Protective Effect Of SOD Mimesis Tempol On Intermittent Hypoxia-induced Vascular Injury In Mice

Background:As a common condition affecting 5%of the general population and 18%of the elderly population over 50 years of age,Obstructive sleep apnea（OSA）is a diseae with ever-increasing incidence and complications.OSA is characterized by repeated apnea during sleep,resulting in intermittent hypoxia（IH）and sleep fragmentation.As independently risk of cardiovascular and cerebrovascular diseases,many diseases are involved.Such as hypertension,ischemic heart disease,atherosclerosis,coronary heart disease,and stroke.IH can lead to vascular endothelial dysfunction through oxidative stress,underutilization of nitric oxide,systemic inflammation,hyperactivation of the sympathetic nervous system,and decreased endothelial repair capacity,thereby increasing the risk of cardiovascular diseases such as hypertension.Tetramethylpiperidine（4-Hydroxy-TEMPO,Tempol）is an antioxidant that acts as a superoxide dismutase（SOD）analogue that penetrates cell membranes and reacts with oxygen radicals inside and outside cells,reducing oxidative stress by scavenging reactive oxygen species（ROS）to protect vascular endothelial function.The aim of this study was to explore the protective effect of oral Tempol on vascular damage caused by intermittent hypoxia in mice.Objectives:1.Establish IH mice model to study the effect of changing ROS levels of blood vessels on vasodilation function in IH mice.2.To explore the protective effect and mechanism of oral Tempole on vascular damage in IH mice.Methods:Male healthy C57 mice were randomly grouped into normal oxygen（Control）group,intermittent hypoxia（IH）group,and intermittent hypoxia Tempol intervention（IH+Tempol）group.The function of blood vessels was detected using an in vivo vascular tone measurement system to observe the soothing function of the thoracic aorta of mice;the fluorescence probe needle-dihydroethidium（DHE）staining method was used to detect reactive oxygen species（ROS）;the concentration of nitric oxide（NO）in the thoracic aorta tissue of mice was measured by ELISA;Hematoxylin-eosin（HE）staining,Masson staining,observed histomorphological changes in the thoracic aorta,and western blot（WB）method to detect the correlation proteins expression of thoracic aortic tissue.1.Effect of changing ROS levels in isolated blood vessels on vasodilation function in IH mice.Male healthy C57 mice were grouped into normal oxygen（Control）group and intermittent low oxygen（IH）group.The IH group was placed in a low-oxygen chamber for intermittent hypoxia treatment for 8 h a day for 4 weeks.They are all reared on a normal diet.After the hypoxia model molding is completed,the thoracic aorta is taken for vitro tension experiment.Incubate Tempol（1 m M）or nadph oxidase inhibitor Apocynin（0.3 m M）in ex vivo vessels to observe the effect of reducing ROS levels in blood vessels on vasodilation function in IH mice.2.Protective effect and mechanism of oral Tempol on vascular damage in IH mice.Male healthy C57 mice were randomly divided into 3 groups:normal oxygen（Control）group,intermittent hypoxia（IH）group,intermittent hypoxia Tempol intervention（IH+Tempol）group,and 3 groups of mice were fed normal diet during molding.Mice in the IH group and IH+Tempol group were placed into a hypoxic chamber for intermittent hypoxia treatment for 8 h per day for 4 weeks.The normal oxygen（Control）group is not placed in the low oxygen tank and is fed normally.The Tempol intervention group administered Tempol(100 mg·kg^-1)gastric lavage daily during the last week of intermittent hypoxia treatment.（1）Effect of oral Tempol on thoracic aortic function in IH mice.After the end of the molding,three groups of mice were taken from the thoracic aorta for vitro vascular tension experiments,and the vascular tone changes in the thoracic aorta of mice after oral Tempol treatment were observed,and the effects of Tempol on vascular endothelial-dependent relaxation and endothelial non-dependent relaxation were explored.（2）Effect of Tempol on weight and heart weight of IH miceRecording the weight values before and after the molding of the three groups mice,and the hearts and tibia of the mice were recorded after the molding was completed.Then analyzing the effects of taking oral Tempol intervention on the hearts of IH mice.（3）ROS content in the thoracic aorta of oral Tempol on IH miceTissue sections were performed on the thoracic aorta of 3 groups of mice,and DHE fluorescent staining of histopathological frozen sections was performed to detect vascular ROS content.（4）Effect of oral Tempol on no content in thoracic aorta of IH miceThe 3 groups of mouse thoracic aorta were stripped clean,ground separately,and the no noo was measured by ELISA method after taking the tissue fluid supernatant.Detection and study of NO concentration and bioavailability in the thoracic aorta of mice of different groups.（5）Effect of oral Tempole on the histology of the thoracic aorta of IH mice on thehistology of the thoracic aorta of miceThe hematoxylin-eosin staining（HE）and Masson staining methods were used to visualize and analyze the histomorphological changes of the thoracic aorta under the microscope of the mouse vascular pathology sections.（6）Western blot method detects associated protein expression in the thoracic aorta of miceBy using Western blot,the protein expression changes of total e NOS,NOX4 and SOD containing the thoracic aorta of three different groups of mice were detected and analyzed.Results:1.IH leads to thoracic aortic function impaired and endothelial-dependent diastolic function weakened in mice.Both vitro incubation and oral administration of Tempol can improve this situation.2.IH caused heart hypertrophy in mice,and oral Tempol can inhibit this phenomenon.3.IH caused remodeling of thoracic aortic vessels in mice,while oral Tempol intervention reversed vascular remodeling to a certain extent.4.IH led to increased ROS and decreased NO content in the thoracic aorta of mice,and oral Tempol intervention increased the concentration of NO in the thoracic aorta,which also reduced the content of ROS;5.IH led to decreased SOD and e NOS expression and increased NOX4 expression;oral Tempol downregulated NOX4 and upregulated SOD and e NOS expression,reducing oxidative stress in IH mice.Conclusion:Tempol may improve cardiovascular dysfunction caused by intermittent hypoxia by reducing ROS,increasing the bioavailability of NO.