The Role Of CGMP-PDE3-cAMP Signaling Pathway In Hyperoxia-induced Lung Injury And Interventional Effect Of CNP

With the improved current survival rate of preterm children,the incidence of bronchopulmonary dysplasia(BPD)in premature infants has increased year by year.Premature infants’ pulmonary vascular endothelial cells are particularly sensitive to oxidative damage induced by hyperoxia and inflammation.Therefore mechanical ventilation and oxygen therapy frequently lead to pulmonary edema,pulmonary lymphatic dilatation and pulmonary fibrosis,which is the main pathological change of BPD.Meanwhile as BPD can bring series of adverse sequelae,BPD have been the burden of children and their families,society,and aroused concern of the majority of pediatricians.Cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate,(cGMP)are the intracellular second messengers,can be hydrolyzed by phosphodiesterase(PDE),involved in regulating physiological signal activity.The mammalian PDE superfamily contains at least 11 functionally distinct,highly regulated,and structurally related gene families.PDE families differ in their primary sequences,substrate affinities and catalytic properties,sensitivity to effectors and inhibitors,responses to regulatory molecules,and cellular functions.By virtue of their distinct intrinsic characteristics,their intracellular targeting to different subcellular locations,and their interactions with molecular scaffolds,cellular structural elements,and regulatory partners,different PDEs integrate multiple cellular inputs and modulate the intracellular diffusion and functional compartmentalization of cyclic nucleotides,as well as the amplitude,duration,termination,and specificity of cyclic nucleotide signals and actions.PDE2 play a locus for cross-talk between cAMP and cGMP pathways,PDE2 play regulatory role in cells that express cGMP-inhibited PDE3,the presence of both cAMP and cGMP allowed locolization,duration,and intensity of signals rugulated by cGMP.C-type natriuretic peptide(CNP),a member of the natriuretic peptide family,selectivelybinds to the transmembrane guanylyl cyclase(GC)-B receptor,leads to a large increase in intracellular cGMP.CNP plays a role in the effect of dilating vessel,local regulation of vascular tone and remodeling,and has been shown to have mainly cardioprotective effects.Therefore,we made hyperoxic lung injury model using neonatal rats to explore the role of the cGMP signaling pathway in the hyperoxic-induced lung injury.Furthermore clarify the role of exogenous CNP on cGMP signal pathway lin hyperoxia-induced lung injuryPart One The role of cGMP pathway in hyperoxia-induced lung injuryObjective:To investigate changes of cGMP signaling pathways in hyperoxia-induced lung injury and its molecular mechanism.Methods:Prepare full-term newborn Wistar rats to make hyperoxia-induced lung injury model,after modeling,wet and dry ratio of brain tissues was detected at the 1st,3rd,7th and 14th days.The lung development and morphopathological changes were observed by HE staining method.The MDA,SOD、CAT,were detected in lung homogenates,Expression levels of TNF-α、IL-1β、IL-6、cAMP、cGMP in the BALF and lung tissue were detected by ELISA,the levels of PDE2A、PDE3A、PDE3B protein expression,and levels of TNF-α,IL-1β,IL-6 protein expression were detected by western blotting method and RT-PCR analysis.Results:1.Compared with the control group,the weight decreased on the 3rd,7th and 14th day after exposure to high oxygen in hyperoxia group,the differences were significant(P<0.05),but there was no significante differences compared with the control group on the 1st day.2.Compared with control group,lung wet/dry(W/D)proportion of hyperoxia group increased on the 3rd,7th and 14th day after exposure to high oxygen(P<0.05),the differences were statistically significant.There were no significante differences compared with control group on the 1st day.3.HE staining results showed that,In hyperoxia group,the small blood vessels in the lungs expanded,bronchi wall mild edema,alveolar epithelium swelling,inflammation cells exudated with mainly neutrophils and lymphocytes were observed.4.Compared with control group,levels of SOD,CAT in hyperoxia group on 1st,3rd,7th and 14th day were significantly decreased(p<0.05).Compared with control group,levels of MDA in hyperoxia group on 1st,3rd,7th and 14th day were significantly increased(p<0.05).5.Compared with control group,levels of TNF-α,IL-1β,IL-6 in hyperoxia group on 1st,3rd,7th and 14th day were significantly increased(p<0.05).With high oxygen exposure time prolonged,levels of TNF-α,IL-1β IL-6 gradually increased,also reached its peak on 7th day.Meanwhile,we detected these levels reduced to the 14th day.6.Compared with control group,levels of cAMP cGMP in high oxygen group on 1st,3rd,7th and 14th day were significantly decreased(p<0.05).With high oxygen exposure time prolonged,levels of cAMP,cGMP gradually decreased(p<0.05),also reached its lowest point on 14th day.7.Western blotting results showed that,PDE2A protein expression in hyperoxia group on 1st,3rd 7th and 14th day have no differecese than that of control group(p>0.05).PDE3A,PDE3B protein expression.in hyperoxia group on 1st,3rd,7th and 14th day were higher than control group(p<0.05).p-VASP protein expression in hyperoxia group on 1st,3rd,7th and 14th day were lower than control group(p<0.05).Compared with control group,levels of TNF-α,IL-1β,IL-6 protein expression in hyperoxia group on 1st,3rd,7th and 14th day were significantly increased(p<0.05).8.RT-PCR results showed that,PDE2A mRNA expression in hyperoxia group on 1st,3rd 7th and 14th day have no differecese than that of control group(p>0.05).PDE3A mRNA,PDE3B mRNA expression in hyperoxia group on 1st,3rd 7th and 14th day were higher than control group(p<0.05),with high oxygen exposure time prolonged,expression of PDE were gradually increased.Conclusion:1.High concentrations of oxygen can induce pulmonary edema,oxidative stress response and inflammatory response.2.High concentrations of oxygen can damage cGMP and PKG activity,upregulate PDE3A,PDE3B expression,which indicate that cGMP signal pathway involved in hyperoxia-induced lung injury.Part Two Protective role of CNP-cGMP-PDE3-cAMP signaling pathway in hyperoxia-induced lung injuryObjective:To observe molecular mechanism of cGMP-PDE3-cAMP signaling pathway and interventional effect of CNP in hyperoxia-induced lung injury.Methods:Prepare full-term newborn Wistar rats to make hyperoxia-induced lung injury model,then divided into three groups:(1)room air control group,(2)H yperoxia group(85%O2);③Hyperoxia+CNP group.Wet and dry ratio of brain tissues was detected at the 1st,3rd,7th and 14th days.The brain development and morphopathological changes was observed using by HE staining.The MDA,SOD、CAT,were detected in lung homogenates,Expression levels of TNF-α、IL-1β、IL-6、cAMP、cGMP in the BALF and lung tissue were detected by ELISA,the levels of PDE2A、PDE3A、PDE3B protein expression,and the levels of TNF-α、IL-1β、IL-6 protein expression were detected by western blotting method and RT-PCR analysis;different PDE inhibitors were used to detect the inhibition of cGMP-PDE activity.Results:1.Compared with hyperoxia group,weight of hyperoxia+CNP group increased on the 3rd,7th and 14th day(P<0.05),the differences were statistically significant.Compared with control group,weight of hyperoxia+CNP group decreased on the 3rd,7th and 14th day(P<0.05),but there were no statistical significance compared with control group on the 1st day.2.Compared with hyperoxia group,lung wet/dry(W/D)proportion of hyperoxia+CNP group decreased on the 3rd,7th and 14th day,the differences was statistically significant(P<0.05),Compared with control group,brain W/D proportion of hyperoxia+CNP group increased on the 3rd,7th and 14th day(P<0.05).There was no statistical significance compared with control group on the 1st day.3.HE staining results showed that,compared with hyperoxia group,we observed that pulmonary cell swelling waer reduced and cell morphology were significantly improved in hyperoxia+CNP group.4.Compared with hyperoxia group,levels of SOD,CAT in hyperoxia+CNP group on 1st,3rd,7th and 14th day were significantly increased(p<0.05).Compared.with hyperoxia group,levels of MDA in hyperoxia+CNP group on 1st,3rd,7th and 14th day were significantly decreased(p<0.05).5.Compared with hyperoxia group,levels of TNF-α,IL-1β,IL-6 in hyperoxia+CNP group on 1st,3rd,7th and 14th day were significantly decreased(p<0.05).With high oxygen exposure time prolonged,levels of TNF-α,IL-1β,IL-6 gradually decreased.6.Compared with hyperoxia group,levels of cAMP cGMP in hyperoxia+CNP on 1st,3rd,7th and 14th day were significantly in creased(p<0.05).7.Western blotting results showed that,PDE2A,p-VASP protein expression in hyperoxia+CNP group on 1st,3rd 7th and 14th day have no differece than that of hyperoxia group(p>0.05);PDE3A,PDE3B protein expression in hyperoxia+CNP group on 1st,3rd,7th and 14th day were lower than hyperoxia group(p<0.05).Compared with hyperoxia group,levels of TNF-α,IL-1β,IL-6 protein expression in hyperoxia+CNP group on 1st,3rd,7th and 14th day were significantly decreased(p<0.05).8.RT-PCR results showed that,PDE2A mRNA,PDE3A mRNA,PDE3B mRNA expression in hyperoxia+CNP group onlst,3rd 7th and 14th day were lower than control group(p<0.05).PDE2A mRNA expression in hyperoxia+CNP group on 1st,3rd 7th and 14th day have no differece than that of hyperoxia group(p>0.05).9.Added CNP in the presence of IBMX compared with only CNP,observed that CNP can change the level of cGMP in the presence of IBMX,but not cAMP.Added CNP in the presence of EHNA compared with only CNP,observed that CNP can change the level of both cGMP and cAMP.Added CNP in the presence of Milrinone compared with only CNP,observed that CNP can change the level of cGMP in the presence of IBMX,but not cAMP.Inhibition of cGMP-PDE activities were highest in the IBMX group,and then in the Milrinone group.Conclusion:1.CNP reduced hyperoia induced pulmonary edema,oxidative stress response and inflammatory response.2.CNP modulated cGMP concentration by affecting PDE3 activity.and then regulate intracellular cAMP concentration,which indicated CNP-cGMP-PDE3-cAMP signaling pathway have protective effect in hyperoia induced lung injury.