Molecular Pathways Related To The Longevity Promotion And Cognitive Improvement Of Cistanche Tubulosa in Drosophila

for more information:ali.ma@wecistanche.com

Wei-Yong Lin et al

Abstract:

Background: The aging process, including physical dysfunction and age-related memory impairment (AMI), are considered to be correlated with cumulative oxidative damages and the insulin/IGF-1 signaling pathways. Purpose: The present study was to elucidate the in vivo effects on delaying aging and ameliorating AMI and underlying molecular mechanisms of Cistanche tubulosa (CT), a herb used in traditional Chinese medicine to improve sexual function and treat kidney dysfunction

Methods: The flies, treated and untreated with CT (Cistanche Tubulosa), were observed for lifespan, resistance to oxidative stress with H2O2 or paraquat, starvation assay, cognitive behaviors with T-maze, and transcript levels of target genes with quantitative RT-PCR.

Results: Administering CT (Cistanche Tubulosa) extended the mean and maximum lifespan and increased resistance to oxidative stress in flies. CT (Cistanche Tubulosa) supplementation also enhanced memory formation in young flies and suppressed AMI upon aging. Several genes and signaling pathways, including the mechanistic target of rapamycin (mTOR) and Notch networks, have been identified as causing these pharmacological effects and alterations in the gene expression of glutamate receptors.

Conclusion: Our results indicate that CT (Cistanche Tubulosa) supplementation may contribute to slowing aging phenotypes and alleviating cognitive-behavioral decline in flies, indicating potential applicability for enhancing human health and reducing susceptibility to age-related disorders.

Keywords: Aging, Cistanche tubulosa, Cognitive behavior, Herb

Click to Cistanche stem products

Introduction

As the aged population increases worldwide, facilitating healthy aging and longevity is an increasingly crucial public health concern for science, society, and the medical care field. The aging process has been proposed as being controlled by complex mechanisms, driven by a broad interplay of genetic and environmental factors. Because of gradual damages at the cellular and tissue levels, age is a risk factor for and highly correlated with the susceptibility to chronic diseases such as cancer as well as cardiovascular and neurodegenerative diseases (Kenyon, 2005). Aging also reduces synaptic activity, new protein synthesis, and cognitive function. These functional changes contribute to age-related memory impairment (AMI) (Burke and Barnes, 2010; Schimanski and Barnes, 2010). Therefore, developing drugs for delaying age-related pathology and cognitive aging might be beneficial for the quality of human life. In this study, we used Drosophila melanogaster, which is an ideal organism for in vivo aging studies because of its short lifespan, to evaluate drug efficacy and explore the molecular mechanisms underlying the amelioration of aging and AMI.

Herba cistanche, also known by its Chinese name Rou Cong Rong, is a parasitic plant that is mainly distributed in the deserts of Northwest China. Two Cistanche species, Cistanche deserticola Y. C. Ma and Cistanche tubulosa (Schrenk) Wight are included in the Chinese Pharmacopoeia (2005 edition) for their similar chemical ingredients and pharmacological activities. Their stem extract has been widely used for thousands of years as a tonic remedy, mainly for treating various symptoms, including deficient kidney function, impotence, infertility, and for facilitating catharsis, as described in ancient Chinese medical books. Numerous chemical constituents, including essential oils, phenylethanoid glycosides (PhGs), iridoids, lignans, and saccharides, have been isolated from Cistanche species, and two major PhG components, acteoside, and echinacoside are typically selected as markers for the quality evaluation of Herba cistanche because of their significant pharmacological actions (Wang et al., 2012).

Cistanche tubulosa (CT) extracts possess various biological functions that include modulating immune activity, enhancing sexual function, promoting hair growth, and other benefits on antioxidation, hepatoprotection, antiosteoporotic activity, and anti-tumor (Li et al., 2016; Seok et al., 2015; Wang et al., 2012). Additionally, in vitro study, revealed that Herba cistanche extract can protect dopamine-like neurons from H2O2-induced oxidative damage and significantly increase the levels of nerve growth factor and brain-derived neurotrophic factor (Lin et al., 2013). Echinacoside prevented dopamine neuron death in the MPTP-lesioned mouse model of Parkinson’s disease (Zhao et al., 2010). Moreover, CT (Cistanche Tubulosa) extract ameliorated the cognitive dysfunction through decreasing amyloid deposition and increasing acetylcholine and dopamine levels in a rat model of Alzheimer's disease (AD) (Wu et al., 2014). Although the therapeutic effect of Herba cistanche is still required to confirm, a small size, non-placebo-controlled clinical trial on CT (Cistanche Tubulosa) glycoside extracts showed that the drug has the potential to treat patients with mild to moderate AD and is with relatively mild adverse reactions, even after long-term administration (Guo et al., 2013). These data suggest that glycoside extracts in Herba cistanche have neuroprotective and anti-dementia effects. Despite various benefits of CT (Cistanche Tubulosa), its molecular mechanisms and effects on delaying aging and ameliorating AMI have not been elucidated. In this study, the effects of CT (Cistanche Tubulosa) on a significant extension of lifespan improved resistance to environmental stress, and suppressed age-related cognitive dysfunction were examined to determine the possible clinical utility of CT (Cistanche Tubulosa) in slowing the aging process.

Materials and method

Herbal medicine

The origin identification of Cistanche tubulosa (Schrenk) Wight (Orobanchaceae) (CT) was verified in the Ko Da Pharmaceutical Co. Ltd. (Taoyuan, Taiwan), where voucher specimens (10,603,205) have been kept and the CT concentrated herbal medicine is manufactured. Briefly, the dried fleshy stem of CT was extracted in 12- fold boiled water for 1 h followed by filtration. The filtrates were collected and subjected to vacuum and reduced-pressure concentration for 1 h to obtain extracts. The CT extracts were mixed with starch at a 1:1 ratio (w/w) and subjected to spray granulation for obtaining CT powder.

Fly stock maintenance and lifespan measurement

The wild-type fly line used in this study was of the Canton S, obtained from the Bloomington Drosophila Stock Center. Flies were raised and maintained in cornmeal standard media at 25 °C in 50–60% relative humidity under a 12-h light-dark cycle. Emerging adult flies were collected within 24 h and separated by sex. From early adulthood, flies were supplemented with CT. CT (Cistanche Tubulosa) powder was dissolved in water and added to cornmeal media at 5.4, 10.8, and 21.6 mg/ml concentrations, which were proportional to the effective daily dosage for human oral administration. In a lifespan analysis, twenty to thirty flies were raised in a food vial with or without CT (Cistanche Tubulosa); at least 250 flies were conducted for each treatment. Food vials were replaced every 3 to 4 days, and dead flies were counted at that time

Stress resistance assays

Adult flies were collected, and then pretreated with a control diet without CT (Cistanche Tubulosa) and experimental diets supplemented with 5.4, 10.8, and 21.6 mg/ml of CT for 20 days at 25 °C. For starvation analysis, flies were kept in vials with wet papers only and evaluated their stamina against starvation-induced oxidative stress. For oxidative stress assays, the flies were transferred to food-free vials containing a filter paper with hydrogen peroxide (H2O2) (SigmaAldrich) in a 5% glucose solution to gauge the flies' resistance against H2O2-induced hydroxyl radical (OH−) stress. Similarly, flies were transferred to vials containing a filter paper saturated with 10 mM paraquat (Sigma-Aldrich), which generates superoxide anions (O2 −) that cause oxidative damage to animals. Flies were scored for mortality at least three times daily until all were dead. At least 250 flies per treatment were performed. For climbing assay, flies exposed to paraquat for 0 and 16 h were placed in a plastic vial and gently tapped to the bottom. The number of flies at the top of the vial was counted after 18 sec of climbing.

Fecundity assessment

Both virgin males and females fed 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa) for 20 days served as experimental groups. Ten experimental females were mated with an equal number of virgin males, which were 9- day-old and had not received CT. Similarly, the experimental males were mated with virgin females in the same condition. The mated female flies were then transferred to a new vial for egg-laying for 24 h, and the total eggs laid in the vials were counted manually. An index of fecundity was calculated as the mean daily egg production per female. At least five vials per treatment were performed, and total flies n = 50–80.

Olfactory associative learning and memory

Flies were collected after eclosion and pretreated with 5.4, 10.8, and 21.6 mg/ml of CT (Cistanche Tubulosa) served as experimental groups, and the control flies had not received CT (Cistanche Tubulosa) for 5 and 20 days at 25 °C. A single training trial was performed according to a previous study (Dubnau et al., 2001). Briefly, approximately 100 flies were exposed sequentially to two odors, either 3-octanol (OCT, Sigma-Aldrich) or 4-methyl cyclohexanol (MCH, Sigma-Aldrich). Flies exposed to the first odor (conditioned stimulus, CS+) were paired with 80 V electric shocks and then received a second odor (CS-) without shocks. Learning ability was determined immediately after training and the abilities of 3- and 24-h memory has conducted the tests after training procedure for 3 and 24 h, respectively. To perform the test, the trained flies were trapped into the choice point of a T-maze in which they were exposed simultaneously to OCT and MCH. A performance index (PI) was calculated to represent the conditioned odor avoidance. A PI of zero indicated a 50:50 distribution, and a PI of 100 showed a 0:100 distribution away from the CS+ odor.

Measurements of transcript levels through real-time quantitative PCR (qPCR)

Flies were collected after eclosion and pretreated with 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa) for 20 days at 25 °C served as experimental groups, and the control flies had not received CT. The total RNA was extracted using an RNeasy Mini kit (Qiagen), and cDNA was synthesized from the total RNA through reverse transcription reaction by using a High-Capacity cDNA Reverse Transcription (Thermo Fisher Scientific). The transcript levels of target genes were quantified through real-time quantitative polymerase chain reaction (Applied Biosystems ViiATM 7 Real-Time PCR System) by using FastStart SYBR Green Master (Roche) according to the manufacturer's instructions. Relative transcript abundances were normalized using the comparative cycle threshold method with a housekeeping gene, RpLP0, as the internal standard. The relative expression differences in the target genes of the experimental groups were normalized to those of a control group and presented as a fold change. The primers for the target genes are listed in Supplementary Table 1.

Network construction and analysis

To analyze the functional interpretation and biological processes mediated by the differentially expressed genes we identified, the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to annotate and analyze the enrichment of the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway and Gene Ontology for these genes (Jiao et al., 2012). To analyze the direct and indirect interactions of differentially expressed genes, the gene list was also uploaded to the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) to retrieve the interaction information and generate protein interaction networks (Franceschini et al., 2013).

Statistical analysis

All experiments were conducted with at least three independent replicates, and statistical analyses were performed with SigmaStat v3.5 software (Systat Software, Inc., IL). Data for lifespan extension and oxidative stress challenges were performed with a total number of flies ranging from 250–350, and the log-rank test was assessed for different analyses. Group comparisons for fecundity assessment and cognitive test were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett's post-hoc test. Other experimental comparisons were made by performing a Student’s t-test compared with the control group. Data are represented as the mean ± SEM, and a p-value of < 0.05 was considered significant.

Results

CT (Cistanche Tubulosa) supplementation increases fly lifespan

To examine whether CT (Cistanche Tubulosa) supplementation can extend fly lifespan, flies were supplemented with 5.4, 10.8, and 21.6 mg/ml of CT immediately after eclosion at 25 °C throughout the experimental procedures. Our data indicated that CT (Cistanche Tubulosa) supplementation can extend the lifespan of both male and female flies. As shown in Fig. 1A, the lifespan of the male flies was extended by all doses of CT (Cistanche Tubulosa). The mean lifespan of the groups supplemented with 5.4, 10.8, and 21.6 mg/ml of CT significantly increased by 18.9%, 10.3%, and 9.9%, respectively. CT (Cistanche Tubulosa) also significantly increased the maximum lifespan of all the CT groups compared with the control group from 88 to 102, 98, and 102 days upon treatment with 5.4, 10.8, and 21.6 mg/ml of CT (Cistanche Tubulosa), respectively. In females, CT (Cistanche Tubulosa) significantly increased the mean lifespan of the CT groups compared with the control group (Fig. 1B). The mean lifespan increased up to 18.8% with 5.4 mg/ml of CT (Cistanche Tubulosa) and 5.5% and 6.7% with 10.8 and 21.6 mg/ml of CT, respectively. We also observed that the maximum lifespan was significantly extended from 91 days (controls) to 109, 98, and 102 days upon treatment with 5.4, 10.8, and 21.6 mg/ml of CT (Cistanche Tubulosa), respectively; the maximum magnitude of increase in the maximum lifespan was 19.8% at 5.4 mg/ml of CT (Cistanche Tubulosa). Our results suggest that CT (Cistanche Tubulosa) diet supplementation significantly extends the lifespan of both male and female flies. Curcumin (10 μM) was used as a positive control and effective in lifespan extension, as shown in Supplementary Fig. 1.

CT supplementation increases resistance to starvation and oxidative stress

Lifespan extension is often associated with enhanced resistance to oxidative stress during the aging process. We assessed whether CT (Cistanche Tubulosa) supplementation increases resistance to oxidative stress. Male and female flies were pretreated with different doses of CT (Cistanche Tubulosa) for 20 days and evaluated for their survival after starvation (Fig. 2A). The mean survival time and maximum lifespan of both sexes, compared with those of the control group, exhibited significant increases in the 5.4 and 10.8 mg/ml CT groups but not in the 21.6 mg/ml CT (Cistanche Tubulosa) group. The pretreated flies were also evaluated for their survival after challenges with H2O2 and paraquat, which produce reactive oxygen species (ROS) to trigger cellular senescence and cell death. We found that CT (Cistanche Tubulosa) protected both male and female flies against H2O2 toxicity and extended their mean and maximum lifespan (Fig. 2B, p < 0.01 and 0.001, at 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa), respectively). A similar trend showing increased resistance to the paraquat challenge was also observed. Male flies fed with all doses of CT, and females, particularly those supplemented with 5.4 mg/ml of CT (Cistanche Tubulosa) (Cistanche Tubulosa), survived longer than did the controls (Fig. 3A, p < 0.001). Regarding climbing ability, both male and female flies administered 10.8 mg/ml of CT (Cistanche Tubulosa) exhibited attenuated paraquat-induced locomotor dysfunction at 16 h after the paraquat challenge (Fig. 3B, all p < 0.05). These results reveal that CT supplementation improves resistance to some oxidative stress, suggesting that CT (Cistanche Tubulosa)-induced lifespan extension is associated with increased antioxidative defense.

Effects of CT (Cistanche Tubulosa) on reproductive capacity

Because the theory of aging indicates that the aging process negatively affects reproduction, we examined whether CT (Cistanche Tubulosa) affected reproductive fitness by analyzing the egg-laying ability of adult flies. Adult flies were collected after eclosion, separated by sex, and administered 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa) for 20 days. Virgin male or female flies administered CT (Cistanche Tubulosa) were mated with 9-day-old flies not administered CT (Cistanche Tubulosa). We observed dose-dependent increases in the daily number of eggs laid per female, namely 30.3% and 57.2% in aged flies treated with 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa), respectively, compared with the controls (Fig. 4, p < 0.05). These data suggest that CT (Cistanche Tubulosa) supplementation could facilitate the reproductive capacity in aged females but likely does not in aged males.

CT (Cistanche Tubulosa)supplementation improves cognitive ability

We determined whether CT (Cistanche Tubulosa) supplementation has beneficial effects on learning and memory abilities, a single cycle of olfactory associative training was conducted. We evaluate learning performance as well as 3- and 24-h memory, which has been considered to be the intermediate-term memory and long-term memory, respectively. First, flies were supplemented with various doses of CT (Cistanche Tubulosa) for 5 days. Learning ability was similar in both the CT and control groups (Fig. 5A). Regarding 3-h memory, the 10.8 mg/ml CT (Cistanche Tubulosa) group tended to have a higher performance index (PI) than did the controls (Fig. 5B, 51.5 ± 3.2 versus 42.6 ± 2.1), although there is no significant difference. Moreover, 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa) significantly improved the 24-h memory of flies compared with the controls (Fig. 5C, PI = 41.6 ± 3.6 and 45.9 ± 3.0 versus 29.6 ± 2.9, respectively, p < 0.05 and 0.01). These results indicated that CT supplementation improves memory formation in young flies. Second, while CT (Cistanche Tubulosa) supplementation increased the longevity and oxidative resistance of flies, we further determined whether the supplementation could also improve cognitive behavior in aged flies. Flies were administered standard food with or without 5.4, 10.8, and 21.6 mg/ml of CT (Cistanche Tubulosa) for 20 days to mimic aging; the flies supplemented with these doses of CT (Cistanche Tubulosa) had significantly higher PIs of learning (67.6 ± 1.9, 72.5 ± 0.5, and 75.2 ± 1.3, respectively) than did the controls (58.0 ± 2.0; Fig. 5D, p < 0.001), suggesting that CT (Cistanche Tubulosa) has a beneficial effect on delaying the age-related decline in learning. Similar results also revealed that the 5.4 mg/ml CT (Cistanche Tubulosa) group had more efficient memory retention in 3-h (Fig. 5E, 47.5 ± 1.9 versus control 37.0 ± 2.9, p < 0.05) and 24-h memory tests (Fig. 5F, 31.8 ± 3.8 versus control 19.3 ± 2.4, p < 0.01). These results indicate that CT (Cistanche Tubulosa) supplementation not only enhances memory formation in young flies but also delays cognitive behavior impairment and maintains more efficient learning and memory ability with age.

Molecular pathways related to the longevity promotion and cognitive improvement of CT supplementation

CT (Cistanche Tubulosa) supplementation both suppresses AMI and extends lifespan. Therefore, we reasoned that AMI- and longevity-related genes might be determined by assessing differential expression between the control and 5.4 and 10.8 mg/ml CT groups through qPCR. The changes in gene expression of the CT-treated flies were compared with those of the control group was shown in Fig. 6A. The upregulated expression of several target genes, including Sirt6, Sir2, Sod, p38b, and Sirt2, might be involved in the aging process. Transcript levels of S6k, raptor, Psn, Rpd3, and N also increased, suggesting that CT (Cistanche Tubulosa) supplementation changes the signal transduction of the mTOR and Notch pathways. In addition, CT supplementation significantly affected the mRNA expression of Nmdar1, Nmdar2, and SkpA. To highlight the CT-relevant protein interactions that extend lifespan and improve learning and memory, a literature-based protein interaction network of qPCR-screened genes was created (Fig. 6B). Each node represents one protein, whereas each edge denotes one literature-based interaction. Nodes are clustered according to the KEGG pathway and gene ontology biological process (GOBP). As shown in Fig. 6B, the GOBP neuroactive ligand-receptor interaction, which affects learning and memory, and another GOBP of aging, which is correlates with lifespan, are both connected with the Notch signaling pathway. In addition, the GOBP of aging is influenced by the mTOR signaling pathway. The significance of CT -induced gene overexpression on these GOBPs and pathways is described in the Discussion section.

Fig. 6. Network analysis of differentially expressed genes. Flies pretreated with 5.4 and 10.8 mg/ml of CT (Cistanche Tubulosa) and control diets at 25 °C for 20 days were collected for quantifying differential gene expression. (A) The relative transcript levels of target genes to the controls were quantified through qPCR. n = 4 for each group. (B) Upregulated genes involved in cell signal transduction and their interaction network. Nodes represent genes. Genes with overexpression are circled in green. Edges represent interactions among gene products. Interaction types are denoted by different colors in the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Discussion

An imbalance between the generation and elimination of free radicals by endogenous antioxidative defense systems may cause oxidative damage to tissues with age; thus, free radicals are speculated to play a critical role in senescence (Dato et al., 2013). Here, we showed that CT (Cistanche Tubulosa) supplementation in flies extends lifespan and results in a high tolerance to oxidative damage caused by H2O2 and paraquat in both sexes. The studies, correlated with CT (Cistanche Tubulosa) and antiaging, are less explored, but extracts from C. deserticola, can extend life span in senescence-accelerated mouse prone 8 (SAM-P8) mice (Zhang et al., 2014) and echinacoside, one of the major compounds that exist in CT, has beneficial effects on the lifespan extension and stress tolerance in C. elegans (Wang et al., 2015).

Recent animal studies demonstrated that echinacoside has protective effects against bisphenol A-induced testicular toxicity and enhances testosterone biosynthesis by induction of testicular steroidgenic enzymes, including CYP11A1, 3β-HSD, and CYP17A1 (Jiang et al., 2016). CT (Cistanche Tubulosa) ethanol extract also promotes the viability and quality of sperm in rats (Wang et al., 2016). Although the correlation between CT (Cistanche Tubulosa) and estrogen still needs to explore, one study reported that echinacoside significantly increases the expression of estrogen receptors, similar to the estradiol-treated group in ovariectomized rats (Li et al., 2013). Because reproductive capacity occurs negatively correlated with age (Kenyon, 2005), we examined whether CT has benefits in aged flies. Reproductive fitness impacted by CT (Cistanche Tubulosa) was only observed in females, suggesting that female flies might be more responsive to CT (Cistanche Tubulosa) supplementation. These results reveal that CT might possess a sex hormone-like effect and delay reproductive senescence, consistent with the clinical use of CT (Cistanche Tubulosa) for treating sexual dysfunction in humans.

Acteoside can elucidate the protective effects to decrease neuron apoptosis and memory loss in a senescent mouse model induced by a combination of D-galactose and AlCl3, suggesting that CT might have the potential to have neuroprotective and memory enhancement properties (Gao et al., 2015; Peng et al., 2015). Our data indicated that memory formation improved in CT-supplemented young flies. Therefore, we assessed whether CT (Cistanche Tubulosa) supplementation is beneficial for memory retention upon aging because cognitive functions are known to decline in normal aging. Our results revealed that several types of AMIs, including learning, 3- and 24-h memory, can be ameliorated by CT (Cistanche Tubulosa) supplementation for 20 days. According to the aforementioned results, CT (Cistanche Tubulosa) supplementation may have beneficial effects on longevity and enhancement of memory formation as well as sufficiently delay AMI.

Using animal models facilitates the identification of the potential mechanisms and the path dependency of CT (Cistanche Tubulosa) to decelerate brain aging and postpone AMI at the cellular level. We focused on molecular mechanisms underlying aging, neuronal transmission, and oxidative stress and reasoned that AMI- and longevity-related genes might be identified by examining differential expression between control and CT groups through qPCR. The Notch signaling pathway is highly conserved among species from Drosophila to humans; it expresses in the adult central nervous system for neural plasticity and regulates neural differentiation along with its inhibitor numb during development (Presente et al., 2001). Mice carrying a heterozygous mutation of Notch are deficient in spatial learning and memory (Costa et al., 2003). Presenilin (Psn) is required for activating the cleavage of Notch into the Notch intracellular domain and knockout of Psn causes the impairment of synaptic plasticity and memory formation (Saura et al., 2004). Our data revealed a dose-dependent upregulation of Notch and Psn in CT (Cistanche Tubulosa) supplemented flies, with an unchanged expression level of numb, which implies the Notch signaling pathway as a potent mechanism of memory improvement by CT supplementation. In addition to Notch signaling, N-methyl-D-aspartate receptor (NMDAR), glutamate- and D-serine-driven ion channel in neurons, controls synaptic plasticity and memory function (Furukawa et al., 2005). The Drosophila genome contains two NMDAR subunits, Nmdar1 and Nmdar2, and both are crucial in memory function. Nmdar1 knockdown in Drosophila impairs learning and memory (Xia et al., 2005), whereas Nmdar2 overexpression in mice enhances learning and memory (Tang et al., 2001). Our data showed a dose-dependent increase in Nmdar1 and Nmdar2 upregulation in CT-supplemented flies, suggesting another potential mechanism of memory improvement by CT (Cistanche Tubulosa) supplementation.

The class O of the forkhead box transcription factor (FoxO) pathway is conserved among species and critical in metabolism, stress tolerance, and the extension of lifespan (Lehtinen et al., 2006). Posttranslational modifications, including phosphorylation, acetylation, and ubiquitylation, regulate the activity of FoxOs (van der Horst and Burgering, 2007). Silent information regulator 2 (Sir2; the homolog in mammals is known as SIRT1) regulates the activity of FoxO through the deacetylation and affects animals’ lifespan related to the insulin-like growth factor (IGF) pathway (Lemieux et al., 2005). Sirt6 dephosphorylates FoxO to cause lifespan extension in male mice (Kanfi et al., 2012). Our data revealed Sir2 and Sirt6 upregulation in CT-supplemented flies, implying that FoxO regulation by Shirts may be a potent mechanism of the extension of lifespan by CT (Cistanche Tubulosa) supplementation. Free radicals produced during metabolic processes cause damage to DNA and proteins, thus leading to a shortened lifespan. Sir2 is also an oxidative stress response gene (Whitaker et al., 2013), and overexpressed superoxide dismutase (SOD), which converts O2 − to H2O2 and finally to H2O by using catalase and glutathione peroxidase, has been reported to extend lifespan in flies (Le Bourg, 2001). Our data revealed dose-dependent Sod and Sir2 upregulation in CT-supplemented flies, suggesting that antioxidative pathways may be a potent mechanism of the extension of lifespan by CT (Cistanche Tubulosa) supplementation.

Another key pathway in aging is regulated by the mTOR complexes mTORC1 and mTORC2, both of which promote organism aging in an evolutionary conservative manner. Signals from the insulin receptor activate mTORC1 through the phosphoinositide 3-kinase–Akt pathway to promote protein synthesis and inflammation and to inhibit autophagy and glycolysis by using distinct downstream proteins (Johnson et al., 2013). Protein synthesis is activated by ribosomal protein S6 kinase (S6K), which forms a negative feedback loop to inhibit insulin receptor signaling (Hands et al., 2009). An inhibitory target of mTORC1 is the Drosophila protein similar (sima), whose homolog in mammals is known as hypoxia-inducible factor 1 alpha (HIF1 α). HIF1 plays a protective role against age-dependent damages to blood vessels and motor neurons (Oosthuyse et al., 2001; Rivard et al., 2000). Our data revealed an unchanged expression level of mTOR complex subunits, including Tor, rictor, and raptor, whereas CT (Cistanche Tubulosa) supplementation dose-dependently upregulated S6K and sima, implying a protective role of CT (Cistanche Tubulosa) against mTOR activity.

Conclusions

Despite some implicated pathways being identified, longevity and high intelligence contributed by CT (Cistanche Tubulosa) intervention may be a polygenic trait affected by the interplay of multiple genes, and other genetic factors that we have not elucidated may also modulate the aging process. For the elderly, maintaining desirable physical fitness and cognitive abilities in order to postpone or prevent dementia and chronic diseases should be a crucial acquirement from studies on aging. Because CT (Cistanche Tubulosa), known as the “ginseng of the desert,” has a marked curative effect on the reinforcement of kidney yang, which nourishes all organs and replenishes the vital essence in traditional Chinese medicine, we believe that our study provides evidence supporting the clinical treatment of CT(Cistanche Tubulosa) and might provide valuable insight for developing new drugs or food additives to not only delay aging and age-related diseases but also to improve the quality of human life.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the China Medical University (CMU103-S-25); China Medical University Hospital (DMR-101-065, DMR-103-099); the Ministry of Science and Technology in Taiwan (NSC 102-2632-B-039-001-MY3, MOST 104-2632-B-039-002), and in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212-133019).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2017.01.006.

From: 'Molecular pathways related to the longevity promotion and cognitive improvement of Cistanche tubulosa in Drosophila' by Wei-Yong Lin et al

--W.-Y. Lin et al. / Phytomedicine 26 (2017) 37–44

References

Burke, S.N., Barnes, C.A., 2010. Senescent synapses and hippocampal circuit dynamics. Trends Neurosci. 33, 153–161.

Costa, R.M., Honjo, T., Silva, A.J., 2003. Learning and memory deficits in Notch mutant mice. Curr. Biol. 13, 1348–1354.

Dato, S., Crocco, P., D’Aquila, P., de Rango, F., Bellizzi, D., Rose, G., Passarino, G., 2013. Exploring the role of genetic variability and lifestyle in oxidative stress response for healthy aging and longevity. Int. J. Mol. Sci. 14, 16443–16472.

Dubnau, J., Grady, L., Kitamoto, T., Tully, T., 2001. Disruption of neurotransmission in Drosophila mushroom body blocks retrieval but not the acquisition of memory. Nature 411, 476–480.

Franceschini, A., Szklarczyk, D., Frankild, S., Kuhn, M., Simonovic, M., Roth, A., Lin, J., Minguez, P., Bork, P., von Mering, C., Jensen, L.J., 2013. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815.

Furukawa, H., Singh, S.K., Mancusso, R., Gouaux, E., 2005. Subunit arrangement and function in NMDA receptors. Nature 438, 185–192.
Gao, L., Peng, X.M., Huo, S.X., Liu, X.M., Yan, M., 2015. Memory enhancement of Acteoside (Verbascoside) in a Senescent Mice Model induced by a combination of D-gal and AlCl3. Phytother. Res. 29, 1131–1136.
Guo, Q., Zhou, Y., Wang, C.J., Huang, Y.M., Lee, Y.T., Su, M.H., Lu, J., 2013. An open-label, non-placebo-controlled study on Cistanche tubulosa glycoside capsules (Memoregain((R))) for treating moderate Alzheimer’s disease. Am. J. Alzheimer's Dis. Other Demen. 28, 363–370.
Hands, S.L., Proud, C.G., Wyttenbach, A., 2009. mTOR’s role in aging: protein synthesis or autophagy? Aging (Albany NY) 1, 586–597.
Jiang, Z., Wang, J., Li, X., Zhang, X., 2016. Echinacoside and Cistanche tubulosa (Schenk) R. Wight ameliorate bisphenol A-induced testicular and sperm damage in rats through gonad axis-regulated steroidogenic enzymes. J. Ethnopharmacol 193, 321–328.
Jiao, X., Sherman, B.T., Huang da, W., Stephens, R., Baseler, M.W., Lane, H.C., Lempicki, R.A., 2012. DAVID-WS: a stateful web service to facilitate gene/protein list analysis. Bioinformatics 28, 1805–1806.
Johnson, S.C., Rabinovitch, P.S., Kaeberlein, M., 2013. mTOR is a key modulator of aging and age-related disease. Nature 493, 338–345.
Kanfi, Y., Naiman, S., Amir, G., Peshti, V., Zinman, G., Nahum, L., Bar-Joseph, Z., Cohen, H.Y., 2012. The sirtuin SIRT6 regulates lifespan in male mice. Nature 483, 218–221.
Kenyon, C., 2005. The plasticity of aging: insights from long-lived mutants. Cell 120, 449–460.
Le Bourg, E., 2001. Oxidative stress, aging, and longevity in Drosophila melanogaster. FEBS Lett. 498, 183–186.
Lehtinen, M.K., Yuan, Z., Boag, P.R., Yang, Y., Villen, J., Becker, E.B., DiBacco, S., de la Iglesia, N., Gygi, S., Blackwell, T.K., Bonni, A., 2006. A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell 125, 987–1001.
Lemieux, M.E., Yang, X., Jardine, K., He, X., Jacobsen, K.X., Staines, W.A., Harper, M.E., McBurney, M.W., 2005. The Sirt1 deacetylase modulates the insulin-like growth factor signaling pathway in mammals. Mech. Ageing Dev. 126, 1097–1105.
Li, F., Yang, X., Yang, Y., Guo, C., Zhang, C., Yang, Z., Li, P., 2013. The antiosteoporotic activity of echinacoside in ovariectomized rats. Phytomedicine 20, 549–557.
Li, Z., Lin, H., Gu, L., Gao, J., Tzeng, C.M., 2016. Herba Cistanche (Rou Cong-Rong): one of the best pharmaceutical gifts of traditional Chinese medicine. Front Pharmacol. 7, 41.
Lin, S., Ye, S., Huang, J., Tian, Y., Xu, Y., Wu, M., Wang, J., Wu, S., Cai, J., 2013. How do Chinese medicines that tonify the kidney inhibit dopaminergic neuron apoptosis? Neural Regen. Res. 8, 2820–2826.

Oosthuyse, B., Moons, L., Storkebaum, E., Beck, H., Nuyens, D., Brusselmans, K., Van Dorpe, J., Hellings, P., Gorselink, M., Heymans, S., Theilmeier, G., Dewerchin, M., Laudenbach, V., Vermylen, P., Raat, H., Acker, T., Vleminckx, V., Van Den Bosch, L., Cashman, N., Fujisawa, H., Drost, M.R., Sciot, R., Bruyninckx, F., Hicklin, D.J., Ince, C., Gressens, P., Lupu, F., Plate, K.H., Robberecht, W., Herbert, J.M., Collen, D., Carmeliet, P., 2001. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat. Genet. 28, 131–138.

Peng, X.M., Gao, L., Huo, S.X., Liu, X.M., Yan, M., 2015. The mechanism of memory enhancement of Acteoside (Verbascoside) in the Senescent Mouse Model induced by a combination of D-gal and AlCl3. Phytother, Res. 29, 1137–1144.
Presente, A., Andres, A., Nye, J.S., 2001. Requirement of Notch in adulthood for neurological function and longevity. Neuroreport 12, 3321–3325.
Rivard, A., Berthou-Soulie, L., Principe, N., Kearney, M., Curry, C., Branellec, D., Semenza, G.L., Isner, J.M., 2000. Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity. J. Biol. Chem. 275, 29643–29647.
Saura, C.A., Choi, S.Y., Beglopoulos, V., Malkani, S., Zhang, D., Rao, Shankaranarayana, Chattarji, B.S., Kelleher, S., 3rd, R.J., Kandel, E.R., Duff, K., Kirkwood, A., Shen, J., 2004. Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron 42, 23–36.
Schimanski, L.A., Barnes, C.A., 2010. Neural protein synthesis during aging: effects on plasticity and memory. Front Aging Neurosci. 2. Seok, J., Kim, T.S., Kwon, H.J., Lee, S.P., Kang, M.H., Kim, B.J., Kim, M.N., 2015. Efficacy of Cistanche Tubulosa and Laminaria Japonica extracts (MK-R7) supplement in preventing patterned hair loss and promoting scalp health. Clin. Nutr. Res. 4, 124–131.
Tang, Y.P., Wang, H., Feng, R., Kyin, M., Tsien, J.Z., 2001. Differential effects of enrichment on learning and memory function in NR2B transgenic mice. Neuropharmacology 41, 779–790.
van der Horst, A, Burgering, B.M., 2007. Stressing the role of FoxO proteins in lifespan and disease. Nat. Rev. Mol. Cell. Biol. 8, 440–450.
Wang, T., Chen, C., Yang, M., Deng, B., Kirby, G.M., Zhang, X., 2016. Cistanche tubulosa ethanol extract mediates rat sex hormone levels by induction of testicular steroidgenic enzymes. Pharm. Biol. 54, 481–487.
Wang, T., Zhang, X., Xie, W., 2012. Cistanche deserticola Y. C. Ma, “Desert ginseng”: a review. Am. J. Chin. Med. 40, 1123–1141.
Wang, X., Zhang, J., Lu, L., Zhou, L., 2015. The longevity effect of echinacoside in Caenorhabditis elegans mediated through daf-16. Biosci. Biotechnol. Biochem. 79, 1676–1683.
Whitaker, R., Faulkner, S., Miyokawa, R., Burhenn, L., Henriksen, M., Wood, J.G., Helfand, S.L., 2013. Increased expression of Drosophila Sir2 extends life span in a dose-dependent manner. Aging (Albany NY) 5, 682–691.
Wu, C.R., Lin, H.C., Su, M.H., 2014. Reversal by aqueous extracts of Cistanche tubulosa from behavioral deficits in Alzheimer’s disease-like rat model: relevance for amyloid deposition and central neurotransmitter function. BMC Complement. Altern. Med. 14, 202.
Xia, S., Miyashita, T., Fu, T.F., Lin, W.Y., Wu, C.L., Pyzocha, L., Lin, I.R., Saitoe, M., Tully, T., Chiang, A.S., 2005. NMDA receptors mediate olfactory learning and memory in Drosophila. Curr. Biol. 15, 603–615.
Zhang, K., Ma, X., He, W., Li, H., Han, S., Jiang, Y., Wu, H., Han, L., Ohno, T., Uotsu, N., Yamaguchi, K., Ma, Z., Tu, P., 2014. Extracts of Cistanche deserticola can Antagonize Immunosenescence and extend life span in Senescence-Accelerated Mouse
Prone 8 (SAM-P8) Mice. Evid. Based Complement. Alternat. Med. 2014, 601383.
Zhao, Q., Gao, J., Li, W., Cai, D., 2010. Neurotrophic and neurorescue effects of Echinacoside in the subacute MPTP mouse model of Parkinson’s disease. Brain Res. 1346, 224–236.