The Effect Of NF-κB And HIF-1in The Pathogenesis Of Obstructive Sleep Apnea-hypopnea Syndrome

Sleep apnea-hypopnea syndrome (SAHS) has been considered as a systemic disease, characterized as loud snoring, apnea, night repetitive hypoxemia, hypercapnia, changes in sleep architecture and excessive daytime sleepiness and consequences of multi-organ complications, of which the pathophysiological basis is the chronic hypoxia/reoxygenation. It was classified to obstructive, central and mixed, mainly as obstructive (obstructive sleep apnea syndrome, OSAHS).The incidence rate in adults is2%-4%, It is associated with hypertension, coronary heart disease, arrhythmia, congestive heart failure, cerebrovascular disease, dementia, pulmonary hypertension, pneumocardial disease, asthma, liver and kidney dysfunction, insulin resistance, polycystic ovary syndrome, gestational hypertension, acouesthesia loss, impotence.The pathogenesis of OSAHS is not completely understood, especially for cardiovascular diseases,which has become a hot topic in the sleep medicine.Current studies on the pathogenesis of OSAHS with cardiovascular disease are mainly summarized as the following:the sympathetic excitation, oxidative stress, inflammation, hypercoagulability of the erythrocyte, endothelial dysfunction, changes in intrathoracic pressure, endocrine and metabolic dysregulation. Chronic intermittent hypoxia, as the pathophysiological basis of OSAHS, is the central part of vascular endothelial damage. More research about two pathways has brought the study of OSAHS complicated with cardiovascular disease a new process. Two pathways caused by intermittent hypoxia may be included in the pathogenesis of OSAHS:One is the nuclear factor-kappa B (NF-kappa B)-dependent inflammatory pathway through increased inflammatory cytokines (IL-6, IL-8and TNF-alpha) and adhesion molecules (ICAM-1, VCAM-1, selectin), exacerbating the inflammatory response to atherosclerosis.The other is hypoxia inducible factor-1(HIF-1)-dependent adaptive pathway through elevated EPO, iNOS and VEGF, facilitating vascular endothelial cells proliferation, vascular permeability and angiogenesis.Nuclear factor-kappa B (NF-κB) is a protein which can specifically bind to promoter-κB sequence (GGG ACT TTC) and activate gene transcription. It plays an important role in immune response, inflammation, cell differentiation and growth, cell adhesion, and apoptosis.NF-KB and IκB non-covalently combine into inactive dimers in the cytoplasm. When exposure to activation signals (TNF-alpha, LPS and oxidants, oxygen free factor, radiation, ultraviolet radiation, viruses, and its metabolites), NF-κB dissociates with IκB, transfers into the nucleus, binds to DNA promoter of response element, and regulates the transcription of inflammatory gene, inducing upregulation of adhesion molecules and downstream cytokines (TNF-alpha, IL-6, IL-8, MMP-9, IACM-1, VCAM-1). Thus NF-κB is involved in the start of the inflammatory response.Hypoxia-inducible factor-1(HIF-1) is a specific combination into hypoxia-inducible gene protein composed of two subunits:alpha, beta. HIF-1α is a transcription factor of the oxygen balance induced by hypoxia, causing the hypoxic tissue to maintain oxygen homeostasis tolerance to hypoxia. It widely expresses in tissues and cells under hypoxia. Normoxia, HIF-1α, HIF-1βwith structural expression have no DNA binding activity, the increase of HIF-1α protein levels and HIF-1DNA binding activity are induced by hypoxia. The increased HIF-1α combines with HIF-1β as a stable dimer into the nucleus, binds to the HIF-1binding sites of hypoxia response genes to promote transcription, making the downstream cytokines as EPO, VEGF, iNOS, ET-1, COX, IGFBP-1increase. It participates in the hypoxic-ischemic injury and repair process involved in organ or tissue.Currently the mechanisms of cardiovascular disease in OSAHS remain unclear, By activating the inflammatory pathways, intermittent hypoxia may plays an important role, blood mononuclear cells, especially of CD8+T cells are important inflammatory cells, which acts on vascular endothelial cells, resulting in endothelial dysfunction and further the formation of atherosclerotic plaques. There is a controversy about whether the two pathways associated with OSAHS or not. The inflammatory pathways is mainly regulated by NF-κB are believed by most scholars; the HIF-1-dependent adaptive pathway can also be induced by intermittent hypoxia also are insisted by some scholars. It is in dispute whether HIF-1-dependent adaptive pathway is activated in the intermittent absence of oxygen. The same inducing factor of the two pathways is hypoxia, the most insist that sustained hypoxic activate the HIF-1-dependent adaptive pathway, but intermittent hypoxia activates only the NF-κB-dependent inflammatory pathway. NF-κB and HIF-1may be associated with chronic sustained hypoxia disease, such as COPD, HIF-1exerts its inflence on COPD possibly by activating NF-κB. Therefore, in order to find out the relationship between the two pathways and the intermittent hypoxic diseases OSAHS, and to explore the different expressions of the two pathways in the intermittent hypoxic environment and sustained hypoxia, we measured NF-κB, HIF-1gene and protein expression in peripheral blood mononuclear cells (PBMCs) of OSAHS (intermittent hypoxia), COPD (sustained hypoxia) patients and the matched control group to investigate the role of them in the pathogenesis of OSAHS.Objective:NF-κB, HIF-1mRNA levels and NF-κB of HIF-1protein levels were detected in peripheral blood mononuclear cells of OSAHS patients (mild, moderate, severe), COPD patients and the matched control groups. The expression of NF-KB-dependent inflammatory pathway and adaptive HIF-1dependent pathway were observed to explore the role of NF-κB and HIF-1in the pathogenesis and the impact on disease severity in OSAHS.Methods:The subjects were divided into four groups:mild and moderate OSAHS group, severe OSAHS group, COPD group, the matched control group. All patients received polysomnography. OSAHS group and control group enrolled from the October2010to August2011, Guangdong General Hospital, Respiratory Sleep Medicine Room patients, the source of the COPD group comes from Guangdong General Hospital Respiratory out-patient patients with stable COPD patients. Exclusion criteria:autoimmune diseases, the use of immunosuppressive agents or cytotoxic drugs, malignancy, acute infectious diseases, acute and chronic renal failure, severe mental disorders, lobectomy and other long-term chronic hypoxia diseases. Inclusion criteria:OSAHS diagnostic criteria developed by the2003Chinese Medical Association Respiratory Diseases Sleep respiratory disease study group, AHI<30was mild and moderate OSAHS group, AHI>30was severe OSAHS group; The stable COPD patients were diagnosed according the2007Chinese Society of Respiratory Diseases Branch developed chronic obstructive pulmonary disease diagnosis and treatment guidelines (2007Revision).The matched control group without OSAHS and COPD, had no differences in gender, age, disease with other groups. The gene group:mild and moderate OSAHS group including16cases (mild12cases, moderate4cases), Severe OSAHS group was consisted of14cases.Both COPD group and control group were30cases.The protein group:mild and moderate OSAHS group including11cases (mild8cases, moderate3cases), Severe OSAHS group was consisted of13cases.Both COPD group and control group were14cases. Arterial blood gas analysis was detected in the COPD group. Fasting peripheral venous blood were taken in all groups the next morning6:00am after the PSG,8ml into sodium heparin anticoagulant tubes to be separated for mononuclear cells. PBMCs were isolated by Ficoll density gradient. Real-time PCR and Western blotting were measured to detect the mRNA and protein levels of the active subunits NF-κB P65, HIF-1α.Trizol extracted the total RNA in PBMCs, mRNA expression levels were detected by real-time quantitative PCR. The relative expression levels of target genes were computed for the2-ΔCT (ΔCT=CT value of target gene-CT value of reference gene). Nuclear protein were extracted from PBMCs. BCA method was applied to measure the protein concentration.30μg denatured protein sample were added to each well,10%polyacrylamide gel electrophoresis, then transferred the membrane, closed by5%nonfat dry milk, the primary antibody of target proteins (abeam, Amecica, mouse anti-human NF-κB P65dilution1:250, HIF-1α dilution1:400) and internal reference protein (GAPDH dilution1:5,000) were incubated overnight, secondary antibody (Merck, Amecica, goat anti-mouse antibody, dilution1:10,000) were incubated for1h at room temperature. Enhanced chemiluminescence agents (ECL) developed PVDF membrane, then tablet exposure. FluorChem8900software was used after the film to ImageScanner scanned to analyse the picture bar band area and gray value which the ratio of to the internal reference GAPDH bands represented relative expression levels of the NF-κB, HIF-1.Results:1. General information of subjuctsThe gene groups:the mild and moderate OSAHS group including13males and 3females, age42-76years, mean62.1±9.7years old, with coronary heart disease in3cases, an average of the AHI=11.8±5.3, BMI24.7±3.3kg/m2consisted of mild patients (12cases), moderate patients (4cases);the severe OSAHS group including14cases,11males and3females, age41-73years, mean61.6±9.5years old, with coronary heart disease in4cases, an average of the AHI=49.4±18.9, BMI25.2±2.9kg/m2; Total of30cases were enrolled in COPD group with arterial blood gas analysis before enrollment, including23males and7females, age55-75years, mean66.5±5.0years,10cases with coronary heart disease, average FEV1%pred43.72±13.51, SaO293.58±2.88%,mean AHI=2.1±1.4; BMI23.4±1.5kg/m2. The matched30cases control group,22cases were male,8cases were females, age46-75years old, average63.3±7.5years old,12cases have coronary heart disease, an average of the AHI=2.6±1.3BMI23.6±2.5kg/m2.The protein groups:mild and moderate OSAHS group including9males and2females, age42-70years, mean58.3±7.6years old, with coronary heart disease in2cases, an average of the AHI=12.0±5.9, BMI25.4±3.4kg/m2consisted of mild patients (8cases), moderate patients (3cases);the severe OSAHS group including13cases,10males and3females, age41-73years, mean57.1±12.1years old, with coronary heart disease in4cases, an average of the AHI=48.4±19.3, BMI25.1±3.0kg/m2; Total of14stable patients were enrolled in COPD group, including12males and2females, age55-72years, mean64.4±4.6years,7cases with coronary heart disease, average FEV1%pred43.61±13.47%, SaO293.44±2.32%, mean AHI=2.1±1.3; BMI23.0±1.6kg/m2. The matched14cases control group,12cases were male,2cases were females, age46-72years old, average59.9±8.0years old,6cases have coronary heart disease, an average of the AHI=3.1±1.2BMI24.0±2.2kg/m2. There were no significant differences in gender, age and disease among the groups (P>0.05).2The levels of NF-κB gene and protein expression 2.1The levels of NF-κB gene in each groupThe NF-κB P65mRNA expression among mild and moderate OSAHS group, severe OSAHS group,COPD group and control group was (0.007±0.005)VS (0.029±0.042)VS(0.013±0.010) VS(0.004±0.003),There were significant differences among them (F=6.438, P=0.001). Severe OSAHS group was significantly higher than control group, mild and moderate OSAHS group,COPD group (P<0.001, P=0.001,P=0.008), while there were no significant differences between mild and moderate OSAHS group and control group or COPD group (P=0.65, P=0.254).2.2The levels of NF-κB protein expression in each groupThe levels of NF-κBP65protein expression among mild and moderate OSAHS,severe OSAHS group, COPD group and control group was(0.90±0.09) VS (1.04±0.12)VS(1.02±0.17)VS(0.85±0.12),there were significant differences among all groups (F=6.457, P=0.001). There were no significant differences between mild and moderate OSAHS group and the control group (P=0.395).NF-κB P65expression was significantly differenct in the severe OSAHS and COPD groups compared to the control group (P=0.001, P=0.002). There were no significant differences between severe OSAHS group and the COPD group (P=0.639).3The correlation of NF-κB gene and protein expression and disease severity3.1The correlation of NF-κB gene expression and disease severityIn the OSAHS group (n=30), NF-κB P65mRNA expression was positively correlated with AHI (r=0.493, P=0.006), and negatively correlated with the night minimum oxygen saturation (LSaO2)(r=-0.488, P=0.006),there were no correlations between NF-κB mRNA and mean oxygen saturation (MSaO2), Oxygen desaturation index (ODI), Excessive sleepiness scale score (ESS)(r=-0.309, P=0.096; r=0.347, P=0.060; r=0.206, P=0.274).3.2The correlation of NF-κB protein expression and disease severity In the OSAHS group (n=24), the expression of NF-κBP65protein was positively correlated with AHI (r=0.669, P<0.001), while had no correlation with LSaO2, MSaO2, ODI and ESS (r=-0.214, P=0.315; r=-0.286, P=0.176; r=0.379, P=0.067; r=-0.038, P=0.859).4The levels of HIF-1gene and protein expression4.1The levels of HIF-1gene in each groupThe expression of HIF-1αmRNA among mild and moderate OSAHS group, severe OSAHS group, COPD group and control group was (0.013±0.007) VS (0.033±0.038)VS(0.030±0.025) VS (0.008±0.006), There were significant differences among them (F=7.737,P=0.001). The severe OSAHS group was significantly higher than the control group, mild and moderate OSAHS group (P<0.001, P=0.011). COPD group was higher than mild and moderate OSAHS group and control group (P=0.012, P<0.001), there was no significant differences between the severe OSAHS group and the COPD group (P=0.633).4.2The levels of HIF-1protein in each groupThe levels of HIF-1α protein expression among mild and moderate OSAHS,severe OSAHS group, COPD group and control group was (0.87±0.17) VS (1.06±0.09) VS(1.09±0.19)V S (0.86±0.18), there were significant differences among all groups (F=7.058, P=0.001). There were no significant differences in HIF-1α expression between mild and moderate OSAHS group and control group (P=0.825), also between severe OSAHS group and the COPD group (P=0.727). HIF-1α expression in the severe OSAHS group was higher than in the mild and moderate OSAHS group and control group(P=0.007, P=0.002).5The correlation of HIF-1gene and protein expression and disease severity5.1The correlation of HIF-1gene expression and disease severityIn the OSAHS group (n=30), HIF-1αmRNA expression was positively correlated with AHI (r=0.508, P=0.004), and was negatively correlated with LSaO2, MSaO2(r=-0.460, P=0.011; r=-0.405, P=0.026), There were no correlation between the HIF-1mRNA expression and ODI, ESS (r=0.344, P=0.062; r=0.249, P=0.184).5.2The correlation of HIF-1protein expression and disease severityIn the OSAHS group (n=24), HIF-1α protein expression was positively correlated with AHI (r=0.645, P=0.001), also positively correlated with ODI (r0.48, P=0.018), There were no correlation between the HIF-1α protein expression and LSaO2, MSaO2, ESS (r=-0.306, P=0.146; r=-0.378, P=0.069; r=-0.122, P=0.571).6. The correlation of NF-κB gene, protein and HIF-1gene, proteinThere were positive correlation between NF-κB P65mRNA and HIF-1α mRNA (r=0.543, P=0.002), There were also positive correlation between NF-κB P65and HIF-1α protein expression (r=0.716, P<0.001).Conclusions:1. Both NF-KB-dependent inflammatory pathway and HIF-1-dependent adaptive pathway were actived in patients with OSAHS.2. With the disease severity of OSAHS and levels of hypoxia increased, The gene and protein levels of NF-κB and HIF-1were increased, indicating that the gene and protein expression of NF-κB and HIF-1were associated with the severity of the disease.3. The two transcription factors between which there were correlations attributed to the incidence of OSAHS, may be through one transcription factor regulated the other to co-activate the two pathways to cause the occurrence of OSAHS.