[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 (H2O2)-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 Ni2+-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 H2O2 (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 H2O2 administration, cell viability was significantly reduced compared with control group (37.23%+/-5.68% vs. 100%, P<0.05), 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 P<0.05). Preincubation with PEP-1-CAT proteins at various concentrations (0.25-2 micromol/L) significantly attenuated H2O2-induced cell injury.


CONCLUSIONS
The PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.
Yao
Ling-ling
LL
Institute of Clinical Medicine, Renmin Hospital, Yunyang Medical College, Shiyan 442000, China.
Wang
Jia-ning
JN
Huang
Yong-zhang
YZ
Guo
Ling-yun
LY
Kong
Xia
X
D002374:Catalase / Q000378:metabolism*; D002478:Cells, Cultured; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide; D018384:Oxidative Stress / Q000502:physiology*; D010455:Peptides / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
C503924:Pep-1 peptide; D010455:Peptides; D003543:Cysteamine; D006861:Hydrogen Peroxide; D002374:Catalase