Anti-aging Effect And Mechanism Of Sea Cucumber(Acaudina Leucoprocta) Peptides

With the increasing aging population,a rise in the prevalence of age-related degenerative diseases will exert significant pressure on medical care,social services,and labor supply.Consequently,there is a growing interest in strategies to delay the onset of aging and related diseases.Acaudina leucoprocta,an edible sea cucumber species in China,is extensively distributed in the East China Sea.Till now,there are a few research carried on the anti-aging properties of bioactive peptides derived from this species.In this study,we explored the anti-aging effect of sea cucumber peptides(SCP)extracted from Acaudina leucoprocta.Firstly,Drosophila melanogaster was used as a model organism to investigate the effect of SCP on the lifespan and healthspan.Then,the D-galactose(D-gal)induced aging mice was used to explore the effect of SCP on cognitive impairment and underlying mechanism.Finally,the potential peptides of age-related cognitive amelioration in SCP were screened by peptidomics and molecular docking.The main results are as follows:(1)Peptide content accounts for 86.12%in SCP,among which molecular weight below1000 Da peptides are of 94.05%.Also,SCP is rich in glycine,glutamic acid,alanine,aspartic acid,and proline.Different doses of SCP(0.2%,0.4%,0.6%and 0.8%)supplementation significantly increased the survival rate and prolonged the mean and median lifespan of both male and female flies.Notably,the effect was most pronounced in male flies at a dose of 0.6%SCP while at a dose of 0.2%SCP in female.SCP enhanced the healthspan of male flies by improving their climbing ability,intestinal barrier function,and memory.Furthermore,SCP increased the survival rate of flies which were subjected to oxidative,starvation,and heat stress.while not affect the survival rate of flies under cold stress.It should be noted that there are gender differences in the beneficial effects of SCP on flies since the effects on males are more evident than those on females.Moreover,after protein intake restriction,the effects of SCP on the lifespan of flies remained unchanged.However,when antibiotics were used to eliminate gut microbiota,the effect of SCP on prolonging the lifespan of flies was partially inhibited,indicating that the effects of SCP prolonging the lifespan of flies were unrelated to dietary protein restrictions but related to gut microbiota.(2)The underlying mechanism underlying was explored based on microbiome and metabolomics.3-day-old flies were defined as young control group while 30-day-old flies as aging.16s RNA sequencing demonstrated that SCP intervention led to an increase in the diversity of gut microbiota.In genus level,the relative abundance of potential beneficial bacteria such as Lactobacillus,Rhodococcus,and Propioniciclava in flies were increased,while the abundance of potentially harmful bacteria such as Acetobacter,Achromobacter,Pedobacter,and norank＿f＿Saprospiraceae decreased.Furthermore,the metabolomic profile of flies in SCP intervention was altered.13 differential metabolites related to aging were upregulated by SCP.These metabolites were enriched in amino acid metabolic pathways(Arginine biosynthesis,Alanine,aspartate,and glutamate metabolism,beta-Alanine metabolism),nucleotide metabolism(Pyrimidine metabolism,Purine metabolism),and carbohydrate metabolism(Ascorbate and aldarate metabolism,Pentose and glucuronate interconversions).These results suggested that SCP exerts its anti-aging effects by regulating the disorder of gut microbiota and the metabolites in amino acid metabolism,nucleotide metabolism and carbohydrate metabolism on flies.(3)The protective effect of SCP against D-gal-induced cognitive dysfunction in aging mice was investigated.The mice were orally administered with SCP at doses of 0.5 mg/g and1.0 mg/g body weight for 8 weeks.The results indicated that SCP supplementation improved various aspects of cognitive function,including working memory,short-term memory,spatial memory,and long-term memory in aging mice.Besides,SCP supplementation enhanced the activities of antioxidant enzymes,including superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),in both serum and brain of aging mice,and decreased the content of lipid peroxide malondialdehyde(MDA).However,SCP did not affect the inflammatory factors.Notably,SCP did not significantly affect the serum monoamine neurotransmitter level,including 5-hydroxytryptamine(5-HT)and dopamine(DA),and excitatory amino acid neurotransmitters,such as glutamate(Glu),in the brain of aging mice yet increased the choline neurotransmitter level,such as acetylcholine(Ach),and inhibitory amino acid neurotransmitters like gamma-aminobutyric acid(GABA).Furthermore,SCP supplementation improved the neuronal damage in the hippocampal region of aging mice.16s RNA sequencing demonstrated that SCP supplementation increased the diversity of gut microbiota.In genus level,the abundance of beneficial bacteria like Lactobacillus,Enterorhabdus,RF39,and Parvibacter,which were positively correlated with cognitive function,antioxidant enzymes activities,and neurotransmitters were increased.Also,SCP supplementation decreased the abundance of potentially harmful bacteria like Ruminococcaceae,Bacteroides,and Prevotellaceae,which were correlated with cognitive damage.(4)Mechanistically,through q PCR and Western blot analysis SCP significantly inhibited the gene and protein expression of Gabbr1 and GABA_BR which in turn increased the content of second messenger cyclic adenosine monophosphate(c AMP)in cytosol.The c AMP-dependent downstream signal of protein kinase A(PKA)and cyclic adenosine effect element-binding protein(CREB)were found to be phosphorylation.Consequently,the gene expression of brain-derived neurotrophic factor(Bdnf)and neurotrophin-3(Nt3),copper-zinc superoxide dismutase(Sod1),and glutathione peroxidase-1(Gpx1)were increased.The results showed that SCP up-regulated the expression of neurotrophin and antioxidant enzyme genes by regulating GABA_BR-mediated c AMP/PKA/CREB signal to alleviate neuronal injury and oxidative stress damage and increased GABA content which ameliorated the cognitive impairment.Furthermore,when the agonist baclofen of GABA_BR was added to activate the GABA_BR in SH-SY5Y neuroblastoma cell line,the improvement of SCP on D-gal induced neural aging injury was dampened as revealed by the unchanged c AMP and GABA content,the activity of antioxidant enzymes,and brain neurotrophic factors and antioxidant genes expression.The expression of Creb gene and phosphorylation of PKA were also partially inhibited,indicating that the improvement of cognitive impairment in aging mice by SCP was partially dependent on the inhibition of GABA_BR.Therefore,SCP improved cognitive impairment in aging mice by inhibiting GABA_BR activity which subsequently activated c AMP/PKA/CREB signal.(5)A total of 478 peptides were detected by LC-MS/MS in SCP.Through the application of Peptide Ranker score and peptide relative intensity screening,eighteen potentially bioactive peptides with high relative content were obtained.Molecular docking was performed on the binding activity of these 18 peptides to GABA_BR.The results revealed that 14 peptides were able to bind to the GABA_BR via hydrogen bonds,van der Waals forces,hydrophobic interactions,and electrostatic interactions.Among them,KGFP and GPAGPAGLPG exhibited the highest docking energy,and they have the potential to act as bioactive peptides for improve cognitive dysfunction caused by aging.KGFP and GPAGPAGLPG promoted the survival rate,increased the GABA content,SOD and GSH-Px activities of SH-SY5Y senescent cells induced by D-gal through GABA_BR-mediated c AMP/PKA/CREB signaling pathway in vitro.Since the inhibitory effects of SCP on GABA_BR,KGFP and GPAGPAGLPG could binding to the GABA_BR as a GABA_BR-like antagonist.Therefore,KGFP and GPAGPAGLPG may be the potential functional active peptides in SCP to improve the aging-related cognitive impairment.