The Study On The Effect Of Nomilin On Longevity Of Caenorhabditis Elegans And Its Mechanism

Background:Aging is not only a decisive factor in human longevity,but also one of the major risk factors for many age-related diseases,such as cancer,cardiovascular disease,and diabetes.Therefore,the research of anti-aging drugs has been paid more and more attention to all over the world.Nomilin（NML）is one of the major limonoids,which are highly oxygenated triterpenoids present in the Rutaceae family plants.NML is found mostly in citrus fruits and traditional Chinese medicines derived from citrus,such as tangerine seed,tangerine peel,etc.Recent studies have demonstrated that nomilin has many pharmacological activities,including anti-cancer,anti-inflammatory,and antioxidant etc.Our previous studies have proved that NML is an agonist for the human nuclear hormone receptor PXR.It is reported that nuclear hormone receptor PXR participates in the biological regulation and detoxification of endo-and xeno-biotics by activating cytochrome p450oxidase.However,the effect of NML on longevity and whether its activation of the nuclear hormone receptor PXR mediated detoxification is associated with lifespan still remain unclear.Objective:In this study,we used Caenorhabditis elegans（C.elegans）as a model organism to investigate the effect of nomilin on longevity and nuclear hormone receptor mediated detoxification in C.elegans,as well as to elucidate its potential mechanism.Methods:1.Antibacterial tests of DMSO and NML on OP50,and chemotaxis assays were carried out to evaluate whether DMSO and/or NML could influence the food and drug intake of C.elegans;2.Lifespan assays:Wild type N2 or C.elegans mutants,including daf-2,daf-16,sir-2.1,and raga-1 were cultured synchronously to L4 larvae,and then treated with DMSO（as control）or NML at 25,50,and 100μM to investigate the effect of NML on lifespan extension in C.elegans and to determine the most effective dosage of NML;3.Reproduction assay:The period of reproduction as well as the brood size in NML-treated and untreated worms were tested;4.Evaluation of the lifespan-related physiological parameters:Using stereo microscope to observe and record the swallowing frequency,head swing frequency,body bending frequency,and lipofuscin in NML-treated and untreated worms;5.Detection the body fat content in L4 C.elegans:The body fat content in NML-treated and untreated L4 C.elegans was measured by oil red O staining and spectrophotography;6.Stress assays:The heat stress（35℃）and 0.5‰H₂O₂-induced oxidative stress assays were conducted to examine whether NML could improve the anti-stress ability and extend the lifespan in C.elegans;7.DAF-16 translocation assay:The transgenic daf-16P::daf-16::GFP worms were treated with DMSO or 50μM NML under normal culture conditions,and then observed subcellular DAF-16 localization by using confocal laser microscope;8.Detecting the relationship between the nuclear hormone receptor PXR and lifespan:PXR homologous mutants and h PXR transgenic mutants were cultured synchronously to L4 larvae,and then treated with DMSO or 50μM NML to investigate the effect of NML on lifespan extension in these mutants;9.Toxicity assays:The lifespan assays of NML-treated and untreated N2 C.elegans,nhr-8,daf-12,daf-16,and daf-2 mutants fed with the test chemicals:chloroquine,colchicine,paraquat,and Me Hg Cl were carried out;10.q RT-PCR:The m RNA levels of daf-2,daf-16,sod-3,hsp-16.2,sir-2.1,pha-4,and some detoxifying genes in NML-treated and untreated N2 C.elegans were tested.Results:1.DMSO and NML did not inhibit the growth rate of OP50,and also had no effect on the food and drug intake in C.elegans;2.NML extended the lifespan of N2 C.elegans,and 50μM NML is the most effective dosage;NML prolonged the lifespans of C.elegans treated with 50μM NML in its life-cycles L1-L4,L2-L4,and L3-L4;3.NML mediated lifespan extension is not associated with any significant changes in the reproductive capacity of C.elegans;4.NML significantly alleviated the age-related decline in physical activities,such as swallowing frequency,head swing frequency,and body bending frequency,and also significantly reduced the accumulation of lipofuscin induced by aging in N2 C.elegans;5.50μM and 100μM NML increased the body fat content in L4 C.elegans;6.Compared to the controls,NML could prolong the lifespan in C.elegans under heat stress（35℃）and 0.5‰H₂O₂-induced oxidative stress,and enhanced the resistance in C.elegans to heat stress as well as oxidative stress induced by 0.5‰H₂O₂;7.The results of studies on the longevity mechanisms indicated that NML modulates nuclear localization and transcriptional activity of DAF-16,and then regulates its target genes to promote longevity and to increase stress response in C.elegans;8.Compared with the controls,there was no obvious effect on longevity in NML50-treated PXR homologous mutants,including nhr-241,nhr-242,nhr-8,and daf-12;The mean survival times of NML50-treated h PXR transgenic mutants N2-PXR,nhr-8-PXR,and daf-12-PXR were significantly longer than those of the corresponding controls;9.The results of the toxicity assays shown that compared to the controls,NML could increase the resistance of N2 C.elegans to the xenobiotics:chloroquine,colchicine,paraquat,and Me Hg Cl.However,the resistances of NML-treated nhr-8 and daf-12 mutants to these xenobiotics were significantly lower than those of N2 C.elegans;10.The m RNA expression levels of daf-16,sod-3,hsp-16.2,and some detoxifying genes,including cyp35a3,cyp35a4,cyp35b2,cyp36a1,gst-4,pgp-3,pgp-12,and pgp-13 were up-regulated,while the m RNA levels of daf-2,sir-2.1,pha-4 were down-regulated after treatment with NML.Conclusion:The results obtained in this study reveal that the lifespan extension in C.elegans caused by NML is dependent on Insulin/Insulin-like growth factor-1（IIS）signaling pathway and tightly associated with the increased daf-16 expression.The results also demonstrated that NML extends the lifespan in C.elegans by activating the mammalian PXR homologous nuclear hormone receptors NHR-8 and DAF-12 mediated detoxification of endo-and xeno-biotics in C.elegans.