The Protective Effect Of Adenovirus-mediated Transfer Of Hsp70in Rat Liver Cells Against Oxidative Stress

Animal's liver can be injured due to oxidative stress during the cold stress. Hsp70is a protein which canimprove the body's tolerance for stressors. An exogenous hsp70can be introduced and expressed in the cellsusing an adenoviral vector. Based on this, the effect and mechanism of the protection by adenovirus-mediatedtransfer of hsp70in rat liver cells against oxidative stress were studied by using the techniques of WST-1, insitu staining, qRT-PCR and western blotting, etc. The research consisted of3parts:(I) Determining the optimalconcentration and treatment duration of H₂O₂according to the cells' survival rates. Then treated the cells withthis concentration, detected the protein carbonyl content, SOD, GSH-Px and CAT activities, and thencompared with the indicators from cold stress rat experiments, in order to investigate the feasibility ofmimicking oxidative liver damages in vitro.(II) Transfecting the BRL-3A cells using the adenovirus vectorwith gene tags, then proceed with a preliminary screening of the optimal concentration and treatment durationfor virus transfection according to the transfection efficiency. Then the BRL-3A cells with adenovirus carryinghsp70under those concentration and treatment duration were measured the mRNA abundance of hsp70, anddetermined the optimal transfection concentration and exposure time which enabled the maximum expressionof hsp70. Finally, verified the successful establishment of the overexpression model of Hsp70in the BRL-3Acells at the protein level.(III) Dividing the Ad-CMV-hsp70cells group, the Ad-CMV-Null cells group and thenon-transfected cells group into each one another2groups: stressed and non-stressed, then treated themseparately, and detected, in each of these6groups, the proliferation of cells, the LDH leakage rate, the SOD,CAT and GSH-Px activities, the GSH, MDA and protein carbonyl contents, the hsp70mRNA abundance andthe Hsp70protein expression level. This was to study the protective effects of hsp70gene transfer using anadenoviral vector on rat liver cells against oxidative stress, and thus we hoped to lay a foundation for thefurther researches on Hsp70in animals' resistance to cold stress.The results were as follows:(I) Compared with the control group, H₂O₂of500μmol/L which acted on theBRL-3A cells for3h lead to a decrease of survival rate (P<0.01), an increase of protein carbonyl content(P<0.01) and reductions of activities of SOD (P<0.05), GSH-Px (P<0.05) and CAT (P<0.01). These resultsshowed that the H₂O₂of500μmol/L acting on BRL-3A cells could generate oxidative stress, and the trendsof change on indicators were similar to those of a cold exposed and damaged rat liver.(II) It was screened outthat transfecting the BRL-3A cells with the Ad-CMV-hsp70virus at the concentration of1×107PFU/mL,MOI=20, could better keep the cell morphology than with the other concentrations and action times, and it alsobrought a higher transfection efficiency, and a higher hsp70mRNA abundance (P<0.01). Then it was found,with statistically high significance (P<0.01), that the cells in Ad-CMV-hsp70group had the highest expressionof Hsp70than the Ad-CMV-Null group and the non-transfected control group.(III) Compared with thenon-transfected/stressed group, cells in the Ad-CMV-hsp70/stressed group showed higher proliferation rate(P<0.01), lower LDH leakage rate (P<0.01), higher CAT activity (P<0.01), lower GSH-Px activity (P<0.01),higher GSH content (P>0.05), higher SOD activity (P>0.05), higher protein carbonyl content (P<0.01), higherhsp70mRNA abundance and the protein expression level (P<0.01).The conclusions are as follows:(A) An oxidative stress can be generated when H₂O₂is acting on BRL-3A cells for3h, and the status is similar to a real cold-exposed-and-damaged rat liver.(B) By transfecting theBRL-3A cells with the Ad-CMV-hsp70virus at the concentration of1×10⁷PFU/mL, MOI=20, for48h, anHsp70-overexpressing liver cell model can be established.(C) Adenoviral transfection of Hsp70has someprotective effects for rat liver cells against oxidative stress.