| BackgroundMilitary stress is a sort of psychological and physiological responses to the cognitive evaluation of the military activity circumstances and the military activity forms.As military stress is often unpredictable,and the forms of military stresses such as continuous combat,disruption of work and rest,extreme fatigue,extreme weather and climate,violent terrorist scenarios and even weapons of mass destruction are often intense and compounded,part of soldiers experience depression,anxiety,other emotional disorders and impaired cognitive function,which seriously affects the combat effectiveness of troops and causes non-combat attrition.According to a research on 1100 soldiers participating in a manoeuvre,the positive rate of military stress symptoms was as high as 85.0%,and the degree of depression and anxiety among soldiers gradually increased as the detection rate of stress symptoms increased.It is important to conduct research on chronic unpredictable military stress(CUMS)protection.The medial prefrontal cortex(m PFC)and hippocampus are important areas of the brain that control attention,executive functions,memory,and emotional responses,and patients with mood disorders and cognitive deficits have reduced volume and altered synaptic plasticity in the corresponding brain areas.The pathogenesis of imbalance in cognitive and emotional homeostasis due to chronic stress is complex,with reduced synaptic plasticity,mitochondrial dysfunction,and immune inflammation being associated with its onset and development.Recent studies suggest that disruption of circadian rhythms due to chronic unpredictable stress may be the initiating mechanism of altered cognitive and emotional homeostasis.Silent information regulator 1(SIRT1)is a nicotinamide adenine dinucleotide(NAD~+)-dependent deacetylase implicated in various cellular functions,including cellular senescence,lifespan extension,resistance to oxidative stress and energy metabolism.Recent studies have shown that SIRT1 is highly expressed in the human and rodent central nervous systems,especially in regions such as the prefrontal cortex,hippocampus,and basal ganglia.On the other hand,hippocampal SIRT1 expression and activity are reduced in various rodent models of cognitive impairment and depression.Its expression is upregulated by antidepressant drugs,suggesting that SIRT1 may play an essential role in regulating cognition and emotional dyshomeostasis.Previous studies have shown that SIRT1 regulates the transcription of the synaptic plasticity-related proteins cyclic-AMP response binding protein(CREB)and brain-derived neurotrophic factor(BDNF),thereby affecting synaptic plasticity.On the other hand,SIRT1 also modulates the activity of the peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α)through its deacetylation modification.PGC-1αbinds to its downstream target gene,nuclear respiratory factor 1(NRF-1),to co-transcriptionally activate the expression of mitochondrial transcription factor A(TFAM),thereby regulating mitochondrial transcription and biosynthetic function.An increasing number of studies have demonstrated that the circadian clock regulates the transcriptional expression and activity of SIRT1.The core clock gene circadian motor output cycle kaput(CLOCK)/aromatic hydrocarbon receptor nuclear transporter-like protein 1(BMAL1)influences the intracellular concentration of NAD~+by driving the transcription of the clock-controlled gene NAD~+synthase,nicotinamide phosphate ribosyltransferase(NAMPT),which in turn affects the regulation of NAD~+/NADH ratio dynamics,thereby regulating the rhythmic oscillations of the SIRT1 enzyme.Therefore,we hypothesize that circadian clock disruption and SIRT1abnormalities under chronic stress are important pathological mechanisms that induce changes in cognitive function and emotional homeostasis and that correcting the circadian clock system and thus maintaining in SIRT1 expression and biorhythm may be essential for the regulation of cognition and emotional homeostasis.Pterostilbene(PTE),a phytochemical widely found in berries such as blueberries,blackberries and grapes,possesses lipid-lowering,antidiabetics,anti-inflammatory,antioxidative properties has hypolipidemic,hypoglycemic,antioxidant,anti-inflammatory and other health biological effects.Foreign studies have found that oral administration of PTE can affect neurogenesis in hippocampus and medial prefrontal cortical areas through BDNF/CREB-related signaling pathways,thereby improving behavior and alleviating depression-like symptoms in chronic stress rats and;in addition,In addition,PTE has been shown to improve cognitive function in aging-related diseases such as Alzheimer’s disease,but the exact mechanism underlying is unclear..Our group’s previous study confirmed that PTE could improve endurance in mice under sleep deprivation conditions,and it is speculated that its protective effect may be related to the maintenance of skeletal muscle circadian clock rhythm.Retinoic acid-related orphan nuclear receptors(RORs)are transcriptional regulators that are members of the steroid-thyroid-retinoid receptor superfamily and can be involved in the transcriptional regulation of the core circadian clock gene BMAL1,are also key genes in glucolipid metabolism and plays a critical role in metabolism.Through literature and conformational analysis,PTE is structurally similar to the RORγagonist nobiletin,therefore it is important to confirm whether can PTE participate in SIRT1 rhythm maintenance as a small molecule regulator of circadian clock and thus ameliorate cognitive and emotional dyshomeostasis in CUMS-exposed mice.Based on the above analysis,we propose the hypothesis that"PTE can activate RORγand influence the ROR-BMAL1 pathway to participate in the rhythmic oscillatory regulation of SIRT1 activity and maintain cognitive functions and homeostasis of emotional under unpredictable military stress conditions".In this study,we propose to investigate whether PTE can improve the cognitive function and emotional dyshomeostasis in CUMS-exposed mice by using behavioral experiments,immunoblotting,biorhythm analysis,luciferase reporter gene assay and small animal live imaging techniques,and focus on the role of circadian clock-mediated SIRT1 regulation and its molecular mechanism.This study will lay the theoretical foundation for the use of Pterostilbene in the maintenance of emotional and cognitive functions of military operators under chronic military stress conditions.ObjectiveTo investigate the protective effects of PTE on cognitive function and emotional homeostasis in CUMS-exposed mice and its mechanism,and to provide scientific experimental evidence for the application of PTE in the prevention and treatment of cognitive decline and depression-like mood caused by chronic military stress,and the protection of military groups.Methods1.6-8 week old C57BL/6J male mice were randomly divided into control(CON),chronic unpredictable military stress(CUMS),and pterostilbene intervention(CUMS+PTE)groups.Food consumption and body weight of the mice were regularly recorded during the CUMS exposure phase,and behavioral experiments were performed the next day after CUMS exposure.Mitochondrial and neuronal morphology and excitatory synaptic density in the CA1 and m PFC region were observed by TEM,and dendritic spine density in the hippocampus and m PFC region was detected by Golgi staining;the mitochondrial DNA copy number,Sirt1 gene,mitochondrial biosynthesis-related genes Pgc-1α,Nrf1,and Tfam,and synaptic plasticity-related genes Creb,Bdnf,and Syp were detected by RT-q PCR,and the expression levels of SIRT1,PGC-1α,CREB,BDNF and SYP protein in mouse hippocampus and m PFC region were detected by Western blot.2.The CLOCKLAB biorhythm collection system was used to analyze the circadian rhythms of spontaneous activity of mice;serum and tissue samples were taken at four time points,ZT2(10:00),ZT8(16:00),ZT14(22:00),and ZT20(04:00),and serum corticosterone levels at different time points were measured using ELISA kits;RT-q PCR and western blot were used to detect Clock,Bmal1,Rorγm RNA and protein expression levels in the hippocampus and m PFC region at different time points;ELISA kits were used to detect SIRT1 protein expression levels in the hippocampus and m PFC region at different time points;RT-q PCR and Western blot were used to detect Nampt m RNA and protein expression levels at different time points;colorimetric kit was used to detect the NAD~+/NADH ratio at each time point.The circadian rhythms of the above indicators were assessed by cosine analysis.The Rorγgene adenovirus vector and the Bmal1 gene adenovirus vector with fluorescent tag were transfected into the hippocampus and m PFC region of mice separately by microstereotopic injection technique,using in-vivo imaging system to determine the level of Bmal1 translation in the hippocampus and m PFC of CUMS-exposed mice.Results1.PTE significantly improved cognitive function and emotional dyshomeostasis in CUMS-exposed mice.(1)The physical condition of mice:After 30 days of CUMS exposure,the body weight and food consumption of mice in the CUMS group were significantly reduced compared with those in the CON group(P<0.0001),while the body weight and food consumption of mice in the CUMS+PTE group were significantly higher than those in the CUMS group(P<0.05).Serum corticosterone levels in mice in the CUMS group were significantly higher than those in the CON group,while serum corticosterone levels in mice in the CUMS+PTE group were significantly lower than those in the CUMS group(P<0.05).(2)Behavioral tests about cognition:compared with the CON group,the number of platform crossing in the Morris water maze,the new object recognition index,and the spontaneous alternation rate in the Y-maze were significantly lower in the CUMS group(P<0.05);compared with the CUMS group,the number of platform crossing in the Morris water maze,the new object recognition index,and the spontaneous alternation rate in the Y-maze were significantly higher in the CUMS+PTE group(P<0.05).PTE significantly improved the cognition of CUMS-exposed mice.(3)Behavioural tests about the depressive emotion:Compared with the CON group,the CUMS mice showed significantly lower sucrose preference rate,number of cross-frames and activity distance in the open-field test(P<0.05),and significantly longer immobility time in the open-field test and longer immobility time in the tail suspension test(P<0.05);compared with the CUMS group,the CUMS+PTE mice showed significantly higher sucrose preference rate,number of cross-frames in the open-field test and activity distance to(P<0.05),and significantly lower immobility in the open-field experiment and longer immobility time in the tail suspension test(P<0.05).The PTE intervention significantly improved the depression-like behavior of CUMS-exposed mice.2.PTE regulates the SIRT1-PGC-1αpathway and the SIRT1-CREB-BDNF pathway to promote mitochondrial structure,function,and synaptic plasticity in CUMS-exposed mice.(1)PTE intervention may refine mitochondrial structural damage and respiratory and biosynthetic function in CUMS-exposed mice,possibly through modulation of the SIRT1-PGC-1αpathway.We observed the neuronal subcellular fraction by TEM:neuronal mitochondria in the CA1 region of the hippocampus were less damaged in the CUMS+PTE group than in the CUMS group;Neuronal mitochondria in the m PFC region of the CUMS+PTE group were less damaged than in the CUMS group,which were in accordance with the TEM findings in CA1.Experiments on mitochondrial respiratory function of the isolated hippocampus:Compared to the CON group,basal respiration,maximal respiration,and reserve respiration were significantly increased in the CUMS group(P<0.05),while maximal respiration,ATP-linked respiration,and respiratory control rate were improved considerably in the CUMS+PTE group compared to the CUMS group(P<0.05).Molecular biology experiments:mt DNA copies in the hippocampus and m PFC region were significantly higher in the CUMS+PTE group compared to the CUMS group(P<0.05).Meanwhile,the levels of PGC-1α,Nrf-1,Tfam,Sirt1 m RNA,SIRT1,and PGC-1αprotein in the hippocampus and m PFC region were significantly upregulated the CUMS+PTE group compared to the CUMS group(P<0.05).(2)PTE intervention may maintain synaptic plasticity of CUMS-exposed mice by modulating the SIRT1-CREB-BDNF pathway.Excitatory synaptic densities calculation of TEM imaging:Those of CA1 and m PFC of mice in the CUMS group were lower than those in the CON group(P<0.05),and excitatory synaptic densities in CA1 and m PFC regions of mice in the CUMS+PTE group were higher than those in the CUMS group(P<0.05).The result of Golgi staining indicates that:Compared with the CON group,the dendritic spine density of CA1 and m PFC neurons was significantly lower in the hippocampal and m PFC regions of mice in the CUMS group(P<0.05).Compared with the CUMS group,mice in the CUMS+PTE group had greater dendritic spine density in the CA1 region(P<0.05),and the difference in the m PFC region was not statistically significant.Molecular biology experiments:Compared with the CON group,mice in the CUMS group had significantly lower Creb,Bdnf,and Syp m RNA in the hippocampus and m PFC regions and similarly lower CREB,BDNF,and SYP protein expression;PTE intervention increased Creb,Bdnf,Syp m RNA,as well as CREB,BDNF and SYP protein in the hippocampus and m PFC of CUMS-exposed mice(P<0.05).3.PTE significantly restored circadian rhythms in CUMS-exposed mice,and modulated NAMPT and NAD~+/NADH ratios in the hippocampus and m PFC,therefore significantly restoring circadian oscillations of the SIRT1 enzyme.(1)Mice in the CUMS group showed reduced circadian rhythm of spontaneous activity,serum corticosterone level,and circadian rhythm disruption of Clock,Bmal1,Rorγm RNA and CLOCK,BMAL1,and RORγproteins in the hippocampus and m PFC;PTE intervention significantly restored circadian rhythm oscillation of spontaneous activity,serum corticosterone level,core circadian clock genes Clock,Bmal1,Rorγm RNA,and CLOCK,BMAL1,RORγproteins in the hippocampus and m PFC of CUMS exposed mice(2)In addition,the circadian rhythm of Sirt1,Nampt m RNA,SIRT1,and NAMPT proteins in the hippocampus and m PFC of mice in the CUMS group was disrupted compared with the CON group;PTE intervention significantly restored circadian rhythm oscillations of Sirt1,Nampt m RNA,and SIRT1 and NAMPT proteins in the hippocampus and m PFC of mice in CUMS.(3)Compared with CON group,the circadian rhythm of NAD~+/NADH ratio in hippocampus and m PFC of CUMS group was disturbed,and PTE intervention significantly restored the circadian oscillation of NAD~+/NADH ratio in hippocampus and m PFC of CUMS exposed mice.By modulating circadian clock–NAMPT-NAD~+/NADH ratio,PTE may restore the circadian oscillations of SIRT1.4.PTE is involved in the regulation of the circadian clock as a small molecule regulator of RORγ.After molecular docking and conformational analysis using MGLtoosl 1.5.6 and Auto Dock＿vina 1.1.2 software,it was tentatively determined that PTE may combine with RORγ,and then effect of PTE on RORγand BMAL1 were detected using luciferase reporter gene technology combined with in vivo imaging:The expression levels of BMAL1 in the hippocampus and m PFC of ROR~+-transfected mice in all groups were higher than those in the ROR~--transfected group;the fluorescence intensity in the hippocampal region of ROR~+-transfected mice in all groups was higher at ZT14 than at ZT2;the fluorescence intensity in the hippocampus of the CUMS/ROR~+group was significantly lower than that of the CON/ROR~+group at both ZT2 and ZT14(P<0.05);The fluorescence intensity in the hippocampus of the CUMS/ROR~+group was significantly lower than that of the CON/ROR~+group at ZT14(P<0.05);The fluorescence intensity in the hippocampus and m PFC region of the PTE/ROR~+group was significantly higher than that of the CUMS/ROR~+group at ZT14(P<0.05).PTE upregulates RORγexpression and thus restores circadian oscillations of the core circadian clock molecule BMAL1,as determined by the luciferase reporter gene technology combined with in vivo imaging of small animals;PTE may bind RORγand participate in circadian clock regulation.ConclusionIn conclusion,PTE can effectively improve cognitive and emotional impairment in CUMS-exposed mice;PTE may modulate RORγactivity and thus participate in the circadian clock regulating the circadian oscillatory rhythm of NAMP activity and NAD~+/NADH ratio in the hippocampus and m PFC,which in turn regulates the circadian oscillatory rhythm of SIRT1 and affects mitochondrial function and synaptic plasticity.This study will provide new insights into the prevention and treatment of cognitive and emotional imbalances caused by military stress exposure. |
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