| Fatigue is a research hotspot in the fields of food and medicine.Fatigue not only puts the human body in a sub-healthy state,but is also related to various diseases such as cancer and anemia.Therefore,finding a safe and effective method to prevent fatigue has become an urgent need to maintain health in the current society.Hairtail fish(Trichiurus lepturus)is a low-value economic fish with the highest yield in China’s coastal areas,and Mindong Fishery in Fujian is the second largest hairtail producing area in the country,with advantages in region,breeding and raw materials.Hairtail fish is rich in high-quality protein,polyunsaturated fatty acids,etc.These biologically active ingredients are safe and have no side effects,making them good supplements for the body when it is fatigued.However,the current research on hairtail fish is still mainly focused on rough processing,and the research and development of its biologically active components is an important breakthrough in the upgrading of hairtail resource processing.Based on this,this subject took hairtail fish glycoprotein(HGP)as the research object,deeply studied its anti-fatigue activity in vivo and in vitro,and further combined multi-omics to reveal the possible mechanism of hairtail fish glycoprotein’s anti-fatigue effect.This study aims to lay a theoretical foundation for the development of functional foods or health products with anti-fatigue effects,and has certain theoretical significance and practical value for promoting the development of Fujian hairtail deep processing industry.The main research content and results include:(1)Preparation,basic composition and structure identification of HGP.HGP was separated by ultrasonic-assisted salt extraction and membrane separation technology.The protein and total sugar contents,monosaccharide and amino acid compositions and structural properties of HGP were analyzed.The results showed that the extraction rate of HGP was 3.5%,The yield after membrane separation was 1.4%,and its protein and total sugar contents were 89.41±0.25%and 3.59±0.13%,respectively.And four monosaccharides(fructose,glucose,galactose,and fucose)and17 amino acids were found in HGP.Among them,essential amino acids accounted for 34.08%,hydrophobic amino acids accounted for 36.60%,branched chain amino acids accounted for 17.83%,and aromatic amino acids accounted for 4.0%.The results of the structural properties showed that the molecular weight distribution range of HGP was 0.4×10~3-1.2×10~4g/mol,and the thermal denaturation temperature was 85.2℃.O-glycopeptide bonds,characteristic peaks of proteins and polysaccharides,and sugar residue structures dominated byβ-type pyranose were found in HGP.And theα-helix,β-sheet and random coil structure contents of protein in HGP were 31%,10%and 59%,respectively.In summary,HGP is a protein-based glycoprotein rich in amino acids,which has the potential to develop anti-fatigue functional base materials or health foods.(2)In vitro antioxidant activity and in vivo anti-fatigue activity of HGP.The antioxidant activity of HGP was evaluated in vitro.The anti-fatigue activity of HGP was studied by animal behavior experiments.And the biochemical indicators related to anti-fatigue in BALB/c mice were further determined.The results of antioxidant activity research showed that the antioxidant capacity of HGP gradually increased in the concentration range of 10-100 mg/m L,and when the concentration reached 100 mg/m L,the free radical scavenging effect of HGP was close to that of Vc(1 mg/m L).Behavioral research results showed that,compared with the control group,HGP significantly prolonged the exhausted swimming time.Medium and high doses of HGP prolonged the turning time of the mice,shortened the time for the mice to climb down the bottom of the pole.Mice in the HGP groups were more active in the shuttle box and elevated plus maze tests.The results of anti-fatigue biochemical index showed that HGP significantly reduced the serum levels of lactate dehydrogenase,blood lactic acid,blood urea nitrogen,and creatine kinase,and significantly increased the contents of liver glycogen and muscle glycogen in fatigued mice.The content of MDA in the serum and brain tissue of the mice in the HGP groups was significantly reduced,and the activities of antioxidant enzymes such as CAT,GPX,and SOD were also significantly increased,especially in the middle and high dose groups.In summary,HGP has good free radical scavenging ability,can relieve the exercise and mental fatigue of mice,and has anti-fatigue effect.(3)Study on the anti-fatigue activity of HGP based on the changes of intestinal flora.16s r RNA sequencing technology was used to study the effect of HGP on the diversity,community composition and species differences of the intestinal flora in fatigued mice.And the correlation between the intestinal flora of mice and the relevant indicators of anti-fatigue activity in vivo was constructed.The results of the diversity of the intestinal flora showed that oral administration of HGP could change the structure of the intestinal flora in mice and increase the richness and diversity of the flora.The results of intestinal community composition showed that at the phylum level,HGP could reshape the intestinal flora of tired mice by up-regulating Bacteroidota and down-regulating the abundance of Firmicutes,and the high dose of HGP had the best effect.At the genus level,HGP reduced the production and accumulation of lactate in mice by down-regulating the relative abundance of unclassified＿f＿＿Lachnospiraceae and Lactobacillus.The results of species difference showed that at the phylum level,HGP reshaped the intestinal flora structure of fatigued mice by down-regulating the abundance of Firmicutes.At the genus level,HGP reshaped the gut microbiota structure of fatigued mice by down-regulating the abundance of A2,Bacillus,Anaerotruncus,and Family＿XIII＿UCG-001.The results of functional prediction showed that HGP increased Amino acid transport and metabolism,Carbohydrate transport and metabolism,Energy production and conversion in mice by remodeling the intestinal flora structure of fatigued mice.The correlation analysis results showed that there was a significant correlation between the intestinal flora and the anti-fatigue related indicators in mice.In conclusion,HGP may play an anti-fatigue effect by remodeling the intestinal flora structure of mice.(4)Study on the anti-fatigue activity of HGP based on serum metabolomics.The serum metabolites of fatigued mice were detected and differential metabolites between groups were screened by serum metabolomics technology.The correlation between key differential metabolites and biochemical indicators related to anti-fatigue activity in vivo and intestinal flora of mice were constructed.The results of differential metabolite volcano plot showed that the serum metabolites of mice in each group were significantly different,and HGP significantly changed the serum metabolites of fatigued mice.The results of metabolites with significant differences between groups indicated that HGP exerted anti-fatigue effect by down-regulating Uric Acid,Oxoglutaric acid and DL-Citrulline in the serum of fatigued mice,and Uric Acid may be a potential biomarker for HGP to exert anti-fatigue effect.The enrichment analysis of metabolic pathways showed that HGP released energy and reduced physical fatigue by regulating pyrimidine metabolism,TCA cycle and carnitine metabolism.HGP also increased the content of reduced glutathione in mice by enhancing the Glutathione metabolism pathway,thereby scavenging free radicals and reducing mental fatigue in mice.In addition,HGP enhanced the Phenylalanine metabolism in mice,reduced the content of Gamma-Glutamyltyrosine in mouse serum,promoted the synthesis of L-Tyrosine in the liver,and increased the levels of dopamine and adrenaline in the body,thereby relieving fatigue.The results of correlation analysis showed that the key metabolites in mouse serum were significantly correlated with anti-fatigue-related indicators in vivo and intestinal flora.In conclusion,HGP may play an anti-fatigue effect by regulating serum metabolites and activating related metabolic pathways in mice.(5)Study on the differential expression of genes regulated by HGP in mouse liver based on transcriptomics.Transcriptome technology was used to study the differential expression of genes in the liver of fatigued mice regulated by HGP.The correlation between key differential genes and anti-fatigue-related biochemical indicators,intestinal flora,and serum metabolites was analyzed.The results of volcano plot showed that HGP could affect the expression of liver genes in fatigued mice,and there were significant differences in gene expression among the groups.The results of differential gene between groups also showed that HGP played an anti-fatigue effect by up-regulating Rab43 and Tox genes,and down-regulating Inhba and Ccn2 genes.It was speculated that Inhba might be a marker gene for HGP to exert anti-fatigue effect.The results of correlation analysis showed that there was a significant correlation between mouse liver genes and in vivo anti-fatigue-related indicators,intestinal flora,and key serum metabolites.In summary,HGP may play an anti-fatigue role by regulating the differential expression of liver genes in mice.Based on the research results of each chapter of this paper,the possible"gut-liver"axis mechanism of HGP’s anti-fatigue effect was further revealed.It was speculated that HGP down-regulated the levels of Gamma-Glutamyltyrosine and Uric Acid in serum,reduced the abundance of Lactobacillus and Bacillus in the intestinal tract of mice,and increased the abundance of norank＿f＿＿norank＿o＿＿Clostridia＿UCG-014,Bacteroides,Corynebacterium,norank＿f＿＿Oscillospiraceae,norank＿f＿＿Lachnospiraceae and unclassified＿f＿＿Rikenellaceae by up-regulating Rab43 and inhibiting Inhba genes.Regulating liver glycogen and gastrocnemius muscle in mice through the"gut-liver"cycle to achieve the effect of anti-fatigue.In summary,HGP is a glycoprotein with anti-fatigue activity,which may play an anti-fatigue effect by up-regulating Rab43 and inhibiting Inhba gene,and further activating the"gut-liver"cycle. |
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