatin group with corresponding concentration(all P<0.05).Conclusions(1) The abnormol expression of EPCR may be one of the CSE to cause endothelial cell dysfunction of blood coagulation mechanism.(2) Simvastatin obviously increases the mRNA expression of mEPCR, decreases the protein levels of sEPCR, and attenuates the CSE-induced endothelial injury in vitro.