Study On The Injury Of VEC And The Interaction Between PMN And VEC In Response To The Model Of OS Hypoxia Exposure And The Intervention Measures

Research purposes:Obstructive sleep apnea syndrome(OSAS)and chronic obstructive pulmonary disease(COPD)are the two most common chronic respiratory diseases,and the two diseases coexist as respiratory overlap syndrome(OS).Because both OSAS and chronic obstructive pulmonary disease can cause oxidative stress and activation and dysfunction of leukocytes in the body,it is easy to induce body systemic inflammatory response,and the level of inflammatory factors in the body is increased,which becomes the development and aggravation of overlapping syndrome.The important reason.Patients with COPD and OSAS have pathophysiological processes of hypoxia.COPD patients are mainly chronic continuous hypoxia,while OSAS patients are characterized by intermittent hypoxia during nighttime sleep.Systemic inflammatory reactions caused by hypoxia cause related inflammatory factors.The waterfall reaction causes related complications such as cardiovascular disease,atherosclerosis,and pulmonary hypertension.Both COPD and OSAS are independent risk factors for cardiovascular disease.Therefore,when both COPD and OSAS coexist,the risk of cardiovascular complications is higher and the prognosis is worse.At present,the research on overlap syndrome and its comorbidities mainly focuses on clinical and epidemiological aspects.There are few studies on pathological mechanisms,and it is still rare to study in cytology.This study will investigate the mechanism of vascular endothelial cell injury,the interaction between neutrophils and vascular endothelial cells,and the oxidative agent Tempol and the inflammatory channel blocker PTDC,which are intervened in vascular endothelial cells after intermittent and continuous hypoxia exposure.The protective role is to provide the necessary theoretical and experimental basis for revealing the possible pathogenesis of cardiovascular disease and feasible interventions in patients with overlap syndrome.Research content:1.The mechanism of vascular endothelial cell injury and the correlation between various mechanisms of neutrophil and endothelial cells co-culture under intermittent and continuous hypoxic exposure were studied at the cellular level.2.The importance of cell interaction in vascular endothelial cell injury induced by intermittent and continuous hypoxic exposure and the role of intercellular adhesion molecule-1.3.At the cellular level,explore the protective effect of the antioxidant Tempol and the inflammatory channel blocker PTDC on vascular endothelial cell injury induced by intermittent and continuous hypoxic exposure.Research method:The first part: the fasting peripheral venous blood of healthy volunteers was taken,the neutrophils were separated by double-layer density gradient centrifugation,and the neutrophils were co-cultured with the prepared human umbilical vein endothelial cells for 4 hours,then applied to the Respiratory Simulation System of Department of Respiratory and Critical Care of Tianjin Medical University General Hospital performed group exposures of different hypoxic modes(IN,IH,CH,OS)at a time of2 hours,5 hours,and 8 hours.A total of 10 tests were performed,i.e.,n=10.Inflammatory factors,TNF-α and IL-6,the adhesion molecule,ICAM-1,CAT activity and MDA concentrations in supernatants from the co-culture as well as pro-(Bak)and anti-(Bcl-xl)apoptotic gene expression levels in the endothelial cells were determined.The second part: the OS group directly co-cultured with neutrophils and endothelial cells and the OS(H)group cultured with endothelial cells alone were placed in a cell culture incubator for 4 hours,and then applied to the Respiratory Simulation System of Department of Respiratory and Critical Care of Tianjin Medical University General Hospital to intermittent and continuous low-oxygen exposure for 8 hours,and the supernatant and endothelial cells were collected separately after exposure.The test indicators are the same as the first part.The third part: After co-culture of the extracted neutrophils with endothelial cells,one group was subjected to normoxic exposure to N group,and the other three groups were intermittently and continuous hypoxic exposure.The OS group was added without any reagent.The group of Tempol was added(OS+T),and the group of PTDC was added(OS+P),and each group was exposed to hypoxia for 2 hours,5hours,and 8 hours.Supernatants and endothelial cells were collected separately after exposure.The test indicators are the same as the first part.Research result:The first part:1.Comparison of inflammatory factors,adhesion molecules and oxidative stress in supernatant: The results showed that the trend of hypoxia exposure in 2h,5h and 8h groups was the same and time-dependent: each indicator was compared with the N group.There were significant differences(P<0.05).The levels of TNF-a,IL-6,ICAM-1,and MDA in the OS group were higher than those in the IH group and the CH group.The CAT activity value in the OS group was lower than that in the IH group and the CH group.Academic significance(P<0.05).2.Comparison of endothelial cell apoptosis gene expression levels: The results showed that the trend of hypoxia exposure in 2h,5h and 8h groups was the same:there were statistical differences between the groups in each group(P<0.05);OS group Bcl-x1 The expression level of Bcl-x1/BAK was lower than that of IH group and CH group.The expression level of BAK was higher than that of IH group and CH group,the difference was statistically significant(P<0.05).With the prolongation of hypoxic exposure time,the expression levels of pro-apoptosis genes and anti-apoptotic genes showed an increasing trend,while the level of Bcl-x1/BAK showed a downward trend,suggesting that pro-apoptotic genes play a major role.3.Correlation analysis between inflammatory factors and oxidative stress indicators and apoptosis gene: inflammatory factors IL-6 and TNF-α were negatively correlated with antioxidant index CAT,and positively correlated with oxidative stress index MDA,P<0.05;The inflammatory factors IL-6 and TNF-α were significantly negatively correlated with the ratio of apoptosis gene Bcl-xl/BAK,P<0.05.The antioxidant index CAT was positively correlated with the ratio of apoptosis gene Bcl-xl/BAK.There was a strong negative correlation between the stress index MDA and the apoptosis gene Bcl-xl/BAK ratio,P<0.05.The second part:1.Comparison of OS(H)group and OS group: IL-6,TNF-α,ICAM-1,MDA,BAK mRNA expression level and Bcl-xl mRNA expression level in OS(H)group were lower than OS group,P <0.05,The difference was statistically significant.The activity of CAT and the ratio of Bcl-xl/BAK in OS(H)group were higher than those in OS group.P<0.05,The difference was statistically significant.2.Correlation analysis between ICAM-1 and various variables: ICAM-1 was positively correlated with IL-6,TNF-α and MDA;it was negatively correlated with CAT,Bcl-xl/BAK,P<0.05.The third part :1.Comparison of inflammatory factors,adhesion molecules and oxidative stress in supernatant: The results showed that the trend of hypoxia exposure in 2h,5h and 8h groups was the same: TNF-a,IL-6,ICAM-1 and MDA levels were higher in the OS group than group N,OS+T group and OS+P group.(P<0.05)The difference was statistically significant.OS+T group and OS+P group was higher than that of N group(P<0.05).There was no significant difference between OS+T group and OS+P group.With the prolongation of exposure time,the concentrations of TNF-a,IL-6,ICAM-1and MDA increased.The CAT activity value of OS group was lower than N group,OS+T group and OS+P group,OS+T group and OS+P group were lower than N group,the difference was statistically significant(P<0.05);There was no significant difference between OS+T group and OS+P group;however,with the prolongation of hypoxic exposure time,CAT showed a downward trend.2.Comparison of expression levels of apoptosis gene in endothelial cells: The results showed that the trend of hypoxia exposure in 2h,5h and 8h groups was the same: the expression level of Bcl-x1 and the ratio of Bcl-x1/BAK in OS group were lower than those in N group and OS+T group and OS+P group,the expression level of proapoptotic gene BAK was higher than that in N group,OS+T group and OS+P group,the difference was statistically significant(P<0.05);The expression level of Bcl-x1 and the ratio of Bcl-x1/BAK in the OS+T group and OS+P group were lower than those in the N group.The expression of BAK in the OS+T group and the OS+P group was higher than that in the N group.The difference was statistically significant(P <0.05);There was no significant difference between OS+T group and OS+P group.With the prolongation of hypoxic exposure time,the expression levels of pro-apoptotic genes and anti-apoptotic genes showed an increasing trend;while Bcl-x1/BAK levels showed a downward trend.Analysis conclusion:1.Neutrophils and vascular endothelial cells co-cultured intermittently and continuous hypoxic exposure mode compared with normoxia mode,intermittent hypoxia mode,continuous hypoxic mode,inflammatory reaction and oxidative stress are more severe,and endothelial cell apoptosis accelerates.More obvious,endothelial damage is more serious.It is suggested that inflammatory response,oxidative stress,and apoptotic genes are involved in vascular endothelial cell injury induced by overlapping hypoxia exposure.2.The vascular endothelial cell injury induced by overlapping hypoxic exposure mode is time-dependent.With the prolongation of the action time,the damage of vascular endothelial cells is gradually aggravated.3.Inflammation,oxidative stress,and endothelial cell apoptosis genes are involved in the damage of vascular endothelial cells induced by overlapping hypoxia,and there is a strong correlation between each other,cycle back and forth,and cause vascular endothelial cell damage to cascade and enlarge the waterfall.4.The interaction between neutrophils and vascular endothelial cells under overlapping hypoxia makes inflammation,oxidative stress and endothelial cell apoptosis associated with endothelial cell injury more severe.That is,the interaction between neutrophils and endothelial cells causes the damage of vascular endothelial cells caused by overlapping hypoxic exposure to be more serious.5.The adhesion molecule ICAM-1 has strong correlation with inflammatory factors,oxidation/antioxidation index and apoptosis gene,and plays an important role in the mechanism of endothelial cell injury.6.Increased inflammatory factors and adhesion molecules in vascular endothelial cells induced by overlapping hypoxic exposure patterns,enhanced oxidative stress and increased apoptosis of endothelial cells were partially inhibited by anti-inflammatory and antioxidant agents.Anti-inflammatory and anti-oxidative interventions can partly protect vascular endothelial cells.It has the potential to treat overlapping syndrome with cardiovascular disease.7.Anti-inflammatory and anti-oxidation treatment early application is the effect,advocate early intervention,early treatment.It can avoid adverse consequences,resulting in damage to multiple organ function.8.This part of the study shows that anti-inflammatory and anti-oxidative intervention can only partially protect vascular endothelial cells,and still can not completely reverse endothelial cell damage,considering the multi-molecular pathway of endothelial cell injury mechanism.Further research is expected to completely reverse endothelial cell damage through multi-target precision therapy.9.Hypoxia leads to a strong correlation between inflammation and oxidative stress.The reagents Tempol and PDTC have the anti-oxidation effect of scavenging oxygen free radical ROS and the anti-inflammatory effect of inhibiting inflammatory pathway NF-κB.There was no significant difference in antioxidant activity.