Effects Of7-difluoromethoxy-5,4’-dimethoxy-Genistein On Keratin18Phosphorylation In HUVE-12Cells Induced By Lysophosphatidylcholine

Objective:To observe whether the mechanisms underlying the protective effects of7-difluoromethyl-5,4’-dimethoxygenistein (DFMG), a novel genistein(GEN) analogue, on human umbilical vein endothelial cells (HUVE-12) apoptosis induced by lysophosphatidylcholine (LPC) is involved in the regulation of expression of keratin18phosphorylation.Method:HUVE-12cells were cultured in vitro and treated with10μmol/L LPC for (6h,12h,24hå'Œ48h) to explore and determine the desired time of LPC for making apoptosis model of HUVE-12cells. HUVE-12cells were pretreated with0.3,1.0,3.Oμmol/L DFMG for30minutes,and then incubated with LPC for24hours. Cell apoptosis was evaluated by Armexin V-FITC/PI double staining flow cytometry, the content of the fragments of CK18was detected by CK18-M3O quantitative detection kit the expression levels of CK18, Ser33and Ser52phosphorylation of CK18were detected by western blotting, the expression of K18/14-3-3complex in HUVE-12cells was verified By co-immunoprecipitation.Results:1. After treated with LPC10μmol/L for24h, the CK18-M30release of HUVE-12cells increased and the concentration of CK18-M30was elevated up to798.60±8.43mIU/ml in cell culture medium, the apoptosis of HUVE-12increased to30.98±2.04%,which definited LPC10μmol/L for24h as a desired time for making LPC-induced HUVE-12cell apoptosis model.2. DFMG reduced the release of CK18-M30in LPC-induced HUVE-12cells (P<0.001) in a dose dependent way. The concentration of CK18-M30in cell supernatant after treatment with DFMG at various concentrations of0.3,1.0,3.0μmol/L were701.00±2.65mIU/ml,599.77±2.30mIU/ml,402.47±4.16mIU/ml respectively, which was lower than that of GEN (753.90±5.07mIU/ml,700.97±3.60mIU/ml,548.83±3.98mIU/ml)(P<0.05).3. The apoptosis rate of LPC-induced HUVE-12cell after treatment with DFMG at various concentrations of0.3,1.0,3.0μmol/L for24h dropped to27.50±2.06%,23.52±2.59%,13.70±2.00%respectively, which was lower than that of GEN (29.42±2.09%,27.57±0.76%,24.29±2.49%)(P<0.05).4. Western blotting results showed that:Compared with the normal control group,10μmol/L LPC made the expression of Ser33and Ser52phosphorylation of CK18up-regulated while made the expression of unphosphorylated CK18downregulated.DFMG and GEN antagonized the expression of Ser33and Ser52phosphorylation of CK18in LPC-induced HUVE-12cells in a dose dependent manner and the effect of DFMG is better than that of GEN.5. Co-immunoprecipitation results showed that:In the extraction of HUVEC-12cell total protein, CK18can be detected in protein complexes precipitated by14-3-3antibody,while14-3-3can also be detected in protein complexes precipitated by CK18antibody,which showed that CK18and14-3-3interacted in HUVEC-12cells. Compared with the normal control group, the co-immunoprecipitation of14-3-3protein and CK18was enhanced in10μmol/L LPC model group. DFMG and GEN can reduce the level of14-3-3protein and CK18combination in LPC-induced HUVE-12cells and this effect of DFMG is better than that of GEN.Conclusion:The protective effect of DFMG on HUVE-12cell apoptosis induced by LPC may be associated with the blockage of expression of cytokeratin18phosphorylation.