Tsg Negatively Regulates H₂O₂-Induced Apoptosis In Human Umbilical Vein Endothelial Cells By Induction Of HO-1 Apoptosis

Apoptosis in human umbilical vein endothelial cells(HUVECs)contributes to the development of atherosclerosis. So inhibition of HUVESs apoptosis by drug intervention was an important strategy to prevent atherosclerosis.2,3,5,4'-tetrahydroxystilbene-2-O-?-D-glucoside(TSG), is a two styrene glycosides which is extracted from Polygonum multiflorum Thunb,inhibits the H2O2-induced apoptosis in HUVECs. But the mechanism is still unclear. The aim of this study is to investigate how the TSG regulate the effects of exogenous H2O2 on the expression of HO-1 in HUVECs and discuss the roles of TSG in apoptosis. The HUVECs were cultured in vitro and the apoptosis model was constructed by adding 300 ?mol/L H2O2. The HUVECs were pretreated with different concentration of TSG.The mRNA and protein expression of HO-1 were detected by real time PCR or western blot respectively. Enzyme activity of HO-1 was detected by colorimetric method. Apoptosis of HUVECs cells were determined byflow cytometry analysis. The nuclear translocation of Nrf2 was detected by Western blot. Our results show expression of HO-1 is very low under normal condition(control) and is up regulates very little in response to H2O2. However, expression of HO-1 mRNA was induced by TSG in a dose dependent manner. Increased expression of HO-1 began at 6h and reached the peak level at 12 h then start decreasing. The expression profiles of protein are consistent with the patters of mRNA. At the same time, the use of RNA synthesis inhibitor actinomycin D and protein synthesis inhibitor cycloheximide, the expression of HO- 1 was significantly reduced. In addition,the nuclear translocation of Nrf2 also could be induced by TSG. Transfecting cells with Nrf2 specific siRNA reduce the expression of HO-1. The FCM results indicated the H2O2-induced apoptosis in HUVECs could be suppressed by pretreatment of TSG. The TSG did not suppress the apoptosis in HUVECs when the HO-1 was blocked by siRNA or pretreatment with a HO-1 inhibitor Zinc protoporphyrin(ZnPP). However, induction of HO-1 by cobalt protophoryrin(CoPP) would enhance TSG suppress the apoptosis. These results indicated TSG could induce HO-1 expression via Nrf2 pathway to inhibit apoptosis in HUVECs.