Icariin and its Derivative Icariside II Extend Healthspan via Insulin/IGF-1 Pathway in C. elegans Wai-Jiao Cai 1,2. , Jian-Hua Huang 1. , Su-Qin Zhang 1,3 , Bin Wu 1 , Pankaj Kapahi 2 *, Xin-Min Zhang 1 *, Zi-Yin Shen 1 * 1 Institute of Traditional Chinese and Western Medicine, Huashan Hospital, Fudan University, Shanghai, China, 2 Buck Institute for Research on Aging, Novato, California, United States of America, 3 Clinical Medicine College of Hangzhou Normal University & The Second Hospital of Hangzhou affiliated to Hangzhou Normal University, Hangzhou, China Abstract Compounds that delay aging might also postpone age-related diseases and extend healthspan in humans. Icariin is a flavonol extracted from several plant species of the Epimedium family. The icariin and its metabolic derivatives have been shown to exert wide protective effects in age-related diseases. However, whether icariin and its derivatives have the potency of delaying aging remains unclear. Here, we report that icariin and its derivative icariside II extend C. elegans lifespan. Using HPLC, we found high level of icariside II in the animals treated with icariin, suggesting icariside II is the bioactive form in vivo of icariin. Icariside II also increased the thermo and oxidative stress tolerance, slowed locomotion decline in late adulthood and delayed the onset of paralysis mediated by polyQ and Ab 1–42 proteotoxicity. The lifespan extension effect of icariside II is dependent on the insulin/IGF-1 signaling (IIS) since the daf-16(mu86) and daf-2(e1370) failed to show any lifespan extension upon icariside II treatment. Consistently, icariside II treatment upregulates the expression of DAF-16 targets in the wild-type. Moreover, our data suggests that the heat shock transcription factor HSF-1 has a role in icariside II-dependent lifespan extension further implicating the IIS pathway. In conclusion, we demonstrate a novel natural compound, icariside II as the bioactive form of icariin, extends the healthspan via IIS pathway in C. elegans. Citation: Cai W-J, Huang J-H, Zhang S-Q, Wu B, Kapahi P, et al. (2011) Icariin and its Derivative Icariside II Extend Healthspan via Insulin/IGF-1 Pathway in C. elegans. PLoS ONE 6(12): e28835. doi:10.1371/journal.pone.0028835 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received October 11, 2011; Accepted November 15, 2011; Published December 21, 2011 Copyright: ß 2011 Cai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by 973 program in China (code: 2007CB507400). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pkapahi@buckinstitute.org (PK); xmzhang54@yahoo.com (X-MZ); ziyinshen@126.com (Z-YS) . These authors contributed equally to this work. Introduction A 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 [1,2]. Icariin is the major pharmacologically active flavonol diglycoside of Herba epimedii. Various studies indicate the anti-oxidative effect of icariin on DNA damage, b-amyloid mediated neurotoxicity, and vein endothelial cell oxidative injury [1]. Meanwhile, icariin and its derivatives function as signaling modulators to exert beneficial effects in a multitude of age-dependent disease states, including bone loss, cancer, cardiovascular disease, and neurodegenerative disorders [3,4,5,6]. However, whether icariin and its derivatives own the potency of slowing aging remain elusive. Our previous studies on Epimedium Flavones (EF), the raw extract of Epimedium which contains icarrin as a major constituent, show that EF delays aging in Drosophila melanogaster and Caenorhabditis. elegans [7,8]. Interestingly, EF also resets the age-related metabolites (fatty acids, carnosine, ergothio- neine and deoxycholic acid et al) to the juvenile level in rat plasma and urine [9,10]. These findings further prompted us to investigate the anti-aging potential of icariin and its derivatives. The experimental organism, Caenorhabditis elegans is a well- established aging model, sharing similar aspects of aging with mammals, such as sarcopenia and locomotion decline [11]. C. elegans has provided critical insight on conserved lifespan-regulated pathways, for example insulin/insulin-like growth factor signaling (IIS) [12], making C. elegans a model system to identify novel genetic links and compounds that can promote healthy aging in humans. 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 [12]. In C. elegans, the daf-2 gene encodes an insulin/IGF-1 receptor. Mutations in daf-2 suppress the IIS which lead to the nuclear localization of FOXO/DAF-16 transcription factor. The activated FOXO/DAF-16 regulates a series of genes involved in lifespan control, stress tolerance and protein misfolding suppression [13,14]. Nuclear localization of DAF-16 in the daf-2 mutants 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 [15]. 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. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28835