| Aging is a procedural decay process that organs or tissues gradually deterioratewith ageat the cell level. Aging is common in every species. Aging is generallyassociated with senile diseases such as senile cataracts, heart diseases and so on.However at the same time, aging process is regulated by plentiful transcription factors,anti-aging genes and antioxidant genes. As the study of cell signaling pathways andmolecular mechanisms regarding aging goes further, more and more drugs acting onsome specific targest are also in developing.Currently anti-aging drugs mainly empass natural products and synthetic drugs.Natural products from nature benefit people in return. Research and Development ofnatural products has become the new trend of screening ideal anti-aging drugs.Natural product has a wide range of application, from cosmetics and medicine clinic.It includes herbal drugs, animal drugs, microorganism drugs and so forth. Herbaldrugs have unique advantages in development and therapy, the light side effects, theeffective function on aging and other incurable diseases. In this thesis, the compoundwe studied is convallatoxin, from the lily of the valley. Based on our laboratoryprevious work, we have found convallatoxin can significantly improveSIRT1expression and resistance to oxidative injury for mammal cells. It is wellknown that the high expression of SIRT1retards aging and decreases the occurrencefrequency of chronic age-related diseases. To further determine convallatoxin’santiaging effect, we will explore the specific mechanism on the C. elegans model.In the thesis, we adoptedthe Caenorhabditis elegans (C. elegans) toinvestigateconvallatoxin affect on the lifespan, anti-heat viability and anti-oxidantviability. We mainly inspect the carlorie restriction mediated by sir2.1andinsulin/insulin like growth factor-1signalling(IIS) regarding daf-16. Fluoescencemicrospopic and Real Time PCR are applied to study the molecular regulationmechanism convallatoxin conferred.First, we examined the effect of convallatoxinon C. elegans’ lifespan.Under20℃ laboratory culture condition, for the wild N2type C. elegans,20μM convallatoxinadministration group prolonged C. elegansmean life span16.3%, more than thepositive group resveratrol.2μM convallatxin could not extend C. elegans’ lifespansignificantly, yet high concentration200μM convallatxoin shortened C. elegans’lifespan rather than extension. The result illustrated life extension does not depend ondrug concentration. The convallatoxin’s effect is more like the hormesis effect; Tosir2.1deletion type,20μM convallatxoin still preserved the prolonging lifespan effect;To daf-16deletion type and daf-2/daf-16dual deletion type,20μM convallatxoin wasnot able to extend C. elegans’lifespan. The results related to the mutation type provedconvallatoxin roused lifespan extension through daf-16.To further investigate the convallatoxin-induced genetic mechanism of relayingaging, we mainly examined two age-related genes, daf-16and sir2.1. We used thecorresponding single mutant nematode strain GR1307and VC199to examine thechanges in lifespan, oxidative damage survival and heat shock damage survivalrespectively. Convallatoxin administration can still increase sir2.1(ok434) survival.The result demonstrated lifespan extension by convallatoxin is not associated withsir2.1which mediated calorie restriction pathway. By contrast, administration couldnot prolong daf-16(mgDf50) lifespan, which means that the prolonging effectdepends on daf-16function. In addition, loss of function of daf-16significantly lowthe ability of thermal tolerance and oxidative tolerance, indicating the resistancecapability aligned with the lifespan was also dependent on daf-16. Besides, loss offunction of sir2.1leading to no anti-oxidative stress also explained under oxidativestress the protective capacity is codependent on daf-16and sir2.1. DAF-16is one ofthe FOXO family transcription factors. Its nuclear translocation is the premise of itstranscription activity, activated daf-16in nuclear will start many antioxidant protectionand life extension related genes, including SOD-3, ctl-2, etc. To further detect therelationship between convallatoxin and daf-16, we used TJ356strain, DAF-16::GFPfused expression strain, to detect the gene nuclear translocation status. Convallatoxinpromoted DAF-16nuclear location ratio obviously. We used CF1553strain, SOD-3::GFP fused expression strain, and found after administration SOD-3expression risingover two folds. The upregulated mRNA transcription of ctl-2is detected by Real TimePCR. In the meantime, we also inspected the daf-2in insulin/insulin-like growthfactor II signaling pathway. Singe loss of function of daf-2in CB1370strain could not be extended lifespan by convallatoxin. Because inhibited daf-2activated itsdownstream gene daf-16, when daf-16is in cell nucleus covallatxin could not furtheractivated daf-16and could not extend lifespan. This is in accordance of convallatoxindepending activated daf-16. We also CF1588strain, daf-16&daf-2double mutantstrain to examine the lifespan. Convallatoxin could not change the lifespan of CF1588.Therefore, it revealed the final target of convallatoxin is daf-16rather than othergenes. In addition, we found high concentration of convallatoxin had no longevity andstimulation of daf-16nuclear translocation effect, whereby we inferred convallatoxinprobably activate specific genes through hormesis, eventually acted on daf-16transcription factor and promoted downstream anti-aging genes expression,subsequently prolonged the C. elegans lifespan.Taken together, the thesis determined convallatoxin’s effect of prolonginglongevity and characterized its function features, initially explained the molecularmechanism of the drug. The research can lay a foundation for further studying thecomplete and deep molecular mechanism of convallatoxin and determining its directtarget gene and target signaling pathway. |
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