Establishment Of A Mouse Model For Fatigue Induced By Chronic Restraint Stress And Studies On The Anti-fatigue Effects And Its Mechanism Of Nelumbinis Stamen

Fatigue is a persistent experience of weariness,weakness,or exhaustion that can be physiological,psychological,or both.With the progress and development of society,people’s work style and pace of life have changed dramatically,and the physical and psychological pressures brought by competition,as well as unhealthy lifestyles,can lead to fatigue in the body.Currently,fatigue is a prevalent health issue that seriously affects people’s quality of life and work efficiency.However,the current market for drugs to relieve fatigue is inadequate,and more safe and effective anti-fatigue drugs and health products are urgently needed.In addition,the evaluation method currently used in the evaluation technical specifications for fatigue-relieving health products in China is to directly administer normal animals and then evaluate acute fatigue through weight loaded swimming,which cannot reflect the fatigue caused by long-term stress in people.Nelumbinis stamen is the dried stamen of Gaertn.,and has a long history of medicinal use.It is recorded in the 2020 edition of the Chinese Pharmacopoeia that its function and indications are "reinforcing the kidney and controlling noctural emission,spermatorrhea,morbid vaginal discharge and frequent micturition.reinforcing kidney and astringent essence,for spermatospermia and slippage,and for frequent urination",and it has potential anti-fatigue effects.However,there have been no reports on the anti-fatigue effects of Nelumbinis stamen so far,and further research is needed.In addition,there have been few studies on the chemical composition of Nelumbinis stamen,which limits its future development and application.Therefore,this study established a mouse model of physical fatigue caused by chronic stress in humans by using chronic restraint stress(CRS)and studied its potential mechanisms.The anti-fatigue and muscle protection effects and mechanisms of Nelumbinis stamen were studied using acute exercise fatigue model,CRS mouse fatigue model,corticosterone-induced muscle dysfunction model and dexamethasone-induced C2C12 myotube atrophy model.Meanwhile,the chemical composition of Nelumbinis stamen was analyzed by using ultra-high performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF/MS)and high-performance liquid chromatography-ultraviolet(HPLC-UV)techniques.The main research contents of this thesis include of the following aspects:1.Establishment of fatigue model of mice induced by CRS and its related mechanism researchICR mice were subjected to CRS for 0,5,10,and 15 days,8 hours per day.After the stress,the mice were subjected to an open field test,grip strength test,and weight-loaded swimming test.Biochemical parameters related to fatigue,such as muscle glycogen,liver glycogen,as well as glucose.lactic acid,urea,lactate dehydrogenase,and aspartate transaminase levels in serum were measured.Taken together,the modeling conditions of CRS-induced fatigue was determined.What’ more,this condition was applied to verify its applicability in C57BL/6N and BALB/c mice.Transcriptome sequencing technology was used to analyze the differences in gene expression in the gastrocnemius samples of the control group and CRS-modeled mice after 15 days.Additionally,Gastrocnemius muscle fibers were visualized by H&E staining and immunohistochemical staining.The mitochondrial function,mitochondrial biogenesis,and mitophagy of the gastrocnemius were evaluated using methods such as electron microscopy and Western blot,and the possible upstream AMPK/mTOR signaling pathway was studied to reveal the possible mechanisms of CRS-induced fatigue.And chloroquine was used to verify the relationship between fatigue and autophagy in CRS mice.The results showed that compared with the control group,the fatigue symptoms of ICR mice became more apparent with the prolongation of CRS time.8 hours per day for a total of 15 days could cause mice to exhibit stable fatigue symptoms such as decreased spontaneous activity in an open field,decreased forelimb grip strength,and decreased endurance in weight-loaded swimming.C57BL/6N and BALB/c mice also showed significantly decreases endurance in weight-loaded swimming test under this condition,indicating the applicability of this condition to different strains of mice.15 days of CRS caused muscular atrophy of the gastrocnemius muscle,and led to significantly increases in lactate,lactate dehydrogenase,and aspartate aminotransferase,and significantly decreases in urea,liver glycogen,and blood glucose,indicating the accumulation of fatigue-related metabolic products,depletion of energy substrates,and tissue damage.Transcriptome sequencing study of the gastrocnemius identified 239 differentially expressed genes.KEGG and GO enrichment analyses suggested that CRS caused energy metabolism disorder and may be associated with mitochondrial damage,autophagy inhibition,and the AMPK signaling pathway.In CRS mice,mitochondrial-related proteins CYT-C,TOMM20,OXPHOS,and MYH7 were decreased,and autophagy-related proteins PINK1 and LC3 Ⅱ/LC3 Ⅰ were downregulated while p62 was upregulated,indicating mitochondrial damage and reduction,as well as mitophagy inhibition.Furthermore,giving mice chloroquine,an autophagy inhibitor,revealed that when autophagy is inhibited,endurance also decreases,demonstrating that the fatigue in CRS mice is partly related to autophagy inhibition.Further analysis found that CRS caused a decrease in p-AMPK,PGC-1α,and TF AM in the gastrocnemius muscle of mice,while p-AKT,p-mTOR,and pULK1(Ser757)increased,indicating that CRS inhibited the AMPK/PGC-1α signaling pathway,led to mitochondrial biogenesis blockade,and activated the mTOR/p-ULK1(Ser757)pathway to inhibit mitophagy,thereby causing fatigue.2.Studies on the ameliorating effect and mechanism of Nelumbinis stamen on fatigue induced by CRS in miceFor 15 consecutive days,ICR mice were subjected to gavage with Nelumbinis stamen at doses of 0.5 and 1.0 g/kg,and positive control Rhodiola capsules,respectively,while simultaneously subjected to 15 days of CRS for 8 hours per day.The efficacy of Nelumbinis stamen in improving fatigue induced by CRS and its protective effect on the gastrocnemius were evaluated by assessing forelimb grip strength,weight-loaded swimming endurance,body composition,gastrocnemius H&E staining,and serum levels of lactate,glucose,and lactate dehydrogenase.Furthermore,the mechanism of Nelumbinis stamen in improving fatigue was elucidated by studying mouse gastrocnemius mitochondrial function,mitochondrial biogenesis,mitophagy,and antioxidant stress using transcriptome sequencing,electron microscopy,Western blot,and RT-qPCR techniques.The results showed that Nelumbinis stamen administration significantly protected muscle fibers,reversed the decreased forelimb grip strength and weight-loaded swimming endurance induced by CRS in mice.It also reduced serum lactate and lactate dehydrogenase levels,and increase serum glucose level.Body composition and transcriptome sequencing suggested that this was related to significant changes in multiple pathways,such as metabolic pathways,lipid biosynthesis,and muscle development pathways.Nelumbinis stamen increased the expression of MYH7,OXPHOS,PGC-1α,TFAM,and TOMM20 in the gastrocnemius of CRS mice,thereby improving mitochondrial function and quantity.Nelumbinis stamen reversed the decreased expression of PINK1,LC3 Ⅱ/LC3 Ⅰ,and Beclin-1 in CRS mice,as well as reverse the increased expression of p62,p-ULK1(Ser757),and p-mTOR,thereby promoting mitophagy.These changes are related to the selective promotion of Sesn2 expression by Nelumbinis stamen,which activates Sestrin2 and further activates the downstream AMPK signaling pathway.In addition,Nelumbinis stamen increased Nrf2 and inhibited Keapl in CRS mice,upregulated antioxidant enzymes such as NQO1,HO-1,and Mn-SOD,reduced xanthineoxidase and MDA levels,and reduced oxidative stress damage.In conclusion,the anti-physical fatigue effect of Nelumbinis stamen against CRS may be achieved by activating Sestrin2 to regulate AMPK and Nrf2,promoting mitochondrial biogenesis and mitophagy,enhancing antioxidant stress levels,thereby exerted its effects.2.Studies on the ameliorating effect of Nelumbinis stamen on muscle dysfunction and fatigue induced by glucocorticoidFirstly,the levels of corticosterone of CRS mice after Nelumbinis stamen administration were measured to explore its protective effect.Then,the relationship between endogenous corticosterone disorder and fatigue was demonstrated by intraperitoneal injection of the corticosterone synthesis inhibitor metyrapone in CRS mice.Hypodermic injection of corticosterone was given to normal mice,followed by gavage administration of Nelumbinis stamen(1.0 g/kg).The effect of Nelumbinis stamen on ameliorating fatigue induced by corticosterone injection in mice was studied using grip strength and weight-loaded swimming tests,electron microscopy,H&E staining,and Western blot analysis.In vitro,C2C12 mouse myoblasts were differentiated into myotubes and treated with dexamethasone(80 μM)for 24 hours to induce atrophy.The effect of Nelumbinis stamen(0.1,0.2,and 0.4 mg/mL)on ameliorating atrophy was investigated,and the expression of proteins such as Sestrin2 was studied using Western blot analysis.The results showed that Nelumbinis stamen reversed the fatigue and regulated corticosterone endogenous disorder in CRS mice.Moreover,Nelumbinis stamen(1.0 g/kg)significantly reversed the fatigue induced by exogenous injection of corticosterone in mice,and elevated Sestrin2,TFAM,Nrf2,and Mn-SOD levels in corticosterone-injected mice,thereby promoting mitochondrial biogenesis and antioxidant stress.Nelumbinis stamen(0.1,0.2,and 0.4 mg/mL)had no effect on the viability of C2C12 myotubes induced by dexamethasone(80 μM),but it reversed myotube atrophy.Nelumbinis stamen reversed mitochondrial dysfunction by reversing the reduction of Sestrin2,Nrf2,and TOMM20 induced by dexamethasone.These results suggest that Nelumbinis stamen has a good improvement effect on muscle function disorders and fatigue induced by glucocorticoids.4.Studies on the ameliorating effect of Nelumbinis stamen on fatigue induced by acute exercise in miceMice were subjected to gavage with Nelumbinis stamen at doses of 0.5 and 1.0 g/kg(water extract approximately 90 mg/kg and 180 mg/kg,respectively)and positive control Rhodiola capsules for 15 days.The improvement effect of Nelumbinis stamen on acute exercise-induced physical fatigue was evaluated for the first time through grip strength and weight-loaded swimming tests.The mechanism of its anti-fatigue effect was preliminarily studied by detecting the body composition,muscle and liver glycogen,and urea,lactate,lactate dehydrogenase,and alanine aminotransferase levels of serum in mice.The results showed that after administration of Nelumbinis stamen,the grip strength and weightloaded swimming endurance of mice were significantly improved,and the levels of muscle and liver glycogen were increased,while the levels of serum urea,lactate,lactate dehydrogenase,and alanine aminotransferase were decreased.These results indicate that Nelumbinis stamen enhanced the energy substance reserve,reduced metabolic product accumulation,and reduced myocardial and skeletal muscle damage in mice,thereby exerted an anti-physical fatigue function.5.Chemical composition analysis of Nelumbinis stamenThe chemical composition of Nelumbinis stamen extract was analyzed and identified using UPLC-QTOF/MS technology in negative ion mode.The content of major flavonoids,including rutin,hyperoside,astragalin,quercetin,and kaempferol,were determined by HPLC-UV at a wavelength of 360 nm.A total of 44 components were identified,including 6 organic acid compounds,20 flavonoid compounds,6 monoterpene glycosides,12 long chain fatty acid compounds.The content determination results showed that the contents of rutin,hyperoside,astragalin,quercetin,and kaempferol in Nelumbinis stamen were 86.57,60.13,231.34,62.88,and 146.28 mg/100g,respectively.This study provides a clearer material basis for the future development and application of Nelumbinis stamen.In conclusion,this study established a mouse model of fatigue induced by CRS and elucidated it related to the mitochondrial homeostasis imbalance.By using CRS mouse fatigue model,corticosterone induced mouse muscle dysfunction model,dexamethasone induced C2C12 myotube atrophy model,and acute exercise-induced mouse fatigue model,the anti-fatigue effect of Nelumbinis stamen was first found,and its anti-fatigue mechanism was elucidated from the perspective of mitochondrial homeostasis and antioxidative stress.This study provides a stable animal model and a deeper understanding of the mechanism for screening products that alleviate physical fatigue caused by stress,while also laying a solid foundation for the development of Nelumbinis stamen as a drug or health food for anti-fatigue purposes.