The Roles Of NADPH Oxidase In Mediating 5-HT-induced ROS In Pulmonary Hypertensive Rats

Reactive oxygen species(ROS),produced primarily by the NADPH oxidase(NOX)family of enzymes in the vasculature,induce oxidative stress and contribute to damage in biological organisms.Altered redox signaling and high levels of ROS in the cellular microenvironment have been implicated in chronic diseases,including PH.Recent studies have shown that 5-hydroxytryptamine(5-HT)has been implicated in the pathogenesis of pulmonary arterial hypertension(PAH).It can promote pulmonary vascular smooth muscle proliferation,pulmonary artery vasoconstriction and local microthrombosis.In this study,PH rats were imitated by means of monocrotaline(MCT)intraperitoneal injection and chronic hypoxia(CH)continuous to mirror different PH characteristics.To compare the expression alterations of NOX protein and mRNA in PH rats.Using 5-HT to increase ROS in PASMCs from controls and PH rats,PASMCs from controls were stimulated with 5-HT in the presence of inhibitors of NADPH oxidase 1(NOX1)?NADPH oxidase2(NOX2)and NADPH oxidase 4(NOX4).Objective to explore 5-HT induced ROS through NADPH Oxidase-derived ROS generation and the biological function of NOX enzymes on the pathogenesis of PH separately from both organ function level and cell level.In order to supply as experiment and theory foundations for PH prevention and treatment.Objective:To investigate the expression of NADPH oxidase(NOX)in different PH rats and the role of NOX isoforms in 5-HT induced ROS.Methods:SD rats were induced respectively by single intraperitoneal injection of MCT(50mg/kg)and exposed to hypoxic conditions(10.0%±0.5% partial pressure of oxygen)for 21 days.We measured:(1)hemodynamics methods,measurement the variation of rats right ventricular systolic pressure(RVSP),right ventricular mass index(RVMI);(2)Quantitative real-time PCR and Western blot was used to analyse mRNA and protein expression;(3)5-HT in serum and PASMCs from control and PH-PASMCs,was assessed using the 5-Hydroxytryptamine ELISA Kit;(4)Reactive oxygen species(ROS)of PASMCs was assessed with DCFH-DA,NOX inhibitors decreased 5-HT-induced ROS production,which observed in control PASMCs.Results:In comparison to the control rats,(1)RVMI and RVSP were markedly elevated in MCT-treared and CH-induced rats;(2)In PAs of MCT rats,the expression of Rac1?NOX2?NOX4 mRNA and NOX2?NOX4 protein were significantly increased,in PAs of CH rats,the expression of p22phox?p47phox?Rac1?NOX1?NOX2?NOX4?NOXA1 mRNA and NOX1?NOX4 protein were significantly increased;(3)Basal ROS was higher in PASMCs from PH rats compared with control-PASMCs,suggested that PH may increase the release of ROS in PASMCs;(4)5-HT in serum and PASMCs of MCT and CH rats were significantly increased.5-HT could induce the release of ROS in PASMCs of control rats,also indicated that 5-HT increases ROS production in PH-PASMCs;(5)specific NOX inhibitor effect on ROS release caused by 5-HT in CON-PASMCs,suggesting the role of NOX in 5-HT-induced ROS in PASMCs.Conclusion:(1)The expressions of NOX2,NOX4 and Rac1 mRNAs increase and the expressions of NOX2 and NOX4 protein increased in PAs of MCT.The mRNA expressions of NOX1,NOX2,NOX4,NOXA1,Rac1,p47 phox,p22phox and NOXO1 increased and NOX1,NOX4 protein expression increased in CH-induced PH rats;indicating that NADPH oxidase involved in the occurrence and development of PH,which NOX1 in the CH group is more significant,NOX2 role in the MCT group is more significant,NOX4 plays an important role in two different PH models;(2)The levels of 5-HT in serum and PASMCs were significantly increased in different PH rats,and the ROS level in PASMCs were significantly increased compared with the normal group,5-HT could induce the increase of ROS in PASMCs in the control group.It showed that the increase of ROS in different PH rats could be caused by 5-HT;(3)NOX-specific inhibitors reduce 5-HT-induced ROS release,indicating that 5-HT can play rugulatory role for ROS through the NOX pathway,in whcih NOX4 has the greatest effect;(4)This study provides new research ideas for the research and prevention of PH pathogenesis.