The Behavioral Effects Of Sleep Deprivation On High-fat-fed ApoE^-/- Mice And The Preliminary Investigation Of Its Molecular Mechanism For Promoting Carotid Atherosclerosis

Objective:This paper will construct the chronic sleep deprivation（CSD）model in mice by using gentle stimulation method through sleep deprivation equipment,analyze the behavioral effects of sleep deprivation（SD）on high-fat fed ApoE^-/-mice and preliminarily explore the molecular mechanism of SD to accelerate the progression of Atherosclerosis（AS）based on the changes of inflammatory indexes and proteomic studies.Methods:1.Sixty SPF 7-week-old male ApoE^-/-mice were randomly and equally divided into Control group,SD group,Cannula group,sham-operated（Sham）group and cannula+SD（Cannula＿SD）group,with 5 groups and 12 in each group.The general status and the body weight of the mice was recorded throughout the experiment.After adaptively feeding to 8 week-old,all mice were fed high-fat.The cannula was placed in right carotid artery of Cannula group and Cannula＿SD group cannulated group for carotid AS modeling at 8 week-old.The 12week-old mice in the SD and Cannula＿SD groups completed 28 days of SD modelling with the sleep deprivation equipment.Then,mice were subjected to behavioural experiments（open field test and Y-maze）to analyze changes in emotions（such as anxiety,depression）and the ability of working memory.After high-fat feeding to 16 weeks of age,the mice were executed.We collected blood and carotid artery tissue specimens,weighed the liver,spleen and kidneys to assess metabolic levels.2.Carotid atherosclerotic plaque progression was observed by HE staining and Oil Red O staining to assess the superior method of AS modelling.Lipid levels（total cholesterol（TC）,triglycerides（TG）and low-density lipoprotein cholesterol（LDL））and inflammatory factors（tumour necrosis factor-α（TNF-α）,interleukin-6（IL-6）and IL-1β）were examined by ELISA.Differentially expressed proteins of SD accelerating carotid atherosclerosis were selected by proteomic analysis.Based on the proteomic results,the differentially expressed proteins were verified at the gene level by RT-PCR.Results:1.This study showed that the SD model could be successfully constructed by the sleep deprivation equipment（gentle stimulation method）.In the early stage of SD,the mice in the sleep deprivation group（SD and Cannula＿SD group）changed from the normal status to the aroused status,and later presented a depressed status;with the extension of SD,the hair of mice gradually sparse,greasy and rough.Initially,the weight of the mice in the sleep deprivation group decreased significantly compared with the non-sleep deprivation group（SD group vs Control group,Cannula＿SD group vs Cannula group）;as the mice adapted to the sleep deprivation equipment,the weight in the sleep deprivation group（SD group,Cannula＿SD group）increased slowly,and the rate of increase was lower than that in the non-sleep deprivation group;in the late SD period,the weight of the mice in the sleep deprivation dropped again due to the reduction in food intake.The liver and kidney weight indexes were significantly higher but the spleen weight index was lower（P<0.05）in the sleep deprivation group compared with the non-sleep deprivation group（SD vs Control,Cannula＿SD vs Cannula）.The behavioural experiments indicated that the total distance and the average speed in the open field of the sleep deprivation group were significantly lower（P<0.05）than in the non-sleep deprivation group（SD vs Control,Cannula＿SD vs Cannula）;Similarly,the spontaneous alternation accuracy in the Y-maze was significantly lower in the sleep deprivation group（P<0.05）.2.HE staining and red oil O staining suggested that SD could obviously accelerate the progression of carotid atherosclerotic plaques;high-fat fed ApoE^-/-mice combined with carotid cannulation（genetic engineering+diet+physical injury method）is a superior method for constructing carotid AS model.Lipid levels（LDL,TG,TC）and inflammatory levels（TNF-α,IL-6,IL-1β）were significantly higher（P<0.05）in the sleep deprivation group compared with the non-sleep deprivation group（SD vs Control,Cannula＿SD vs Cannula）.Proteomic analysis showed that SD accelerates AS progression through NOD-like receptor mediated MAPK/NF-κB pathway;The functional annotation of GO and KEGG identified several differentially expressed proteins such as MMP9,Casp1,Rxra,Keap1 and Bcl2 that were enriched in the AS pathway.Conclusion:1.SD makes the mice present the mental state from excitement to depression;the weight goes through a short period of decline and gradually regains,then decreases again in the late SD.SD increases metabolic levels.SD can cause emotional changes（such as anxiety and depression）and impair learning memory in mice.2.SD can accelerate the progression of AS that high-fat fed ApoE^-/-mice combined with arterial cannulation is superior method for building carotid AS model.SD can disrupt the balance of lipid metabolism and enhance the inflammatory response.And SD accelerates AS progression through the MAPK/NF-κB pathway;There are several differentially expressed proteins enriched in the AS pathway such as MMP9,Casp1,Rxra,Keap1 and Bcl2.