Neurobiology of Aging xxx (2015) 1e9

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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging

Tetrahydroxystilbene glucoside extends mouse life span via upregulating neural klotho and downregulating neural insulin or insulin-like growth factor 1 Xuanxuan Zhou a, b,1, Qian Yang a, b,1, Yanhua Xie a, b,1, Jiyuan Sun a, b, Jing Hu a, b, Pengcheng Qiu a, b, Wei Cao a, b, *, Siwang Wang a, b, ** a b

Department of Natural Medicine, School of Pharmacy, Fourth Military Medical, University, Xi’an, China Collaborative Innovation Center for Chinese Medicine in Qinba Moutains, Xi’an, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 December 2013 Received in revised form 26 October 2014 Accepted 4 November 2014

A Chinese herb, Polygonatum multiflorum, has been reported to prolong animal life span, but the relevant molecular mechanism remains unclear. Tetrahydroxystilbene glucoside (TSG) is one main component of P. multiflorum and may contribute to extending life span of mammals. On the other hand, neuronal insulin signaling mediates the life span of mammals. Therefore, we investigated the effects of TSG on memory ability, life span, and the neural insulin signaling in the senescence-accelerated prone mouse (SAMP8). TSG improved the memory ability significantly (p < 0.01, compared with a control group). TSG prolonged the life span of SAMP8 by 17% at the most (p < 0.01, compared with a control group). TSG increased the protein level of neural klotho and reduced the levels of neural insulin, insulin-receptor, insulin-like growth factor-1, and insulin-like growth factor-1 receptor in the brain of SAMP8 (p < 0.01, compared with a control group). All these proteins are key factors of the pathways related to neural insulin/IGF-1 signaling. These findings suggest that TSG has anti-aging effects on mammals. From these results, TSG from P. multiflorum should be developed as a potential anti-age drug. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Aging Insulin Insulin-like growth factor 1 Klotho Senescence-accelerated prone mouse Tetrahydroxystilbene glucoside

1. Introduction Aging, an inevitable biological process, is mainly characterized by a general decline in physiological functions that lead to morbidity and mortality. Many scientists dedicate their whole lives to find the best way to reverse aging process (Mikhelson and Gamaley, 2012; Sirin et al., 2012), but no sure results are available, and the specific causes of the decline remain unknown. The multiple roles of insulin in the central nervous system have been widely reported (Coomans et al., 2013; Duarte et al., 2012; Dupraz et al., 2013; Estrada et al., 2014; Filippi et al., 2013). It seems that the dysregulation of insulin can increase the risk of aging-related disorders (Avogaro et al., 2013; Craft, 2010; Solas et al., 2013; Sonntag et al., 2013; Thambisetty et al., 2013; Zoico et al., 2013). * Corresponding author at: Department of Natural Medicine, School of Pharmacy, The Fourth Military Medical University, Xi’an 710032, China. Tel.: þ86 29 84774352; fax: þ86 29 84774353. ** Alternate corresponding author at: Collaborative Innovation Center for Chinese Medicine in Qinba Moutains, 169 Changle West Road, Xi’an 710032, China. Tel.: þ86 29 84772519; fax: þ86 29 83224790. E-mail addresses: [email protected] (W. Cao), [email protected] (S. Wang). 1 These authors contributed equally to this work. 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.11.002

In mammals, insulin, insulin-like growth factor-1 (IGF-1), and growth hormone (GH) are interdependent molecules (Melmed et al., 2010; Piotrowska et al., 2013). Despite the similarities between mammals and invertebrates in insulin-like proteins and their functions, the mechanism for these signals controlling aging is still controversial (Bartke et al., 2003; Sonntag et al., 2012). Klotho is a 120e135 kDa type-I transmembrane protein and directly linked to aging (Kuro-O, 2008). Enhancing the expression of klotho is another way to delay aging process (Bartke, 2006; Bonafè and Olivieri, 2009). On the other hand, type-I transmembrane proteins are often shedded by the metalloproteases just like ADAM, a kind of disintegrins and metalloproteases (Zhang et al., 2014). The ectodomains of type-1 transmembrane protein can be released by ADAMs and show important physiological functions, such as the release of soluble amyloid precursor protein via ADAM10 (alphasecretase) and BACE1 (beta-secretase, a key enzyme in the pathology of Alzheimer’s disease) (Endres and Fahrenholz, 2012; Prox et al., 2012; Shaikh et al., 2014). The remaining membrane-bound fragments “stubs” can be the substrates of an enzymatic complex called gamma-secretase (Chalaris et al., 2010). Therefore, it is important to explore these enzymes which may affect the functions of type-1 transmembrane proteins, just like klotho.

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Among other proteins, forkhead transcription factor (FOXO) regulated by klotho, insulin, and IGF-1, is well known to inhibit the accumulation of free radicals reactive oxygen species (ROS), which is closely related with aging (Kurz et al., 2004). The maintenance of ROS levels by klotho and insulin/IGF-1 signaling pathway can prevent the progression of cell damage and aging. Because klotho and insulin/ IGF-1 signaling pathway are closely associated with ROS accumulation, the protein levels of klotho and insulin signaling pathway are critical for anti-aging activity (Yamamoto et al., 2005). The highest levels of Klotho have been found in 2 brain regions: the choroid plexus and cerebellar Purkinje cells (German et al., 2012). Klotho can extend animal life span and has been regarded as an “aging suppressor” (Dermaku-Sopjani et al., 2013). IGF-1 has important effects on the cerebrovasculature as well as neurons and glia (Sonntag et al., 2013). Klotho represses intracellular signals of insulin and/or IGF-1 (Kurosu et al., 2005). The reduced IGF-1 is associated with an extension of animal life span (Shimokawa et al., 2003). Gene therapy may be an ideal way to increase the life span of mammals (Fu et al., 2007). However, the early hopes of gene therapy remain unfulfilled (Takeda, 2009). Much pharmaceutical research has focused on the development of some drugs to prevent aging and creates many pharmacologic agents (rapamycin, metformin, resveratrol, and sirtuins) and their targets (Blagosklonny, 2013; Della-Morte et al., 2013; Guarente, 2013; Marchal et al., 2013; Moiseeva et al., 2013; Park et al., 2013), which interfere with rapamycin pathway (Blagosklonny, 2007). However, the side effects of these agents are unacceptable. For instance, the common side effects of metformin include diarrhea, nausea, weakness, indigestion, abdominal discomfort, and headache (Foss and Clement, 2001). It also can cause more serious side effects, such as symptoms of high blood sugar, chest pain, or signs of an allergic reaction (Heinzerling et al., 2008). Some medicinal plants, with fewer side effects, may provide some ideal antiaging compounds (Ndlovu et al., 2013). Many Chinese herbs have been reported to contribute to extending the life span (Chang and So, 2008; Jafari et al., 2007; Yu et al., 2010). For example, a Polygonaceae plant, Polygonatum multiflorum, has been well known to delay the onset of age-related disorders and to extend animal life span (Wang et al., 1988), but the molecular mechanism is still unclear. Tetrahydroxystilbene glucoside (TSG) is one main component of P. multiflorum and may involve in the life span extension. Because neural Klotho and insulin/IGF-1 signaling is crucial for anti-aging activity, we want to know whether the levels of these proteins are affected by TSG.

(pMBMECs) from the senescence-accelerated prone mouse (SAMP8) (Center of Experimental Animals of the Fourth Military Medical University, Xian, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. After 24 hours of serum starvation, PI3K inhibitors, 20 mM LY294002, were added to the medium and incubated for 2 hours. Subsequently, 30 mM glucose, 10, 20, and 50 mM TSG were added and incubated for 48 hours. TAPI-1 (ADAM inhibitor, production No. 163847-77-6) was purchased from Shanghai Weihuan Biological Technology Co, Ltd (Shanghai, China). Calbiochem (BACE1 inhibitor, production no. 444253) was purchased from Shanghai Sunivo Supply Chain Management Co, Ltd (Shanghai, China). DAPT (GSI-IX, gamma-secretase inhibitor, catalog no. A8200) was purchased from ApexBio Technology (Boston, USA). The antibodies for alpha-secretase ADAM10 (ab1997) and beta-secretase BACE1 (ab2077) were purchased from Abcam Trading (Shanghai) Company Ltd (Shanghai, China). Mouse Neuro-2A cells were purchased from ATCC (production No. CCL-131, Manassas, USA). Neuro-2A was cultured in DMEM (cat. no. 09,2211L, Shanghai FuMeng Gene Biotechnology Co, LTD, Shanghai, China) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin. The extracellular domain of klotho was prepared according a previous report, and flag tag was added (Kurosu et al., 2006). Neuro2A cells were used to express flag-tagged extracellular domain of klotho. Anti-Flag was purchased from Sigma (St. Louis, USA). 2.2. Detection of senescence Senescence-associated beta-galactosidase (SA-beta-gal) is a hydrolase enzyme that catalyzes the hydrolysis of beta-galactosides into monosaccharides only in senescent cells (Dimri et al., 1995). Presently, the phenomenon is explained by the overexpression of the endogenous lysosomal beta-galactosidase only in senescent cells. Although beta-galactosidase expression is not required for senescence, it remains the widely used biomarker for detection of senescent cells for its reliability both in situ and in vitro (Lee et al., 2006). Here, the senescent status of pMBMECs was verified with Mammalian beta-galactosidase assay Kit (production no. C0602, Beyotime Institute of Biotechnology, Shanghai, China). The number of SA-b-gal-positive cells was counted using a hemocytometer. 2.3. Animals

2. Methods 2.1. Reagents and cell culture TSG was isolated from P. multiflorum and provided by Shanxi medicinal plant research center (No. 2010ZDGC-19). Other materials were purchased as follows: acetonitrile (No. 10071743, SK chemicals, Gyeonggi-do, Korea), ethanol (No. 120793301, SK chemicals, Gyeonggi-do, Korea), Trizol (Dalian TaKaRa Biotechnology Co, Ltd., Dalian, China), lysate containing a proteinase inhibitor cocktail (P8340, Sigma-Aldrich (Shanghai) Trading Co, Ltd, Shanghai, China), anti-klotho (No. ab69208, Abcam), anti-betaactin (CST, Danvers, MA, USA), and horseradish peroxidaselabeled goat anti-mouse IgG (No. a0216, Beyotime Institute of Biotechnology, Shanghai, China). Mouse Klotho kit (production no. E03K0027), mouse insulin kit (production no. 03I0004), and mouse insulin receptor kit (insulin-R, production no. 03I0074) were from Shanghai BlueGene Biotech Co, LTD (Shanghai, China). Mouse IGF-1 kit (production no. 5400) was from Shanghai Westang Bio-Tech Co, Ltd (Shanghai, China), and mouse insulin growth factor-1 receptor (IGF-1R) kit (production No. 16,001) was from antibodies-online Inc (Atlanta, USA). Primary mouse brain microvascular endothelial cells

SAMP8 aged 18 days were purchased from the Center of Experimental Animals of the Fourth Military Medical University (Xian, China). Experiments were initiated after ethical committee approval from the Fourth Military Medical University (Xi’an, China). SAMP8 mice were housed in the vivarium facility with 12 hours light and/or 12 hours dark cycle and maintained under standard conditions (22  C  1  C, 45% humidity, food and water ad libitum). After 2 weeks of acclimatization, the mice underwent the experiments. A total 32 SAMP8 were divided into 4 groups randomly: control group treated with saline, TSG group treated with 2, 20, and 50 mM (according to preliminary experiment’s data) TSG respectively each day. Twelve animals were killed after blood donation under anesthesia on the 30th day. 2.4. Mirrors In an open circle pool with a-214 cm diameter, a circular platform (diameter 7.4 cm; 1 cm under the water surface) was placed at a fixed point. The pool was filled with water (22  C  1  C). Before the experiment, mice were trained to climb onto a platform (4 trials a day) on the 24the27th and 52nde55th day, and tested on the

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28th and 56th day. Mice were kept standing on the platform for 10 seconds. The mice would be guided to swim to the platform when they failed to find the platform in 60 seconds and were kept standing on the platform for 10 seconds too. During the tests, the platform was removed. The mice were put into the water from 4 different points. The number of times of crossing the platform was recorded, because the mice were put into the water to swim around the pool for the same times. 2.5. Sample collection SAMP8 brain was resected, and cerebral cortex was fixed with paraformaldehyde and stored at 80  C. Parts of the organ were homogenized, treated with Trizol, and kept in paraformaldehydes. All the samples were ground into powder using liquid nitrogen. A total 200 mL PBS (50 mM Potassium Phosphate Buffer pH 7.0) was added, and the mixture was then centrifuged (3000 rpm for 15 minutes), and supernatant was discarded. The lysate containing a proteinase inhibitor cocktail was added to the pellets and stored at 4  C for 30 minutes. The supernatant was collected after centrifuging at 15,000 rpm for 45 minutes at 4  C. 2.6. Enzyme-linked immunosorbent assay kits analysis and hematoxylin-eosin staining The protein levels of klotho, insulin, insulin receptor (insulin-R), IGF-1, and IGF-1R were measured in the brain using sandwich enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was performed according to previous reports (Drachenberg et al., 1997). The paraffin blocks were stained after being embedded. 2.7. Western blot analysis Brain cortex samples were lysed in lysis buffer. Proteins were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes and washed 3 times with Tris-buffered saline buffer. After washing, the membranes were blocked with 5% skimmed milk at room temperature for 2 hours and stained with the primary antibodies in Tris-buffered saline overnight at 4  C. Blots were washed and incubated with appropriate horseradish peroxidaseconjugated secondary antibodies for 1 hour at room temperature. The blots were detected using Supersignal Ultra-sensitive enhanced chemiluminescent kit, and the membranes were exposed to X-ray film. Signal intensities were quantified by densitometry. 2.8. Shedding assay Shedding assay was performed according to a previous report (Bloch et al., 2009). Briefly, Neuro-2A cells were cultured in 6-well plates and grown up to 70% confluency. Twenty-four hours after treatment with 20 mM TSG or 16 hours after drug treatment as following mentioned, cells were washed in PBS and then incubated with DMEM without serum for 6 hours. Immunoblot analysis for klotho was conducted from the media after trichloric acid treatment. Cells were lysed in STEN-lysis buffer, and proteins were separated with SDSePAGE or Tris-Tricine gels (Bloch et al., 2009). The separated proteins were transferred to PVDF membranes. Membranes were blotted with antibodies as previously indicated. An LAS-4000 Luminescent Image Analyzer (Fujifilm Life Science, Stamford, USA) was used for quantitation. The protein in media was also examined using the previously mentioned method. To inhibit gamma-secretase transfected cells were treated overnight with 1 mM DAPT. To inhibit ADAM-mediated shedding, TAPI-1 was used at 25 mM for 16 hours. To inhibit BACE1, Calbiochem was dissolved in DMSO and used at 2 mM for 16 hours.

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2.9. Statistical analysis The SPSS10.0 analysis statistics software was used. KaplanMeier plots were constructed to estimate the relationship between the life span of SAMP8 and TSG concentration. Results are expressed as means  standard deviation. Student t test was used for determining the significance of differences between 2 groups in cells, and statistical differences were tested by 1-way analysis of variance between 2 groups of SAMP8, and the accepted level of significance is p < 0.05. 3. Results 3.1. TSG attenuates SA-beta-gal activity induced by glucose In humans, 30 mM of plasma glucose result in approximately 9 mM of brain glucose. Therefore, 30 mM glucose is quite high for the incubation of the pMBMECs. Here, the high concentration glucose can accelerate senescence of pMBMECs (Shuang et al., 2014). Meanwhile, high concentration of glucose (30 mM) significantly enhanced SA-beta-gal activity in pMBMECs after 48 hours culture compared with a control group. Although TSG had a little effect on SA-beta-gal activity in the cells without glucose introduction (p < 0.05), it significantly reduced the activity in the cells treated with 30 mM glucose in a dose-dependent manner (p < 0.01) (Fig. 1A). In addition, the effect of TSG on attenuating SA-beta-gal activity was inhibited by adding the PI3K/Akt inhibitor LY294002 (p < 0.01) (Fig. 1B). These results suggest that TSG attenuates glucose-induced senescence may be mediated by the PI3K/Akt signaling pathway, which is associated with insulin action (Taniguchi et al., 2006). The effects of 20 and 50 mm TSG on the pMBMECs showed no difference (p > 0.05) (Fig. 1). 3.2. TSG increases the phosphorylation of Akt and reduced the protein level of IGF-1R IGF-1R has been identified to regulate animal life span (Holzenberger et al., 2002) and interacted with phosphorylation of Akt (pAkt) (Yu et al., 2013). To explore the possible mechanism of the anti-senescent effect of TSG on glucose-induced pMBMECs, the effects of TSG on the expression of IGF-1R and pAkt were evaluated. As shown in Fig. 2, treatment with TSG alone significantly changed the protein levels of pAkt when compared with a control group. On the other hand, 30 mM glucose decreased the ratio of the pAkt in an experiment group to the pAKt in a control group by 30%. TSG increased the pAkt levels in glucose-induced pMBMECs in a dose-dependent manner. Furthermore, TSG inhibited the IGF-1R expression in glucose-induced pMBMECs in a dose-dependent manner (Fig. 2). 3.3. TSG improves the memory ability of mice The time for mice finding and climbing up to the platform was written on a time sheet, and the roadmap of mice swimming was recorded. After the platform was removed, the number that mice passed through the platform was recorded. The results showed that the number that the mice passed through the platform was significantly increased in TSG-treated groups compared with those from the controls without TGS treatment although the training time in TSG-treated groups was less that in the controls (Table 1). The results also applied that TSG improved the memory ability of mice. 3.4. TSG prolongs the life span of SAMP8 Overall survival of SAMP8, as presented in the Kaplan-Meier plot (Fig. 3), was improved in TSG-treated groups compared with that

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Fig. 1. Effect of TSG on the senescence of pMBMECs. (A) Glucose (30 mM, 48 hours)-induced senescence of endothelial cells was inhibited by TSG in a concentration-dependent manner. (B) Glucose (30 mM, 48 hours)-induced senescence of endothelial cells was inhibited by TSG in a concentration-dependent manner. The inhibition could be removed when PI3K inhibitors LY294002 was added. Values are mean  SD (n ¼ 4). Abbreviations: pMBMECs, primary mouse brain microvascular endothelial cells; SD, standard deviation; TSG, tetrahydroxystilbene glucoside. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

from the controls without TSG treatment. As Fig. 1 showed, 20 mM TSG could prolong the life span of SAMP8 male by 17% (p < 0.01, compared with a control group), which was similar with that from 50 mM TSG group. 3.5. TSG reduces the weight of SAMP8 SAMP8 in TSG group had significantly reduced their weight (p < 0.01, compared with a control group). The results were shown in Fig. 4A. The thickness and area of abdominal visceral fat cells in TSG was smaller than those in a control group (p < 0.01). The SAMP8 in TSG had fewer fat cells than those in a control group (p < 0.01) (Fig. 4B). The results were consistent with body weight comparison and shown in Fig. 3B. Weight loss is closely related with the insulin status (Bigornia et al., 2013), which can be affected by the level of klotho (Château et al., 2010). 3.6. The effect of TSG on the levels of neural insulin, insulin-R, IGF-1, IGF-1R, and klotho The mutations that interfere with GH biosynthesis and actions, or sensitivity to IGF-1, lead to extended longevity (Kenyon, 2011).

There are considerable evidences to suggest that the genetic and endocrine mechanisms that influence aging and longevity in mice may play a similar role in other mammals, including human beings (Bartke, 2005). Here, there were obvious difference in the protein levels of insulin and insulin-R in 3 groups (Fig. 5A and B), suggesting that TSG can increase the life span via reducing the amounts of insulin and insulin-R. Caloric restriction has been shown to increase longevity in organisms from yeasts to mammals. In some organisms, this has been associated with a decreased fat mass and alterations in the insulin/ IGF-1 pathways (Bluher et al., 2003). To further explore these associations with enhanced longevity, a fat-specific insulin-R was knockout in mice. These animals reduced fat mass and protected against age-related obesity (Bluher et al., 2003). Major extension of life span in mice may be related with IGF-1 deficient. Reduced level of IGF-1 and life span extension of GH-deleted mice is associated with caloric restriction (Bartke et al., 2003). Suspected mechanisms of IGF-1 action in aging, including reduced insulin signaling, enhanced sensitivity to insulin, and reduced thermogenesis with diminished oxidative damage of macromolecules, is the likely final common pathway of these effects (Bartke et al., 2003). The other controversial theory of IGF-1 is that elevated IGF-1 results in an

Fig. 2. The effects TSG on the protein expression of IGF-1R and the phosphorylation of Akt in pMBMECs. The cells were treated with glucose, TSG, and TSG plus glucose for 48 hours. Whole-cell lysates were prepared, and IGF-1R, p-Akt, and Akt levels were measured by Western blot. Band intensities were quantified by densitometry. The data are presented as means  SD (n ¼ 4). Abbreviations: IGF-1R, insulin growth factor-1 receptor; pMBMECs, primary mouse brain microvascular endothelial cells; SD, standard deviation; TSG, tetrahydroxystilbene glucoside. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

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Table 1 Effects of TSG on the memory ability of mice 4 wk

8 wk

Training time (s) Control 30 mM glucose 2 mM TSG 20 mM TSG 2 mM TSG þ 30 mM glucose 20 mM TSG þ 30 mM glucose

14.1 33.3 12.5 10.0 16.5 14.5

     

1.0 4.0 1.0 1.0 3.0 1.0

Times of crossing the platform 4.2 2.5 5.0 7.0 4.0 6.0

     

0.5 0.4 0.5 0.9 0.5 1.0

Training time (s) 13.2 45.5 12.5 10.0 16.5 14.5

     

0.7 8.0 1.0 1.0 1.0 1.0

Times of crossing the platform 3.7 2.0 6.5 8.0 5.5 7.0

     

0.5 0.5 0.8 1.0 0.6 0.7

Note: the total times for that mice swam around the pool were same, so a mouse crossing the platform less times suggested they missed the platform more times just like below showed. Key: TSG, tetrahydroxystilbene glucoside.

increase of 15% in muscle and a 14% increase in strength in mice, and remarkably, prevents aging-related muscle changes in old mice, leading to a 27% increase in strength compared with the muscles in controls (Barton-Davis et al.,1998). The results suggest that IGF-1 can prevent the process of aging significantly. Here, our results support the former theory. TSG can reduce the protein levels of IGF-1 in brain (Fig. 5C). Higher dosage of TSG shows higher inhibitory activity, compared with low dosage of TSG. IGF-1R is a protein found on the surface of mammalian cells. It is a transmembrane protein and activated by IGF-1. IGF-1R mediates the effects of IGF-1, which is a kind of hormone similar to insulin in molecular structure. Here, TSG can reduce the protein levels of IGF-1R brain (Fig. 5D). Klotho mutant in mice promotes the degeneration of many agesensitive traits. The high level of Klotho extends the life span of mice (Kurosu et al., 2005). Here, we show that TSG increases the protein levels of klotho in brain (Fig. 5E). 3.7. Klotho is shedded by ADAM10, 17, BACE1, and gamma-secretase Klotho is processed into extracellular fluids, suggesting that ADAM or BACE1 is involved. To make sure that these proteases could cleave klotho, ADAM10 was first analyzed in Neuro-2A cells (Fig. 6A). The ADAM10 inhibitor TAPI-1 decreased the shedding of klotho by up to 90%. More specifically, TSG treatment promoted the overexpression of klotho compared with controls (Fig. 6A). Next, we analyzed if BACE1 could shed klotho. BACE1 increased the shedding of klotho (Fig. 6A). Additionally, using a BACE1 inhibitor, Calbiochem, decreased the shedding of Klotho by 90%. To investigate if gamma-secretase could cleave klotho stub, gamma-secretase inhibitor DAPT was used. In the presence of the inhibitor, a 5-kDa fragment was found in transfected

Fig. 3. Kaplan-Meier plot of the life span of SAMP8 affected by TSG. Abbreviations: SAMP8, senescence-accelerated prone mouse; TSG, tetrahydroxystilbene glucoside.

cells treated with inhibitor, which was not seen in wild-type cells and transfected cells without inhibitor (control) (Fig. 6B). Taken together, all these results suggested that alpha-, beta- and gamma-secretases were involved in the shedding of klotho. 4. Discussion The root of P. multiflorum is widely used in traditional Chinese medicine (Yi et al., 2007) and can be used to treat age-related diseases (Chan et al., 2003; Li et al., 2005). TSG is one of the main active ingredients of P. multiflorum and has many pharmacologic effects such as antioxidative and anti-inflammatory effects as well as improving memory and learning ability (Zhang et al., 2006). A previous report suggested that TSG affords a significant neuroprotective effect against 1-methyl-4-phenylpyridinium-induced damage in cells through activation of the PI3K/Akt signaling pathway. Furthermore, TSG was also found to be associated with increasing Akt phosphorylation (Zhang et al., 2013). TSG is one main component of P. multiflorum, which has been well known to delay the onset of age-related disorders and extend the life span of multiple species (Wang et al., 1988). TSG is a free radical scavenger and a strong antioxidant (Xu et al., 2014; Zhang et al., 2012). However, the mechanism for TSG causing AKt phosphorylation is widely unknown. In the present study, TSG was investigated in SAMP8. Our findings also revealed that TSG mediated Akt phosphorylation.

Fig. 4. The effects of TSG on the weight of mouse. (A) TSG reduced the weight of mouse. TSG group compared with control groups, p < 0 01. (B) TSG reduced the thickness, area, and count of abdominal visceral fat cells (200). Fat cavities were showed by red arrows proved that the 2 TSG groups had slimmer and less fat cells than the control group. Abbreviation: TSG, tetrahydroxystilbene glucoside. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

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Fig. 5. Effects of TSG on the protein levels of klotho, insulin, insulin-R, IGF-1, and IGF-1R. (A) TSG reduces the protein level of insulin receptor in the brain. (B) TSG reduces the protein level of insulin receptor in the brain. (C) TSG reduces the protein level of IGF-1 protein in the brain. (D) TSG reduces the protein level of IGF-1R in the brain. (E) TSG increased the level of klotho in the brain; n ¼ 8 in each group. Abbreviations: IGF-1, insulin-like growth factor-1; IGF-1R, insulin growth factor-1 receptor; TSG, tetrahydroxystilbene glucoside. *p < 0.05 and **p < 0.01 via the control group.

Improved understanding of the mechanisms of action of TSG should provide insights to how TSG upregulated Akt phosphorylation. Thus, further work will be conducted to meet the needs in future. Here, we determined the anti-aging effects of TSG via upregulating neural klotho and downregulating neural insulin and/or IGF1. The klotho is the central point in the insulin signaling pathway and the insulin signaling is one of the pathways that have been implicated in aging (Rincon et al., 2004). TSG is physiologically distributed in the kidney, the brain, and the heart (Lv et al., 2011) and has been shown to be resistant to radicals in vitro and in vivo (Duchnowicz et al., 2012). It has been shown that aged mouse have reduced klotho and increased IGF-1 (Junnila et al., 2013; Kurosu et al., 2005). Reduced level of Klotho has also been observed in the brains of aged mammals (Duce et al., 2008). Klotho ablates the effect of the insulin/IGF-1 signaling pathway’s signals with aging process and can be mutually regulatory (Bartke, 2006). Consistent with the theory, TSG delays aging by increasing the levels of klotho in aged mice (Xu et al., 2009). The insulin/IGF-1 signaling pathway signaling inhibits the degradations of the physiological functions because the anti-aging effects are characterized by extended life spans (Clancy et al., 2001; Tatar et al., 2001). Normally, high fat will

increase ROS production, whereas insulin reduces fat production and thereby decreases ROS levels. In the case of aged mice, TSG reduces the levels of insulin and insulin-R in brain (Fig. 5A and B), suggesting other mechanisms are also existed. PI3K influences apoptosis by inhibiting assembly of deathinducing signaling complex (Ehrenschwender et al., 2010). Insulin signaling is a vital pathway in the regulation of protein synthesis and glucose metabolism, whereas PI3K/Akt is derived from insulin signaling pathway and the insulin/IGF-1/PI3K/Akt pathway is often considered together (Yamamoto et al., 2005). There is striking evidence indicating that the IGF-1/PI-3K/Akt signaling enhances growth of animals during development but later in life can potentiate the aging process. This conserved pleiotropy has been called insulin/IGF-1 paradox (Salminen and Kaarniranta, 2010). Here, PI3K/Akt was found to be upregulated in the aged pMBMECs treated with TSG, suggesting the anti-aging was also related with the insulin/IGF-1 signaling pathway. However, we also found the paradox of TSG regulating the level of insulin, which reduced the adiposity (Guerre-Millo et al., 2000). Aging pMBMECs could be detected with Mammalian BetaGalactosidase Assay Kit. After that, IGF-1R and the phosphorylation

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pathway. It will be more interesting to elucidate the mechanisms also existed in human beings. With the work going forward, TSG from P. multiflorum should be developed as a novel resourceful drug against aging. Disclosure statement The authors declare that they have no actual or potential conflicts of interest. This project was approved by the F.M.M.U. Committee (10/21/2012 project #1987, Fourth Military Medical University). Acknowledgements This study was supported by the project of Shanxi Medicinal Plant Effect Components Research and New Drug Create Engineering Technology Research Center (No. 2010ZDGC-19). The authors would like to thank Mr. Zhongmin He for his technical assistance. References Fig. 6. Immunoblot analysis for the shedding of Kl by ADAM10, BACE1, and gammasecretase. (A) Neuro-2A cells were treated with 20 mM TSG or TSG plus TAPI-1 or TSG plus DMSO, and or TSG plus Calbiochem for 16 hours. The protein levels of ADAM10 and BACE1 were analyzed using ADAM10 and BACE1 antibodies. (B) The Neuro-2A cells (transfected with gene fragments for the extracellular domain of klotho) were treated with DAPT for 16 hours. The transfected Neuro-2A without ADPT treatment was assigned as control. Untransfected Neruo-2A was assigned as wt. The protein levels of klotho and klotho (sub) from cells lysate were analyzed using klotho and flag antibody, respectively. Abbreviations: TSG, tetrahydroxystilbene glucoside; wt, wild type.

of Akt were mostly investigated in pMBMECs. TSG inhibited the protein expression of IGF-1R and increase the phosphorylation of Akt in the aged pMBMECs, suggesting the anti-aging effects of TSG for mice via the insulin/IGF-1R signaling pathway. Apple polyphenols (AP) are rich and commonly called dietary antioxidants. The previous work explored the functions of AP on the life span of fruit fly and interaction with expression of superoxide dismutase, catalase, methuselah, Rpn11, and cytochrome c oxidase subunits III and VIb. The findings showed the mean life span was prolonged by 10% in the fruit flies fed with AP (Peng et al., 2011). TSG exhibits antioxidative and anti-inflammatory effects and protects against cerebral ischemia and/or reperfusion injury through multifunctional cytoprotective pathways (Wang et al., 2009). Klotho is a single-pass transmembrane protein that is predominantly found in distal convoluted tubules in the kidney and choroid plexus in brain (German et al., 2012). The transmembrane proteins are directly linked to aging and often shedded by some secreatase (Kuro-O, 2008; Munter et al., 2013; Zhang et al., 2014), which may affect the function of klotho. Therefore, we explored that what secretases were involved in cleaving klotho to generate the secreted form of klotho using the inhibitors of alpha-, beta- and gammasecretases. Our results showed that alpha-, beta- and gammasecretases participated in the shedding of klotho, which suggested that all these enzymes were critical for the normal functions of klotho. On the other hand, the thing is more complex than these findings and further work is still needed to clarify more factors affecting the functions of klotho. For example, the intramembranous cleavage of klotho is likely to be influenced by the membrane phospholipid composition as has been suggested by Pettegrew et al. (2001) for the intramembranous cleavage of amyloid precursor protein. Taken together, the results indicate that TSG has anti-aging effects in mammals, which are associated with the insulin signaling

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