The Role Of Macrophage-derived Heme Oxygenase-1 In Septic Cardiac Dysfunction And Its Mechanism

Background:Sepsis is a systemic inflammatory disease that can lead to cardiac dysfunction and high mortality.Macrophages are important inflammatory cells and play an important role in sepsis.Inducible nitric oxide synthase(iNOS)is highly expressed in activated macrophages and is closely related to inflammation and oxidative stress.The traditional concept is that inflammation and oxidative stress can activate heme oxygenase-1(HO-1)and catalyze the production of metabolites of heme with anti-inflammatory and anti-oxidative stress effects.However,some research suggest that HO-1 in macrophages has pro-inflammatory effects.The role of macrophage-derived HO-1 in septic cardiac dysfunction has not been reported.By applying intraperitoneal injection of lipopolysaccharide(LPS)to induce septic cardiac dysfunction in mice,our preliminary experiments found that intraperitoneal injection of LPS significantly activated the expression of HO-1 in infiltrated macrophages in the septic hearts.In vitro,HO-1 gene silencing significantly reduced iNOS protein expression levels in LPS-induced macrophages.Therefore,we propose the scientific hypothesis that HO-1 in macrophages derives the occurrence of septic cardiac dysfunction.The purpose of our study is to elucidate the role of macrophage-derived HO-1 in septic cardiac dysfunction and its mechanism,and to provide a scientific basis for clinical search for new targets.Methods:Myeloid-specific HO-1 knockout mice were used to establish a model of LPS-induced septic cardiac dysfunction,echocardiography,immunofluorescence staining,western blot,and quantitative PCR were used to observe the effects of myeloid HO-1 deletion on LPS-induced septic cardiac function and survival rate in mice;application of HO-1 gene silencing or its inhibitors,western blot,quantitative PCR,and confocal microscopy to observe the effect of HO-1 on the regulation of iNOS in LPS-induced macrophages;by using techniques of plasmid construction,plasmid transfection and co-immunoprecipitation(Co-IP),culturing HEK293T and RAW 264.7 cells with overexpressed plasmids,the underlying molecular mechanism of HO-1-regulated iNOS protein expression was further investigated.Results:We found that myeloid-specific HO-1 deletion significantly improved LPS-induced septic cardiac dysfunction and reduced the mortality in vivo,which was accompanied by reduced cardiomyocyte apoptosis in the septic hearts and decreased peroxynitrite production and iNOS protein expression in the cardiac infiltrated macrophages,with no significant change in the production of reactive oxygen species(ROS)and the mRNA levels of iNOS.Further,we found that inhibition of HO-1 promoted the lysosomal degradation of iNOS protein rather than proteasomal degradation in macrophages.HO-1-regulated iNOS protein expression in macrophages was independent of its metabolites:ferrous iron,carbon monoxide,and bilirubin.Suppression of the iNOS lysosomal degradation by bafilomycin A1 reversed the protective effect of myeloid HO-1 deletion on septic cardiac dysfunction in mice.With the application of Co-IP and other techniques,we demonstrated the molecular mechanism that HO-1 interacted with iNOS at ’the flavin mononucleotide(FMN)domain,which further prevented the iNOS conjugation with light chain 3(LC3)and subsequent lysosomal degradation in macrophages.Conclusions:Our results indicate that HO-1 in macrophages drives septic cardiac dysfunction via suppressing lysosomal degradation of iNOS,and further elucidate the molecular mechanism of HO-1-regulated lysosomal degradation of iNOS.