Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder, which ia associated with age and is characterized by progressive memory loss and cognitive dysfunction. Epidemiological studies have shown that the incidence of AD double increases every 5–10 years after the age of 65, indicating that globally the total number of patients with AD will rise to 973 million by 2030 (1). Clinically, AD is characterized by progressive impairments in behavior, cognition and memory (2). The presence of extracellular amyloid plaques, which develop as a result of the deposition of progressively increasing amyloid β (Aβ) peptide levels in the brain, are a key marker of AD (3). The Aβ peptides are important in the pathogenesis of AD. Therefore, inhibiting the generation of Aβ and increasing the rate of clearance of this protein, may be potential therapeutic strategies with which to delay the development of AD (4). While the Aβ hypothesis states that the β-amyloid protein is involved in the progression of AD pathology, the etiology remains unclear. Aβ is a 39–43 residue amyloidogenic peptide, which is derived from β-amyloid pro-protein (APP). APP may be cleaved by β-secretase and γ-secretase sequentially (5,6), and the resulting hydrolysate is ~4 KDa of Aβ peptide (5). With increasing age, the efficiency of organisms to eliminate Aβ is reduced, and the concentration of Aβ in the brain is increased, resulting in the formation of plaques. In addition, a previous study indicated that the soluble Aβ peptide causes severe toxicity to nerve cells, including tau hyperphosphorylation, axonal transport disorder and the disruption of organelle trafficking (7). Therefore, reducing the generation of Aβ (8) or increasing its clearance (9) may be beneficial in the treatment of AD.
Autophagy (macroautophagy in the current study) is the primary approach by which cells remove abnormal proteins and damaged organelles. When autophagy is activated, the substrates requiring removal are surrounded by double-membrane structures, termed autophagosomes (10,11). Autophagosomes fuse with lysosomes to form autolysosomes for substrate degradation. Deregulation of autophagy in AD has become an increasing focus of research (12). The enhancement of autophagy may slow the ageing process and reduce age-associated diseases, such as AD (13). For example, a previous study indicated that plaques were reduced, and cognitive deficits significantly improved, at an early age in 3xTg-AD mice following the administration of rapamycin, which induced autophagy (14).
Mammalian target of rapamycin (mTOR), a 289 KDa serine/threonine protein kinase, is the principle negative regulatory kinase of autophagy, and is also involved in cell growth, proliferation, metabolism and survival (15,16). In addition, mTOR may inhibit autophagy by regulating its downstream target, p70s6 kinase (17). Adenosine monophosphate-activated protein kinase (AMPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) are the two upstream regulators of mTOR, each of which are associated with autophagy (18,19). The metabolic sensor, AMPK, has been reported to inhibit mTOR via an effect on its downstream target, Raptor (9). Akt is the positive regulatory kinase of mTOR.
Radix Polygalae is the root of Polygala tenuifolia Willd. and its extract appears to be capable of improving memory (20). In addition, Polygala tenuifolia has been reported to improve cognitive impairment in rat AD models (21). Radix Polygalae extract was used to protect rat neuronal cells in vitro, which were induced by N-methyl-D-aspartate (22). In addition, Tenuifolin, extracted from tenuigenin, has been reported to inhibit Aβ secretion in COS-7 cells expressing APP (23). In the present study, the molecular mechanism underlying the induction of autophagy by Radix Polygalae extract was investigated, as this process was hypothesized to be associated with reductions in Aβ secretion.
Materials and methods
Reagents and antibodies
3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT), dansylcadaverine (MDC) and the rabbit monoclonal anti-light chain 3 (LC3) B antibody (1:1,000; cat. no. L7543) were purchased from Sigma-Aldrich (Shanghai, China). The rabbit monoclonal anti-mTOR (1:1,000; cat. no. 2983), anti-phospho-mTOR (Ser2448; 1:1,000; cat. no. 2971), anti-p70s6k (1:1,000; cat. no. 2708), anti-phospho-p70s6k (Thr389; 1:1,000; cat. no. 9205), anti-AMPK (1:1,000; cat. no. 2532), anti-phospho-AMPK (Thr172; 1:1,000; cat. no. 2535), anti-Raptor (1:500; cat. no. 2280), anti-phospho-Raptor (Ser792 (1:500; cat. no. 2083), anti-Akt (1:1,000; cat. no. 4685) and anti-phospho-Akt (Ser473; 1:1,000; cat. no. 4058) antibodies were obtained from Cell Signaling Technology, Inc. (Shanghai, China). The sheep anti-rabbit IgG antibody conjugated with horseradish peroxidase (IgG-HRP), SDS-PAGE, phenylmethanesulfonyl fluoride, loading buffer, chemiluminescence kit, and penicillin and streptomycin were purchased from Beyotime Institute of Biotechnology (Haimen, China). Fetal bovine serum (FBS), G418 sulfate, DMEM/F12 and F12 basic culture medium were purchased from Life Technologies (Shanghai, China). Paraformaldehyde, Tris-buffered saline (TBS) and TBS supplemented with 0.05% Tween-20 (TBST), were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Preparation of Radix Polygalae extract
Radix Polygalae (Pudong New District Medicine and Medicinal Materials Co., Ltd., Shanghai, China) was extracted using a spin steaming process with a rotary evaporator (Yarong Biochemical Instrument Plant, Shanghai, China) (24).
Cell culture
The SH-SY5Y human neuroblastoma cell line and Chinese hamster ovary (CHO) cells were obtained from Shanghai Institute of Materia Medica (Shanghai, China). SH-SY5Y cells were cultured in DMEM/F12, supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. CHO cells, stably transfected with APP and BACE1, were cultured in F12 supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin and 400 μg/ml G418 sulfate. Cells were maintained in an incubator at 37°C, with an atmosphere of 95% air and 5% CO2.
MTT assay for drug toxicity
SH-SY5Y cells were seeded in 96-well plates and treated with different concentrations (100, 40, 20, 10, 5 or 0 μg/ml) of Radix Polygalae. Following incubation for 24 h, 10 μl MTT (5 mg/ml) was added, and the cells were maintained at 37°C for an additional 4 h. The liquid was then discarded, crystals were dissolved in 100 μl DMSO and the light absorbance was read at 570 nm using a Multiskan FC Microplate Reader (Thermo Fisher Scientific, Shanghai, China), with 620 nm as the reference wavelength. Cells treated with 0 μg/ml Radix Polygalae served as controls (CTL). Radix Polygalae was dissolved in DMSO and while preparing a working solution, isometric DMSO was used in accordance with the drug treatment group.
Measurement of Aβ1-40 secretion in the supernatant
CHO-APP/BACE1 cells were seeded into 24-well plates at a density of 80,000 cells/well. Following treatment with (100, 40, 20, 10, 5 or 0 μg/ml) Radix Polygalae for 24 h, the Aβ1-40 ELISA kit (Shanghai ExCell Biology, Inc., Shanghai, China) and the Bicinchoninic Acid Protein Assay Reagent (Thermo Fisher Scientific) were used to measure the Aβ1-40 concentration in the supernatant and the total protein respectively. The experiments were conducted in accordance with the manufacturer’s instructions. The ratio of Aβ1-40 level and total protein in the DMSO group was similar to the control.
MDC-labeled autophagosomes detected in SH-SY5Y cells
SH-SY5H cells were treated with 3 different doses (0, 10 and 100 μg/ml) of Radix Polygalae with in the 6-well plate for 24 h, followed by incubation with 50 μmol/l MDC at 37°C for 30 min. Following incubation, the cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde. Autophagosomes were observed by fluorescence photometry (Olympus, Tokyo, Japan) at the excitation wavelength of 380 nm and emission filter of 525 nm (25).
Western blot analysis
SH-SH5Y cells were treated with different doses of Radix Polygalae (100, 40, 20, 10, 5 and 0 μg/ml) for 24 h in a 6-well plate. The total proteins were collected by the addition of radioimmunoprecipitation assay buffer containing 1 mM phenylmethanesulfonyl fluoride, mixed with loading buffer and boiled at 95°C for 15 min. Total proteins were separated by SDS-PAGE (20 μg/lane) and transferred to nitrocellulose membranes (Merck Millipore, Boston, MA, USA). The membranes were blocked for 2 h in TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 5% non fat milk, then incubated with LC3,(p)-mTOR, (p)-p70s6k, (p)-AMPK, (p)-Raptor and (p)-Akt primary antibodies at 4°C overnight. Membranes were washed three times with TBST, and incubated with horseradish peroxidase-linked anti-rabbit IgG for a further 2 h, followed by washing with TBST again. Blots were detected by enhanced chemiluminescence (ECL) and band intensity was analyzed by ImageJ software 1.48 (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
Data are expressed as the mean ± standard deviation. Data for multiple variable comparisons were analyzed by one-way analysis of variance using GraphPad Prism v5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Radix Polygalae exerts no significant effects on the viability of SH-SY5Y cells
The cell viability of human neuroblastoma cells with and without exposure to Radix Polygalae, was measured using an MTT assay. Following 24 h treatment, cell viability was not significantly different between the control group and the Radix Polygalae-treated groups (P>0.05). These results implied that the drug is not cytotoxic at doses <100 μg/ml (Fig. 1).
Aβ1-40 peptides level is markedly lower in the supernatant of cells treated with Radix Polygalae
The level of the Aβ1-40 peptide in the supernatant of CHO-APP/BACE1 cells was measured using an ELISA assay. The data showed that there was a reduction in the level of Aβ1-40 following treatment with Radix Polygalae, and that this effect occurred in a dose-dependent manner. The Aβ1-40 level in the 5 μg/ml group was reduced compared with DMSO group, although this result was not statistically significant. However, the reduction in Aβ1-40 was significantly different from the control group at doses of ≥10 μg/ml (P<0.01; Fig. 2).
Autophagy is induced by Radix Polygalae
In order to demonstrate that the Aβ peptide is degraded by autophagy, autophagy vesicles were detected using the MDC method, and he autophagy protein marker, LC3, was measured by western blotting analysis. Intracellular autophagy vesicles were labeled by MDC, and were observed as green dots. Few vesicles were observed in the control group (Fig. 3A), while an increase in the number of labeled vesicles occurred in the 10 μg/ml group (Fig. 3B) and a marked increase was observed in the 100 μg/ml group (Fig. 3C). LC3I and LC3II are the two forms of the microtubule-associated protein light chain 3 (LC3). When autophagy occurs, LC3I, located in the cytoplasm, is modified and processed by the ubiquitin-proteasome system and converted into LC3II (9,26). Western blot analysis indicated that LC3II/LC3I gradually increased, in accordance with the concentration of Radix Polygalae (Fig. 4).
Radix Polygalae induces autophagy by activating AMPK/mTOR signaling
In order to investigate the molecular mechanisms underlying the autophagy induced by Radix Polygalae in neurones, the negative regulator of autophagy, mTOR, in addition to its associated kinases, was examined by western blot analysis. The phosphorylation level of mTOR was reduced in a dose-dependent manner in the Radix Polygalae-treated groups. This indicates that Radix Polygalae may increase the level of autophagy. In addition, the downstream signaling kinase of mTOR, p70s6k, which also participates in the regulation of autophagy, was also inhibited by Radix Polygalae (Fig. 5).
In order to investigate the upstream pathway of mTOR, which may be affected by Radix Polygalae, the expression of AMPK and its downstream target, Raptor, was measured (27). With increasing doses of Radix Polygalae, the phosphorylation level of AMPK at Thr172 was increased, as was the phosphorylation of Raptor at Ser792 (Fig. 6).
Akt is another kinase that is known to activate mTOR (15). The results of the present study demonstrated that the phosphorylation level of Akt at Ser473 in the different groups was not significantly altered (Fig. 7).
Discussion
Aβ is an important protein in the pathogenesis of AD, and has become an increasing focus of the research into the development of novel anti-AD treatments (28–31). Reducing the levels of Aβ in the brain, with the aim of improving cognitive function and quality of life for patients with AD, are currently key aims in AD research. However, a specific and effective drug with which to treat AD remains to be developed, although the understanding of AD has advanced rapidly (20,32). In previous years, traditional Chinese medicine (TCM) products have gained attention for their potential use in AD treatment, due to the low toxicity and the fact that they are easy to obtain (20). Radix Polygalae, a TCM that is used to improve memory, has been previously studied, however the mechanisms involved remain to be elucidated (21–23). In the current study, the effect of Radix Polygalae on Aβ peptide levels was investigated in vitro.
Serious damage to neurons by Aβ peptides results in the formation of amyloid plaques and also leads to induction of the inflammatory response (33) and hyperphosphorylation of Tau (34,35), resulting in neuronal death. In the present study, secreted Aβ was detected in the CHO-APP/BACE1 supernatant, and the levels were significantly reduced following treatment with Radix Polygalae, suggesting that this compound simulates the removal of the Aβ peptide. Autophagy, which is the primary method of removing abnormal proteins and organelles from cells, was hypothesized to have been involved in this process. In order to confirm this hypothesis, MDC staining was used to detect autophagosomes. In addition, levels of the autophagy marker protein, LC3, were measured. The results from these two experiments supported the hypothesis.
In order to investigate the signaling mechanism underlying the induction of autophagy by Radix Polygalae, the mTOR pathway was assessed, due to its association with autophagy. In a previous study, mTOR was shown to be inhibited by rapamycin, while autophagy was increased, with the result that β amyloid plaque in a mouse model of AD decreased and cognitive function improved (14,36). p70s6k is the downstream target of mTOR. It is regulated by mTOR and participates in the inhibition of autophagy (17,37). In the current study, it was observed that the levels of mTOR and p70s6k were reduced by the addition of Radix Polygalae, indicating that autophagy had been induced. AMPK and PI3K/Akt are the two upstream regulators of mTOR, and AMPK is the key energy-sensing kinase that regulates cellular energy homeostasis in eukaryotes (27). AMPK regulates mTOR by phosphorylating Raptor at Ser792 (9,38). The Akt pathway is an additional upstream regulator of mTOR, exerting its action by phosphorylating Akt at Ser473 (16). In the current study, the phosphorylation of AMPK/Raptor increased. By contrast, Akt was not observed to be activated. These results support the hypothesis that Radix Polygalae induces autophagy via the activation AMPK/Raptor/mTOR signaling.
In conclusion, autophagy is involved in the removal of Aβ peptide by Radix Polygalae. The basic signaling mechanism underlying this effect was detected, and the results of the current study may provide a basis for further investigation into the use of this compound in the treatment of AD.