The Effect Of Calycosin On Caenorhabditis Elegans Lifespan And Its Molecular Mechanism

Calycosin is a naturally flavonoid,mainly occurring extracted from Mongholicus Bunge,has been widely utilized as a traditional medicine for treatment of hypertensions and diabetes in China.Given its well known antioxidative and anti-inflammatory properties,organisms treated with calycosin often exhibit a significant reduction of peroxides species,potent members of ROS,while activities of antioxidant enzymes such as superoxide dismutases(SOD)are up-regulated.The antioxidant activity of calycosin has attracted more and more attention.However,there is no report on the study of anti-aging effects of clycosin now.Objective: To investigate the effects of calycosin on the lifespan and the molecular mechanism in C.elegans(Caenorkabditiselegans).Methods: 1.Lifespan assay: Synchronized L4 larvae were treated with 0,50,100,200,and 400 ?M calycosin to evaluate its anti-aging effect.2.Stress resistance assays: To determine whether the lifespan extension induced by calycosin was associated with its anti-oxidadant effect,treated and untreated worms were subjected to oxidative stress and heat shock stress conditions.3.Analysis of reactive oxygen species(ROS): To explore whether calycosin treatment could delay the accumulation of aging-related cellular damage in C.elegans,we evaluated the free radical-scavenging abilities of calycosin.4.SubcellularDAF-16 ang SKN-1 localization:Transgenic strain TJ356 and LD001 were treated with 200 ?M of calycosin and then detected the subcellular localization of DAF-16 and SKN-1.5.Mutant lifespan assay:We used kinds of loss-of-function mutant worms to investigate whether the calycosin-modulated longevity in C.elegans were acting through relative genes.6.Molecular mechanism: We examined the effect of calycosin on the expression of daf-16,sod-3,hsp-16.2 and ctl-1 in N2 worms.7.Pharyngeal pumping assay: Comparing the pharyngeal pumping of calycosin-treated worms and untreated worms on the 3rd,6th,and 9th day of adulthood,to determine whether calycosin could leading to reduced food intake(calorie restriction,CR).Results: 1.Calycosin can extended the lifespan of C.elegans in a certain concentration range,and 200 ?M of calycosin treatment displayed the largest extension.2.Calycosin improves the stress resistance and reduces the intracellular ROS level in C.elegans.3.Extending lifespan by calycosin requires insulin signaling.4.Calycosin likely activates the core transcription factor DAF-16 and promotes its nuclear localization,and that the calycosin-induced longevity process is independent of the factor SKN-1.5.The longevity enhanced by calycosin isn?t dependent on calorie restriction.6.The longevity effect of calycosin does not require the presence of unc-43.7.Calycosin induces lifespan extension may not proceed through the CR mechanism.Conclusion: In summary,we have reported here the longevity effect of calycosin and elucidated its potential underpinning mechanisms using Caenorhabditis elegans as a model organism.Given that the insulin signaling pathway is highly conserved from nematodes to mammals,this study will be utilized in anti-aging food and drugs.