Mechanism Actions Of D-glucosamine Regulating Circadian Rhythms

Background:The circadian rhythm refers to the biological rhythms of life activities with a cycle of approximately 24 hours.The circadian rhythm is closely related to human life.It maintains a variety of physiological functions and adjusts the body to respond to changes in the external environment.In mammals,circadian rhythms are controlled by the transcription/translation feedback loop(TTFL)of clock genes.The circadian rhythm is regulated by internal factors(like hormone secretion,etc.)and external factors(such as light conditions,temperature changes,etc).When the body’s endogenous factors or external environmental factors suddenly change,such as cross-time zone flight and shift work caused by the change of external light conditions,the balance of the biological clock will be disturbed,resulting in circadian rhythm disorder.The phase misalignment of circadian rhythm is the main cause of the occurrence and development of circadian rhythm-related diseases.Therefore,the adjustment of the circadian rhythm phase is an important means to treat diseases related to circadian misalignment.At present,the small molecule modulators used to regulate circadian rhythm disorder,such as melatonin,have obvious toxic and side effects,which cannot meet the needs of our soldiers to adjust new rhythm,improve military operation ability and medical support.Therefore,it is critical to discover new drugs that specifically regulate the phase of circadian clock.D-glucosamine(Glc N)is a common amino carbohydrate with immune activity,and has the effects of osteoarthritis treatment,antioxidant,anti-aging,antibacterial and antiviral.The experiments confirmed that it has the function of regulating circadian rhythm phase.At present,there is no report on the application of this compound in regulating circadian rhythm disordes.Objective:To study the regulatory effect of D-glucosamine on circadian rhythm in vivo and in vitro,and explore its related mechanism.It is expected to provide new therapeutic candidate drugs and interventions for the treatment of circadian rhythm disorders caused by shift work and cross-time zone flight.Methods:1.Firstly,at the cellular level,Lumicycle equipment was used to investigate the effects of different concentrations of Glc N on the circadian clock of human osteosarcoma cell lines(Per2-d Luc U2 OS,D15)transfected with Per2 luciferase.The effect of Glc N administration at different time points on the circadian clock was also studied.2.In animals,the above experimental results were verified through the wheelrunning behavior experiments in mice.After controlling the light time of day and night light to form a stable 12h/12 h light/dark circadian rhythm in C57BL/6J mice,the time of turning on the light on was shifted back 8h.The phase shift of mice every day and the time needed to synchronize to a new circadian cycle were analyzed by recording the wheel running activities of mice.3.At the molecular level,the mechanism of Glc N regulating circadian phase shift was studied.The traditional view is that Glc N may affect biological rhythms through Olink-N-acetyl glycosylation modification(O-Glc NAc).Using intermediate metabolites in Hexosamine-biosynthesis pathway(HBP)of Glc N,O-Glc NAcylation inhibitors DON and si RNA of O-linked N-acetylglucosamine Transferase(OGT),key enzymes in the O-Glc NAc pathway,were used to determine the effects of O-Glc NAc level changes on biological rhythms.4.Western Blot was used to detect the changes of m TOR signaling pathway in U2 OS cells,mouse SCN tissues and liver tissues after Glc N treatment.The AMPK antagonist MRT199665 and m TOR agonist MHY1485 was used to explore whether Glc N action would be blocked.5.Cycloheximide(CHX)was used to study the effect of Glc N on BMAL1 stability.Next,proteasome inhibitor(MG132)and lysosome inhibitor(Chloroquine)were used to investigate the mechanism of Glc N down-regulating BMAL1 stability.6.CUT&Tag assay was used to discover the regulation of Glc N on the transcription of E-box gene.The q PCR experiments were used to verify.7.The competition experiment between Glc N and glucose was conducted to uncover the relationship between Glc N and glucose.The possible target of Glc N was found using the network pharmacology methods.DARTS and CETSA experiments were performed to identify whether GLUT1 was the target of Glc N.Results:1、The phase shift of 5 mmol/L,10 mmol/L and 15 mmol/L Glc N on D15 cells was 5.57 ± 0.25 h,13.23 ± 0.39 h and 17.44 ± 0.41 h,respectively.The effect of Glc N on regulating the phase of rhythm on D15 cells was dose-dependent.The results of Glc N administration at different time points showed that the phase shift reached its peak at CT0 or CT24 and fell to trough at CT12,indicating that the effect of Glc N on regulating the circadian phase was time-dependent.2 、 Compared with the control group(7.40 ± 0.24 days),Glc N significantly promoted the circadian phase shift of mice,and 600 mg/kg Glc N(5.00 ± 0.00 days)was better than 300 mg/kg Glc N(5.50 ± 0.22 days),indicating that,Glc N also promotes phase shift in a dose-dependent manner at the animal level.3、The results showed that only Glc N changed the phase of the biological rhythm in the HBP pathway.Nerther O-Glc NAc inhibitors nor OGT si RNA could block the phase delay of circadian rhythm caused by Glc N.4、The results looking upstream of m TOR showed that p-AMPK/AMPK was upregulated and p-AKT/AKT and p-ERK/ERK were almost unchanged,indicating that Glc N selectively activated tha AMPK/m TOR pathway.The changes of m TOR downstream pathway showed that p-m TOR/m TOR,P70S6 K,p-BMAL1/BMAL1 were down-regulated after Glc N treatment.The combination of Glc N and MRT199665 or MHY1485 attenuates the phase shift effect of Glc N.5、After CHX treatment,the BMAL1 half-life of Glc N was shorter(4.362 h)than that of the control group(7.441 h),indicating that Glc N promoted the degradation of BMAL1.The BMAL1 level after MG132 and Glc N administration was significantly higher than that after Glc N administration,while the BMAL1 level after chloroquine and Glc N administration was lower,suggesting that Glc N promoted the degradation of BMAL1 through the proteasome pathway.6、After Glc N administration,the peak value of E-box gene decreased,indicating that Glc N inhibited E-box gene transcription.After Glc N administration,the phase of Ebox gene transcription rhythm shifted backward7、Slc2a1,a gene encoding glucose transporter 1(GLUT1),was identified as one of the rhythm-related target genes of Glc N by network pharmacology.In the competitive experiment,the higher the concentration of Glc N,the lower the concentration of glucose,indicating the competitive relationship between Glc N and glucose.DARTS and CETSA experiments showed that GLUT1 remained at a high level after Glc N administration under different concentrations of protease and different temperatures,confirming that GLUT1 was a potential target for Glc N.Conclusions: Based on the above results,we draw the following conclusions: 1.Glc N can promote phase delay of biological rhythm at the cellular level,and this effect is dose-and time-dependent.2.At the animal level,Glc N can accelerate rapid reentrainment in 8-hour phase delay mouse jet-lag model,and the effect is also dosedependent.3.Glc N affects circadian phase through AMPK/m TOR pathway rather than O-Glc NAcylation.4.Glc N promotes the degradation of BMAL1 through the proteasome pathway and down-regulates the stability of BMAL1 protein.The above conclusions suggest that Glc N can play a role in regulating circadian rhythm.In view of the current situation of the urgent need for drugs regulating circadian rhythm disorder,Glc N,as a widely used health care product,has a good application prospect in the treatment of circadian rhythm disorder.