The Study On Lymphocytes Apoptosis And Interaction Mechanisms Between Lymphocytes And Vascular Endothelial Cells Induced By Intermittent Hypoxia Of Sleep Apnea Mode In Rats

Objective and Background:Obstructive sleep apnoea syndrome (OSAS) is an increasingly common disorder and characterized by periodic collapse of the upper airway during sleep. Intermittent hypoxia (IH) that is characteristic of OSAS. OSAS is an independent risk factor for cardiovascular morbidity and mortality. Accumulated evidence implicates that oxidative stress and inflammation play an important role for its pathogenesis. Leukocyte-endothelial cell adhesion can induce endothelial injury and has been implicated in the pathogenesis of cardiovascular complications. It is well-recognized that IH mimicking sleep apnea can activate neutrophils and neutrophil-endothelial interaction, triggering inflammatory responses. However, the effect of IH on lymphocyte has not been studied in detail so far. And lymphocyte-mediated endothelial injury mechanisms also remain unknown. IH can activate monocytes, leading to increased adhesion molecules expression and production of reactive oxygen species. Consequently, monocyte adhesion to endothelial cell is elevated. So monocyte is an important factor for OSA-induced endothelial injury. Based on the above results, we hypothesize that IH can induce lymphocyte activation in circulating blood of rats. Activated lymphocyte can readily attach to endothelial cell. Activated endothelial cells release proinflammatory cytokines, TNF-a, IL-8, CRP and ICAM-1. Oxidant/antioxidant imbalance also occurs. Inflammatory mediators induced endothelial cell injury in a contact-dependent manner and many transcription factors are involved in the injury and apoptosis of endothelial cells. In this study, we established an animal model mimicking OSAS and observed the apoptosis lymphocyte subsets in circulating blood of rats. We also determined the levels of inflammatory cytokines, oxidative stress, and signal proteins involved in apoptosis of endothelial cells after lymphocyte-endothelial cells co-culture. Finally, we investigated the effect of antioxidant tempol on IH induced events.Content:1. Effect of IH on lymphocyte apoptosis in circulating blood of rats and the effect of antioxidant tempol intervention on lymphocyte apoptosis.2. The level of inflammatory responses and oxidative stress and the possible apoptosis mechanisms of endothelial cells after lymphocyte-endothelial cells co-cultureMethods:1. A total of 56 male Wistar rats were randomly assigned to seven groups, with 8 rats per group:(1) the normal oxygen control group (NC); (2) 4-week-IH (oxygen content:5%; AHI 30/h) group (IH4); (3) 6-week-IH group (IH6); (4) 6-week-IH group treated with tempol injections for 6 weeks (IH6T); (5) 6-week-IH group treated with saline injections for 6 weeks; (6) 6-week-IH group treated with tempol injections only for latter 2 weeks; (7) 6-week-IH group treated with saline injections only for latter 2 weeks. The abdominal skin was retracted to expose the abdominal aorta. Blood sample was obtained by antecubital venepuncture into acid citrate dextrose. Lymphocytes were purified and the lymphocyte apoptosis was determined using flow cytometry assay.2. Rat aortic endothelial cells were seeded in 6-well plate and cultured exposed to IH for 5 h. Lymphocyte cells from the above groups NC, IH6 and IH6T were used and seeded in 6-well plate containing endothelial cells. This experiment were assigned to six groups:(1) lymphocyte cells from group NC plus endothelial cells exposed to the normal oxygen (NC+NE); (2) lymphocyte cells from group NC plus endothelial cells exposed to IH (NC+IHE); (3) Lymphocyte cells from group IH6 plus endothelial cells exposed to the normal oxygen (TH6+NE); (4) lymphocyte cells from IH6 plus endothelial cells exposed to IH (IH6+IHE); (5) lymphocyte cells from group IH6T plus endothelial cells exposed to the normal oxygen (IH6T+NE); (6) lymphocyte cells from group IH6T plus endothelial cells exposed to IH(IH6T+IHE). Then lymphocyte cells and endothelial cells were co-incubated for another 4 h. Cell supernatants were centrifuged and stored until use. Proteins and mRNA were extracted from endothelial cells.3. The ELISA method was used to determine the levels of CRP, TNF-a, IL-8, ICAM-1, MDA, CAT and SOD. The endothelial cell apoptosis-related proteins caspase3, NF-kB P65, Bcl-2 and Bax were measured by western blotting. Real-time PCR were performed for the detection and quantification of mRNA of NADPH P22, C-FOS, HIF-la and MAPK P38 from endothelial cells.Results:Section 1:Compared with group NC, the apoptosis of CD4 and CD8 were significantly decreased, while the apoptosis of B and NK were significantly increased for groups IH6 and IH4. Six-week IH challenge showed more potential effect than four-week treatment. The antioxidant tempol can attenuate the IH-induced alterations in populations of CD4, CD8, B and NK lymphocyte subsets. We also compared the apoptosis of lymphocyte subsets continuously injected with tempol for 6 weeks with counterparts exposed to IH for 4 weeks followed by 2 weeks in the presence of tempol. We observed that tempol did moderate the regulating apoptosis of lymphocyte subsets when injected after IH exposure for 4 weeks, although not as efficiently as when tempol is given from the start of the experiment.Section 2:1. Compared with group NC+NE, supernatant levels of CRP, TNF-a, IL-8, ICAM-1 and MDA were significantly increased in groups IH6+IHE, H6+NE, NC+IHE and IH6T+NE, while the levels of SOD and CAT were significantly decreased. And the same is the case for group IH6+IHE when compared with group IH6+NE and NC+IHE. The supernatant levels of CRP, TNF-a, IL-8, ICAM-1 and MDA were significantly decreased in groups IH6T+NE (compared with group IH6+NE) and IH6T+IHE (compared with group IH6+IHE), while the levels of SOD and CAT were significantly increased.2. The levels of NF-kB P65, Caspase-3 and Bax were significantly increased in groups in groups IH6+IHE and H6+NE(compared with group NC+NE), IH6+IHE (compared with group IH6+NE and NC+IHE), NC+IHE (compared with group NC+NE), IH6T+NE (compared with group NC+NE), and IH6T+IHE (compared with group NC+IHE), while the levels of BCL-2were significantly decreased. The case is opposite for groups IH6T+NE (compared with group IH6+NE) and IH6T+IHE (compared with group IH6+IHE).3. The mRNA levels of NADP P22, C-FOS, HIF-1α and MAPK P38 were significantly in creased in srouos IH6+IHE. IH6+NE. NC+IHE. IH6T+NE (comDared with group NC+NE), IH6+IHE (compared with groups IH6+NE and NC+IHE), IH6T+IHE (compared with group NC+IHE), while significantly decreased in groups IH6T+NE (compared with group IH6+NE) and IH6T+IEH (compared with group IH6+IHE).Conclusion:1. IH modified the apoptosis in circulating blood lymphocyte subsets and therefore may contribute to immunologic imbalance. Treatment with tempol moderates the changes in the lymphocytes apoptosis.2. Lymphocyte cells (exposed to IH) and endothelial cells interaction led to oxidant/antioxidant imbalance, increased proinflammatory cytokines release and evaluated injury and apoptosis of endothelial cells. Lymphocyte cells induced endothelial cells apoptosis and dysfunction, which contributes to the pathogenesis of cardiovascular complications.3. Different apoptosis-related proteins were involved in endothelial cells apoptosis.4. Antioxidants can attenuate IH-induced inflammatory damage and oxidant/ antioxidant imbalance. This effect is more efficient when tempol is given from the start of IH exposure. Antioxidants were potentially beneficial to prevent and treat cardiovascular complications.