The Study On The Mechanism Of Apoptosis And Injury Of PMN And Vascular Endothelial Cells In Overlap Syndrome Rat Model

Objective and Background:Obstructive sleep apnea syndrome(OSAS)is a sleep-disordered disease characterized by intermittent hypoxia.This hypoxic mode can cause multi-systemic damages.And the cardiovascular system injury is the most common and severe.Chronic obstructive pulmonary disease(COPD)is a progressive disease characterized by incomplete reversible airflow limitation.Moderate and severe patients can cause chronic persistent hypoxia and lead to cardiovascular complications.Respiratory overlap syndrome(OS)refers to the coexistence of OSA and COPD,with intermittent hypoxia and persistent hypoxia.It has more serious cardiovascular damage and higher mortality.The occurrence of cardiovascular disease is closely related to the damage of vascular endothelium,and the interaction between inflammatory cells represented by neutrophils(PMN)and vascular endothelial cells becomes a key link in endothelial cell injury.At present,there are few studies on the pathological mechanism of OS.In this study,the intermittent hypoxia with persistent hypoxia animal model was established to simulate the intermittent and persistent hypoxia injury model in OS patients.It focused on the apoptosis and damage of PMN and endothelial cells in OS mode,and its gene regulation mechanisms.Furthermore,it was observed whether anti-oxidation or anti-inflammatory intervention can ameliorate abnormal apoptosis of PMN and endothelial cells,thereby reducing damages caused by hypoxia.This study provides the necessary theoretical and experimental basis for revealing the pathogenesis of cardiovascular complications and feasible interventions in OS patients.It is of great clinical significance to reduce the incidence of cardiovascular complications and mortality in OS patients.Content:1.The establishment of the OS rat model with intermittent hypoxia and cigarette exposure.The research on the apoptosis of PMN and endothelial cells in OS rats and the effects of antioxidant or anti-inflammatory intervention.2.The research on the oxidative stress and inflammatory response in OS rats and the effects of antioxidant or anti-inflammatory intervention.3.The research on the expression of PMN pro-apoptotic gene and anti-apoptotic gene in OS rats and the effects of antioxidant or anti-inflammatory intervention.Methods:Section 1: Male Wistar rats(n=48)were randomly divided into normal oxygen group(NC),intermittent hypoxia group(IH),cigarette exposed group(CH),IH overlapping cigarette exposure group(OS),OS group treated with Tempol(OST),OS group treated with PDTC(OSP).The rats were given IH,CH and OS mode hypoxic exposure for 8 weeks.PMN and CECs were isolated from peripheral blood and the apoptosis of PMN and CECs were detected by flow cytometry.The aortic endothelium were stained by Tunel method,and the apoptosis of aortic endothelial cells were observed under fluorescence microscope.Section 2 :The expression level of the following inflammatory markers in peripheral blood were detected by ELISA methods: TNF-?,IL-6,IL-8,SOD,CAT,MDA and ICAM-1.Section 3: qRT-PCR method was used to detect the expression of PMN proapoptotic gene Bak and anti-apoptotic gene Mcl-1 mRNA in circulating blood.Results:Section 1:1.Compared with NC group,IH group and CH group,the apoptosis of PMN in peripheral blood of rats in OS group reduced.After the intervention of antioxidant Tempol or inflammatory signaling blocker PDTC,the apoptosis rate of PMN in OST and OSP group increased compared with that in OS group(P<0.001).2.Compared with NC group,IH group and CH group,apoptosis of circulating blood endothelial cells in OS group increased.Compared with OS group,the apoptosis rate of circulating blood endothelial cells in OST and OSP groups decreased after the intervention of Tempol or PDTC(P<0.001).Compared with NC group,IH group and CH group,the apoptosis rate of aortic endothelial cells in OS group increased after Tunel staining.Compared with OS group,the apoptosis rate of circulating blood endothelial cells in OST and OSP groups decreased after the intervention of Tempol and PDTC(P<0.001).3.Bivariate correlation analysis showed that the apoptosis rate of PMN was significant negatively correlated with that of endothelial cells(r=-0.854,P=0.000).Section 2:1.Compared with NC group,IH group and CH group,the expression levels of ICAM-1,TNF-?,IL-6,IL-8 and MDA were increased in OS group,while the expression levels of SOD and CAT were decreased.After the intervention of Tempol or PDTC,the expression levels of ICAM-1,TNF-?,IL-6,IL-8 and MDA were reduced in OST and OSP groups,while the expression levels of SOD and CAT were increased(P<0.001).2.Bivariate correlation analysis showed that there was a significant negative correlation between the apoptosis rate of PMN and the expression levels of TNF-?,IL-6,IL-8,MDA,and ICAM-1(r values were 0.767,0.849,0.660,0.644,and 0.885,respectively)(P=0.000).The apoptosis rate of PMN was positively correlated with the expression levels of SOD and CAT(r values were 0.794,0.751)(P=0.000).There was a significant positive correlation between the apoptosis rate of endothelial cells and the expression levels of TNF-?,IL-6,IL-8,MDA,and ICAM-1(r values were 0.872,0.933,0.813,0.748,and 0.515,respectively)(P=0.000).The apoptosis rate of endothelial cells was negatively correlated with the expression levels of SOD and CAT(r values were 0.773,0.757)(P=0.000).Section 3:1.Compared with NC group,IH group and CH group,the expression level of Bak mRNA in OS group was the lowest,and the expression level of Mcl-1 mRNA was the highest.After the intervention with Tempol or PDTC,the expression level of Bak mRNA and Mcl-1 mRNA in OST and OSP groups were increased and decreased compared with that in OS group(P<0.01).2.Compared with NC group,IH group and CH group,the expression level of Mcl-1/Bak in OS group was the highest.Compared with the OS group,the expression of Mcl-1 /bak was decreased in OST and OSP groups after the intervention of Tempol or PDTC(P<0.01).Conclusion:1.Compared with the group of intermittent hypoxia and cigarette exposure,the OS rats presented more significant neutrophil apoptosis delay,which could be reduced by the intervention of antioxidant Tempol or inflammatory signaling pathway blocker PDTC.The OS rats showed more obvious apoptosis of endothelial cells and more obvious adhesion of endothelial cells.The intervention of antioxidant Tempol or inflammatory signaling blocker PDTC can delay the apoptosis and damage of endothelial cells.2.More significant inflammatory and oxidative stress responses were observed in the OS rats.Intervention with antioxidant Tempol or inflammatory signaling pathway blocker PDTC can significantly reduce inflammatory and oxidative stress responses.3.The expression of PMN pro-apoptotic genes decreased in OS rats,and the expression of anti-apoptotic genes increased,which was associated with the regulation of PMN apoptosis delay and endothelial cells damage.