Research On The Effect And Mechanism Of EF, Icariin And Icariside â…¡ In Extending Healthspan

IntroductionA major goal of current research on aging is to identify compounds that delay age-related diseases and extend healthspan in humans. Herba epimedii is a popular herbal tonic used in traditional Chinese medicine, with proven efficacy in treating several age-related diseases including osteoporosis, cardiovascular diseases, neurodegenerative diseases and sexual dysfunction. Our previous studies on Epimedium Flavones (EF), the raw extract of Epimedium which contains icarrin (ICA) as a major constituent, show that EF delays aging in Drosophila melanogaster and cell senescence model. Interestingly, EF also resets the age-related metabolites (fatty acids, carnosine, ergothioneine and deoxycholic acid et al) to the juvenile level in rat plasma and urine. These findings prompted us to investigate the anti-aging potential of EF and its major pharmacologically active flavonol diglycoside, icariin. Various studies indicate the anti-oxidative effect of icariin and derivatives on DNA damage, β-amyloid mediated neurotoxicity, and vein endothelial cell oxidative injury. Meanwhile, icariin and its derivatives function as signalling modulators to exert beneficial effects in a multitude of age-dependent disease states, including bone loss, cancer, cardiovascular disease, and neurodegenerative disorders. These researches further strengthen the possibility of the effects for EF, icariin and its derivatives to extend healthspan.The highly conserved IIS pathway plays a key role in aging. It has been demonstrated in multiple species that inhibition of the IIS pathway extends lifespan. In C. elegans, the daf-2gene encodes an insulin/IGF-1receptor. Mutations in daf-2suppress the IIS which lead to the nuclear localization of FOXO/DAF-16transcription factor. The activated FOXO/DAF-16regulates a series of genes involved in lifespan control, stress tolerance and protein misfolding suppression. Nuclear localization of DAF-16in the daf-2mutants requires the heat shock transcription factor (HSF)-1, which modulates the expression of heat shock proteins and protease responsible for the stress tolerance and protein folding. Despite the well established role of a number of genes in the IIS pathway in modulating aging, pharmacological tools that inhibit IIS pathway to extend lifespan are not commonly available, which limits its translation to mammalian model systems.Objectives1. To investigate the delaying aging effects of EF and ICA in the experimental systems from invertebrate to mammalian.2. To measure the effects of ICA in extending healthspan.3. To ascertain the bioactive formation of ICA in vivo for the healthspan extension and investigate the molecular mechanisms of these effects.MethodsIn this study, we employed three experimental models including the cell senescence model MRC-5, the simple animal model C. elegans and the mammalian model C57BL/6mice to investigate the delaying aging effects of the compounds. Lifespan is the major measurement to evaluate the effect of the compounds. Stress-resistant assays, mobility assay, qualification of muscle cells, rotarod experiment, protein-misfolding disease models are used to evaluate the effects of the compounds in healthspan. HPLC was used to measure the level of ICA and its derivatives in vivo. Genetic epistasis experiments were used to study the mechanism. Three major genetic modulate pathways in longevity were tested in this study, the insulin/IGF-1pathway, the dietary restriction and the target of rapamycin (TOR) pathway. Real-time PCR, western blot were used to measure the downstream targets of the pathway to ascertain the molecular mechanism.ResultsIn this study, we tested the anti-aging properties of EF, ICA and its three derivatives, icariside Ⅰ (ICS Ⅰ), icariside Ⅱ (ICS Ⅱ) and icaritin (ICT) in the cell senescence model MRC-5, the simple animal model C. elegans and the mammalian model C57BL/6mice. We found that EF has a conserved role in extending lifespan from simple animal to mammalian model. ICA is the major pharmacological element of EF to extend lifespan. Additionally, ICA promotes the stress resistance and mobility in late life of the animals. Meanwhile it ameliorates protein aggregation and protetoxicity-mediated paralysis phenotype. And one of its derivatives, ICS Ⅱ prolonged adult lifespan. Chemical profiles of ICA treated animals revealed that ICS Ⅱ was the predominant bioactive form of ICA in vivo. Furthermore, we found that ICS Ⅱ treated animals have delayed age-associated phenotypes suggesting ICS Ⅱ enhances the healthy aging significantly. Finally, our genetic analysis indicates that Icariside Ⅱ may act through the IIS pathway to affect lifespan.ConclusionOur findings reveal a novel role for EF, ICA, along with its bioactive form ICS II in extending healthspan via the well conserved pathway IIS. Given the extensive protective effects and safe long term use of ICA and ICS II in humans they may serve as promising anti-aging candidates in the future.