Study On The Protective Effect Of Adenovirus-mediated Transfer Of Hsp70in BRL Cells Against Apoptosis By Hydrogen Peroxide

Liver cell apoptosis will be caused by encounting oxidative stress. Hsp70is a protein whichcan improve stress tolerance of cells to protecte cells from oxidative damage and apoptosis,behaving as molecular chaperone. Based on this, an exogenous hsp70was introduced andexpressed in the cells using an adenoviral vector to research the affectes on H2O2-inducedapoptosis in BRL cells by over-expressive hsp70. First, different concentrations of H2O2wereused to stimulate BRL cells2h, then the viability and apoptosis of BRL cells were assayed byWST-1, Annexin-V/FITC-PI double staining, and the protein expression of Caspase-3wereanalyzed using Western blotting, which was to chose the optimal concentration of H2O2. Thentreated the cells with this concentration, detected the viability of SOD and protein carbonylcontents to evaluate oxidative damage of cells. Second, dividing cells into six groups: controlgroups, Ad-CMV-Null cells groups, Ad-CMV-hsp70cells groups, H2O2groups, Ad-CMV-Null+H2O2groups, Ad-CMV-hsp70+H2O2groups, then treated them separately, and screenedhsp70mRNA abundance by qRT-PCR, the protein expression of Hsp70, Bcl-2, Bax, Cyt-c,cleaved Caspase3, Fas, Caspase8were analyzed using Western blotting technology, andapoptosis rate by flow cytometry.The results were as follows:(Ⅰ) Comparing with the control groups, the cell proliferationrate was significantly decreased (P<0.01), an increase in protein expression level of Caspase3(P<0.01) and apoptosis (P<0.01) were observed after treatment by150μmol/L H2O2actingon BRL cells2h, but cell necrosis were lower than other H2O2treatment, reductions ofactivities of SOD (P<0.05) and an increase of protein carbonyl content (P<0.05) werefound.These results showed that the H2O2of150μmol/L acting on BRL cells could induceapoptosis and oxidative damage.(Ⅱ)Comparing with the H2O2groups, cells in the Ad-CMV-hsp70+H2O2groups showed higher hsp70mRNA abundance and protein expression level ofHsp70(P<0.01), higher protein expression level of Bcl-2(P<0.01), no difference of Baxprotein expression (P＞0.05), lower Cyt-c leakage rate (P<0.01), lower protein expressionlevel of cleaved Caspase3(P<0.01), no difference of Fas protein expression (P＞0.05), lowerprotein expression level of cleaved Caspase8(P<0.01), lower apoptosis rate (P<0.01).The conclusions were as follows:(a) The H2O2of150μmol/L acting on BRL cells couldinduce apoptosis and oxidative damage.(b) Adenoviral transfection of over-expresive Hsp70protect BRL cells from apoptosis, inhibiting Caspase8/3activation, inceasing anti-apoptosive protein expression, as Bcl-2, preventing cytochrome c transferring from mitochondria tocytoplasm.