Regulation Of Hypothalamic Main Clock BMAL1 On Anxiety Like Behavior Induced By Sleep Deprivation In Adolescent Mice

Objective:Lack of sleep time is common among children and adolescents around the world,and in recent years,the sleep time of children and adolescents has gradually shortened,and the decline in sleep time is worrying.Lack of sleep has been reported to cause oxidative damage to the brain and anxiety.The prefrontal cortex（PFC）,a key part of the brain,plays an important role in mood regulation.Sleep duration is controlled by the circadian system,a 24-hour physiological oscillation driven by the circadian clock that regulates hormones,oxidative stress,and metabolism.Studies have shown that the high expression of brain and muscle ARNT-like 1（BMAL1）in the peripheral system can affect metabolism and damage repair,and enhance the body’s antioxidant capacity,but the regulation of the central nervous system has not been clarified.In this study,we intend to establish an animal model of sleep deprivation（SD）in adolescent Kunming mice to explore the regulatory effect of BMAL1,the main circadian clock of the hypothalamus,on the anxiety-like behavior of SD-induced adolescent mice.Methods:In this study,32 3-week-old male Kunming mice were randomly divided into CON^GFPgroup,CON^AAVgroup,SD^GFPgroup and SD^AAVgroup after one week of adaptive feeding,with 8 mice in each group.After grouping,mice in CON^GFPgroup and SD^GFPgroup were labeled with GFP.Mice in CON^AAVgroup underwent BMAL1overexpression adenovirus injection.Mice in the SD^AAVgroup underwent EEG,EMG electrode implantation and BMAL1 overexpression adenovirus injection.Recovery week after surgery begins SD experiments lasting 4 weeks.In the last 4 days of the experiment,the anxious-like behavior and social ability of each group of mice were evaluated by the elevated cross maze experiment and the three-box social experiment.At the end of the experiment,mice were subjected to blood from the eye after isoflurane inhalation anesthesia,and the total protein and total RNA of PFC were extracted.Western blot（WB）method detects PFC circadian protein 1（PER1）,PER2,circadian locomotor output cycles kaput（CLOCK）,p-BMAL1,BMAL1,cryptochrome1（CRY1）and CRY2,nuclear factor erythroid-2-related factor 2（Nrf2）,kelch’s ECH-like ECH-associated protein 1（Keap1）and heme oxygenase-1（HO-1）of protein levels.The RT-qPCR method determines the mRNA levels of the PFC circadian clock Per1,Per2,Clock,Bmal1,Cry1and Cry2,and the oxidative stress signaling pathways Nrf2,Keap1 and HO-1.The biochemical kit detects PFC serum superoxide dismutase（SOD）activity and lipid malondialdehyde（MDA）content.Results:1.Compared with the CON^GFPgroup,the expressions of PFC circadian clock PER1,PER2,CLOCK,p-BMAL1,CRY1 and CRY2 proteins decreased significantly in the SD^GFPgroup（P<0.05）,and the mRNA levels of the circadian clock Per1,Per2,Clock,Bmal1,Cry1 and Cry2 decreased significantly（P<0.05）;Compared with SD^GFPgroup,the expressions of PFC circadian clock PER1,PER2,CLOCK,p-BMAL1,CRY1and CRY2 proteins were significantly increased in SD^AAVgroup（P<0.05）,and the mRNA levels of the circadian clock Per1,Per2,Clock,Bmal1,Cry1 and Cry2 were significantly increased（P<0.05）.2.In the elevated cross maze experiment,compared with the CON^GFPgroup,the number of times and residence time of mice in the SD^GFPgroup entered the open arms were significantly reduced（P<0.05）;Compared with the SD^GFPgroup,mice in the SD^AAVgroup had a significant increase in the number of times and residence time entered the open arms（P<0.05）.3.In the three-box social experiment,compared with the CON^GFPgroup,the number of times and residence time of mice in the SD^GFPgroup entering the E chamber were significantly increased（P<0.05）;Compared with SD^GFPgroup,mice in SD^AAVgroup had a significant increased the number of times and residence time entered S1 chamber（P<0.05）.At the experimental stage of social novelty preference,compared with the CON^GFPgroup,mice in the SD^GFPgroup significantly increased the number of times and residence time of entering the S1chamber（P<0.05）;Compared with SD^GFPgroup,mice in SD^AAVgroup had a significantly increased in the number of times and residence time of entering S2 chamber（P<0.05）.4.Compared with the CON^GFPgroup,the SOD activity of PFC in the SD^GFPgroup decreased significantly,and the MDA content increased significantly（P<0.05）.Compared with the SD^GFPgroup,SOD activity increased and MDA content decreased significantly in SD^AAVgroup（P<0.05）.5.Compared with the CON^GFPgroup,the expression and mRNA levels of Nrf2,Keap1 and HO-1 proteins of PFC in the SD^GFPgroup decreased significantly（P<0.05）.Compared with the SD^GFPgroup,Nrf2,Keap1and HO-1 protein expression and mRNA levels were significantly increased in the SD^AAVgroup（P<0.05）.Conclusion:SD causes decreased expression of the main circadian clock BMAL1 and other circadian clocks PER1,PER2,CLOCK,CRY1 and CRY2,oxidative stress in PFC,imbalance of Nrf2/HO-1 antioxidant system,and anxiety-like behavior in mice.Overexpression of the hypothalamic main circadian clock BMAL1 can rescue PFC oxidative stress,Nrf2/HO-1 antioxidant system repair,and improve anxiety-like behavior caused by SD.