Roles And Change Of Noxs On Pulmonary Artery Constriction Induced By Agonists In The Process Of Pulmonary Hypertension

Reactive oxygen species(ROS) generally indicates single oxygen-containing electron substances with reducibilty in organisms, and participates in the mobilization of intracellular calcium associated with signal transduction of vascular contractibility and remodeling. It has been clear that in the pulmonary vasculature, ROS primarily induced by nicotinamide adenine dinucleotide phosphate(NADPH oxidase, Nox). Pulmonary hypertension(PH) display a functional organic contraction abnormalities and vascular lumen stenosis pipeline.Research show that changes of cytosolic Ca2+ concentration([Ca2+]i) in pulmonary arterial smooth muscle cells(PASMCs) in the process of pulmonary hypertension are closely related, as well as calcium and reactive oxygen species signaling pathways are interactions.In chronic hypoxia(CH), monocrotaline(MCT) and lamb ductus ligation-induced PH, pulmonary aterials(PAs) appear an increasing ROS of basal level, and intravascular agonist can also cause a significant elevation of ROS.In addition, PH patients exhibit high oxidative stress, such as increasd edlipid peroxidation and DNA oxidation, and redued antioxidant enzymes. In this study, through the establishment of CH and MCT PH model,we compared agonist-induced contraction effect with PH by pretreatment nonspecific and specific Nox inhibitors, which determine which agonist and Nox isoforms play an important role in PH.It is verified that Nox inhibitors descend the agonist-induced ROS in PASMCs, providing a new theoretical basis for the study of the pathogenesis of PH.Objective: We observed agonists(5-HT, Ang II and ET-1)contraction effect on rat PAs of CH and MCT-induced PH via applying different Nox inhibitors in contrast to control, dtermind agonists and Nox isoforms in the pathogenesis of PH. Methods: SD rats were utilized MCT intraperitoneal injection(50mg/kg) and CH-induced(10.0%±0.5% partial pressure of oxygen) for 21 days to establish PH rat models,using:(1)hemodynamic, measure rat pulmonary aterial systolic pressure(PASP), right ventricular mass index(RVMI);(2)vascular circle tone detection methods, measured dose curve of 5-HT, Ang II and ET-1 in PAs after preincubation different Nox inhibitors in CH and MCT PAs compared with control.(3)ROS of PASMCs with DCFH-DA,Nox inhibitors decreased agonist-induced ROS production,which observed in control rats PASMCs. Results: 1. Compared with control group, RVMI and RVSP of corresponding PH rats were significantly increased, suggesting that the model prepared well;2. Contraction reaction induced by 5-HT, Ang II and ET-1 in control rat PAs pretreatment with Nox inhibitors.(1)two non-specific Nox inhibitors(APO and DPI) can reduce the 5-HT-induced contractile response of PAs, the maximum shrinkage rate decreased and EC50 increased, suggesting Nox involved in the reaction process; with three specific Nox inhibition,5-HT-induced contractile response of PAs were reduced, the inhibition is specific Nox1 inhibitor(ML171)> Nox2 inhibitor(gp91 ds-tat)> Nox1/4 inhibitor(GKT137831), prompted that Nox1,Nox2 and Nox4 subtypes are involved in contraction reaction induced by 5-HT, and Nox1 isoform plays an important role in this reaction.(2)APO and DPI can reduce Ang II-induced PAs contractile response, the maximum shrinkage rate decrased and EC50 increased, suggesting Nox involved in the process; preincubation with ML171 and gp91 ds-tat, Ang II-induced contractile response decreased, inhibition is gp91 ds-tat> ML171, while GKT137831 is no significant inhibition, suggesting Nox1 and Nox2 subtype involved in Ang II-induced contractile responses, Nox2 isoform plays an important role in this reaction, and Nox4 isoforms not involved in the reaction process.(3)in ET-1 induced contractile response, only the APO and ML171 inhibit the response, other inhibitors had no effect, suggesting that Nox1 subtype involved in ET-1-induced contractile responses, but Nox2 and Nox4 subtypes did not participate in the reaction process.3. Compared with the CON group,PAs contraction reaction of 5-HT-induced two PH model pretreated with Nox inhibitors.(1)in both PH model(CH and MCT), 5-HT-induced contractile response were increased, suggesting that in the PH, 5-HT showed high reactivity;(2)in the CH group,APO can reduce the 5-HT-induced contractile response,three specific Nox inhibitors may reduce the 5-HT-induced contractile response, the same as CON group, the ML171> gp91 ds-tat> GKT137831, among these three Nox isoforms are involved in the CH group highly reactive process of 5-HT, Nox1 subtype play a major role in this process.(3)in the MCT group, APO decreased 5-HT-induced contractile response, three kinds of inhibitors can reduce 5-HT-induced contractile response,three kinds of specific inhibition of similar inhibitory agents, but less inhibition than the CH group, suggesting the three Nox isoforms are partially involved 5-HT highly reactive process in MCT group.4. Non-specific Nox inhibitor effect on ROS release caused by 5-HT in PASMCs. In the control PASMCs,5-HT can increase ROS,after prtreated with APO, intracellular ROS production was significantly reduced, suggesting Nox involved in 5-HT-induced ROS production process. Conclusion: 1. In the control rats,Nox involved in 5-HT, Ang II and ET-1 induced PAs contractile response,three kinds of Nox isoforms are involved in contraction reaction induced by 5-HT and Nox1 subtypes in this reaction from an important role; Nox1 and Nox2 subtypes are involved in contraction reaction induced by Ang III, Nox2 subtype play a major role in this reaction.2. In both models PH, PAs ratio 5-HT-induced contractile response showed high; the CH group, play a major role in the high Nox1 reaction of 5-HT; MCT group in three sub-Nox MCT type are part of the group involved in the process of high reactivity of 5-HT.3. Nox involved in ROS 5-HT-induced increased production process.4. The results of this study may provide new ideas for prevention and treatment of PH and PH clarify pathogenesis.