The Study On PMN Apoptosis And Pathogenesis Of The Interaction With Vascular Endothelial Cell With Intermittent Hypoxia Exposed Rats Of OSAS Model

Obstructive sleep apnea syndrome (OSAS), characterized by frequently recurrent intermittent hypoxia/reoxygenation (IH/ROX) as its obvious pathological trait, is clearly associated with an increased risk of multiple system damage, particularly cardiovascular and cerebrovascular events. IH can induce a series of oxidative stress and the changes of apoptosis cycle, affect the cellular function and the interaction with peripheral cells, and result in organs damage and dysfunction eventually. Endothelial dysfunction is the pathological basis of vascular diseases such as hypertension, atherosclerosis and stroke which related to OSAS. Circulating blood cells is the main component involved in the interaction and contract with vascular endothelial cells. Polymorphonuclear neutrophil(PMN) is the most important inflammatory cell, and its survival, function and interaction with vascular endothelial cells will affect vascular endothelial function directly. Our study explored the effect on polymorphonuclear (PMN) apoptosis with different duration of intermittent hypoxia and the interaction effect on cellular adhesion and the apoptosis of vascular endothelial cell between PMN and vascular endothelial cell though direct or indirect co-culture, and further studied the physiopathologic mechanism of vascular comorbidity related to OSAS, and provide potential therapeutic strategy and experimental basis for clinical treatment.Objectives1. To explore the effect of different duration of intermittent hypoxia on PMN apoptosis in rats model2. To explore the changes of ICAM-1, E-selectin, bcl-2/bax, caspase-3of endothelial cell which is co-cultured with PMN isolated from rats exposed to intermittent hypoxia for6weeks directly or indirectly, and the effect of antioxidant tempol.MethodsPart oneForty eight male Wistar rats were randomly divided into six groups of eight each according the different duration of4,6&8weeks, including three normal oxygen control groups (NIH4, NIH6, NIH8) and three intermittent hypoxia groups (IH4, IH6and IH8). After exposure, blood (approximately8to10mL per rat) was collected (exsanguination) from the aorta abdominalis, PMN were isolated from rat blood by Ficol-Histopaque double density gradient method (Histopaquel.083and Histopaque1.119), and PMN apoptosis were detected by flow cytometry method (AnnxinV/7-AAD double staining).Part twoThirty two male Wistar rats were randomly divided into four groups of eight each according the different treatment, including NIH group, IH group, IH+saline(NS) group (IHN group), IH+Tempol group (IHT group). Rats were exposed to normoxic compressed air or IH for6weeks. After exposure, PMNs were isolated from blood and co-cultured directly and indirectly using Transwell with aortic endothelial cells or4hours respectively. The levels of ICAM-1, E-selectin culture medium were measured by ELISA and aortic endothelial cell bcl-2, bax and caspase-3expression levels were measured by western blot analysis.ResultsPart one1. PMN apoptotic rate in IH4, IH6, IH8groups are all lower than those in NIH4, NIH6, NIH8groups (22.35±2.26vs16.23±3.00,23.56±1.26vs16.71±1.97,25.48±1.93vs20.16±1.11respectively, all P values<0.05).2. PMN apoptotic rate were continuously increased along with IH exposure time: PMN apoptotic rate in NIH4, NIH6and NIH8groups were different statistically (F=4.297, P=0.033), PMN apoptotic rate in NIH8group is higher in NIH4group (P=0.011); PMN apoptotic rate in IH4, IH6and IH8groups were different statistically (F=5.845, P=0.013), PMN apoptotic rate in IH8group is higher than those in IH4and IH6groups (P=0.007and0.015).Part two1. The effect of co-culturing vascular endothelial cell and PMNs from rats directly on intercellular adherent and endothelial apoptosis.1). The concentrations of ICAM-1in supernatant of directly co-culture medium increased, and the concentrations in IHE, IHNE and IHTE groups are higher than those in NIHE group (all P values<0.05); The concentrations of E-selectin increased, and the concentrations in IHE and IHNE groups are all higher than those in NIHE group (all P values<0.001), when the concentrations in IHTE are all lower than those in IHE and IHNE groups (all P values<0.001), but there is no statistical difference between IHNE and NIHE group (P=0.557).2). The expression level of endothelial cell Bcl-2in directly co-culture increased, the levels in IHE, IHNE and IHTE groups are higher than those in NIHE group (all P values<0.05); The expression level of endothelial cell Bax in directly co-culture increased, the levels in IHE and IHNE groups are higher than those in NIHE group (all P values<0.001); The value of Bcl-2/Bax reduced, the levels in IHE, IHNE and IHTE groups are lower than those in NIHE group (all P values<0.05); The expression level of Caspase-3increased, the levels in IHE and IHNE groups are higher than those in NIHE group (all P values<0.001).2. The effect of co-culturing vascular endothelial cell and PMNs from rats indirectly on intercellular adherent and endothelial apoptosis.1) The concentrations of ICAM-1and E-selectin in the supernatant of lower chamber of the Transwell increased, the concentrations in T-IHE and T-IHNE groups are higher than those in T-NIHE group (P<0.001) when the concentrations in T-IHTE group are lower than those in T-IHE and T-IHNE group (P<0.05), there is no statistical difference compared with T-NIHE group (P>0.05).2) The expression level of endothelial cell Bcl-2in indirectly co-culture increased, the levels in T-IHE and T-IHNE groups are higher than those in T-NIHE group (all P values<0.05), but there is no statistical difference between T-IHTE and T-NIHE group (P=0.649); The expression level of endothelial cell Bax increased, the levels in T-IHE, T-IHNE and T-IHTE groups are higher than those in T-NIHE group (all P values<0.05); The value of Bcl-2/Bax reduced, the levels in T-IHE, T-IHNE and T-IHTE groups are lower than those in T-NIHE group (all P values<0.05); The expression level of Caspase-3increased, the levels in T-IHE and T-IHNE groups are higher than those in T-NIHE group (all P values<0.001). But there is no statistical difference between T-IHTE and T-NIHE group (P=0.602).3. The levels of ICAM-1, E-selectin, Bcl-2, Bax and Caspase-3in groups which cells were co-cultured directly higher than those in groups which cells were co-cultured indirectly (all P values<0.05); The value of Bcl-2/Bax in groups which cells were co-cultured directly lower than those in groups which cells were co-cultured indirectly (all P values<0.05).Conclusions1. Different duration of IH exposure can delay PMN apoptosis; and PMN apoptosis showed increasing tendency along with time. 2. The ways of direct and indirect coculturing of vascular endothelial cell and PMNs both can enhance intercellular adhesion and compared with the way of indirect coculturing, direct coculturing can cause more strong effects.3. The ways of direct and indirect coculturing of vascular endothelial cell and PMNs both can induce endothelial apoptosis and compared with the way of indirect coculturing, direct coculturing can cause more strong effects.4. The antioxidant tempol can partly improve the damage of PMN to endothelial adhesion and apoptosis, and it will be more obvious when controlled the adherent effect.5. These are important measures that anti-adhesion, antioxidant, accelerate inflammatory cells apoptosis and protect endothelial cells for vascular related comorbidity caused by IH from OS AS.