Role Of BMAL1 In Chronic Sleep Deprivation Induced Appetite Dysregulation In Adolescent Mouse Hypothalamus

Objective:To investigate the effect of chronic sleep deprivation on the imbalance of central appetite in developing mice,to clarify the role of the expression of the biological clock gene BMAL1 on the regulation of the appetite-related pathway JAK2/STAT3,to reveal the mechanism of imbalance of central appetite regulation in developing mice due to sleep deprivation,and to further investigate the causes of childhood obesity and provide experimental reference data for its prevention and treatment targets.Methods:32 Kunming mice aged three weeks were randomly divided into a null control group(CON^GFP,n=8),a BMAL1 overexpression control group(CON^BMAL1,n=8),a sleep deprivation+null control group(SD^GFP,n=8),and a sleep deprivation+BMAL1 overexpression group(SD^BMAL1,n=8)after 7 days of adaptive feeding.In the CON^GFPgroup,adeno-associated viral vectors containing only the green fluorescent protein GFP were injected into the SCN region of the hypothalamus;in the CON^BMAL1group,adeno-associated viruses containing the biological clock gene bmal1 were injected into the SCN region of the hypothalamus;in the sleep deprivation group,sleep deprivation was performed daily（19:00-13:00）using a rodent sleep deprivation device,with 6 hours of free sleep;both empty and bmal1-containing adeno-associated viruses were injected at the same time before electrode implantation in the sleep deprivation group.Mice were fed a maintenance diet,deionized water,and given a12-hour light-dark cycle with lights on at 7:00 am.The mice in each group were placed in the Sleeplab sleep chamber and fed and watered freely.The amount of food and body weight of each group of mice were recorded every day and night.The software recorded all electrophysiological signals of the mice,and after 4 weeks of sleep deprivation,the mice were executed by uniform isoflurane anesthesia,and blood and brain tissues were collected.Fresh serum was used to detect Leptin,NPY andα-MSH levels by ELISA assay,and brain tissues were stored in deep-freeze refrigerator at-80℃.The infection effect of bmal1 overexpression was detected by immunofluorescence assay,and the in situ expression of p-STAT3,AGRP,NPY,and POMC proteins were detected.The protein expression levels of CLOCK,BMAL1,CRY1,CRY2,PER1,PER2,Lep Rb,JAK2,p-JAK2,STAT3,p-STAT3,SOCS3,AGRP,NPY,and POMC were detected by Western Blot.The m RNA levels of clock,bmal1,cry1,cry2,per1,per2,agrp,pomc were detected by Realtime RT-PCR.Results:1.BMAL1 overexpression in the SCN region of the hypothalamus ameliorated the overeating behavior induced by chronic sleep deprivation.mice in the SD^GFPgroup gained significantly more weight than the CON^GFPand SD^BMAL1groups（P<0.05）.Compared with the CON^GFPand SD^BMAL1groups,food intake was significantly increased in the SD^GFPgroup mice starting from the third week of sleep deprivation（P<0.05）.2.BMAL1 overexpression in the SCN region of the hypothalamus rescued the chronic sleep deprivation-induced decrease in biological clock gene expression.The levels of BMAL1,CLOCK,CRY1,PER1 and PER2proteins were significantly lower in the hypothalamus of SD^GFPgroup mice compared to the CON^GFPgroup（P<0.05）.changes in clock,bmal1,cry1,per1 and per2 m RNA levels were consistent with the changes in proteins（P<0.05）.3.BMAL1overexpression in the hypothalamic SCN region ameliorated chronic sleep deprivation-induced impairment of JAK2/STAT3 signaling pathway transduction.Compared with the CON^GFPgroup,the SD^GFPgroup showed reduced levels of JAK2and STAT3 phosphorylation,upregulated expression of the pro-appetite peptides AGRP and NPY protein（P<0.05）,and downregulated expression of the appetite suppressing POMC protein in the hypothalamus.changes in POMC,AGRP and NPY m RNA levels were consistent with changes in protein（P<0.05）.p-STAT3 inhibition factor,SOCS3,was significantly upregulated during chronic sleep deprivation（P<0.05）.The SD^BMAL1group reversed the changes in protein and m RNA levels of the JAK2/STAT3 signaling pathway under sleep deprivation conditions.Conclusions:The imbalance of central appetite regulation caused by sleep deprivation in developing mice caused an increase in dietary weight which may be related to the down-regulation of biological clock protein expression and the blockage of JAK2/STAT3 signaling pathway transduction;the chronic sleep deprivation-induced blockage of JAK2/STAT3 signaling pathway transduction and appetite regulation disorder was improved after overexpression of the biological clock gene BMAL1 in the SCN region of hypothalamus in sleep deprived mice.