The Expression And Regulation Of GSNO Reductase In Adult Mice Model Of Hypoxic Pulmonary Hypertension

Persistent pulmonary hypertension of the newborn(PPHN) is one of the critical diseases.Hypoxia is an important factor contributing to the formation and deterioration of PPHN.The disorders in the nitric oxide(NO)-cyclic guanosine monophosphate (cGMP),prostacyclin-cyclic adenosine monophosphate(cAMP) and endothelin signaling pathways play an important role in the vascular abnormalities associated with PPHN.The NO-cGMP pathway has been a topic of particularly intense investigation over the past decade.Nitric oxide(NO) is an important endothelial relaxing factor.Recent studies have paid more attention to the nitrosylation of the proteins in the regulation of nitric oxide homeostasis,which is a cGMP-undependent signaling pathway.Nitrosylation of cysteine groups in proteins has now become established as the redox-based post-translational modification.Nitrosylation and denitrosylation can control the bioactivity of many proteins with cysteine groups.It can further change the signaling pathways and the function of some proteins.Nitrosothiols(RSNO) are the product of nitrosylation of protein and low molecular thiols and represent a means either for the storage or transport of NO.The change in nitrosothiols concentration is associated with a series of pathophysiology processes. Recent study has shown that classâ…¢alcohol dehydrogenase,also known as glutathione-dependent formaldehyde could selectively degrade nitrosoglutathione (GSNO).The end product is ammonia not NO.It is also known as GSNO reductase (GSNOR).GSNO is equilibrated with other RSNO through transnitrosation,so GSNOR can regulate the concentration of RSNO in vivo indirectly.Three kinds of nitric oxide synthesis take part in the synthesis of NO or RSNO.It is suggested that the concentration of NO/GSNO/RSNO is under the double gate control of nitric oxide synthase(NOS)-GSNOR.Hypoxic pulmonary hypertension was associated with reactive oxygen species overproduction,redox imbalance and NO/RSNO deficiency. NADPH oxidase,especially for NOX2 and NOX4,was the main sourece of reactive oxygen species(ROS) in pulmonary vessels and important for redox signalling.NaHS injection could supplement the endogenous hydrogen sulfide(H₂S) and relaxed blood vessels.H₂S was proved to be an important antioxidant and could change the redox state in vivo.However,the role of GSNOR in regulating pulmonary vascular tone and remodeling has not yet been clearly addressed in hypoxic pulmonary hypertension.In this study,we select C57BL/6J wild-type(WT) and endothelial nitric oxide synthase (eNOS) knockout(KO) mice as study subjects to investegate the change of GSNOR in chronic hypoxic pulmonary hypertension.We also observed the change of NOX2,4 and ROS after NaHS administration to explore the regulatory mechanism of GSNOR in chronic hypoxia.The aim of this study was to explore a possible new therapy pathway for the treatment of neonatal pulmonary hypertension.Partâ… The expression of GSNOR in hypoxic pulmonary hypertensionAim:1.To investigate the changes of GSNOR on mRNA and protein levels,and GSNOR enzymatic activities during the development of hypoxic pulmonary hypertension.2.To analyze the interactions among GSNOR,eNOS and inducible nitric oxide synthase(iNOS) in hypoxic pulmonary hypertension.3.To investigate the role of redox equilibration in the pathogenesis of hypoxic pulmonary hypertension by evaluating glutathione(GSH)/glutathione disulfide(GSSG) and nitrite/nitrate concentration.Methods:1.Pulmonary hypertension model and groupsHypoxic group:C57BL/6 mice were exposed to 10%oxygen in a forced-air environmental chamber for 21 days.Normoxic group:mice were exposed to 21%oxygen(room air) for 21 days.2.Assessment of pulmonary hypertensionThe right ventricular systolic pressure(RVSP) was detected in mice after 21 days' hypoxic exposure.The change of blood pH and hematocrit was analyzed at different time point of hypoxic exposure.The degree of muscularization of pulmonary vessels was assessed by immunohistochemistry staining ofα-SMA.The ratio of the right ventricle(RV) to the left ventricle plus septum(LV+S) was used to evaluate the hypertrophy of right ventricle.3.Real-time PCR was used to analyze the GSNOR,eNOS and iNOSmRNA expression in mice lungs.Immunohistochemistry was used for the localization and expression of these proteins.Western blot analysis were used for the evaluation of GSNOR and eNOS protein levels in mice lungs.Biochemistry methods was used to assay the GSNOR enzymatic activity and the concentration of nitrite/nitrit in mice lungs.Results:1.RVSP increased significantly in mice after 21 days' hypoxic exposure than that of normoxic group(N:21.41±1.92mmHg;H21:32.64±3.56mmHg,P＜0.01).Blood pH and hematocrit decreased significantly with the duration of exposure to hypoxia (P＜0.01,respectively).The number of muscular pulmonary vessels increased significantly in mice following 21 days' hypoxic exposure than in normoxic group.The ratio of RV/(LV+S) was also significantly increased after hypoxic exposure(P＜0.01).2.The change of GSNOR:The mRNA expression of GSNOR decreased after exposure to hypoxia for 1 day(P＜0.05),but increased significantly as high as 1.36-fold on day 7 compared to normoxic group(P＜0.01).The protein expression of GSNOR and its bioactivities in the lung were also increased significantly after 7 days' hypoxic exposure(P＜0.05,P＜0.01 respectively).3.The change of NOS:The mRNA expression of eNOS increased significantly in mice after 3 day' hypoxic exposure than in normoxic group(P＜0.01).The peak expression was observed in day 14 of hypoxic exposure and decreased gradually thereafter.The protein expression of eNOS increased significantly in mice after 3 day' hypoxic exposure than in normoxic group.The change of iNOS mRNA expression was correlated with that of eNOS.4.The ratio of GSH to GSSG and the concentration of nitrite/nitrate decreased significantly in mice lungs following hypoxic exposure than in normoxic group(P＜0.05, P＜0.01,respectively).Conclusions:1.The increased expression of GSNOR is responsible for the depletion of RSNO in mice after hypoxic exposure.2.Increased eNOS or iNOS expression and decreased nitrite and nitrate concentration following hypoxic exposure suggest that hypoxia may lead to the deficiency of NO in vivo.3.The redox balance was impaired in mice after hypoxic exposure,which may be related to the pathogenesis pulmonary hypertension. Partâ…¡The mechanism of hypoxia induced GSNOR overexpressionAim:1.To investigate the change of pulmonary vascular remodeling and right ventricle hypertrophy in wild-type or eNOS knockout mice after 21 days' hypoxic exposure and the effect of NaHS administration.2.To explore the mRNA expressions of GSNOR,eNOS,iNOS and NADPH oxidase 2,4 in wild-type or eNOS knockout mice after hypoxic exposure and the effect of NaHS administration.3.To investigate the role of redox in the pathogenesis of hypoxic pulmonary hypertension by evaluating the change of reactive oxygen species(ROS) concentration in lung tissues in wild-type or eNOS knockout mice after hypoxic exposure and effect of NaHS administration.Methods:1.Pulmonary hypertension model and groupsNormoxic group:mice were exposed to 21%oxygen(room air) for 21 days.Hypoxic group:C57BL/6 mice were exposed to 10%oxygen in a forced-air environmental chamber for 21 days.Treatment group:NaHS,which was dissolved in normal saline at a dosage of 14μmol/kg,was administrated daily by intraperitoneal injection from day 0 to day 21.Control group:The same dosage of saline as treatment group was administrated daily by intraperitnoeal injection from day 0 to day 21.2.The morphological changes of the small pulmonary arteries were examined by media wall thickness(MT%).The ratio of RV/(LV+S) was calculated to evaluate the hypertrophy of right ventricle.3.Real-time PCR was used to analyze the GSNOR,eNOS,iNOS and NOX2,4 mRNA expression in mice lungs after hypoxic exposure and the effect of NaHS administration.4.Biochemistry methods were used to determine the concentration of ROS in mice lungs after hypoxic exposure and effect of NaHS administration.Results:1.The mRNA expression of NOX2,4 increased significantly in WT mice after 21 days' hypoxic exposure than in normoxic group(P＜0.01,respectively) while the concentration of ROS decreased significantly(P＜0.01).The expression of GSNOR increased significantly on day 7 in hypoxic exposure than in normoxic group(P＜0.05) and decreased gradually thereafter.2.The mRNA expression of NOX2 decreased significantly after 21 days' hypoxic exposure in KO mice than in normoxic group(P＜0.01).There was no significant change in the expression of NOX4 and the concentration of ROS in KO mice after 21 days' hypoxic exposure.The expression of iNOS increased significantly(P＜0.01).The expression of GSNOR decreased significantly in KO mice after 21 days' hypoxic exposure than in normoxic group(P＜0.05).3.The media width of small pulmonary vessels and the ratio of RV/(LV+S) were reduced significantly after NaHS administration in WT mice than in hypoxic group (P＜0.05,P＜0.01 respectively).The mRNA expression of eNOS or iNOS decreased significantly in mice after NaHS administration for 7 days compared to in mice of 7 days' hypoxic exposure(P＜0.01,respectively).The expression of NOX2,4 also decreased significantly in mice after NaHS administration for 21 days compared to in mice of 21 days' hypoxic exposure(P＜0.01,respectively).The concentration of ROS had no change.The expression of GSNOR increased significantly after NaHS administration for 21 days compared to in mice of 21 days' hypoxic exposure(P＜0.05).4.The ratio of RV/(LV+S) decreased significantly(P＜0.05) while there was no significant change in MT%in KO mice after NaHS administration than in hypoxic group.The expression of iNOS,NOX2 and NOX4 had no significant change in KO mice after NaHS administration for 21 days than in mice of 21 days' hypoxic exposure. The concentration of ROS and the expression of GSNOR increased significantly (P＜0.01,respectively).Conclusion:1.Pulmonary hypertension model was successfully established in WT and KO mice after 21 days' hypoxic exposure.There was no significant difference in response to hypoxia between the two kinds of mice.There may be other pathways to compensate for the deficiency of eNOS.2.The expression of NOX2 and GSNOR decreased significantly in KO mice after hypoxic exposure while there was no significant change in the expression of NOX4 compared to normoxic group.It suggests that eNOS could induce the expression of NOX and GSNOR.3.Remodeling of pulmonary vessels and right ventricle hypertrophy induced by hypoxia can be reversed after NaHS administration in WT mice.eNOS may take part in the process of vascular relaxation of NaHS.The concentration of ROS in lungs increased and the abnormal expression of GSNOR was corrected partly after NaHS administration in WT and KO mice.The expression of GSNOR may be associated with an imbalanced redox status.