The Protective Effects Of Active Halophenols Against The Hypoxia/Reoxygenation Injury On Cardiomyocytes And Their Correlations With HO-1 Protein

Background:In our previous works we designed and synthesized four new skeleton type halophenol compounds,which are benzophenone,diphenylmethane,phenyl furan-2-ketone and phenyl thiophen-2-ylmethanone,respectively.We obtained several new halophenols with strong activity in vitro by system screening.Among them,5,2’-dibromo-2,4’,5’-trihydroxy-diphenyl-methanone（LM49）,2,3-dibromo-4,5-dihydroxy-diphenyl-methanone（LM48）and 3’-chloro-3,4-dihydroxy-diphenyl-methanone（LM46）showed the highest protective effects against vascular endothelial cells oxidative injury,and their EC50 are 0.4μmol/L,0.8μmol/L and 5.0μmol/L,respectively.The further in vivo studies showed that the three compounds exihibited significant protective effects on myocardial ischemia-reperfusion injury in rats,and LM49 was the best for pharmacological activity,followed by LM48 and LM46.In addition,the preliminary mechanism of action studies have shown that HO-1 is a key protein for the protection of vascular endothelial cells by halogenols.Objective:To investigate the protective effects of halophenol LM46,LM48 and LM49 on Na₂S₂O₄ induced hypoxia/reoxygenation injury in H9c2 cells,and to explore the correlation between the protective effect and HO-1 protein.Methods:1.H9c2 cells hypoxia/reoxygenation model was established byNa₂S₂O₄ oxygen consumption method.2.The cell viability was detected by MTT assay.ROS and apoptosis were detected by flow cytometry.LDH,MDA,SOD and CK were detected by the specific kit,respectively.The protective effect of halophenols on hypoxia/reoxygenation cardiomyocytes was thus studied.3.Levels of HO-1 protein expression and gene expression affected by halophenols were detected respectively by western blot and PCR.4.To clarify the relation between the protective effect of halophenols to cardiomyocytes and the key protein HO-1,the specific HO-1 inhibitor ZnPP and MTT assay were used.And the HO-1 activity was measured by the activity fluorescent quantitative kit.5.To evaluate whether this HO-1 protein induction by halophenols was dependent on new HO-1 mRNA transcription,an mRNA synthesis inhibitor actinomycin D（ActD）was added into the medium before the addition of halophenols,and then HO-1 protein expression was detected by Western blot.And to evaluate whether this HO-1mRNA induction by halophenols was dependent on de novo protein synthesis,the protein synthesis inhibitor cycloheximide（CHX）was added into the medium before the addition of halophenols,and then HO-1mRNA expression was studied by PCR.6.Fluorescence spectroscopy was used to study the relation between the halophenols and the HO-1 protein,thereby to investigate whether the interaction is consistent with the previous cytoprotective activity and the induction sequence of HO-1 gene level.Results:1.Halophenols LM46,LM48 and LM49（2.5,5,and 10 μmol/L）,concentration-dependently increased cell viability and SOD activity,decreased cell apoptosis rate and ROS levels,and inhibited LDH,CK and MDA release.And the protective activity order for cardiomyocytes was LM49>LM48>LM46.2.All three halophenols causes a dose-and time-dependent increase levels of HO-1 protein and mRNA level in H9c2 cells,and the induction order of HO-1 protein and gene expression was LM49>LM48>LM46.3.All three halophenols could increase the activity of HO-1 in cardiomyocytes in a dose-dependent manner,the induction order was LM49>LM48>LM46.With ZnPP block,HO-1 activity and cytoprotection rate of administration group significantly reduced.4.After addition of the transcription inhibitor ActD,the induction of HO-1 protein expression by three halophenols was significantly lower than that of the single administration;after addition of the protein synthesis inhibitor CHX,HO-1 mRNA levels of three halophenol administration group compared with single administration group was significantly increased.5.All three halophenols LM46,LM48 and LM49 are able to quench the fluorescence of HO-1 in static quenching mechanism.There is little difference in the binding constants of the three compounds and HO-1 protein.Judged by their Thermodynamic Parameters,the interaction between LM46 and HO-1 is mainly van der Waals force,hydrogen bond and protonation while that of LM48 and LM49 is electrostatic and hydrophobic forces.With the increasing concentration of halophenol LM48 and LM49,the maximum emission wavelength of the tryptophan in their Synchronous fluorescence spectrumwas slightly red shifted,which showed the hydrophobicity decreased around the Trp residues on HO-1 while the polarity increased.Combined with three-dimensional fluorescence spectroscopy Contour map,we could conclude that the conformation of HO-1 changed.Conclusions:1.All three halophenols showed the obvious protective effects on Na₂S₂O₄-induced hypoxia/reoxygenation injury in H9c2 cardiomyocytes,with the order of LM49>LM48>LM46,and the protective effects are related to their anti-oxidative stress abilities and the inhibition of apoptosis.2.HO-1 maybe a key protein for the protective effects of halogenols on cardiomyocytes.The halogenols protect cardiomyocytes from hypoxia/reoxygenation injury by inducing HO-1 gene transcription and translation to improve HO-1 protein expression and HO-1 activity.3.HO-1 protein induction by halogenols is dependent on an increase of HO-1 gene transcription.Halogenols-stimulated HO-1 gene transcription isn’t dependent on new protein synthesis.4.The interaction between the halogenol compounds and the HO-1 protein could be observed by fluorescence spectroscopy.