Study On The Pharmacodynamics And Mechanism Of Tangeretin In Extending The Lifespan Of Caenorhabditis Elegans

Object:Aging is a process in which various physiological functions of the body gradually decline and lead to death.It is often accompanied by the occurrence of age-ralated diseases such as dementia,heart disease,and arthritis,which brings heavy medical and economic burdens to the elderly and the society.Therefore,effective anti-aging methods have always been expected.Citrus fruits have been proven to have good anti-aging effects,but it is not clear whether tangeretin,the main active ingredient of citrus fruits and citrus original Chinese medicinal herbs,has anti-aging effects.This project uses Caenorhabditis elegans（C.elegans）as an animal model to study the pharmacodynamics of tangeretin to prolong the lifespan of C.elegans and the underlying mechanism.Methods:1.N2 wild-type nematode was used to investigate the effect of various concentrations of tangeretin on the lifespan of the nematode.2.Aging-related phenotype experiments（motility,pharyngeal pumping rate and reproductive ability,etc.）were conducted to explore whether tangeretin can promote healthspan of C.elegans.3.Stress assays（anti-oxidative assay in vitro,H₂O₂-induced oxidative stress tolerance assay and high temperature-induced heat stress tolerance assay）were used to determine whether there is any correlation between the lifespan extending and stress response.4.The lifespan assay of mutant C.elegans（raga-1,aak-2,daf-2,age-1 and daf-16）,real-time fluorescent quantitative PCR experiment（RT-PCR）,nuclear localization of DAF-16,fluorescent quantitative experiment of HSP-16.2,cellular immunofluorescence experiment and Western Blot experiment were conducted to explore whether tangeretin the effects of on the lifespan of C.elegans is related to insulin/insulin-like growth factor signaling pathway（ⅡS pathway）and heat shock protein pathway.Results:1.Compared with the control group,tangeretin at the concentrations of 3μM-100μM prolonged the lifespan of wild-type C.elegans in a concentration-dependent manner,and 100μM tangeretin increased the average lifespan of the worms by 19.8%.2.Compared with the control group,30μM and 100μM tangeretin had no effect on the reproductive capacity and pharyngeal pumping rate of the nematodes,but improved the motility of the worms.3.Anti-oxidative stress experiments in vitro showed that tangeretin had no effect on antioxidant stress,and did not enhance the ability of nematodes to resist oxidative stress,while tangeretin prolonged the lifespan of nematodes under high temperature conditions.4.Compared with that in the control group,the lifespan of mutant nematodes（raga-1,aak-2,daf-2,age-1 and daf-16）was not increased by different concentrations of tangeretin.RT-PCR results showed that tangeretin enhanced the m RNA expression of daf-16,hsp-16.2,hsp-14.9 and other genes.Compared with that in the control group,DAF-16 in the tangeretin treated worms was mainly expressed in the nucleus,and the fluorescence intensity of HSP-16.2 was significantly enhanced.Compared with that in the control group,the fluorescence intensity of autophagy spots in He La-GFP-LC3 cells in the tangeretin group did not change significantly,and the protein expression of P62,LC3Ⅱ and AMPK（AMP-Activated Protein Kinase）did not change significantly.Conclusions:Tangeretin can extend the lifespan of wild-type C.elegans,improve the aging-related phenotypes,and enhance the ability to resist heat stress,but it cannot enhance the oxidative stress tolerance.The lifespan extending effect of tangeretin may be related to ⅡS pathway and heat shock protein（HSPs）,but not autophagy and AMPK signaling pathway.