The Relationship Between HIF-1and ROCK-1in Hypoxia/reoxygenation Of H9C2Cells

Objective:To study the relationship between hypoxia inducible factor-1(HIF-1) andRho-associated kinase (ROCK-1), and their effects in myocardial hypoxia/reoxygenation (H/R).Methods:(1). H9c2cells were cultured in vitro and the H/R model (95%N2and5%CO2) ofH9c2cells was established.(2). At first, a preliminary experiment was done to find out the adequate time of H/Rby Western blotting to measure the levels of protein of HIF-1and ROCK-1.(3). Then, the cells were intervened by adding each inhibitor in the select time of H/R(hypoxia10h/reoxygenation6h,8h,10h). Cutured cells were randomly dividedinto four groups: the normal group, the only H/R group, the H/R plus YC-1(theinhibitor of HIF-1; final concentration was10μmol/L) group, and the H/R plusY-27632(the inhibitor of ROCK-1; final concentration was10μmol/L) group.(4). The levels of protein and mRNA were measured by Western blotting and RT-PCR.Cells apoptosis rates and survival rates were detected by flow cytometry andMTT assay respectively.Results:(1). Western blotting showed that the level of protein of HIF-1and ROCK-1weresignificantly higher in hypoxia for10h;(2). Among the time of reoxygenation, the Western blotting found that each gene wasassociated with a higher level of protein as compared to normal group in6,8,10h, respectively.(3). In presence of YC-1in the each time of reoxygenation (6,8,10h), the followingresults were found: Firstly, the both levels of protein and mRNA of HIF-1andROCK-1were lower than H/R group.(P <0.05); Secondly, cells apoptosis werefound lesser than H/R group (P <0.05); Thirdly, MTT assay showed that cells viability were higher than H/R group (P <0.05).(4). In presence of Y-27632in the each time of reoxygenation (6,8,10h), thefollowing results were found: Firstly, the levels of protein and mRNA ofROCK-1were lower than H/R group.(P <0.05), but the levels of protein andmRNA of HIF-1were higher than H/R group (P <0.05); Secondly, cellsapoptosis were found higher than H/R group (P <0.05); Thirdly, MTT assayshowed that cells viabilities were lower than H/R group (P <0.05).Conclusion:(1). In hypoxia/reoxygenation of H9c2cells, there are peak times between HIF-1andROCK-1in the hypoxia for10to12h; and also their expressions are higher inoxygenation for4,6,8,10h.(2). Under10h hypoxia and6,8,10h reoxgenation, inhibition of HIF-1in the H9C2cells may reduce the cells apoptosis rates and increase the cells survival rates.(3). Under10h hypoxia and6,8,10h reoxgenation, use the inhibitor of ROCK mayincrease the H9C2cell apoptosis and reduce the cell survival rates.(4). In the hypoxic environment of H9c2cells, there is some relationship betweenHIF-1and ROCK-1. HIF-1may inflence the expression of ROCK-1.