Effects Of 7-difluoromethoxy-5,4’-dimethoxygenistein On Apoptosis And Expression Of Death Receptor 5 In HUVE-12 Cells Induced By Lysophosphatidylcholine

Objective:To evaluate the protective effects of 7-difluoromethyl-5, 4’-dimethoxygenistein (dFMG), a novel genistein(GEN) analogue, on human umbilical vein endothelial cells (HUVE-12) injury induced by lysophosphatidylcholine (LPC) and explore whether its mechanism is involved in blocking induction of apoptosis by mitochondrial and death receptor 5 pathway.Methods:HUVE-12 cells were cultured in vitro and treated with LPC(1.0、3.0、10、30、100μmol/L) for 24 hours to decide the desired concentration of LPC for making injury model of HUVE-12 cells. HUVE-12 cells were pretreated with 0.3、1.0、3.0μmol/L of dFMG before incubation with LPC for 24h. The activity of lactate dehydrogenase(LDH) was determined in cell supernatant, the survival rate of the HUVE-12 cells was determined by trypan blue refusing method, the apoptosis is dectected by flow cytometry of ANNEXIN V/PI staining; the enzymatic activity of caspase-3、8、9 were determined by caspase colorimetric assay kits; DR5 expression of HUVE-12 was determined by direct immunofluorescence flow cytometry.Results:①After treated with LPC 30μmol/L for 24h, the LDH release of HUVE-12 cells was elevated up to 62.59±1.82U/L(P<0.001), the survival rate of the HUVE-12 cell descended to 16.93±0.54% (P<0.001), the apoptosis of HUVE-12 increased to 15.31±1.85% (P<0.001),which definited LPC30μmol/L as a desired concentration for making LPC-induced HUVE-12 cell injury model.②dFMG reduced the release of LDH in LPC-induced HUVE-12 cells (P<0.001). The activity of LDH in cell supernatant after treatment with dFMG at various concentrations of 0.3、1.0、3.0μmol/L were 47.83±2.19 U/L,40.81±1.71 U/L,27.46±1.26 U/L respectively, which was lower than that of GEN (53.71±1.83,44.711±1.75,32.55±2.03 U/L)(P<0.001).③dFMG could improve the survival of the HUVE-12 cells in a concentration dependent manner, the survival rate of the HUVE-12 cells after treatment with dFMG at various concentrations of 0.3、1.0、3.0μmol/L for 24h were 63.39±0.98%,78.58%±1.09%,84.12±1.12% espectively, which were higher than that of GEN (59.67±2.08%,69.74±1.56%,82.21±0.74%) (P<0.05).④The apoptosis of LPC-induced HUVE-12 cell after treatment with dFMG at various concentrations of 0.3、1.0、3.0μmol/L for 24h descended to 8.56±0.96%,6.10±0.71%,4.05±0.63% respectively, which was lower than that of GEN(10.50±0.77%,7.77±0.59%,5.06±0.31%) (P<0.05).⑤Results of the examine for the enzymatic activity of caspase-3、8、9 were as follow:the enzymatic activity of caspase-3、8、9 of LPC-treated HUVE-12 cell was significantly increased in comparison with control group(P<0.001); dFMG and GEN could decreased the enzymatic activity of caspase-3、8、9 in LPC-induced HUVE-12 cells in a concentration dependent manner; the effect of dFMG at a higher concentration(3μmol/L) on the enzymatic activity of caspase-8、9 was stronger than that of a lower concentration(0.3μmol/L) (P<0.001); comparing with GEN(3μmol/L), dFMG (3.0μmol/L) could significantly decreased the enzymatic activity of caspase-3、9(P<0.05).⑥Results of direct immunofluorescence flow cytometry showed that the expression of death receptor 5 was 42.75±1.25% in LPC-induced HUVE-12 cells. The expression of DR5 after treatment with dFMG at various concentrations of 0.3、1.0、3.0μmol/L were 27.76±1.07,18.66±1.13,7.92±0.64%, which were lower than that of GEN (33.49±1.04,24.98±0.79,14.60±1.06%) (P<0.001).Conclusion:1. dFMG possess protective effect from HUVE-12 cell damage induced by LPC.2. The protective effect against HUVE-12 cell damage induced by LPC is a stronger by dFMG than by the lead compound genistein.3. The protective effect of dFMG on HUVE-12 cell damage induced by LPC may be associated with the blockage of mitochondrial and death receptor 5 pathway.