RBTI Slow Down Aging And Its Effects On Age-related Disease In C.elegans

Buckwheat,belonged to small coarse cereal crop,contains rich natural activity composition including proteins,amino acids,vitamins B1 and B2,dietary fiber,minerals and so on.Supplement of buckwheat in daily diet can reduce the risk of some age-related diseases,such as hypertension,hyperlipemia,heart diseases,diabetes and so on.Buckwheat trypsin inhibitor(BTI),derived from buckwheat seeds,is composed of 69 amino acids and has a molecular weight of 7.9 kD.BTI has excellent water-solubility.And it belongs to serine protease inhibitor of the potato inhibitor I family,and could strongly inhibit the activity of trypsin in specific activity assays.For being convenient to research its biological function,BTI gene sequence was obtained by polymerase chain reaction(PCR)using BTI cDNA as the template.BTI cDNA was synthesized using total RNA isolated from buckwheat leaves.And then a recombinant buckwheat trypsin inhibitor(rBTI)was prepared via cloning,expression and one-step affinity purification in our laboratory.rBTI possesses same amino acid sequence and properties with the nature BTI from buckwheat seeds.Function analysis showed that rBTI could inhibit the proliferation in several solid tumor cells,such as EC9706,Hep G2,and HeLa.Moreover,rBTI could induce mitophagy by inducing a transient ROS signal in Hep G2,and induce cell cycle arrest in G0/G1 phase,keep the cell division cycle longer in EC9706.Recently,the study in drosophila melanogaster showed that rBTI could extend lifespan by increasing the antioxidant capacity in drosophila,but the underlying mechanisms are still mysterious.In this paper,we mainly investigate the effects of rBTI on aging,age-related diseases and the mechanisms in the classical model organism caenorhabditis elegans(C.elegans),specific results are as follows:1 The effects of rBTI on the healthspan,food intake and motility in C.elegansThe effect of rBTI on the lifespan was detected by 5-fluoro-2′-deoxyuridine(FudR)lifespan assay method in C.elegans.The results showed that rBTI significantly extended the lifespan in a dose-dependent way within the range of experimental doses(2.5-10 μM)in C.elegans.Detection of aging-related indicators showed that rBTI significantly reduced the activity of senescence-associated β-galactosidase and intestinal lipofuscin accumulation in C.elegans.At the same time,the results of microscopic observation showed that rBTI did not significantly affect the motility and pharyngeal pumping rate in young worms,but improved the motility in old worms.This suggested that rBTI extented the heathspan in C.elegans.2 The underlying mechanism of rBTI-mediatd longevityThe test results of reverse phase high performance liquid chromatography(HPLC),oxygen consumption,fluorescence probe,enzyme activity and oxidative stress induced by juglone showed rBTI increased the AMP: ATP ratio,induced the enhancement of respiration and the production of ROS.And then the ROS signal induced the increase of endogenous oxidative defense system,which in turn reduced the ROS content,finally enhanced anti-oxidative stress resistance in C.elegans.When the ROS signal was inhibited by the ROS inhibitor N-Acetyl-L-cysteine(NAC),the effect of rBTI-mediated longvity was abolished in C.elegans.This indicated rBTI-mediated longevity was dependent on the transient ROS signal.This mechanism is very similar with calorie restriction(CR),thus we assumed that the effect of rBTI-mediated longevity mimicked CR,namely calorie restriction mimetic(CRM).The results of qRT-PCR showed that rBTI down-regulates the transcriptional expression of genes involved in the insulin/IGF-1 signaling(IIS)pathway,namely daf-2,age-1,and akt-1.In addition,the test results of transgenic stains and qRT-PCR revealed that rBTI increased the transcriptional activity of DAF-16 which up-regulated the expression of genes involving in antioxidant defense.However,rBTI did not affect transcriptional activity of HSF-1 and SKN-1 which are the other two transcrptional factors downstream of the IIS pathway.The experiments in mutant strains showed that rBTI did not extend lifespan in daf-2 and daf-16 mutants,suggesting that rBTI-mediated longevity was dependent on daf-2 and daf-16,namely IIS pathway.Meanwhile,detection of fluorescence probe showed that rBTI did not induce transient ROS signaling in daf-2,daf-16 mutants,but rBTI still down-regulated the IIS pathway when the ROS signal was inhibited by ROS inhibitor NAC.These data indicated that the down-regulation of IIS was the upstream event of ROS signal.3 The effects of rBTI on metabolism in C.elegansSimilar with the effects of CR on metabolism,qRT-PCR and enzyme activity analysis showed that rBTI reduced the activity of enzymes involved in glycolytic and enhanced the lipase acivity in C.elegans.At the same time,oil-red staining showed that the fat storage was decreased in rBTI-treated worms.The results indicated that rBTI induced shift from carbohydrate catabolism to lipid catabolism in C.elegans.Additionally,rBTI increased the autophagy level in C.elegans.4 The relation between rBTI-mediated longevity and its molecular structureBased on the structural features and active sites of rBTI,the 45,53,44 residues active sites of rBTI were mutated by site-mutation technique,and rBTI-R45 A,rBTI-R45 F,rBTI-W53 R,rBTI-P44 T mutants whose trypsin inhibitory activity ware changed,were generated.Then the effects of rBTI mutants on lifespan were investigated in C.elegans.The results showed the mutants of rBTI had no significant effect on the lifespan in C.elegans.Consistent with the study of lifespan,rBTI mutants also did not increase the transcriptional activity of DAF-16 which is the main transcriptional factor regulated by wild rBTI to extent lifespan in C.elegans.Overall,these date suggested that rBTI-mediated longvity was depended on its trypsin inhibitory activity.5 The effects of rBTI on age-related diseases and its mechanismAlzheimer’s disease(AD)is an age-related neurodegenerative disease,of which β-amyloid(Aβ)induced toxicity was suggested as a main cause.Some substances with prolongevity effects have been shown to be protective against AD.In this study,the AD model worms CL4176 which express human Aβ3-43 in body wall cells,was used to test the effect of rBTI on AD.And western blot,immunoblotting,fluorescence staining and RNA interference(RNAi)were used to investigate the preliminary mechanism.The results showed that rBTI not only extended lifespan but also reduced Aβ toxicity-triggered body paralysis in AD model C.elegans.Further study found the accumulation of Aβ was decreased and autophagy-lysosomal degradation pathway was activated in AD worms treated with rBTI.Moreover,the inhibition of autophagy reduced rBTI-mediated paralysis delay.Genetic analyses showed rBTI increased the transcriptional activity of DAF-16 and the disruption of daf-16 abolished rBTI-mediated protective effect in AD worms.Taken together,these data indicated that rBTI promoted the autophagy-lysosomal degradation pathway to reduce the Aβ-induced toxicity via DAF-16 in an AD model C.elegans,implying that BTI has the potential to protect against AD.In addtion,the experiment in Huntington’s disease(HD)model C.elegans showed that rBTI also reduced the aggregation of the polyglutamine amide and its induced paralysis phenotype in the HD model.The results suggested that rBTI may have a beneficial effect on another aging-related disease HD.In conclusion,this study demonstrated that rBTI could improve the motor ability of aging worms,and extend the healthspan in C.elegans.Similar to mechanism of CR-mediated longvity,rBTI could down-regulate the IIS pathway,induced a transient ROS signal,and then activated the transcriptional activity of DAF-16 which increased the expression of its target genes and the ability of oxidative stress resistance,finally extend the lifespan.Moreover,rBTI could induce the change of carbohydrate metabolism to the lipid metabolism in C.elegans.The studies in transgenic worms and AD model worms indicated that r BTI not only enhanced autophagy,but also promoted the autophagy-lysosomal degradation pathway,and protected against AD.These results provide a good foundation for further research on the mechanism and application of bioactive substances in anti-aging and anti-Alzheimer’ disease.