Molecular Mechanisms Underlying Extending Healthspan Of Caenorhabditis Elegans By Peptides From Sesame Cake

With the continuous improvement of living standards,people's expectations for health and longevity are rising gradually.Accumulating evidence has shown that aging is mainly caused by the imbalance of intracellular free radicals,which leads to metabolic disorders and occurrence of a variety of diseases with high mortality in the organism.Antioxidants could delay human aging by removing excess free radicals and reducing the invasion of toxic substances in the organism.Hence,an alternative strategy for delaying senescence is to search and develop efficient and accessible antioxidants,which has become an urgent issue in the field of biology and food science.It is a preferred option to use agricultural by-products to produce active peptides,because the materials are inexpensive and safe.Sesame(Sesamum indicum Linn.)is one of the four major oil crops in China and more than 80 % of the sesame in China is used to extract sesame oil.The annual yield of sesame cake is more than 500,000 tons,which is mainly used as agricultural fertilizer or feed.Sesame cake is rich in nutrients and bioactive substances.In order to increase the bioavailability of sesame cake,peptides from sesame cake(PSC)were prepared by buffer extraction,ammonium sulfate precipitation and chromatography in our laboratory.The peptides in PSC were identified by Q-Exactive LC-MS/MS and PSC exhibited good antioxidant activity in vitro.In order to further explore the anti-oxidative stress and anti-aging effects of PSC in vivo,Caenorhabditis elegans(C.elegans),a classic model organism in aging research,were employed to study the effect of PSC on the healthspan of C.elegans and underlying molecular mechanisms.The main research contents of this project are as follows:1.The effect of PSC on the lifespan and aging-related parameters in C.elegansThe effect of PSC on the survival rate of wild-type(N2)C.elegans was tested under normal culture condition.The result showed that PSC could prolong the mean lifespan of C.elegans in a dose-dependent manner in the range of 3.125-12.5 ?g/m L.However,the extent of PSC-mediated longevity is decreasing when PSC concentration exceeds 12.5 ?g/m L.Since aging is a complex physiological process,lifespan is not enough to describe this complicated process.Therefore,three healthy parameters in C.elegans were assayed.The results showed that PSC improved the health parameters of C.elegans,including pharyngeal pumping rate,locomotion and lipofuscin accumulation.2.The molecular mechanism underlying PSC-mediated longevity in C.elegansThe lifespan of C.elegans is positively correlated with stress resistance.The effect of PSC on survival rate of C.elegans under stress condition was tested.Results showed that PSC increased the survival rate of C.elegans by about 38.9 % under 240 ?M juglone induced stress condition.In addition,PSC increased survival rate significantly in the hypersensitive mutant mev-1(kn1),which carries a mutation in cytochrome b of the mitochondrial respiratory chain complex II.Moreover,PSC promoted antioxidant enzyme activity in C.elegans.Furthermore,DCF-DA fluorescent probe was used to detect endogenous reactive oxygen species(ROS)in C.elegans.The result showed that PSC reduced the ROS levels,suggesting that PSC may reduce the ROS levels by increasing the activity of antioxidant enzymes,thereby increased resistance to stress and prolonged lifespan in C.elegans.We subsequently explored the specific molecular mechanism of PSC-mediated longevity.In order to find out whether PSC-mediated longevity was derived from the amino acids of PSC,lifespan assay was performed by using amino acids formed by PSC hydrolysis.The result showed that hydrolysed PSC did not affect the mean lifespan of N2 nematodes compared with the control group,indicating that PSC-induced longevity was independent on the hydrolysed amino acids of PSC.Dietary restriction(DR)is an evolutionarily conserved longevity mechanism.In order to explore whether dietary restriction is involved in PSC-mediated lifespan-extension,food intake and the lifespan of two DR mutants,eat-2 and sir-2.1,were analyzed.The results showed that PSC did not affect food intake and increased the lifespan of eat-2 and sir2.1 mutant,indicating that PSC-mediated lifespan-extension was independent of dietary restriction.The results of q RT-PCR and nuclear translocation of transcription factors showed that the beneficial effects of PSC on C.elegans were dependent on the stress-related transcription factor Nrf-2/SKN-1 and independent of FOXO/DAF-16 and HSF-1 transcription factors.The result of the mutant lifespan further verified PSC-mediated longevity was dependent on activating the Nrf-2/SKN-1 transcription factor.Nrf-2/SKN-1 transcription factor is primarily regulated by the IIS pathway and the p38 MAPK signaling pathway in C.elegans.The lifespan of mutants in these two pathways were examined.The result showed that PSC increased the lifespan of daf-2 mutant and did not affect the lifespan of tir-1,nsy-1,sek-1 and pmk-1 mutants,indicating that the p38 MAPK signaling pathway is necessary for the PSC-mediated lifespan extension.q RT-PCR of mutants further confirmed that the PSC-activated Nrf-2/SKN-1 transcription factor is dependent on the p38 MAPK signaling pathway.In C.elegans,Nrf-2/SKN-1 transcription factor is mainly expressed in two isoforms,which are SKN-1b expressed in ASI neurons and SKN-1c expressed in intestinal cells.We used two SKN-1 transgenic strains to explore the tissue specificity of PSC-activated Nrf-2/SKN-1 transcription factor.The results showed that PSC significantly increased the nuclear translocation of SKN-1c.In addition,PSC could increase the expression of PMK-1.We successfully constructed pmk-1 RNA interference(RNAi)bacteria.The results showed that pmk-1 RNAi abolished PSC-induced SKN-1c nuclear translocation.The results of VP303 lifespan showed that the intestine-specific pmk-1 and skn-1 RNAi abrogated PSC-mediated longevity,further confirming PSC regulated intestine-specific SKN-1c and p38 MAPK signaling pathway.Together,PSC activated the intestine-specific p38 MAPK signaling pathway,inducing nuclear translocation of SKN-1c,and then activating the antioxidant enzyme system,thereby increasing the stress resistance and lifespan of C.elegans.3.The effect of PSC on neurodegeneration diseases and potential molecular mechanism in C.elegansThe prolonging of healthspan mainly includes the extension of functional and disease-free period of life and the shortening of disease duration.Aging is closely associated with chronic diseases including neurodegeneration disease,cancer,cardiovascular disease,and type II diabetes.In this study,the disease model C.elegans were used to explore the beneficial effects of PSC on the most common neurodegeneration diseases and underlying mechanism.Alzheimer's disease(AD),an age-related disease,is the most common neurodegeneration disease.The pathogenesis of AD is complex and not yet fully understood.However,its pathogenesis is believed to be driven by amyloid-?(A?)-induced toxicity and oxidative stress.We employed two A? transgenic AD models C.elegans,which were CL4176 nematodes expressed A? in body wall muscle cells and CL2355 nematodes expressed A? in neurons.The effects of PSC on A?-induced toxicity and molecular mechanism were studied.The results showed that PSC prolonged the lifespan of CL4176 nematodes and improved their movement ability.Moreover,PSC reduced A?-induced paralyzed rate and improved chemotaxis defect.The results of immunoblotting,immunofluorescence,and thioflavin S staining showed that PSC reduced the accumulation of A? species.The chemical kinetics experiment and atomic force microscopy(AFM)results showed that PSC accelerated the aggregation of A? in vitro,indicating that the interaction between PSC and A? in vivo may be indirect.Proteasome activity and autophagy lysosomal assays showed that PSC may reduce the accumulation of A? by increasing the proteasome activity.It needs to be explored how PSC activate the proteasome pathway in the next step.Based on the mutual promotion between A? accumulation and ROS,the ROS levels were examined.The results showed that PSC reduced the ROS levels in CL4176 nematodes and increased the expression of antioxidant enzyme genes.RNAi assay showed that SKN-1 was required for PSC-mediated protection against A?-induced toxicity and oxidative stress.Parkinson's disease(PD)is the second most common neurodegeneration disease.PD is characterized by two pathological conditions: the formation of Lewy bodies [i.e.,the aggregation of ?-synuclein(?-syn)] in the brain and the selective death of dopaminergic(DA)neurons in the substantia nigra of the midbrain.In order to study the effect of PSC on PD-related pathology,two transgenic PD models C.elegans were adopted,which were NL5901 nematode expressed ?-syn::YFP fusion proteins in muscle cells and BZ555 nematode expressed GFP fusion in dopaminergic neurons.The results showed that PSC could promote the longevity of NL5901 nematodes and improve the locomotion.PSC not only reduced ?-syn accumulation,but also reduced MPP+-induced dopaminergic neuron degeneration and dysregulation of food-sensing behaviour.RNAi assay demonstrated that the beneficial effects of PSC on PD are dependent on SKN-1.Huntington's disease(HD)is characterized by the hallmark presence of expanded polyglutamine(poly Q)tracts.To study the effects of PSC on poly Q-induced toxicity and underlying molecular mechanisms,we employed two transgenic C.elegans models,AM140 and HA759,which respectively express poly Q expansions in muscle cells and ASH neurons.PSC extended lifespan and alleviated poly Q-induced toxicity in these nematodes.Strikingly,PSC not only decreased poly Q aggregation,but also reduced poly Q-mediated neuronal death and behavioral dysfunction in ASH neurons.Moreover,PSC increased oxidative stress resistance and restored functional parameters of mitochondria.RNAi assay demonstrated that the beneficial effects of PSC on poly Q-induced toxicity were dependent on SKN-1.In summary,this study showed that PSC could prolong the healthspan of C.elegans through the intestinal-specific p38 MAPK-SKN-1c pathway,reduce the marker proteins aggregation and toxicity of aging-related neurodegenerative diseases,indicating that PSC may be used as nutraceuticals to improve and prevent oxidative stress complications during the course of aging and aging-related diseases.This project has enriched the application of bioactive substances in the aging intervention field and provided an important theoretical basis for improving the economic value of agricultural by-products and the functional development of PSC.