Effects of HSP90 on complement-mediated hypoxia/reoxygenation injury of H9c2 cardiomyocytes during hypoxic postconditioning

AIM To investigate the effects and mechanisms of heat shock protein 90 （HSP90） on complement-mediated hypoxia/reoxygenation （H/R） injury of rat H9c2 cardiomyocytes during hypoxic postconditioning （HPC）. METHODS Rat H9c2 cardiomyocytes were divided into 7 groups according to different treatments： （1） control group （cultured for 10 h under normal oxygen）； （2） H/R group （hypoxia for 4 h and reoxygenation for 6 h）； （3） HPC group （3 cycles of 5 min H/R after hypoxia for 4 h， followed by reoxygenation for 6 h）； （4） HPC+geldanamycin （GA） group （1 μmol/L HSP90 inhibitor GA was added 20 min before HPC）； （5） negative control group （empty plasmid was transfected before HPC）； （6） C3 over-expression group （C3a plasmid was transfected before HPC）； （7） C5 over-expression group （C5a plasmid was transfected before HPC）. Morphological changes of the H9c2 cells were detected by Hoechst 33242 staining. The effects of HPC on the apoptosis of H9c2 cells were examined by flow cytometry. The protein levels of HSP90， C3a， C5a， NF-κB p65， TNF-α， IL-1β， IL-6， Bcl-2 and Bax were determined by Western blot. RESULTS With up-regulation of HSP90， HPC significantly reduced H/R-induced apoptosis of the H9c2 cells， inhibited the expression of C3a， C5a， NF-κB p65， TNF-α， IL-1β， IL-6 and Bax， and increased the expression of Bcl-2. These effects were blocked by GA. The inhibitory effects of HPC on NF-κB p65 expression and H9c2 cell apoptosis were offset after over-expression of C3a or C5a. CONCLUSION HSP90 attenuates H/R injury of H9c2 cardiomyocytes by inhibiting complement-NF-κB signaling pathway during HPC.