The Role Of Reactive Oxygen Species In The Process Of Chronic Intermittent Hypobaric Hypoxia Adaptation And The Underlying Mechanisms

In recent years, the cardioprotective effect of chronic intermittenthypobaric hypoxia (CIHH) has been recognized and attracts the publicattention more and more. The study on the mechanism of CIHHcardioprotection is a hot research topic in clinical medicine, space medicineand high altitude medicine. The cardioprotective effects of CIHH can bemainly summarized as protecting the heart against ischemia injury,antagonizing arrhythmia, and improving the degeneration of myocardial cell.Multiple mechanisms or pathways have been suggested to contribute to thecardioprotection of CIHH, such as increase of myocardial capillaryangiogenesis and coronary flow, activation of ATP-sensitive K+channels,inhibition of mitochondrial permeability transition pores and suppression ofthe adrenergic receptor activity on cardiac membrane. However, these findingsare the end effects of CIHH treatment, that is to say, the observations of thefinal results of CIHH. What happened during CIHH exposure and how theseprotective effects of CIHH produced have not been reported. It is generallyaccepted that reactive oxygen species (ROS) exert both deleterious andbeneficial actions depending on the amount of ROS. A lot of evidence showsthat ROS are protective at low level but detrimental at high level on the heart.Intermittent hypoxia increases ROS generation both in cell culture and inintact animals and ROS-mediated signaling mechanisms contribute to cellularand systemic responses to intermittent hypoxia. The nuclear factor erythroid2-related factor2-thioredoxin1-hypoxia-inducible factor (Nrf2-Trx1-HIF-1α)signal transduction pathway was identified as ROS-induced mechanisms inresponse to intermittent hypoxia. However, it is still unknown whether ROSplay a role in the dynamic formation of CIHH cardiac protection. This study aimed to investigate the role of ROS in adaptivecardioprotection of CIHH dynamically and the mechanism underlying thisprocess in conscious rats through physiological, pharmacological,morphological and molecular biology techniques. Our study consisted of threeparts:(1) To clarify the dynamic changes of the cardiovascular activity and therole of ROS during CIHH adaptation.(2) To investigate the Nrf2-Trx1-HIF1αsignaling pathway with its main cardioprotective molecules in adaptivecardioprotection.(3) To determine the effect of CIHH on the sensitization ofcarotid body chemoreceptor.Ⅰ Reactive oxygen species contribute to the dynamic changes ofcardiovascular responses during the process of chronic intermittenthypobaric hypoxia adaptation in conscious ratsObjective: CIHH has been reported to have cardioprotective effects.Cumulating evidences have indicated that ROS released during hypoxia assecond messengers may help to trigger various adaptive responses. The aim ofpresent study was to clarify the dynamic changes of the cardiovascular activityand the role of ROS during CIHH adaptation in conscious ratsMethods: Adult male Sprague-Dawley rats were randomly divided intothe four groups: control group (Control), CIHH treatment group (CIHH),antioxidant N-Acetylcysteine group (Control+NAC), CIHH plus NACtreatment group (CIHH+NAC). The rats in CIHH group were exposed tohypoxia simulating5000m high altitude (oxygen11.1%) in a hypobaricchamber for42days,6hours each day. Control+NAC rats accepted ROSscavenger NAC(80mg/kg)injection subcutaneously each day for42days.CIHH+NAC rats were given NAC before the hypobaric hypoxic exposure.Control rats were living in the same environment as CIHH rats with freeaccess to food and water, except no CIHH and NAC treatment. The meanarterial pressure (MAP) and heart rate (HR) were recorded and analyzedcontinuously in conscious rats by using the PhysioTelTMTelemetry system.The activity of total superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), lipofuscin-like pigments (LFP),protein carbonyl (PCL) and the ratio of glutathioneto glutathione disulfide(GSH/GSSG) in ventricular myocardium were measured with ELISA kit.Results:(1) CIHH enhanced oxidative stress and ROS generation in myocardialtissue with the increased GSH-PX, MDA and LFP (P<0.05), and decreasedGSH/GSSG (P<0.05).(2) CIHH did not affect MAP, but abolished the age-dependent decreaseof HR under normobaric normoxia condition (P<0.05).(3) During CIHH exposure in hypobaric chamber, MAP and HRdisplayed adaptive changes. The MAP and HR increased rapidly from thebaseline to the peak level, and gradually returned to the lowest level within theearly period (first hour)(P<0.05). In late period (from second to six hours),MAP and HR were keeping in plateau level. Along with hypobaric hypoxiaexposure, the peak and plateau level of MAP gradually increased and the timeto reach the peak gradually shortened (P<0.05), but the peak HR graduallydecreased and the time to reach the peak gradually prolonged (P<0.05) in earlyperiod, while plateau HR gradually increased in late period (P<0.05). Suchadaptive changes of MAP and HR in CIHH rats were partly abolished by theROS scavenger NAC (P<0.05).(4) During acute normoxia hypoxia, MAP and HR of rats were increasedsignificantly (P<0.05), but the increasing of MAP and HR was much smallerin CIHH rats than those in Control and NAC rats (P<0.05). This anti-acutehypoxia effect of CIHH lasted for two weeks and could be partly abolished byNAC (P<0.05).Conclusion: The result of this study demonstrated for the first time thatappropriate ROS, produced during CIHH adaptation, contribute to theadaptive protection against acute hypoxia-induced changes of MAP and HR inconscious rats. Moreover, this ROS-dependent adaptive protection needs atleast28days CIHH to trigger, and can maintain effectively for two weeksafter CIHH schedule. Ⅱ The Nrf2-Trx1-HIF1pathway activated by reactive oxygen speciesparticipates the formation of chronic intermittent hypobaric hypoxiaadaptation in ratsObjective: The aim of this study was to investigate the activation ofNrf2-Trx1-HIF1α signaling pathway with its main cardioprotective moleculesand the role of ROS in the activation during the process of CIHH adaptation.Methods: The experimental groups and CIHH exposure were the sameas those in Part. The expression of Nrf2, Trx1, HIF1α, inducible nitric oxidesynthase (iNOS), vascular endothelial growth factor (VEGF) anderythropoietin (EPO) in ventricular myocardium were detected by WesternBlot analysis and IHC staining.Results:(1) ROS-induced Nrf2-Trx1-HIF1α signaling pathway was activatedduring CIHH. The expression of Nrf2, Trx1and HIF1α were increased after28days of CIHH exposure (P<0.05). Consistently, CIHH significantlyenhanced nucleus Nrf2and increased the positive nuclei number in the LVmyocardial cells (P<0.05). NAC treatment abrogated these responses(P<0.05).(2) ROS induced the translocation of Nrf2from the cytosolic fraction tothe nucleus, i.e. the activation of Nrf2. Nrf2expression was significantlyenhanced in the nucleus fraction and the positive nuclei number was increasedafter one day CIHH exposure (P<0.05). NAC treatment delayed thistranslocation to2days after CIHH as well as significantly abrogated theactivation of Nrf2(P<0.05).(3) ROS mediated the activation of Nrf2-Trx1-HIF1α signaling pathwayduring CIHH. CIHH enhanced Nrf2protein in the whole-cell extraction after2days of CIHH exposure while increased Trx1and HIF1α after3days CIHHexposure (P<0.05). Chronic NAC injection attenuated the effects of CIHHmarkedly (P<0.05), and also prolonged the activation of Nrf2, Trx1and HIF1αto the3days and5days after CIHH exposure, respectively.(4) CIHH significantly promoted the expression iNOS, VEGF and EPO, the downstream protective molecules of HIF (P<0.05). Among which theexpression of iNOS and VEGF were increased after7days of CIHH exposure(P<0.05) while the expression of EPO (P<0.05) was enhanced after14days ofCIHH exposure. NAC partially blocked these effects (P<0.05).Conclusion: We found in this part of study that the Nrf2-Trx1-HIF1αpathway with its downstream genes iNOS, VEGF and EPO was activatedduring the formation of CIHH adaption. According to the fact thatenhancement of iNOS, VEGF and EPO attenuated by ROS scavenger NAC, itsuggests that activation of iNOS, VEGF and EPO genes induced byNrf2-Trx1-HIF1α pathway contribute to the ROS-depended adaptivecardiovascular protection.Ⅲ Chronic intermittent hypobaric hypoxia facilitates the chemoreceptoractivity in carotid body via reactive oxygen species during the process ofCIHH adaptationObjective: To investigate the effect of CIHH on carotid body (CB)sensitization and the respiration frequency (RF). And to verify the role of ROSplay on the CB sensitization during CIHH adaptation.Methods: The experimental groups and CIHH exposure were the sameas those in Part. The RF was recorded and analyzed continuously inconscious rats through the PhysioTelTMTelemetry system. The carotid sinusnerve activity (CSNA) was recorded by the conventional whole nerve bipolarplatinum electrodes recording techniques.Results:(1) The RF of control rats did not show age-dependent changes. Therewas no significant difference of RF among control, CIHH, Control+NAC andCIHH+NAC rats under normoxia conditions.(2) During CIHH exposure in hypobaric chamber,RF shown adaptivechanges. The RF increased rapidly from the baseline to reach the peak level,and gradually returned to the lowest level within the early period (first hour)(P<0.05). In late period (from second to six hours), RF were keeping in plateau level (P<0.05). Along with hypobaric hypoxia exposure, the peak RFgradually decreased and the time to reach the peak gradually prolonged inearly period (P<0.05), while plateau HR gradually increased in late period(P<0.05). Such adaptive changes were partly abolished by the ROS scavengerNAC (P<0.05).(3) During acute normoxia hypoxia, RF of rats was increasedsignificantly (P<0.05), but the increasing of RF was much smaller in CIHHrats than those in Control and NAC rats (P<0.05). This anti-acute hypoxiaeffect of CIHH lasted for two weeks and could be partly abolished by NAC(P<0.05).(4) Compared with the control rats, CSNA response to hypoxia in CIHHrats significantly increased (P<0.05). Chronic NAC treatment completelyblocked augmentation effect of CIHH on CSNA (P<0.05).Conclusion: CIHH can induce ROS-dependent adaptive respiratoryresponse and anti-acute hypoxia effect which can be preserved for two weeksat least. Also CIHH augments the carotid body chemoreceptor response tohypoxia and this CIHH-induced sensitization of CB activity is mediated byROS generated during CIHH exposure.