Abstract
Background:
Dendrobium nobile is a well-known traditional Chinese herbal medicine used for age-related diseases. Dendrobium nobile Lindl. alkaloid (DNLA) is the active ingredient to improve learning and memory deficits in laboratory animals.
Objective:
The aim of the present study was to examine the anti-aging effects of long-term administration of DNLA and metformin during the aging process in senescence-accelerated mouse-prone 8 (SAMP8) mice.
Methods:
SAMP8 mice were orally given DNLA (20 and 40 mg/kg) or metformin (80 mg/kg) starting at 6 months of age until 12 months of age. Age-matched SAMR1 mice were used as controls. DNLA and metformin treatments ameliorated behavioral deficits of 12-month-old SAMP8 mice, as determined by Rotarod, Y-maze, and Open-field tests.
Results:
DNLA and metformin treatments prevented brain atrophy and improved morphological changes in the hippocampus and cortex, as evidenced by Nissl and H&E staining for neuron damage and loss, and by SA-β-gal staining for aging cells. DNLA and metformin treatments decreased amyloid-β1–42, AβPP, PS1, and BACE1, while increasing IDE and neprilysin for Aβ clearance. Furthermore, DNLA and metformin enhanced autophagy activity by increasing LC3-II, Beclin1, and Klotho, and by decreasing p62 in the hippocampus and cortex.
Conclusion:
The beneficial effects of DNLA were comparable to metformin in protecting against aging-related cognitive deficits, neuron aging, damage, and loss in SAMP8 mice. The mechanisms could be attributed to increased Aβ clearance, activation of autophagy activity, and upregulation of Klotho.
INTRODUCTION
Aging, a natural phenomenon, is the risk factor for the functional decline in most organs [1]. The brain is susceptible to aging. Brain aging is often accompanied by cognitive and motor dysfunction, neuronal loss, amyloid-β (Aβ) and lipofuscin deposition, and brain atrophy, seriously affecting the life quality of the elderly [2]. The pathogenesis of brain aging has not been fully understood, but it is believed to be related to immune disorder, genetic mutation, telomere loss, Aβ cascade reaction, and autophagy impairment [3]. Previous studies have found that the Aβ cascade hypothesis is one of the most important pathogenesis, and autophagy contributes to the clearance of Aβ. Moreover, Klotho is an anti-aging factor that can induce autophagy activity and clear Aβ [4–6].
Senescence accelerated mouse (SAM), a mouse strain of accelerated aging, was generated from AKR/J mice by Takeda at the University of Kyoto in 1990s [7]. Senescence accelerated mouse prone 8 (SAMP8) mice present the aging-related neurodegenerative characteristics, such as cognitive and motor dysfunction, neuronal cell loss, shortened lifespan, tau protein hyperphosphorylation, Aβ deposition, and dementia [8]. The age-matched senescence accelerated mouse resistant 1 (SAMR1) mice are commonly used as a background control with normal aging phenotype.
Dendrobium nobile is a well-known traditional Chinese herbal medicine widely used for treatment of age-related diseases in China. Dendrobium nobile Lindl. alkaloids (DNLA) is one of the active ingredients extracted from Dendrobium nobile [9]. Our previous studies have found that DNLA could improve learning and memory deficits in several Alzheimer’s disease (AD) animal models, such as in APP/PS1 AD transgenic mice [10], in LPS-induced AD rats [11], and in Aβ25–35-induced neuronal damage in rat primary cortical and hippocampal cultures [12, 13]. The mechanisms may be related to inhibition of Aβ deposition by improving impaired autolysosomal proteolysis [10] and inhibition of tau protein hyperphosphorylation [11]. In addition, DNLA prevents Aβ25–35-induced axonal degeneration via activation of autophagy [12]. These studies suggest that DNLA may have anti-aging effects, and the mechanism may be related to the regulation of autophagy.
Therefore, in the present study, we used SAMP8 mice to confirm the anti-aging effect of DNLA and further analyzed whether its anti-aging effects are related to inhibition of Aβ deposition, activation of autophagy, and upregulation of Klotho.
MATERIALS AND METHODS
Animals
Male SAMP8 mice were purchased from Hua Fu Kang Biological Technology Company (Chongqing, China). Male SAMR1 mice were from Peking University Medical College. All mice were specific pathogen free (SPF) grade (Beijing, China) and their certificate number are SCXK2014-0004 and SCXK2011-0012, respectively. Animals were housed in SPF grade (Certificate No.: SYXK 2014-003) animal room with a 12 h light/dark cycle at 22∼24°C and were given free access to food and water. Animal experiment procedures follow the Animal Care and Use Guidelines in China and were approved by Animal Use and Care Committee of Zunyi Medical University.
Drugs
DNLA was isolated from Dendrobium nobile, in which 6 alkaloids account for 86.2%, including Dendrobium alkaloids, Dendrobine, Dendrobine-N-oxide, Nobilonine, Dendroxine, 6-Hydroxy-nobilonine, and 13-Hydroxy-14-oxodendrobine. as determined by LC-MS/MS [12, 14]. We consider DNLA (total alkaloids) as a whole to be a single agent, consistent with our prior studies [10–14]. Metformin (Met, purity ≥97%) was purchased from Sigma Chemical Company (St. Louis, MO, USA).
Experimental designs
Six-month-old male SAMP8 mice were randomly divided into four groups: SAMP8 model group, DNLA low-dose group (20 mg/kg), DNLA high-dose group, and Met group (80 mg/kg); age-matched SAMR1 mice were used as background control group (n = 11–18). The dose selection of DNLA was based on our prior publications [10–14], and the inclusion of Met as the positive control was based on our recent study in SAMP8 mice [10]. DNLA and Met were dissolved in 1% Tween 80 and were given in a volume of 0.1 mL/10 g, and the SAMR1 and SAMP8 controls were administered with equal volume of vehicle. All animals were orally administrated once a day for 6 months. The duration of the treatment was aimed to match aging process in SAMP8 mice as shown in our recent publications [10, 14].
Y maze
Y maze is a spontaneous alternation test for working memory. Rodents typically prefer to investigate a new arm of the maze rather than returning to one that was previously visited. Y maze consists of three blue Plexiglas arms (40 cm length×8 cm width×15 cm height), which was placed in a quiet room with fixed brightness. Animals were allowed explored the maze for 10 min. A triad was defined as a standard entry when the four paws of the animal were fully entered into one arm and entering into all three arms consecutively was defined as successful alternation behavior. Total number of arm entries and alternation behavior were measured. The scores are calculated as follows: [(number of alternation)/(total arm entries –2)] × 100 [15, 16]. Top View Animal Behavior Analyzing System (Version 3.00) was used to record and analyze data.
Open field test
General locomotion and anxiety-like behavior was measured by the open field test (OFT). Animals were placed in an OFT box (25 cm length×25 cm width×35 cm height) for 10 min. The 12 cm×12 cm region in the center of the arena was defined as the center, and the other region was defined as the peripheral area. Mice were put in the corner of the arena facing the wall, and allowed to freely move throughout the arena for 10 min. This procedure could not be repeated, and the apparatus was thoroughly cleaned with 70% alcohol before the test of another animal. Total distance traveled in the arena was recorded. The Top View Animal Behavior Analyzing System (Version 3.00) recorded the behavioral responses in the open field test [17].
Rotarod
Rotarod test is widely used to evaluate the motor coordination of rodents. Rotarod consisted of four standard channels were used to evaluate the motor function of mice. The mice were trained to stay on the rotarod for three days (once per day) before performing the experiments. Then all animals were tested simultaneously at a speed of 10 rpm/min, followed by an increase of 5 rpm/min. The maximum time was 300 s per trial. The time at which the mice fall off the rod is recorded. If the mice remained on the rod for more than 300 s, the stay time was recorded as 300 s. The latency to falling was recorded automatically by photo-cells and the total latencies on the rod were analyzed [18].
Hematoxylin–eosin (H&E) staining
All animals were sacrificed after the behavior tests, three mice of each group were perfused transcardially with 0.1 M PBS and fixative solution of ice-cold 4% paraformaldehyde, and brains were removed and fixed in 4% paraformaldehyde for 24 h at 4°C. Frozen sections of 5μm thickness were made by using Cryomicrotome (Thermo Scientific, USA) for histological analysis according to the instructions. Light microscopy was used to observe the histomorphology of neurons. Hippocampus and prefrontal cortex were selected to examine for morphological alterations (scale bar = 50μm).
Nissl staining
Brains were immersion in 4% paraformaldehyde, embedded by paraffin, and cut into coronal sections of 5μm thick for Nissl staining. Briefly, the sections were deparaffinized in xylene and rehydrated using gradual alcohol, treated with Nissl staining solution (Solarbio, Beijing, China) for 5 min. Then, the sections mounted with neutral balsam [19]. The sections were examined under a light microscope (Leica Microsystems Ltd., Wetzlar, Germany). The numbers of Nissl bodies were captured in the three fields of the CA1 region of the hippocampus and prefrontal cortex. For quantification, we used the Image J open source software to analyze.
SA-β-gal staining
After sectioning, tissues were covered with 500μL cold fixative solution for 15 min at room temperature. Then, rinsed twice with PBS, and incubated with β-galactosidase staining solution in an incubator (without CO2) overnight at 37°C. The stained tissues were examined under a light microscope (KS300, Zeiss-Kontron, Germany). We used the Image J open source software to perform digital slide image counts for quantification of senescent cells in the prefrontal cortex [20].
Western blot
The hippocampal and cortical tissues were homogenized in cold lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. The supernatant was acquired and the whole protein levels were quantified by BCA assay kit. Proteins lysates were separated in 8% SDS-PAGE, bands were transferred to PVDF membranes after electrophoresis. PVDF membranes were blocked with 5% nonfat milk for 2 h. Then, membranes were incubated with rabbit amyloid-β1–42 (Aβ1–42, 1 : 1,000, Abcam, Cambridge, MA, USA), amyloid-β protein precursor (AβPP, 1 : 1,000, BBI Life Sciences, Shanghai, China), presenilin-1 (PS1, 1 : 1,000, Abcam), β-site APP-cleaving enzyme 1 (BACE1, 1 : 500, BBI Life Sciences), autophagy marker Light Chain 3 (LC3, 1 : 1000, Abcam), nucleoporin p62 (P62, 1 : 1000, Abcam), autophagy-related protein (Beclin-1, 1 : 1000, Proteintech, Wuhan, China), neprilysin (NEP, 1 : 1000, Proteintech), insulin degrading enzyme (IDE, 1 : 1000, BBI Life Sciences), Klotho (1 : 1000, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 2000, Proteintech) at 4°C overnight. Membranes were incubated with secondary antibodies for 1 h. Finally, blots were detected using ECL Western blotting detection kit (7 Sea Biotech, Shanghai, China), and scanned to BIO-RAD Gel Imaging and the results were analyzed using Quantity One software v4.52 analysis (BIO-RAD, Hercules, CA, USA).
Statistical analysis
All data were analyzed by SPSS software version 18.0, and data were presented as mean±SD. Data were analyzed by one-way analysis of variance (ANOVA), followed by a Least Significance Difference Method (LSD) test or Dunnett’s t-test. p < 0.05 was considered statistically significant.
RESULTS
Effects of DNLA on cognitive and motor in SAMP8 mice
Short-term spatial working memory was evaluated by spontaneous alternation behavior in Y-maze test. As illustrated in Fig. 1B, compared with the SAMR1, SAMP8 mice showed working memory dysfunction, and 6-month DNLA treatment improved working memory deficits in SAMP8 mice (p < 0.05), which suggested that DNLA could improve the spatial working memory deficits of SAMP8 mice in the Y-maze test.
Effects of DNLA on cognitive and motor deficits in SAMP8 mice. A) Schematic drawing of experimental schedule. B) Alternation (%) in the Y-maze. C) Assessment of the behaviors in the Rotarod test. D) Assessment of the behaviors in the Open field test. (mean±SD, n = 6–9), *p < 0.05 versus SAMR1, #p < 0.05 versus SAMP8 vehicle.
Rotarod is a method for quantitative evaluation of motor function in rodents. Figure 1C showed that SAMP8 mice exhibited the shorter time than that of SAMR1 mice (p < 0.05). The latency was obviously increased in SAMP8 mice treated with DNLA and Met (p < 0.05), indicating that DNLA could alleviate the motor function impairment in SAMP8 mice.
Open field test was used to evaluate spontaneous activity and exploration behavior of mice. As illustrated in Fig. 1D, compared with the SAMR1 mice, total distance between the central and peripheral region was significantly decreased in the SAMP8 mice, which was recovered in DNLA and Met groups to certain extent, although the difference was not statistically significant. In addition, no clear dose-response of DNLA was evident. Nonetheless, both DNLA and Met were effective in ameliorate cognitive decline in SAMP8 mice.
Effects of DNLA on the brain coefficient of SAMP8 mice
At the end of the experiment, the brains of surviving mice were collected to calculate brain coefficient. At the 12 months of age, the surviving mice/rates were SAMR1, 9/64%; SAMP8, 8/44%; SAMP8 + DNLA20, 9/55%; SAMP8 + DNLA40 6/54% (several mice died at 6–7 months of age due to gavage accident), and SAMP8 + Met 7/56%, respectively. The brain coefficient is the ratio of brain weight to body weight and is often used as an important indicator to evaluate brain atrophy. Brain atrophy is an indicator of brain aging. As shown in Fig. 2, the brain coefficient of SAMP8 mice was significantly lower than SAMR1 mice (p < 0.05), which was significantly increased with DNLA and Met treatment (p < 0.05).
Effect of DNLA on brain coefficient in SAMP8 mice. (mean±SD, n = 6–9) *p < 0.05 versus SAMR1, #p < 0.05 versus SAMP8 vehicle.
Effects of DNLA on morphological changes of cortex and hippocampus tissues in SAMP8 mice
SA-β-gal staining
SA-β-gal staining was used to verify the degree of brain aging. The results revealed that the positive expression of SA-β-gal in the brain of SAMP8 mice was significantly increased compared with SAMR1, which was decreased with DNLA and Met treatment (Fig. 3).
Effect of DNLA on SA-β-gal in SAMP8 mice. A) Representative images of SA-β-gal staining in the prefrontal cortex (magnification 200×, scale bar = 50μm); B) Quantitation of senescent positive cells. (
Nissl staining
Nissl is a neural characteristic structure; the number of Nissl bodies reflects the growth and development of neurons. Compared with the SAMR1 control group, the neuronal Nissl bodies of the cortex and hippocampus in the SAMP8 mice were slightly stained and sparsely arranged. The number of Nissl bodies in the CA1 region of hippocampus was decreased 23% compared to SAMR1 mice, and number of Nissl bodies in the cortex decreased 20%. All these pathological changes were reversed by DNLA and Met treatment, and Nissl bodies were increased (Fig. 4).
Effect of DNLA on neuron morphology in SAMP8 mice. A) Representative images showing Nissl bodies in the hippocampal CA1 (magnification 200×, scalebar = 50μm). B) Quantitation of pyramidal cells in the CA1 hippocampal region. C) Representative images showing Nissl bodies in prefrontal cortex (magnification 200×, scalebar = 50μm). D) Quantitation of pyramidal cells in prefrontal cortex. (
Effect of DNLA on Aβ and AβPP protein levels
As indicated in Fig. 5, the results of western blot showed that, compared with SAMR1 mice, the protein levels of Aβ1–42 and AβPP were increased in SAMP8 mice compared with SAMR1 mice in the hippocampus (p < 0.05) and cortex (p < 0.05), which were clearly decreased with DNLA and metformin treatment (p < 0.05).
Effects of DNLA on Aβ1–42, AβPP, BACE1, and PS1 protein levels in the hippocampus and cortex in SAMP8 mice. A) Representative bands of protein expression in hippocampus. B) Representative bands of protein expression in cortex. C, E, G, I) The level of Aβ1–42, AβPP, BACE1, and PS1 protein in hippocampus. D, F, H, J) The level of Aβ1–42, AβPP, BACE1, and PS1 protein in cortex. (
BACE1 and PS1 protein levels in the hippocampus and cortex are presented in Fig. 5. Compared with SAMR1 mice, the BACE1 and PS1 protein levels in the hippocampus and cortex of SAMP8 mice were increased (p < 0.05), which were decreased with DNLA and Met treatment (p < 0.05).
Effect of DNLA on Aβ degrading enzymes
In order to investigate the effects of DNLA on the Aβ level, Aβ degrading enzymes was used to detect the Aβ degrading. As shown in Fig. 6, the increased insulin degrading enzyme (IDE) and neprilysin (NEP) levels were decreased in the hippocampus and cortex of SAMP8 mice compared with that of SAMR1 mice (p < 0.05), which were increased with DNLA and Met treatment (p < 0.05).
Effect of DNLA on IDE and NEP protein levels of hippocampus and cortex in SAMP8 mice. A) Representative bands of protein expression in hippocampus. B) Representative bands of protein expression in cortex. C) The level of IDE and NEP protein in hippocampus. D) The level of IDE and NEP protein in cortex. (
Effects of DNLA on LC3, Beclin1, P62, and Klotho protein levels
As shown in Fig. 7, the protein expression of LC3 and Beclin1 were decreased, accompanied by an increase of the expression of P62 protein in the hippocampus and cortex of SAMP8 mice compared with that of SAMR1 mice (p < 0.05), all of which were reversed with 6-month DNLA and Met treatment (p < 0.05). As shown in Fig. 7, the protein expression of Klotho in the hippocampus and cortex were lower in SAMP8 mice compared with SAMR1 mice, and DNLA-treated and Met-treated mice showed that increased protein expression of Klotho relative to SAMP8 mice (p < 0.05).
Effects of DNLA on LC3, Beclin1, P62, and Klotho protein levels in the hippocampus and cortex in SAMP8 mice. A) Representative bands of protein expression in hippocampus. B) Representative bands of protein expression in cortex. C, E, G, I) The level of LC3, Beclin1, P62, and Klotho protein in hippocampus. D, F, H, J) The level of LC3, Beclin1, P62, and Klotho protein in cortex. (
DISCUSSION
The current study using SAMP8 and SAMR1 mice clearly demonstrated that 6-month treatment with DNLA and Met is effective against brain aging process in senescence accelerated SMAMP8 mice, as evidenced by improved cognitive and motor defects, increased Nissl bodies, ameliorated Aβ cascades, but importantly, by induction of autophagy machinery and upregulation of the levels of Klotho. This is among the first study to reveal anti-aging effects of DNLA and highlights the Aβ cascades and autophagy as the therapeutic targets for aging.
SAMP8 mice, an excellent aging model, are characterized by progressive motor and cognitive dysfunctions with age. The cognitive ability of SAMP8 mice was impaired as early as 4 months of age [21]. Besides, SAMP8 mice at 12 months of age are close to death, and have severe cognitive and motor deficits [22, 23]. In this study, we selected 6-month-old SAMP8 mice to study the effect of DNLA administration continuously for 6 months during the aging process. The result of Y maze showed that DNLA significantly improved spatial working memory impaired in 12-month-old SAMP8 compared with the SARM1 mice. The other hallmarks of aging were the decline in muscle function [18, 24]. Our present experiments demonstrated that DNLA treatment for 6 months could ameliorate the impaired motor functions in 12-month-old SAMP8 mice. However, we did not use “delay period” in the Y-Maze test, and thus the results reflect frontal cortex dependent working memory rather than hippocampus dependent working memory. Nonetheless, these behavioral tests show that DNLA could improve both the cognitive ability and the motor function, suggesting that DNLA may have a potential to improve daily life qualities of the elderly.
It has been reported that cognitive and motor deficits may be associated with brain atrophy. Brain atrophy is the situation that the senescent brain reduces in volume, quality, and the number of normal brain cells [25]. We found that the brain wet weight ratios of SAMP8 mice were significantly decreased, indicating the occurrence of brain atrophy. Fortunately, treatment with 40 mg/kg DNLA prevented brain atrophy in SAMP8 mice. In addition, SA-β-gal staining assay has been widely used as biological indicators for aging cells [26]. Administration of DNLA also reduced brain SA-β-gal activity in SAMP8 mice, consistent with the literature that senescent cells might exist and accumulate with SA-β-gal [26, 27]. It should be mentioned that cell aging is not the only factor that can increase senescence-associated SA-β-gal, DNA damage response could also increase SA-β-gal staining [28]. DNLA was found to be effective to suppress endoplasmic reticulum stress and reduce DNA damage (manuscript submitted), all these factors could contribute to anti-aging effects of DNLA.
It has been reported that typical aging characteristics of SAMP8 mice appear nearly twice as fast as those of SAMR1 mice, especially the aging symptoms in the central nervous system [29]. Numerous studies have reported that memory disorders in SAMP8 mice were attributed to early neurological damage [30]. Neurons are the basic functional unit of the intricate network inside the brain, excessive neuronal loss can destroy cognitive and other functions [31]. Nissl body is a characteristic cell structure that reflects the function of neurons and helps the nerve cells to synthesize proteins. The results of H&E (Supplementary Figure 1) and Nissl staining showed that the hippocampal and cortical neurons of SAMP8 mice were damaged, the vertebral body cells were arranged disorderly, and the number of normal cells were reduced. All of these alterations were reversed by DNLA and Met treatment, suggesting that the neuroprotective effect of DNLA might be related to the anti-aging effects in SAMP8 mice.
The levels of Aβ is related to the damage degree of neurons; therefore, decreasing production of neurotoxic Aβ is beneficial to reduce neuronal damage and improve cognitive and motor impairment [32]. Aβ is generated from the AβPP and is produced via sequential proteolytic cleavage of AβPP by BACE1 and PS1 [33, 34]. BACE1, an important cleavage enzyme of AβPP, is responsible for overexpression of neurotoxic Aβ [35]. Additionally, PS1, an essential component of the active γ-secretase complex, liberates the Aβ peptides from AβPP [36]. Therefore, the dysregulation of BACE1 and PS1 leads to overproduction of neurotoxic Aβ [37]. Furthermore, in the clean-up process of Aβ, increased IDE and NEP are major proteases responsible for the degradation of Aβ. IDE mainly cleans up soluble Aβ, and neprilysin mainly cleans up insoluble Aβ. Increasing evidence indicates that overexpression of IDE can reduce Aβ levels and senile plaques in IDE transgenic mice [38]. Similarly, upregulation of neprilysin can reduce the production of Aβ, delay the formation of Aβ plaques, prevent related pathological changes, and even prolong the lifespan of AβPP transgenic mice [39]. In this study, we found that DNLA not only inhibited Aβ production, but also promoted Aβ clearance in SAMP8 mice. DNLA downregulated the protein levels of BACE1 and PS1, and upregulated the protein levels of IDE and neprilysin, which may be important against Aβ etiology.
As we know, the aggregation of Aβ is correlated with abnormal autophagy, and alterations in the autophagy occur with increasing age [40]. Autophagy is executed by several multi-step processes. In the first step of forming a double membrane structure, Beclin1 is necessary to combine with different proteins to regulate autophagy activity [41]. Then, the soluble form of LC3-I is converted to LC3-II, which remains on mature autophagosomes until fusion with lysosomes and is commonly used as an important marker of autophagy [42]. Finally, p62 as an autophagy specificity substrate interacts with LC3 to deliver ubiquitinated proteins to autolytic lysozymes for degradation [43]. It should be noted that autophagy activity in SAMP8 mice changed with age [44]. Our previous research found that compared with SAMR1, the hippocampal neurons of SAMP8 mice harbored more autophagosomes at the age of 8-month-old [45]. However, autophagy activity decreased in 12-month-old SAMP8 mice in the present study. Activation of autophagy at 12-month-old SAMP8 mice could be an efficient way to facilitate the clearance of Aβ aggregates [46]. Fortunately, DNLA was able to activate autophagy via increasing the expression of Beclin-1, LC3-II. Autophagy deficient mice are accompanied by accumulation of p62, suggesting that the level of p62 is negatively correlated with autophagy activity [47]. In the present study, we find the protein level of p62 was significantly reduced in DNLA treatment SAMP8 mice, which further confirmed our previous study in primary neurons [12]. All above results demonstrated that DNLA can delay aging in SAMP8 mice by modulating autophagic flux.
Klotho is related to the development of aging in SAMP8 mice. Klotho was discovered in 1997 and named by the ancient Greek goddess [48]. Overexpression of Klotho extends lifespan [49] and increases survival time in SOD mutant ALS mice [50]. A literature review indicates that Klotho can clear Aβ, induce autophagy activity and increase LC3 expression [51]. Overexpression of Klotho by intracerebroventricular injection of lentiviral vector that encodes Klotho [6], or by 14-week oral treatment with natural phthalide Ligustilide [4], all ameliorated AD symptoms in APP/PS1 transgenic mice, with increased Aβ clearance and activation of autophagy. The results of this study showed that the level of Klotho protein was decreased in the brain of 12-month-old SAMP8 mice. Interestingly, treatment with DNLA and Met could increase Klotho protein level. It is suggested that the protective effect of DNLA on cognitive and motor deficits in brain aging of SAMP8 mice might be associated with Klotho protein upregulation.
In summary, the present study revealed the beneficial anti-aging effects of DNLA and the underlying mechanisms. The anti-aging mechanisms of DNLA appear to be mediated, at least in part, via decreasing Aβ aggregation and enhancing autophagy activity, as well as the upregulation of Klotho.
Footnotes
ACKNOWLEDGMENTS
This study was financially supported by the National Natural Science Foundation of China, Grant Number 81773739; Guizhou Province Synergy Innovation Center Grant Number, CJ-926; Guizhou Provincial Department of Education “125” Major Science and Technology Projects, Grant Number 2012012; Guizhou Provincial Department of Education Tutor Studio, Grant Number 99-030; Shijingshan’s Tutor Studio of Pharmacology [GZS-2016(07)]; Science and Technology Foundation of Guizhou Province (NO. JZ [2014]2016).
