[The protective effect of PEP-1-CAT fusion protein on hydrogen peroxide-induced oxidative stress injury in human umbilical vein endothelial cells].

Objective To investigate the transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell(HUVECs)and the effects on hydrogen-peroxide(H_2O_2)-induced oxidative stress injury in these cells.Methods With the use of TA-cloning program and isocaudamer technique,the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)and the protein expression was induced by IPTG.The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni~(2+)-NTA-resin column.The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro.The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro.The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity.The cells were treated with H_2O_2(0.5 mmol/L)alone and in combination with PEP-1-CAT fusion protein for 4 h.Then,the cell viability,lactate dehydrogenase(LDH)and malondialdehyde(MDA)contents were measured.Results The PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose-and time-dependent manner and be stable for at least 48 h. After H_2O_2 administration,cell viability was significantly reduced compared with control group(37.23%± 5.68% vs.100%,P0.05),while LDH leakage(849.3 U/L±95.1 U/L)and MDA(8.23 nmol/L± 1.58 nmol/L)content were significantly higher than that in control group(540.6 U/L±65.7 U/L and 2.46 nmol/L±1.42 nmol/L,respectively,all P0.05).Preincubation with PEP-1-CAT proteins at various concentrations(0.25-2μmoL/L)significantly attenuated H_2O_2-induced cell injury.Conclusion The PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H_2O_2.The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.