The Preventive Effect Of Asperuloside In Eucommia Ulmoides Male Flower On Age-related Muscle Dysfunction And Its Mechanism

Muscle aging,especially the decline of its function,has a profound impact on health,independence,and quality of life of the elderly.As a multifunctional signal transduction and energy metabolism center,mitochondria link cell function,metabolism and age-related diseases.Mitochondrial dysfunction in muscle is closely related to the occurrence and development of aging.Specific autophagy-mitophagy is a quality control modality for the targeted clearance of dysfunctional or damaged mitochondria,and its activity is particularly important for muscle health and mitochondrial homeostasis during aging.Therefore mitophagy enhancers have good application prospects in the prevention and treatment of muscle aging.Studies have found that many plants have potential mitochondrial autophagy regulation and anti-muscle aging functions,among which edible flowers are a large class of emerging food resources with anti-aging value.Domestic edible flowers are rich in resources,and the industry has gradually developed and became a new growth point of the flower industry.Our laboratory has been carrying out research on edible flowers for a long time,and constantly promoted the flower intensive processing industry by mining and utilizing the health function factors in edible flowers.Based on the above background,it is of great significance to explore the anti-muscle aging effect of edible flowers.In this study,30 kinds of edible flowers were selected as the research objects,and the species and active components of edible flowers with anti-muscle aging effect were screened and verified by Caenorhabditis elegans and mouse models,and the mechanism of action of the active components was revealed by cell model.The main research contents and results are as follows:1.Screening and composition analysis of anti-aging edible flowersUsing nematode model,12 species of flowers were screened,including Eucommia ulmoides male flower,roselle,safflower,etc.,which had significant in vivo longevity-promting activity,and E.ulmoides male flower had outstanding effect.Further,100 μg extract equivalent/m L male flower of E.ulmoides improved the muscle health-related indices of pharyngeal pumping rate,motility,and muscle fiber morphology of aged nematodes.Through correlation analysis of total components(including total phenols,total flavonoids,and total terpenes),qualitative analysis by ultra-performance liquid chromatography-mass spectrometry,longevity-promting efficacy verification and quantitative analysis by high performance liquid chromatography,aucubin,geniposide,asperuloside,geniposidic acid and chlorogenic acid were determined as the main active components of E.ulmoides male flower,and the content reached 3.54%.Using the model of age-related muscle dysfunction of C.elegans,asperuloside(ASP)was screened as the best active component of anti-muscle aging.2.Effect of asperuloside on age-related muscle dysfunction in miceUsing C57BL/6N mice model of age-related muscle dysfunction induced by high fat diet,it was shown that ASP(50 mg/kg BW/day)intragastric intervention for 3 months could reduce the weight gain and fat accumulation on epididymis and gastrocnemius muscle of mice,and inhibit the increase of triglyceride content in serum.ASP prevented instantaneous explosive,persistent and autonomy-related decline of muscle function in aging mice,and slowed down the pathological changes such as the reduction of cross-sectional area of gastrocnemius muscle,collagen deposition,increased apoptosis of muscle cells,transformation of muscle fiber types,and increased levels of inflammation and oxidative stress.The intervention effects of ASP were similar to that of urolithin A.Western blot analysis showed that ASP decreased the expression of senescence-related β-galactosidase and pro-aging factors P21,P16,but increased the expression of anti-aging factor Klotho in gastrocnemius muscle.The expression of MAFbx and Mu RF-1 was also decreased by ASP.Luciferase luminescence assay,transmission electron microscopy and real-time fluorescence quantitative PCR showed that ASP could maintain the ATP content and mitochondrial morphology in gastrocnemius muscle,but did not increase the number of mitochondria.3.Effect of asperuloside on muscle aging based on mitochondrial homeostasisUsing the high-fat-induced nematode model of age-related muscle dysfunction,it was confirmed that 100 μM ASP intervention for 9 days could improve the body bending frequency and the morphology of body wall muscle fibers of C.elegans.Metabonomics data show that ASP regulated mitochondria-related metabolism,including lifespan regulation,citric acid cycle,glucose and lipid metabolism.Oil red O staining reflected the effect of ASP on improving lipid metabolism.The results of fluorescent strain and probe,Seahorse and other experiments fully reflected that ASP could maintain the normal morphology of mitochondria in the body wall muscle of aged nematode,increase its basic,maximum and spare respiratory capacity,and reduce the production of reactive oxygen species.Combined with the mitochondrial dysfunction of mev-1 nematode strain,it is suggested that the anti-aging effect of ASP was closely related to mitochondrial homeostasis.The changes of gene expression levels related to mitochondrial quality control network showed that ASP mainly affected mitochondrial dynamics and mitophagy.Using IR1511 nematode strain,ASP had mitophagy activation activity in vivo.4.Effects of asperuloside on aging myoblasts and mitochondrial homeostasis based on mitophagyUsing the mouse C2C12 myoblast senescence model induced by palmitic acid sodium,it was confirmed that 2.5 μM ASP intervention for 5 days could control the enhancement of senescence-related-β-galactosidase activity,lipid accumulation and the increase of P21 and P16 expression,and up-regulate the expression of Klotho.For mitochondrial health,ASP maintained the mitochondrial respiratory function of aging myoblasts,and slowed down the decrease of mitochondrial membrane potential and the generation of reactive oxygen species.Transmission electron microscopy also showed that ASP was beneficial to mitochondrial homeostasis,and the autophagosome structure surrounding mitochondria increased.The mtKeima 293 T cell line constructed by lentivirus showed that ASP also had the activity of activating mitophagy in vitro.Using western blotting,ASP was found to increase autophagy flux,as reflected by a decrease in P62 protein expression and an increase in LC3 II/I ratio.When autophagy activity was inhibited,the effect of ASP on mitochondrial respiration of aging myoblasts disappeared.In addition,when bnip3 or lc3 expression was inhibit by si RNA that anti-myoblast senescence effects of ASP also disappeared.This confirms that these effects of ASP were dependent on BNIP3-specific receptor-mediated activation of mitophagy.5.Effect of asperuloside on upstream regulatory factor of mitophagy and its mechanismUsing the above myoblast senescence and nematode models of age-related muscle dysfunction,the effect and mechanism of ASP on forkhead box protein O(FOXO)/DAF-16,an upstream regulator of mitophagy,were investigated.Molecular docking and western blot experiments confirmed that the FOXO subtype affected by ASP was FOXO3.2.5μM ASP intervention promoted the expression and nuclear penetration of FOXO3 protein in aging myoblasts.The nuclear entry activity may be related to the decrease of phosphorylation at Ser253 residue of FOXO3 by ASP.When the expression of foxo3 was inhibited by si RNA,the effect of ASP on regulating the expression of senescence and mitophagy-related proteins disappeared,which confirmed that ASP played the above roles dependent on foxo3 gene.Using TJ456 and DA2123 fluorescent labeling nematode strains,combined with western blotting,it was shown that 100 μM ASP increased the expression of DAF-16 protein and promoted the entry of the protein into the nucleus.At the same time,the expression of LGG-1 protein associated with autophagy was also increased.By feeding p L4440-daf-16-HT115 interfering bacteria,it was found that when daf-16 expression was inhibited,the effects of ASP on maintaining normal body bending frequency,improving mitochondrial respiratory capacity and increasing mitophagy-related gene expression of aged nematode disappeared,indicating that daf-16 gene mediated these effects of ASP.In conclusion,we found that asperuloside in E.ulmoides male flower,has anti-muscle aging activity,and further revealed the mechanism of asperuloside activating BNIP3-dependent mitophagy by regulating FOXO3/DAF-16 transcription factor,thereby maintaining mitochondrial homeostasis and preventing muscle aging.This study expanded the anti-aging potential of edible flower resources,provided possible natural dietary components for the development of muscle health products for middle-aged and elderly people,and was beneficial to the deep processing and further application of E.ulmoides male flowers.