| Highland barley tea(HBT)is a kind of cereal tea made from roasted barley seeds,which is rich in natural active ingredients and can effectively reduce the risk of metabolic diseases after long-term consumption.At present,China has officially entered into an aging society,and the degeneration of body organ functions and the resulting chronic metabolic diseases are widely seen in elderly individuals,among which the decline of skeletal muscle functions has seriously affected the quality of life of the elderly and restricted the social and economic development.Therefore,it is of great scientific and social significance to investigate the effects of HBT on the skeletal muscle health of the elderly population to improve their health status and reduce the social medical burden.In this thesis,we investigated the effects of long-term HBT consumption on aging and skeletal muscle function through a natural aging mouse model,combined with an in vitro skeletal muscle aging cell model to screen for potential functional components,and used separation and purification and mass spectrometry techniques to enrich,identify and analyze the activity of functional components,and further investigated the potential molecular mechanism of functional components to inhibit skeletal muscle aging through animal and cell models.Finally,the potential regulatory genes in mouse skeletal muscle were identified and specifically knocked out using molecular biology techniques to further validate and clarify the potential mechanism of HBT in improving skeletal muscle aging.The main studies are as follows:(1)To investigate the effects of long-term HBT consumption on the physiological status and skeletal muscle function of aging mice.The effects of long-term HBT consumption on aging were investigated by replacing drinking water with HBT for 5 months,after which the basal physiological parameters,inflammation,plasma metabolites,skeletal muscle function and exercise capacity were measured.The results showed that long-term consumption of HBT reduced body weight and spleen weight,increased the weight percentage of skeletal muscle and reduced plasma inflammation levels in aging mice.The plasma levels of short-chain acylcarnitine,a mitochondrial marker,and some amino acids were increased,and the levels of some bile acids and long-chain acylcarnitine were decreased.The level of fibrosis in the gastrocnemius muscle of aging mice was suppressed,and the percentage of type I muscle fibers was increased,thus improving the endurance of mice.Therefore,long-term consumption of HBT can reduce inflammation levels and mitigate mitochondrial damage in aging mice,and increase mitochondrial activity and exercise capacity in skeletal muscle(2)Identification and investigation of the potential components of HBT that regulate skeletal muscle senescence through in vitro models.A mouse skeletal muscle senescence model with in vitro mitochondrial damage was constructed using palmitic acid(PA)treated mouse skeletal muscle myogenic cells(C2C12).The mitochondrial respiratory capacity of the cells detected by Seahorse and the relative expression levels of acylcarnitine measured by LC-MS were utilized as essential basis to test the validity of the cellular model.The HBT broth was divided into two fractions,alcohol soluble(AS)and water soluble(WS),and the basic components and in vitro antioxidant activities of the two fractions were determined,as well as the modulating effects of skeletal muscle mitochondrial injury of AS and WS using the skeletal muscle in vitro mitochondrial injury model.The results showed that the main components of AS were small molecule compounds dominated by polyphenolic flavonoids,while the main components of WS were soluble carbohydrates.As a result,AS exhibited higher antioxidant activity and could effectively mitigate PA-induced skeletal muscle mitochondrial damage.Further studies revealed that AS could inhibit intracellular protein acetylation levels,mitochondrial oxidative damage and mitochondria-mediated apoptosis via the AMPK/SIRT3/Fox O3 a pathway.Based on the above results it is clear that phenolics are functional components of HBT in controlling skeletal muscle aging.(3)The phenolics in HBT were enriched,purified,qualitatively and quantitatively analyzed,as well as their mitochondrial regulatory activity.The crude polyphenols(CHBP)of barley tea were purified using macroporous adsorption resin combined with dynamic adsorption,and the purified polyphenols were qualitatively and quantitatively analyzed using UPLC-TOFMS,as well as the activity of the phenolics was investigated.The results showed that the total phenolic content of barley tea phenolics(HBP)was significantly increased after purification using dynamic adsorption on AB-8 macroporous adsorption resin,and the 28 phenolic monomers were identified in the purified phenolics.Among the purified polyphenols,procyanidin B3 was the most abundant compound,followed by gallic acid,protocatechuic acid,p-hydroxybenzoic acid,epicatechin,caffeic acid,p-coumaric acid,eugenol,ferulic acid,benzoic acid,rutin,kaempferol,catechin,and lignan.The results of mitochondrial regulatory activity revealed that HBP,CHBP,and procyanidin B3 could mitigate PA-induced mitochondrial damage,while catechin,protocatechuic acid,p-coumaric acid,and ferulic acid could improve some mitochondrial functions,with HBP activity being higher than CHBP and monomeric polyphenols.(4)To investigate the inhibitory effects of different doses of HBP on aging-induced muscle atrophy and advanced glycosylation end products(AGEs),as well as the effects of AGEs on skeletal muscle during the aging process.The results showed that D-galactose-induced aging increased AGEs content in serum and flounder muscle,decreased flounder muscle mass and mitochondrial functional protein SIRT3 expression,and increased the expression of the myasthenia gravis-associated gene Atrogin-1 in mice.In ageing mice,a medium to high dose of HBP intervention might effectively mitigate the above and decrease the level of oxidative stress.Further study revealed that AGEs could lower SIRT3 expression in C2C12 cells,leading to myasthenia and mitochondrial oxidative stress,whereas HBP could effectively inhibit the production of AGEs in vitro and in vivo,and the inhibition rate was positively correlated with HBP concentration.The rate of inhibition was correlated with the concentration of HBP.Medium and high doses of HBP could alleviate skeletal muscle aging by inhibiting the formation of AGEs.In addition,the optimal intervention concentration of HBP in animal experiments was shown to be 100 mg/kg/bw.(5)To investigate the effect of HBP on senescence-induced skeletal muscle fibrosis and to identify the potential mediating genes.In vivo and in vitro skeletal muscle fibrosis models in mice were constructed using D-galactose and skeletal muscle blunt contusion to further investigate the effects and potential mechanisms of HBP on aging-induced skeletal muscle fibrosis.The results showed that D-galactose increased skeletal muscle aging and fibrosisrelated indicators in mice,including oxidative stress level,β-galactosidase content,inflammation level,cellular acetylation level,expression level of skeletal muscle fibrosis markers represented by α-SMA,and decreased SIRT3 expression.The intervention of HBP could effectively alleviate D-galactose-induced senescence and fibrosis in skeletal muscle cells.Significantly,the silencing of SIRT3 in C2C12 cells enhanced D-galactose-induced skeletal muscle damage and fibrosis,and reduced the effect of HBP intervention,suggesting that SIRT3 is an important gene mediating HBP to improve skeletal muscle senescence and fibrosis.(6)To investigate the effect of SIRT3-mediated HBP on skeletal muscle senescence.Gene editing and recombination techniques were used to create skeletal muscle SIRT3 knockout mice(mSIRT3 KO).We also measured adult mSIRT3 KO mice’s body weight and skeletal musclerelated indexes and found that the body weight of adult mSIRT3 KO mice was reduced,the mass of gastrocnemius and flounder muscles was decreased,the gastrocnemius fiber type was converted from oxidative muscle fibers(type I)to glycolytic muscle fibers(type II),the expression levels of genes related to myasthenia and fibrosis were increased,the strength and endurance of mice decreased,and the level of inflammation and oxidative stress increased levels of inflammation and oxidative stress.Furthermore,HBP intervention could not alleviate skeletal muscle senescence caused by skeletal muscle-specific knockdown of SIRT3,but it could alleviate inflammation in the organism.In summary,SIRT3 has an important role in regulating the skeletal muscle aging process and is a key gene that mediates the inhibition of skeletal muscle aging by HBP.In summary,phenolics in barley are important functional components of barley tea in regulating skeletal muscle aging,which can alleviate skeletal muscle aging by inhibiting AGEs,inflammation and oxidative stress,among which SIRT3 has an important role in regulating skeletal muscle aging process.This study provides new ideas and theoretical support for the efficient utilization of barley resources and the development of cereal health drinks. |
