The Effects And Underlying Mechanism Of Pterostilbene On Improving Endurance And Circadian Misalignment Of Mice Subjected To Sleep Restriction

BackagroudWith the fast pace of modern life and the changing pattern of work,insomnia or lack of sleep has become a social norm.Millions of people worldwide suffer from sleep restriction(SR)or be in potential SR status which has led to many health problems.The sleep-wake cycle interacts with circadian system to control the endogenous time and regulate physiological functions and behaviors.Endurance is one of the most basic elements of physical fitness.It is essential to maintain health and guarantee the quality of life.Recent studies have found that circadian misalignment caused by SR is an independent risk factor for the decrease of endurance.Therefore,how to correct the decline in endurance caused by SR has attracted more and more attention,and it is urgent to find a way to deal with it.The sleep-wake cycle is the basic rhythm of body.Adequate sleep and sleep quality play important roles in eliminating fatigue,regaining strength,and enhancing immunity.SR changes the normal sleep-wake cycle.The most direct and earliest effect is disrupting the normal circadian rhythm.Increasing evidences indicate that SR have adverse effects on health.SR is closely related to the occurrence and development of metabolic diseases,which can increase the risk of obesity and metabolic syndrome characterized by elevated blood glucose and triglycerides,insulin resistance,type 2 diabetes,and cardiovascular disease.Previous studies have shown that SR enhances the expression of genes related to protein degradation signaling pathways in skeletal muscle tissue,leading to reductions in skeletal muscle mass and the exercise capacity.Meanwhile,SR impairs immunity,promotes the secretion of a large number of inflammatory cytokines,impedes the damage repair of muscles,and causes autonomic nerve system dysfunction.In addition,studies have shown that SR impaired brain function and affected judgment and decision-making during exercise.Therefore,sleep deficiency will disturb metabolic balance,affect functions of immune system and exercise system,and so on,which would ultimately affect exercise capacity.However,the mechanism of circadian misalignment caused by SR and the decline of exercise tolerance remains unclear,and there are no effective prevention measures at present.Mitochondria are �energy factories� of the body and produce ATP for movements.A lot of studies have shown that impaired mitochondrial biogenesis caused mitochondrial dysfunction,leading to decreased exercise tolerance.Peroxisome proliferator-activated receptor gamma coactivator 1?(PGC-1?)is a key regulator of mitochondrial biogenesis.Adenosine 5'-monophosphate activated protein kinase(AMPK)and Silent Information Regulator 1(SIRT1),energy sensors,can regulate PGC-1? expression and promote mitochondrial biogenesis in skeletal muscle tissue.AMPK and SIRT1 can not only regulate each other but also interact with circadian locomotor output cycles kaput(CLOCK)/brain and muscle-ARNT-like1(BMAL1)dimers(CLOCK/BMAL1)to establish a functional and molecular link between energy metabolism and circadian rhythm.Recently,growing number of studies have shown that polyphenol phytochemicals could regulate the expression of circadian genes in peripheral clock tissues,and even reset peripheral clocks.Pterostilbene(PTE)is a polyphenol phytochemical widely found in berries such as grapes,and has a wide range of health benefits such as hypolipidemic,hyperglycemic,anti-oxidation,and anti-inflammatory.It has been reported that PTE can also act on AMPK and SIRT1 to exert biological effects.Therefore,we speculate that PTE might improve endurance in SR mice by regulating the AMPK/SIRT1/PGC-1? signaling pathway,increasing mitochondrial biogenesis and regulating the circadian oscillation of skeletal muscle core clock gene expression.ObjectiveThe purpose was to clarify the protective effects of PTE on exercise endurance in SR mice and its underlying mechanism in order to provide an experimental basis for its application in the prevention and treatment of exercise intolerance and circadian misalignment caused by SR.Methods1.Male C57BL/6 mice(6-8 weeks old)were randomly assigned into control group,sleep restriction group,sleep restriction+low-dose-Pterostilbene(10 mg/(kg?d))group,sleep restriction+medium-dose-Pterostilbene(50 mg/(kg?d))group,sleep restriction + high-dose-Pterostilbene(100 mg/(kg?d))group.SR mice were kept awake using a new sleep deprivation apparatus with their daily sleep time limited to 4 hours for 5 consecutive days.Meanwhile,the mice received PTE treatment by gavage.Weight-loaded forced swimming test was performed at the observation end point.The exhausted swimming time and serum biochemical parameters of mice in each group were compared to evaluate the protective effects of different doses of PTE on exercise endurance in SR mice and to screen the optimal intervention dose.2.Based on the successful establishment of SR mouse model,we continued to study the protective mechanism of 100 mg/(kg?d)intervention dose of Pterostilbene for the exercise endurance of SR mice.Hematoxylin and eosin staining was applied to observe the pathological changes of skeletal muscle tissue.Transmission electron microscopy was used to observe morphological and quantity changes of mitochondria.Mitochondrial oxidative phosphorylation system(OXPHOS)related genes expression,mitochondrial DNA(mtDNA)copy number,mitochondrial biogenesis-related genes expression were determined by real-time PCR and immunoblot in order to evaluate the effects of PTE on mitochondrial biogenesis of skeletal muscle in SR mice.Furthermore,the changes of AMPK,p-AMPK,SIRT1,and PGC-1? protein expression levels were detected by immunoblot to determine whether PTE functions by regulating AMPK/SIRT1/PGC-1? signaling pathway.3.To observe the effects of Pterostilbene 100 mg/(kg?d)intervention on the skeletal muscle circadian clock of SR mice and its possible mechanism.Mice were randomly divided into control group,sleep restriction group,sleep restriction+Pterostilbene(100 mg/(kg?d))group.Samples were taken at 5 time points,including ZT0(08:00),ZT6(14:00),ZT12(20:00),ZT18(02:00,the next day)and ZT24(08:00,the next day).ELISA kits were used to detect AMPK phosphorylation activity and SIRT1 deacetylation activity in skeletal muscle tissue at five time points.Immunoprecipitation was applied to detecte deacetylated PGC-1? levels at five time points.Real-time PCR and immunoblotting were used to detect the m RNA and protein expression levels of core clock gene CLOCK and BMAL1 in skeletal muscle at five time points.Cosine analysis was used to evaluate the circadian rhythm of the above indicators to identify the effects of PTE on the circadian rhythm expression of the skeletal muscle clock genes in SR mice.The main results and conclusions1.The exhaustive swimming time of SR mice was significantly shortened,and the exercise endurance was significantly reduced.PTE effectively improved exercise endurance and increased anti-fatigue ability in mice subjected to SR.The exhausted swimming time of SR group mice was 7.60 � 1.42 minutes,which was significantly shortened by 31.55% compared with CON group,and the levels of serum biochemical parameters LDH,CK,and BUN were significantly increased,suggesting that SR resulted in the decrease of exercise endurance and anti-fatigue ability of mice.PTE prolonged the time in SR mice,and reduced the concentration of serum exercise fatigue biochemical parameters to some extent,indicating that PTE could improve the exercise endurance and anti-fatigue activity of mice subjected to SR.Among them,the 100 mg/(kg?d)dose group had the most significant intervention effect.The exhausted swimming time of the mice was 10.71 � 1.40 min,which was significantly longer than the SR group by 40.96%,and the levels of LDH and CK were significantly reduced.Meanwhile,PTE inhibited the decrease in body weight and the abnormal changes in serum TG,TC,HDL-C,LDL-C and FPG levels,reduced HOMA-IR,and ameliorated glucose and lipid metabolism disorder in SR mice.2.PTE significantly attenuated mitochondrial swelling,increased mitochondrial biogenesis and improved mitochondrial function in skeletal muscle of SR mice.PTE significantly attenuated mitochondrial swelling,and increased the number of intermyofibrillar mitochondria and the percentage of normal mitochondria in skeletal muscle tissue of SR mice.Also,it increased OXPHOS-related genes expression,mtDNA copy number and mitochondrial biogenesis-related genes(PGC-1?,NRF-1,ERR?,and TFAM),indicating that PTE can significantly improve mitochondrial biogenesis and mitochondrial function in SR mice.3.PTE regulated AMPK/SIRT1/PGC-1? signaling pathway in skeletal muscle of SR mice and significantly restored circadian rhythm of AMPK phosphorylation activity and SIRT1 deacetylation activity.PTE significantly increased the p-AMPK/AMPK ratio and upregulated the expression of SIRT1 and PGC-1?.Also,PTE significantly restored circadian rhythm of AMPK phosphorylation activity,SIRT1 deacetylation activity in SR mice as well as the circadian rhythm oscillation of deacetylation level of PGC-1?.It indicated that PTE can regulate AMPK/SIRT1/PGC-1? signaling pathway to improve mitochondrial biogenesis in skeletal muscle of SR mice.4.PTE significantly restored circadian oscillation of core clock genes Clock and Bmal1 mRNA levels,BMAL1 protein expression,and regulated CLOCK protein expression in skeletal muscle of SR mice.PTE significantly restored circadian oscillation of the mRNA expression of clock and bmal1 and the protein expression of BMAL1 in skeletal muscle of SR mice.Compared with the SR group,clock mRNA levels in SR+PTE group were significantly increased at ZT0,ZT6,and ZT24;bmal1 m RNA levels were no significantly changed at all time points;BMAL1 protein expression was higher at ZT0,ZT6,ZT18,and ZT24.In addition,the expression of CLOCK protein in skeletal muscle of each group did not show circadian rhythm oscillations.The expression of CLOCK protein in SR group was significantly decreased at ZT0 and increased at ZT12.The expression of CLOCK protein in SR+ PTE group was significantly increased at ZT0 and ZT18,and decreased at ZT12.The results showed that PTE can regulate circadian clock in skeletal muscle and ameliorate circadian misalignment of core clock genes expression of mice subjected to SR.In summary,by regulating the integration of circadian rhythm and mitochondrial biogenesis key regulatory pathway AMPK/SIRT1/PGC-1?,PTE can improve mitochondrial biogenesis,regulate the rhythmic expression of core clock genes in skeletal muscle,to ameliorate circadian misalignment and improve the decrease of exercise endurance of SR mice.The purpose of the study was that elucidating the protective effects of PTE on exercise endurance in SR mice and its mechanism to provide scientific experimental evidences for the application of PTE to improve the decline of exercise endurance caused by SR and the protection of special working people.