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APP695 cells
Author’s Accepted Manuscript Ginsenoside Re reduces Aβ production by activating PPARγ to inhibit BACE1 in N2a/APP695 cells Guoqiong Cao, Ping Su, Shuai Zhang, Limin Guo, Haijing Zhang, Yuexia Liang, Chunxia Qin, Wensheng Zhang www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(16)30695-1 http://dx.doi.org/10.1016/j.ejphar.2016.11.006 EJP70911
To appear in: European Journal of Pharmacology Received date: 21 July 2016 Revised date: 23 October 2016 Accepted date: 3 November 2016 Cite this article as: Guoqiong Cao, Ping Su, Shuai Zhang, Limin Guo, Haijing Zhang, Yuexia Liang, Chunxia Qin and Wensheng Zhang, Ginsenoside Re reduces Aβ production by activating PPARγ to inhibit BACE1 in N2a/APP695 c e l l s , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page Title: Ginsenoside Re reduces Aβ production by activating PPARγ to inhibit BACE1 in N2a/APP695 cells Author names: Guoqiong Cao
a,b,c
, Ping Su
a,b,c
, Shuai Zhang
a,b,c
, Limin Guo
a,b,c
, Haijing Zhang
a,b,c
Yuexia Liang a,b,c, Chunxia Qin a,b,c, Wensheng Zhang a,b,c,d,* a
Beijing Area Major Laboratory of Protection and Utilization of Traditional Chinese Medicine, Beijing Normal University, Beijing 100088, China;
b
Engineering Research Center of Natural Medicine, Ministry of Education, Beijing Normal University,
Beijing 100088, China; c
College of Resources Science Technology, Beijing Normal University, Beijing 100875, China;
d
National & Local United Engineering Research Center for Sanqi Resources Protection and Utilization
Technology, Kunming 650000, China * corresponding author.
Corresponding author with complete address: Wensheng Zhang, Complete address: C Building, Beijing Normal University Science Park, No. 12, Xueyuan Southern Street, Haidian District, Beijing, P.R.China, 100088. Tele: +86 010 62200669. Email: [email protected].
Abstract Alzheimer’s disease (AD) is a neurodegenerative disease characterized by β-amyloid protein (Aβ) deposition. Reducing the Aβ load may be a new perspective for AD treatment. Ginsenoside Re is an extract from Panax notoginseng, which is a well-known traditional Chinese medicine that has been used for the treatment of various diseases for years. Ginsenoside Re has been reported to decrease Aβ in Alzheimer’s 1
,
disease animal models, but the mechanism has not been fully elucidated. In the present study, we investigated the mechanism of ginsenoside Re. Our results showed that ginsenoside Re decreased the Aβ levels in N2a/APP695 cells. Aβ peptides are generated by β-secretase (β-site amyloid precursor protein cleaving enzyme 1 (BACE1)) and γ-secretase. We found that ginsenoside Re decreased the BACE1 mRNA and protein levels and inhibited BACE1 activity in the N2a/APP695 cells. Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that regulates the activity of the BACE1 promoter, and activating PPARγ can inhibit BACE1. The results also showed that ginsenoside Re significantly increased the PPARγ protein and mRNA levels. These effects of ginsenoside Re on BACE1 could be effectively inhibited by the PPARγ antagonist GW9662. These findings indicate that ginsenoside Re inhibits BACE1 through activation of PPARγ, which ultimately reduces the generation of Aβ1-40 and Aβ1-42. Therefore, ginsenoside Re may be a promising agent for the modulation of Aβ-related pathology in AD. Key words: Ginsenoside Re, Alzheimer´s disease, Aβ, BACE1, PPARγ. 1. Introduction Alzheimer´s disease is a chronic neurodegenerative disorder that affects millions of elderly people worldwide. β-amyloid protein deposition is regarded as the pathological hallmark of AD. In the brain, Aβ accumulation can lead to neurofibrillary tangles, inflammation, axonal injury, and synapse loss (Pimplikar, 2009). Aβ peptides are generated by APP (amyloid-β precursor protein) via amyloidogenic pathways by β-secretase and γ-secretase. BACE1 is the initiating and rate-limiting enzyme in Aβ generation (Chen et al., 2012; Lin et al., 2015).Thus, BACE1 inhibition represents an important intervention that may prevent and/or cure AD (Ohno, 2016). 2
BACE1 gene transcription is regulated by many transcription factors, including NF-κB (Buggia-Prevot et al., 2008), Sp1 (Christensen et al., 2004), and PPARγ (Wang et al., 2016). Studies have shown that PPARγ regulates the expression of the genes involved in adipogenesis, lipid metabolism, inflammation, and the maintenance of metabolic homeostasis (Wang et al., 2014) as well as components of Aβ metabolism and toxicity (Mandrekar-Colucci et al., 2012; Mandrekar-Colucci and Landreth, 2011). The binding of the PPARγ response element (PPRE) to the promoter region of the BACE1 gene suppresses BACE1 expression and inhibits Aβ production (Lin et al., 2015). Panax notoginseng (Burk) F.H. Chen (Araliaceae) is a well-known herb that has been used in China for many years. Saponins are the major constituents of P. notoginseng and are also considered its main active compounds (Wang et al., 2016a). The five main saponins (ginsenosides Rb1, Rg1, Rd and Re and the notoginsenoside R1) constitute up to 90 % of the total P. notoginseng saponins (PNS) used in pharmacological experiments (Liu et al., 2014). Among them, ginsenosides Rb1 and Rd belong to the 20(S)-protopanaxadiol-type, whereas ginsenosides Rg1 and Re and notoginsenoside R1 belong to the 20(S)-protopanaxatriol-type (Li et al., 2015). Previous studies reported that ginsenoside Rg1 and notoginsenoside R1 decreased the Aβ level by upregulating PPARγ (Li et al., 2015; Quan et al., 2013). Ginsenoside Re, which also belongs to the 20(S)-protopanaxatriol type, exhibited many beneficial effects, such as anti-inflammatory (Lee et al., 2012), anti-oxidation (Huang et al., 2016), and neuroprotective effects (Chen et al., 2008). Ginsenoside Re activated PPARγ in 3T3-L1 adipocytes (Han et al., 2006) and reduced insulin resistance through activation of the PPARγ pathway (Gao et al., 2013). Additionally, ginsenoside Re reduced the Aβ1-40 and Aβ1-42 levels in the brains of Tg2576 mice (Chen, 2006). However,
3
whether ginsenoside Re reduces the Aβ level through the PPARγ pathway is unknown. Therefore, in this study, we investigated the effect of ginsenoside Re on BACE1 and Aβ through the promotion of PPARγ. 2. Materials and Methods 2.1. Chemicals and Regents Ginsenoside Re (purity > 98 %) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The molecular structure of ginsenoside Re is shown in Fig. 1A. DMSO and GW9662 were purchased from Sigma-Aldrich (St. Louis, MO, USA). For cell culture, penicillin, streptomycin, Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). G418 was ordered from Amresco (Solon, OH, USA). The Aβ1-40 and Aβ1-42 ELISA kits were purchased from Invitrogen (Carlsbad, CA, USA). The Beta-Secretase Activity Fluorometric assay kit was purchased from BioVision (San Francisco, USA). The primary antibodies used in this study were purchased as follows: APP (Merck, Germany), sAPPβ (IBL, Japan), sAPPα (IBL, Japan), APP-CTF (Calbiochem, USA), BACE1 (Abcam, USA), PPARγ (Santa Cruz CA, USA), and GAPDH (Cell Signaling Technology, USA). The PPARγ agonist pioglitazone was obtained from TaiYang Pharmaceutical Co., Ltd. (Beijing, China). 2.2. Cell Culture The N2a/APP695 cell line that stably expressed Swedish mutant human APP695 was constructed and stored by the Beijing Key Laboratory for the Protection and Utilization of Traditional Chinese Medicine Resources of Beijing Normal University. The cells were maintained in DMEM/Opti-MEM (1:1, v/v; containing 200 μg/ml of G418, 5 % FBS, 100 units/ml of penicillin, and 100 mg/ml of streptomycin) and
4
kept at 37 °C in a humidified 5 % CO2/95 % air environment. The cells were passaged every 3 days when they reached 80 % confluence. 2.3. Cell viability assay To explore the cytotoxic effect of ginsenoside Re on N2a/APP695 cells, cell proliferation was assessed using the MTT assay. The cells were treated with increasing doses of ginsenoside Re (0, 25, 50, 100, 150, and 200 μM) for 24 h. The MTT assay was performed after the treatments. The cells were incubated for 4 h at 37 °C with 0.5 mg/ml of MTT dissolved in fresh complete medium. The dark blue formazan crystals were dissolved in DMSO, and the absorbance was measured on a microplate reader (Thermo Multiskan MK3, USA) using a reference wavelength of 630 nm and a test wavelength of 490 nm. The data were expressed as the mean percentages of viable cells versus the control. 2.4. Aβ ELISA assay The cells were incubated with ginsenoside Re (0-100 μM) for 24 h. After treatment, conditioned media from the treated and untreated cells were collected to detect secreted Aβ1-40 and Aβ1-42. The Aβ1-40 and Aβ1-42 concentrations were quantified using ELISA kits following the manufacturer’s protocol. The optical densities of each well at 450 nm were readed on a Multiskan MK3 microplate reader (Thermo Labsystems, Waltham, MA, USA), and the sample Aβ1-40 and Aβ1-42 concentrations were determined by comparison with the Aβ1-40 and Aβ1-42 standard curves. All readings were in the linear range of the assay. 2.5. Western blotting analysis After treatment, the cells were washed with ice-cold PBS and then lysed in RIPA lysis buffer containing a cocktail of complete protease inhibitors (Roche Diagnostics). The protein concentrations were determined by the BCA method. Equal amounts of proteins were loaded and separated by 10 % SDS-PAGE and 5
transferred to a nitrocellulose membrane (Millipore). After blocking with 5 % nonfat dry milk, the membranes were subsequently incubated with gentle shaking overnight at 4 °C with primary antibodies against APP (1:500), sAPPα (2 μg/ml), sAPPβ (2 μg/ml), APP-CTF (1:500), BACE1 (1:500), PPARγ (1:150), and GAPDH (1:3000) in PBST containing 5 % nonfat dry milk. These membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature and then washed four times with PBST. The membranes were scanned with the Odyssey 9120 (LI-COR, Inc.). The bands were analyzed with the Odyssey software (LI-COR, Inc.). 2.6. RNA preparation and real-time RT-PCR Total RNA was extracted from the N2a/APP695 and N2a/WT cells using the RNA Isolation Kit (TIANGEN BIOTECH, Beijing, China). The quantity of the total RNA was measured with the Nanodrop 2000 spectrophotometer (Thermo, USA). The total RNA (2 μg) was reversed transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Molecular Biology, Waltham, MA, USA). PPARγ, BACE1 and GAPDH expression was examined by real-time PCR using SYBR Premix Ex TaqTM (Perfect Real-Time; Takara Biotechnology Co., Ltd.). The primers were 5'-AGAACCTGCATCTCCACCTTAT-3' (PPARγ, sense), 5'-CCACAGACTCGGCACTCAAT-3' (PPARγ, antisense), 5'-ACCTATGCGATGCGAATGTT-3' (BACE1, sense), 5'-AGATGGGCTTCTGTCTTGGAG-3' (BACE1, antisense), 5'- GGTTGTCTCCTGCGACTTCA-3’ (GAPDH, sense), and 5'-TGGTCCAGGGTTTCTTACTCC-3' (GAPDH, antisense). Dissociation curve analysis of the target gene showed a single peak. The real-time PCR conditions were as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The experiments were performed in triplicate. The relative quantification of gene expression was calculated using the 2-ΔΔCt method. 6
2.7. β-secretase activity assay β-secretase activity was measured using a commercial kit from BioVision following the manufacturer’s protocol. Briefly, the cells were harvested and centrifuged. Then 0.1 ml of ice-cold Extraction Buffer was added. The cell lysates were incubated on ice for 10 min and centrifuged at 10,000 ×g for 5 min. The supernatant was transferred to a new tube and kept on ice. The protein concentration was determined by the BCA method. Next, 50 μl of the 2×reaction buffer and 2 μl of the β-secretase substrate were added to 50 μl of the supernatant, and the sample was incubated in the dark at 37 °C for 1 h. Fluorescence was recorded using a microplate reader (Biotek FLx800, USA). 2.8. Statistical Analysis The data were analyzed with the SPSS (version 20.0) software and shown as the mean±S.E.M. of at least three independent experiments. Significant differences between values were determined by ANOVA followed by the post hoc LSD test. The significance level was accepted as P < 0.05. 3. Results 3.1. Effect of Ginsenoside Re on the Survival of the N2a/APP695 Cells To prevent ginsenoside Re from having a cytotoxic effect on the N2a/APP695 cells, the cell viability was first determined by the MTT assay (Fig. 1 B and C). The N2a/WT and N2a/APP695 cells were treated with increasing concentrations of ginsenoside Re (0-200 μM) for 24 h. Ginsenoside Re concentrations under 100 μM did not affect the viability of the N2a/WT and N2a/APP695 cells, whereas the 150 μM ginsenoside Re concentration markedly decreased the survival rate of the N2a/WT and N2a/APP695 cells. Incubation with ginsenoside Re at a 200 μM concentration for 24 h reduced the viability of the N2a/WT and N2a/APP695
7
cells by 15.58 % and 26.82 %, respectively. These data indicate that ginsenoside Re treatment within the range of 0-100 μM for 24 h is safe for the N2a/WT and N2a/APP695 cells (P > 0.05). 3.2. Ginsenoside Re Attenuates Aβ1-40 and Aβ1-42 Secretion from the N2a/APP695 Cells To confirm the effect of ginsenoside Re on Aβ production, we treated the N2a/APP695 cells with ginsenoside Re (0-100 µM) for 24 h. The Aβ1-40 and Aβ1-42 levels in the cell medium were measured by ELISA. The levels of secreted Aβ1-40 and Aβ1-42 in the conditioned medium of N2a/WT cells were undetectable (data not shown) . As shown in Fig.2A and B, ginsenoside Re decreased the extracellular Aβ1-40 and Aβ1-42 levels in a dose-dependent manner in the 25-100 μM range. Ginsenoside Re at the 50 μM concentration significantly decreased the Aβ1-40 and Aβ1-42 levels (P < 0.05). Ginsenoside Re at the 100 μM concentration decreased the Aβ1-40 level to 81.35 % of the basal level (P < 0.05) and the Aβ1-42 level to 66.64 % of the basal level (P < 0.01). The results indicate that Aβ1-40 and Aβ1-42 secretions were suppressed by ginsenoside Re. 3.3. Effect of Ginsenoside Re on APP Expression in the N2a/APP695 Cells The β-amyloid precursor protein (APP) is cleaved by β-secretase and γ-secretase to form Aβ. To confirm whether ginsenoside Re affected Aβ by affecting the APP protein level, the APP protein level was determined by the western blotting method (Fig. 3). The result showed that the APP protein level in the N2a/APP695 cells was higher than the level in the N2a/WT cells (P < 0.001). Ginsenoside Re (25-100 μM) treatment did not change the APP protein level (P > 0.05) compared with the N2a/APP695 control (0 μM) group. The result showed that ginsenoside Re did not decrease APP protein expression in the N2a/APP695 cells. 3.4. Effect of Ginsenoside Re on sAPPα , sAPPβ and C99 Expression in the N2a/APP695 Cells 8
APP processing involves two pathways: the non-amyloidogenic and the amyloidogenic pathways. In the non-amyloidogenic pathway, APP is cleaved within the Aβ domain by α-secretase to generate the soluble form of APP (sAPPα) and p3, thereby preventing the formation of Aβ (Mousavi and Hellstrom-Lindahl, 2009). In the amyloidogenic pathway, APP is cleaved by β-secretase and γ-secretase. β-secretase processing of APP generates secreted APP-β (sAPPβ) and a β-C-terminal fragment (β-CTF) that is further cleaved by γ-secretase to generate Aβ (Tramutola et al., 2016). First, we investigated whether ginsenoside Re regulated the activity of α-secretase by determining the sAPPα protein level in the N2a/APP695 cells. As shown in Fig. 4A, the sAPPα protein level due to APP cleavage by α-secretase in the N2a/WT cells was lower than the protein level in the N2a/APP695 cells. Ginsenoside Re (25-100 μM) treatment did not change the sAPPα protein level compared with the N2a/APP695 control (0 μM) group (P > 0.05). Second, we investigated whether ginsenoside Re could regulate the β-secretase activity by determining the sAPPβ and β-CTF (C99) level in the N2a/APP695 cells. As shown in Fig. 4 B and C, the sAPPβ and C99 protein level in the N2a/APP695 cells was higher than the protein level in the N2a/WT cells but was noticeably reduced by treatment with ginsenoside Re (50-100 μM) for 24 h. These results indicate that ginsenoside Re reduce Aβ production through the amyloidogenic pathways. 3.5. Effect of Ginsenoside Re on BACE1 Expression in the N2a/APP695 Cells Based on the above results, we found that ginsenoside Re (50-100 μM) treatment for 24 h significantly changed the sAPPβ expression level. BACE1 controls the first and rate-limiting step of Aβ formation from sAPPβ. Thus, we investigated the anti-BACE1 action of ginsenoside Re in the N2a/APP695 cells. As shown in Fig. 5A and B, the western blotting and real-time PCR analyses revealed that the BACE1 protein and mRNA levels in the N2a/APP695 cells were higher than the levels in the N2a/WT cells (P < 0.001). 9
Ginsenoside Re in the range of 25-100 μM decreased BACE1 protein and mRNA expression in a dose-dependent manner. Ginsenoside Re at the 100 μM concentration significantly decreased the BACE1 protein level (P < 0.01). Additionally, ginsenoside Re (50-100 μM) significantly decreased BACE1 mRNA expression in the N2a/APP695 cells (P < 0.001). Inhibiting the activity of β-secretase is also very important. As shown in Fig. 5C, the β-secretase activity in the N2a/WT cells was significantly decreased relative to the activity in the N2a/APP695 cells. The β-secretase activity in the ginsenoside Re (25-100 μM) treated groups was also significantly decreased. 3.6 Effect of Ginsenoside Re on PPARγ Expression in the N2a/APP695 Cells PPARγ is a transcription factor that is present in the BACE1 promoter and reduces BACE1 activity. Ginsenoside Re reduces insulin resistance through activation of PPARγ. To determine whether ginsenoside Re suppresses BACE1 expression and inhibits Aβ production through PPARγ activity, we determined the PPARγ protein and mRNA levels in the N2a/WT and N2a/APP695 cells. As shown in Fig. 6A and B, the PPARγ protein and mRNA levels were not changed in the N2a/APP695 control (0 μM) group compared to the N2a/WT cells. Ginsenoside Re (50-100 μM) significantly increased the PPARγ protein level (P < 0.05). Ginsenoside Re (50-100 μM) also significantly increased PPARγ mRNA expression (P < 0.01, and P < 0.001, respectively). These data demonstrated that ginsenoside Re promoted PPARγ protein and mRNA expression in the N2a/APP695 cells. 3.7. Ginsenoside Re Modulates BACE1 Activity via PPARγ To determine whether PPARγ was involved in the anti-BACE1 effects of ginsenoside Re, the cells were exposed to the PPARγ agonist pioglitazone and antagonist GW9662. We compared different concentrations of ginsenoside Re and the PPARγ agonist pioglitazone (10 μM) affected BACE1 protein and mRNA 10
expression and activity. Ginsenoside Re (100 μM) and pioglitazone (10 μM) significantly decreased the BACE1 protein level (P < 0.01) (Fig. 6A). Ginsenoside Re (50-100 μM) and pioglitazone (10 μM) significantly decreased BACE1 mRNA expression and activity (P < 0.001) (Fig. 7B and C). Furthermore, we determined the involvement of PPARγ in the inhibition of BACE1 in the N2a/APP695 cells. The cells were treated with 30 μM GW9662 for 1 h prior to incubation with ginsenoside Re. As shown in Fig. D and E, ginsenoside Re did not decrease the BACE1 protein and mRNA levels. Pretreatment with GW9662 significantly blocked the effect of ginsenoside Re on the BACE1 activity and the sAPPβ protein level in the N2a/APP695 cells (Fig. 7F and G). These data demonstrated that ginsenoside Re inhibited BACE1 transcription, translation and activity through PPARγ activation. 4. Discussion Alzheimer’s disease (AD) is an irreversible neurodegenerative brain disorder and is the most common cause of dementia that affects the elderly (Tramutola et al., 2016). Aβ deposits are involved in Alzheimer's disease like memory impairment; thus, reducing the amount of Aβ might be a potential therapeutic strategy for AD (Song et al., 2013). Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP). APP is processed through two pathways (non-amyloidogenic and amyloidogenic). In the non-amyloidogenic pathway, APP is cleaved to generate sAPPα and p3, thereby preventing the formation of Aβ. In the amyloidogenic pathway, APP cleavage generates sAPPβ and the Aβ peptide (Sathya et al., 2012). Thus, BACE1 gene expression plays an essential role in regulating the APP processing pathways. PPARγ is a transcription factor that regulates BACE1 gene expression (Sastre et al., 2006). The thiazolidinedione (TZD) PPARγ agonists, including rosiglitazone and pioglitazone, are widely used for the treatment of type 2 diabetes and have been tested as potential treatments for AD (Escribano et al., 11
2010; Gupta and Gupta, 2012; Pedersen et al., 2006; Toba et al., 2016). However, the thiazolidinediones have severe adverse effects, which has led to their withdrawal from the market or restricted their clinical applications. Thus, TZDs may not be suitable for the treatment of AD. Natural products have been recognized as sources of new lead compounds for the treatment of various diseases, including AD (Williams et al., 2011). Panax notoginseng (Burk.) F.H. Chen (P. notoginseng), which is known as Sanqi or Tianqi in East Asian countries, is one of the primary herbs in traditional Chinese medicine (Wang et al., 2016b). This herb has been used to treat a wide array of diseases, such as cerebrovascular disorders (He et al., 2015), cancer (Hsieh et al., 2016), and neurodegenerative diseases (Cho, 2012). P. notoginseng saponins (PNS) are the most important active ingredients. Previous studies have shown the beneficial effect of PNS on AD (Huang et al., 2012; Zhong et al., 2005). Ginsenoside Re, which is an important component of PNS, also plays a protective role in AD in vivo and in vitro. Ginsenoside Re attenuates β-amyloid and serum-free induced neurotoxicity in PC12 cells (Ji et al., 2006) and decreases the Aβ1-40 and Aβl-42 levels in the brains of Tg2576 mice (Chen, 2006). Interestingly, the notoginsenoside R1 and the ginsenosides Rg1 and Re belong to the 20(S)-protopanaxatriol-type. The ppt-type ginsenosides have a hydroxyl group at C-3 and sugar moieties at C-6 and/or C-20. A previous study showed that ginsenoside Rg1 and notoginsenoside R1 significantly upregulated PPARγ mRNA and protein expression in N2a/APP695 cells (Chen et al., 2012; Li et al., 2015). Additionally, research confirmed that ginsenoside Re enhanced PPARγ mRNA expression in 3T3-L1 cells. Thus, we speculated that the decrease in Aβ levels in response to ginsenoside Re might be related to PPARγ. In the present study, we investigated the mechanism underlying the ginsenoside Re induced decrease in 12
Aβ levels in N2a/APP695 cells. Based on our results, we drew three main conclusions. First, evaluation of the Aβ1-40 and Aβ1-42 levels demonstrated that ginsenoside Re remarkably decreased the Aβ levels in N2a/APP695 cells (Fig. 2). Second, we confirmed that ginsenoside Re decreased Aβ via the amyloidogenic pathway. The APP, sAPPα, sAPPβ, C99 and BACE1 protein levels and the BACE1 activity and mRNA level were evaluated. Ginsenoside Re did not change the APP and sAPPα protein levels but significantly decreased the sAPPβ, C99 and BACE1 protein levels and BACE1 activity and mRNA level (Figs. 3-5). Third, we confirmed that PPARγ was involved in the decrease in Aβ induced by ginsenoside Re. Ginsenoside Re treatment increased PPARγ protein and mRNA expression in the N2a/APP695 cells. Furthermore, we found that blocking PPARγ using GW9662 reversed the effects of ginsenoside Re decreased the BACE1 protein level, activity and mRNA level. The above evidence demonstrates that ginsenoside Re decreases Aβ through a mechanism that is dependent on PPARγ activation.
However, our study has two limitations. First, we use western blot method to detect the protein level. This method is a semi-quantitative method. The experimental results have certain error. So in Fig 5 A, N2a/WT has around 60% of the expression of BACE1 comparing to N2a/APP695, while in Fig 7 A around 80%. The similar phenomenon was congruent with previous study (Lin et al., 2015). About the change range of BACE1 protein and activity in N2a/APP695 cells and N2a/WT cells, similar study results shown that the change range is different (Li et al., 2010). The phenomenon may be caused by the error of the test method. Second, PPARγ is a transcription factor that regulates BACE1 gene expression. However, other transcription factors are also involved in the regulation of BACE1 gene expression, including NF-κB and Sp1. Therefore, we cannot rule out the possibility that ginsenoside Re may regulate the activity of other transcription factors that affect BACE1 gene promoter activity. This possibility may represent another 13
mechanism through which ginsenoside Re suppresses Aβ generation. Furthermore, it is not clear how Re regulates PPARγ expression. These issues will be explored in future experiments. In summary, our results clearly demonstrated that ginsenoside Re significantly attenuated Aβ generation in N2a/APP695 cells. The effect of ginsenoside Re on Aβ generation was mediated by PPARγ activation and BACE1 inhibition. Our study suggests that ginsenoside Re may be a therapeutic agent for Alzheimer's disease.
Conflict of interests
The authors confirm that this article content has no conflict of interest.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No.81274118), the Key New Drug Creation and Development Program of China (No.2012ZX09103-201) and the Fundamental Research Funds for the Central Universities.
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R1 on an APP/PS1 Mouse Model of Alzheimer's Disease by Up-Regulating Insulin Degrading Enzyme and Inhibiting Abeta Accumulation. CNS Neurol Disord Drug Targets 14, 360-369. Lin, N., Chen, L., Pan, X., Zhu, Y., Zhang, J., Shi, Y., Chen, X., 2015. Tripchlorolide Attenuates β-amyloid Generation via Suppressing PPARγ-Regulated BACE1 Activity in N2a/APP695 Cells. MOL NEUROBIOL. Lin, N., Chen, L.M., Pan, X.D., Zhu, Y.G., Zhang, J., Shi, Y.Q., Chen, X.C., 2015. Tripchlorolide Attenuates beta-amyloid Generation via Suppressing PPARgamma-Regulated BACE1 Activity in N2a/APP695 Cells. MOL NEUROBIOL. Liu, J., Wang, Y., Qiu, L., Yu, Y., Wang, C., 2014. Saponins of Panax notoginseng: chemistry, cellular targets and therapeutic opportunities in cardiovascular diseases. Expert Opin Investig Drugs 23, 523-539. Mandrekar-Colucci, S., Karlo, J.C., Landreth, G.E., 2012. Mechanisms underlying the rapid peroxisome proliferator-activated receptor-gamma-mediated amyloid clearance and reversal of cognitive deficits in a murine model of Alzheimer's disease. J NEUROSCI 32, 10117-10128. Mandrekar-Colucci, S., Landreth, G.E., 2011. Nuclear receptors as therapeutic targets for Alzheimer's disease. Expert Opin Ther Targets 15, 1085-1097. Mousavi, M., Hellstrom-Lindahl, E., 2009. Nicotinic receptor agonists and antagonists increase sAPPalpha secretion and decrease Abeta levels in vitro. NEUROCHEM INT 54, 237-244. Ohno, M., 2016. Alzheimer’s therapy targeting the β-secretase enzyme BACE1: Benefits and potential limitations from the perspective of animal model studies. BRAIN RES BULL. Pedersen, W.A., McMillan, P.J., Kulstad, J.J., Leverenz, J.B., Craft, S., Haynatzki, G.R., 2006. Rosiglitazone attenuates learning and memory deficits in Tg2576 Alzheimer mice. EXP NEUROL 199, 17
265-273. Pimplikar, S.W., 2009. Reassessing the amyloid cascade hypothesis of Alzheimer's disease. Int J Biochem Cell Biol 41, 1261-1268. Quan, Q., Wang, J., Li, X., Wang, Y., 2013. Ginsenoside Rg1 decreases Abeta(1-42) level by upregulating PPARgamma and IDE expression in the hippocampus of a rat model of Alzheimer's disease. PLOS ONE 8, e59155. Sastre, M., Dewachter, I., Rossner, S., Bogdanovic, N., Rosen, E., Borghgraef, P., Evert, B.O., Dumitrescu-Ozimek, L., Thal, D.R., Landreth, G., Walter, J., Klockgether, T., van Leuven, F., Heneka, M.T., 2006. Nonsteroidal anti-inflammatory drugs repress beta-secretase gene promoter activity by the activation of PPARgamma. Proc Natl Acad Sci U S A 103, 443-448. Sathya, M., Premkumar, P., Karthick, C., Moorthi, P., Jayachandran, K.S., Anusuyadevi, M., 2012. BACE1 in Alzheimer's disease. CLIN CHIM ACTA 414, 171-178. Song, X., Hu, J., Chu, S., Zhang, Z., Xu, S., Yuan, Y., Han, N., Liu, Y., Niu, F., He, X., Chen, N., 2013. Ginsenoside Rg1 attenuates okadaic acid induced spatial memory impairment by the GSK3β/tau signaling pathway and the Aβ formation prevention in rats. EUR J PHARMACOL 710, 29-38. Toba, J., Nikkuni, M., Ishizeki, M., Yoshii, A., Watamura, N., Inoue, T., Ohshima, T., 2016. PPARγ agonist pioglitazone improves cerebellar dysfunction at pre-Aβ deposition stage in APPswe/PS1dE9 Alzheimer's disease model mice. BIOCHEM BIOPH RES CO 473, 1039-1044. Tramutola, A., Lanzillotta, C., Perluigi, M., Butterfield, D.A., 2016. Oxidative Stress, Protein Modification and Alzheimer Disease. BRAIN RES BULL. Wang, L., Waltenberger, B., Pferschy-Wenzig, E.M., Blunder, M., Liu, X., Malainer, C., Blazevic, T., 18
Schwaiger, S., Rollinger, J.M., Heiss, E.H., Schuster, D., Kopp, B., Bauer, R., Stuppner, H., Dirsch, V.M., Atanasov, A.G., 2014. Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARgamma): a review. BIOCHEM PHARMACOL 92, 73-89. Wang, T., Guo, R., Zhou, G., Zhou, X., Kou, Z., Sui, F., Li, C., Tang, L., Wang, Z., 2016a. Traditional uses, botany, phytochemistry, pharmacology and toxicology of Panax notoginseng (Burk.) F.H. Chen: A review. J ETHNOPHARMACOL 188, 234-258. Wang, T., Guo, R., Zhou, G., Zhou, X., Kou, Z., Sui, F., Li, C., Tang, L., Wang, Z., 2016b. Traditional uses, botany, phytochemistry, pharmacology and toxicology of Panax notoginseng (Burk.) F.H. Chen: A review. J ETHNOPHARMACOL 188, 234-258. Wang, X., Wang, Y., Hu, J.P., Yu, S., Li, B.K., Cui, Y., Ren, L., Zhang, L.D., 2016. Astragaloside IV, a Natural PPARgamma Agonist, Reduces Abeta Production in Alzheimer's Disease Through Inhibition of BACE1. MOL NEUROBIOL. Williams, P., Sorribas, A., Howes, M.J., 2011. Natural products as a source of Alzheimer's drug leads. NAT PROD REP 28, 48-77. Zhong, Z., Qu, Z., Wang, N., Wang, J., Xie, Z., Zhang, F., Zhang, W., Lu, Z., 2005. Protective effects of Panax notoginseng saponins against pathological lesion of cholinergic neuron in rat model with Alzheimer' s disease. Zhong Yao Cai 28, 119-122.
Figure legends˖ Fig. 1. Effects of ginsenoside Re on the viability of N2a/WT and N2a/APP695 cells. (A) The molecular structure of ginsenoside Re. (B and C) Cell viability was assessed by the MTT reduction assay. As shown,
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ginsenoside Re is safe for cells in the 0-100 μM range. Values are expressed as the mean±S.E.M. of three independent experiments. *P < 0.05, **P < 0.01 compared with the control group.
Fig. 2. Ginsenoside Re reduces the Aβ1-40 and Aβ1-42 levels in N2a/APP695 cells. The results showed that ginsenoside Re attenuated the release of Aβ 1-40 and Aβ 1-42 in a dose-dependent manner. Ginsenoside Re at the 50-100 μM doses significantly reduced the Aβ 1-40 and Aβ 1-42 levels. The data are expressed as the mean±S.E.M. n=3. *P < 0.05 and ** P < 0.01 compared with the N2a/APP695 control (0 μM) group.
Fig. 3. Effect of ginsenoside Re on the APP protein level in N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. The APP protein level was measured by western blotting. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P<0.01 and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 4. Effect of ginsenoside Re on the sAPPα, sAPPβ and C99 protein levels in the N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. (A , B, C) The sAPPα, sAPPβ and C99 protein levels were measured by western blotting. The data are expressed as the mean±S.E.M. n=3. *P < 0.05 , **P < 0.01 and ***P < 0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 5. Effect of ginsenoside Re on the BACE1 mRNA and protein levels and activity in N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. (A) The 20
BACE1 protein level was measured by western blotting. (B) BACE1 mRNA expression was measured by real-time PCR. (C) β-secretase activity was measured using a microplate reader. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P <0.01, and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 6. Effect of ginsenoside Re on the PPARγ protein and mRNA expression levels in N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. (A) PPARγ protein expression was measured by western blotting. (B) PPARγ mRNA expression was measured by real-time PCR. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P <0.01, and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 7. Effect of ginsenoside Re on BACE1 through PPARγ in the N2a/WT and N2a/APP695 cells. The cells were exposed to the PPARγ agonist pioglitazone (10 μM) and antagonist, GW9662 (30 μM). (A-C) The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) and pioglitazone (10 μM) for 24 h. (D-G) The cells were treated with 30 μM GW9662 for 1 h before incubation with ginsenoside Re. (A, D and G) BACE1 and sAPPβ protein expression was measured by western blotting. (B and E) BACE1 mRNA expression was measured by real-time PCR. (C and F) β-secretase activity was measured using a microplate reader. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P <0.01, and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
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PII: DOI: Reference:
S0014-2999(16)30695-1 http://dx.doi.org/10.1016/j.ejphar.2016.11.006 EJP70911
To appear in: European Journal of Pharmacology Received date: 21 July 2016 Revised date: 23 October 2016 Accepted date: 3 November 2016 Cite this article as: Guoqiong Cao, Ping Su, Shuai Zhang, Limin Guo, Haijing Zhang, Yuexia Liang, Chunxia Qin and Wensheng Zhang, Ginsenoside Re reduces Aβ production by activating PPARγ to inhibit BACE1 in N2a/APP695 c e l l s , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page Title: Ginsenoside Re reduces Aβ production by activating PPARγ to inhibit BACE1 in N2a/APP695 cells Author names: Guoqiong Cao
a,b,c
, Ping Su
a,b,c
, Shuai Zhang
a,b,c
, Limin Guo
a,b,c
, Haijing Zhang
a,b,c
Yuexia Liang a,b,c, Chunxia Qin a,b,c, Wensheng Zhang a,b,c,d,* a
Beijing Area Major Laboratory of Protection and Utilization of Traditional Chinese Medicine, Beijing Normal University, Beijing 100088, China;
b
Engineering Research Center of Natural Medicine, Ministry of Education, Beijing Normal University,
Beijing 100088, China; c
College of Resources Science Technology, Beijing Normal University, Beijing 100875, China;
d
National & Local United Engineering Research Center for Sanqi Resources Protection and Utilization
Technology, Kunming 650000, China * corresponding author.
Corresponding author with complete address: Wensheng Zhang, Complete address: C Building, Beijing Normal University Science Park, No. 12, Xueyuan Southern Street, Haidian District, Beijing, P.R.China, 100088. Tele: +86 010 62200669. Email: [email protected].
Abstract Alzheimer’s disease (AD) is a neurodegenerative disease characterized by β-amyloid protein (Aβ) deposition. Reducing the Aβ load may be a new perspective for AD treatment. Ginsenoside Re is an extract from Panax notoginseng, which is a well-known traditional Chinese medicine that has been used for the treatment of various diseases for years. Ginsenoside Re has been reported to decrease Aβ in Alzheimer’s 1
,
disease animal models, but the mechanism has not been fully elucidated. In the present study, we investigated the mechanism of ginsenoside Re. Our results showed that ginsenoside Re decreased the Aβ levels in N2a/APP695 cells. Aβ peptides are generated by β-secretase (β-site amyloid precursor protein cleaving enzyme 1 (BACE1)) and γ-secretase. We found that ginsenoside Re decreased the BACE1 mRNA and protein levels and inhibited BACE1 activity in the N2a/APP695 cells. Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that regulates the activity of the BACE1 promoter, and activating PPARγ can inhibit BACE1. The results also showed that ginsenoside Re significantly increased the PPARγ protein and mRNA levels. These effects of ginsenoside Re on BACE1 could be effectively inhibited by the PPARγ antagonist GW9662. These findings indicate that ginsenoside Re inhibits BACE1 through activation of PPARγ, which ultimately reduces the generation of Aβ1-40 and Aβ1-42. Therefore, ginsenoside Re may be a promising agent for the modulation of Aβ-related pathology in AD. Key words: Ginsenoside Re, Alzheimer´s disease, Aβ, BACE1, PPARγ. 1. Introduction Alzheimer´s disease is a chronic neurodegenerative disorder that affects millions of elderly people worldwide. β-amyloid protein deposition is regarded as the pathological hallmark of AD. In the brain, Aβ accumulation can lead to neurofibrillary tangles, inflammation, axonal injury, and synapse loss (Pimplikar, 2009). Aβ peptides are generated by APP (amyloid-β precursor protein) via amyloidogenic pathways by β-secretase and γ-secretase. BACE1 is the initiating and rate-limiting enzyme in Aβ generation (Chen et al., 2012; Lin et al., 2015).Thus, BACE1 inhibition represents an important intervention that may prevent and/or cure AD (Ohno, 2016). 2
BACE1 gene transcription is regulated by many transcription factors, including NF-κB (Buggia-Prevot et al., 2008), Sp1 (Christensen et al., 2004), and PPARγ (Wang et al., 2016). Studies have shown that PPARγ regulates the expression of the genes involved in adipogenesis, lipid metabolism, inflammation, and the maintenance of metabolic homeostasis (Wang et al., 2014) as well as components of Aβ metabolism and toxicity (Mandrekar-Colucci et al., 2012; Mandrekar-Colucci and Landreth, 2011). The binding of the PPARγ response element (PPRE) to the promoter region of the BACE1 gene suppresses BACE1 expression and inhibits Aβ production (Lin et al., 2015). Panax notoginseng (Burk) F.H. Chen (Araliaceae) is a well-known herb that has been used in China for many years. Saponins are the major constituents of P. notoginseng and are also considered its main active compounds (Wang et al., 2016a). The five main saponins (ginsenosides Rb1, Rg1, Rd and Re and the notoginsenoside R1) constitute up to 90 % of the total P. notoginseng saponins (PNS) used in pharmacological experiments (Liu et al., 2014). Among them, ginsenosides Rb1 and Rd belong to the 20(S)-protopanaxadiol-type, whereas ginsenosides Rg1 and Re and notoginsenoside R1 belong to the 20(S)-protopanaxatriol-type (Li et al., 2015). Previous studies reported that ginsenoside Rg1 and notoginsenoside R1 decreased the Aβ level by upregulating PPARγ (Li et al., 2015; Quan et al., 2013). Ginsenoside Re, which also belongs to the 20(S)-protopanaxatriol type, exhibited many beneficial effects, such as anti-inflammatory (Lee et al., 2012), anti-oxidation (Huang et al., 2016), and neuroprotective effects (Chen et al., 2008). Ginsenoside Re activated PPARγ in 3T3-L1 adipocytes (Han et al., 2006) and reduced insulin resistance through activation of the PPARγ pathway (Gao et al., 2013). Additionally, ginsenoside Re reduced the Aβ1-40 and Aβ1-42 levels in the brains of Tg2576 mice (Chen, 2006). However,
3
whether ginsenoside Re reduces the Aβ level through the PPARγ pathway is unknown. Therefore, in this study, we investigated the effect of ginsenoside Re on BACE1 and Aβ through the promotion of PPARγ. 2. Materials and Methods 2.1. Chemicals and Regents Ginsenoside Re (purity > 98 %) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The molecular structure of ginsenoside Re is shown in Fig. 1A. DMSO and GW9662 were purchased from Sigma-Aldrich (St. Louis, MO, USA). For cell culture, penicillin, streptomycin, Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). G418 was ordered from Amresco (Solon, OH, USA). The Aβ1-40 and Aβ1-42 ELISA kits were purchased from Invitrogen (Carlsbad, CA, USA). The Beta-Secretase Activity Fluorometric assay kit was purchased from BioVision (San Francisco, USA). The primary antibodies used in this study were purchased as follows: APP (Merck, Germany), sAPPβ (IBL, Japan), sAPPα (IBL, Japan), APP-CTF (Calbiochem, USA), BACE1 (Abcam, USA), PPARγ (Santa Cruz CA, USA), and GAPDH (Cell Signaling Technology, USA). The PPARγ agonist pioglitazone was obtained from TaiYang Pharmaceutical Co., Ltd. (Beijing, China). 2.2. Cell Culture The N2a/APP695 cell line that stably expressed Swedish mutant human APP695 was constructed and stored by the Beijing Key Laboratory for the Protection and Utilization of Traditional Chinese Medicine Resources of Beijing Normal University. The cells were maintained in DMEM/Opti-MEM (1:1, v/v; containing 200 μg/ml of G418, 5 % FBS, 100 units/ml of penicillin, and 100 mg/ml of streptomycin) and
4
kept at 37 °C in a humidified 5 % CO2/95 % air environment. The cells were passaged every 3 days when they reached 80 % confluence. 2.3. Cell viability assay To explore the cytotoxic effect of ginsenoside Re on N2a/APP695 cells, cell proliferation was assessed using the MTT assay. The cells were treated with increasing doses of ginsenoside Re (0, 25, 50, 100, 150, and 200 μM) for 24 h. The MTT assay was performed after the treatments. The cells were incubated for 4 h at 37 °C with 0.5 mg/ml of MTT dissolved in fresh complete medium. The dark blue formazan crystals were dissolved in DMSO, and the absorbance was measured on a microplate reader (Thermo Multiskan MK3, USA) using a reference wavelength of 630 nm and a test wavelength of 490 nm. The data were expressed as the mean percentages of viable cells versus the control. 2.4. Aβ ELISA assay The cells were incubated with ginsenoside Re (0-100 μM) for 24 h. After treatment, conditioned media from the treated and untreated cells were collected to detect secreted Aβ1-40 and Aβ1-42. The Aβ1-40 and Aβ1-42 concentrations were quantified using ELISA kits following the manufacturer’s protocol. The optical densities of each well at 450 nm were readed on a Multiskan MK3 microplate reader (Thermo Labsystems, Waltham, MA, USA), and the sample Aβ1-40 and Aβ1-42 concentrations were determined by comparison with the Aβ1-40 and Aβ1-42 standard curves. All readings were in the linear range of the assay. 2.5. Western blotting analysis After treatment, the cells were washed with ice-cold PBS and then lysed in RIPA lysis buffer containing a cocktail of complete protease inhibitors (Roche Diagnostics). The protein concentrations were determined by the BCA method. Equal amounts of proteins were loaded and separated by 10 % SDS-PAGE and 5
transferred to a nitrocellulose membrane (Millipore). After blocking with 5 % nonfat dry milk, the membranes were subsequently incubated with gentle shaking overnight at 4 °C with primary antibodies against APP (1:500), sAPPα (2 μg/ml), sAPPβ (2 μg/ml), APP-CTF (1:500), BACE1 (1:500), PPARγ (1:150), and GAPDH (1:3000) in PBST containing 5 % nonfat dry milk. These membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature and then washed four times with PBST. The membranes were scanned with the Odyssey 9120 (LI-COR, Inc.). The bands were analyzed with the Odyssey software (LI-COR, Inc.). 2.6. RNA preparation and real-time RT-PCR Total RNA was extracted from the N2a/APP695 and N2a/WT cells using the RNA Isolation Kit (TIANGEN BIOTECH, Beijing, China). The quantity of the total RNA was measured with the Nanodrop 2000 spectrophotometer (Thermo, USA). The total RNA (2 μg) was reversed transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Molecular Biology, Waltham, MA, USA). PPARγ, BACE1 and GAPDH expression was examined by real-time PCR using SYBR Premix Ex TaqTM (Perfect Real-Time; Takara Biotechnology Co., Ltd.). The primers were 5'-AGAACCTGCATCTCCACCTTAT-3' (PPARγ, sense), 5'-CCACAGACTCGGCACTCAAT-3' (PPARγ, antisense), 5'-ACCTATGCGATGCGAATGTT-3' (BACE1, sense), 5'-AGATGGGCTTCTGTCTTGGAG-3' (BACE1, antisense), 5'- GGTTGTCTCCTGCGACTTCA-3’ (GAPDH, sense), and 5'-TGGTCCAGGGTTTCTTACTCC-3' (GAPDH, antisense). Dissociation curve analysis of the target gene showed a single peak. The real-time PCR conditions were as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The experiments were performed in triplicate. The relative quantification of gene expression was calculated using the 2-ΔΔCt method. 6
2.7. β-secretase activity assay β-secretase activity was measured using a commercial kit from BioVision following the manufacturer’s protocol. Briefly, the cells were harvested and centrifuged. Then 0.1 ml of ice-cold Extraction Buffer was added. The cell lysates were incubated on ice for 10 min and centrifuged at 10,000 ×g for 5 min. The supernatant was transferred to a new tube and kept on ice. The protein concentration was determined by the BCA method. Next, 50 μl of the 2×reaction buffer and 2 μl of the β-secretase substrate were added to 50 μl of the supernatant, and the sample was incubated in the dark at 37 °C for 1 h. Fluorescence was recorded using a microplate reader (Biotek FLx800, USA). 2.8. Statistical Analysis The data were analyzed with the SPSS (version 20.0) software and shown as the mean±S.E.M. of at least three independent experiments. Significant differences between values were determined by ANOVA followed by the post hoc LSD test. The significance level was accepted as P < 0.05. 3. Results 3.1. Effect of Ginsenoside Re on the Survival of the N2a/APP695 Cells To prevent ginsenoside Re from having a cytotoxic effect on the N2a/APP695 cells, the cell viability was first determined by the MTT assay (Fig. 1 B and C). The N2a/WT and N2a/APP695 cells were treated with increasing concentrations of ginsenoside Re (0-200 μM) for 24 h. Ginsenoside Re concentrations under 100 μM did not affect the viability of the N2a/WT and N2a/APP695 cells, whereas the 150 μM ginsenoside Re concentration markedly decreased the survival rate of the N2a/WT and N2a/APP695 cells. Incubation with ginsenoside Re at a 200 μM concentration for 24 h reduced the viability of the N2a/WT and N2a/APP695
7
cells by 15.58 % and 26.82 %, respectively. These data indicate that ginsenoside Re treatment within the range of 0-100 μM for 24 h is safe for the N2a/WT and N2a/APP695 cells (P > 0.05). 3.2. Ginsenoside Re Attenuates Aβ1-40 and Aβ1-42 Secretion from the N2a/APP695 Cells To confirm the effect of ginsenoside Re on Aβ production, we treated the N2a/APP695 cells with ginsenoside Re (0-100 µM) for 24 h. The Aβ1-40 and Aβ1-42 levels in the cell medium were measured by ELISA. The levels of secreted Aβ1-40 and Aβ1-42 in the conditioned medium of N2a/WT cells were undetectable (data not shown) . As shown in Fig.2A and B, ginsenoside Re decreased the extracellular Aβ1-40 and Aβ1-42 levels in a dose-dependent manner in the 25-100 μM range. Ginsenoside Re at the 50 μM concentration significantly decreased the Aβ1-40 and Aβ1-42 levels (P < 0.05). Ginsenoside Re at the 100 μM concentration decreased the Aβ1-40 level to 81.35 % of the basal level (P < 0.05) and the Aβ1-42 level to 66.64 % of the basal level (P < 0.01). The results indicate that Aβ1-40 and Aβ1-42 secretions were suppressed by ginsenoside Re. 3.3. Effect of Ginsenoside Re on APP Expression in the N2a/APP695 Cells The β-amyloid precursor protein (APP) is cleaved by β-secretase and γ-secretase to form Aβ. To confirm whether ginsenoside Re affected Aβ by affecting the APP protein level, the APP protein level was determined by the western blotting method (Fig. 3). The result showed that the APP protein level in the N2a/APP695 cells was higher than the level in the N2a/WT cells (P < 0.001). Ginsenoside Re (25-100 μM) treatment did not change the APP protein level (P > 0.05) compared with the N2a/APP695 control (0 μM) group. The result showed that ginsenoside Re did not decrease APP protein expression in the N2a/APP695 cells. 3.4. Effect of Ginsenoside Re on sAPPα , sAPPβ and C99 Expression in the N2a/APP695 Cells 8
APP processing involves two pathways: the non-amyloidogenic and the amyloidogenic pathways. In the non-amyloidogenic pathway, APP is cleaved within the Aβ domain by α-secretase to generate the soluble form of APP (sAPPα) and p3, thereby preventing the formation of Aβ (Mousavi and Hellstrom-Lindahl, 2009). In the amyloidogenic pathway, APP is cleaved by β-secretase and γ-secretase. β-secretase processing of APP generates secreted APP-β (sAPPβ) and a β-C-terminal fragment (β-CTF) that is further cleaved by γ-secretase to generate Aβ (Tramutola et al., 2016). First, we investigated whether ginsenoside Re regulated the activity of α-secretase by determining the sAPPα protein level in the N2a/APP695 cells. As shown in Fig. 4A, the sAPPα protein level due to APP cleavage by α-secretase in the N2a/WT cells was lower than the protein level in the N2a/APP695 cells. Ginsenoside Re (25-100 μM) treatment did not change the sAPPα protein level compared with the N2a/APP695 control (0 μM) group (P > 0.05). Second, we investigated whether ginsenoside Re could regulate the β-secretase activity by determining the sAPPβ and β-CTF (C99) level in the N2a/APP695 cells. As shown in Fig. 4 B and C, the sAPPβ and C99 protein level in the N2a/APP695 cells was higher than the protein level in the N2a/WT cells but was noticeably reduced by treatment with ginsenoside Re (50-100 μM) for 24 h. These results indicate that ginsenoside Re reduce Aβ production through the amyloidogenic pathways. 3.5. Effect of Ginsenoside Re on BACE1 Expression in the N2a/APP695 Cells Based on the above results, we found that ginsenoside Re (50-100 μM) treatment for 24 h significantly changed the sAPPβ expression level. BACE1 controls the first and rate-limiting step of Aβ formation from sAPPβ. Thus, we investigated the anti-BACE1 action of ginsenoside Re in the N2a/APP695 cells. As shown in Fig. 5A and B, the western blotting and real-time PCR analyses revealed that the BACE1 protein and mRNA levels in the N2a/APP695 cells were higher than the levels in the N2a/WT cells (P < 0.001). 9
Ginsenoside Re in the range of 25-100 μM decreased BACE1 protein and mRNA expression in a dose-dependent manner. Ginsenoside Re at the 100 μM concentration significantly decreased the BACE1 protein level (P < 0.01). Additionally, ginsenoside Re (50-100 μM) significantly decreased BACE1 mRNA expression in the N2a/APP695 cells (P < 0.001). Inhibiting the activity of β-secretase is also very important. As shown in Fig. 5C, the β-secretase activity in the N2a/WT cells was significantly decreased relative to the activity in the N2a/APP695 cells. The β-secretase activity in the ginsenoside Re (25-100 μM) treated groups was also significantly decreased. 3.6 Effect of Ginsenoside Re on PPARγ Expression in the N2a/APP695 Cells PPARγ is a transcription factor that is present in the BACE1 promoter and reduces BACE1 activity. Ginsenoside Re reduces insulin resistance through activation of PPARγ. To determine whether ginsenoside Re suppresses BACE1 expression and inhibits Aβ production through PPARγ activity, we determined the PPARγ protein and mRNA levels in the N2a/WT and N2a/APP695 cells. As shown in Fig. 6A and B, the PPARγ protein and mRNA levels were not changed in the N2a/APP695 control (0 μM) group compared to the N2a/WT cells. Ginsenoside Re (50-100 μM) significantly increased the PPARγ protein level (P < 0.05). Ginsenoside Re (50-100 μM) also significantly increased PPARγ mRNA expression (P < 0.01, and P < 0.001, respectively). These data demonstrated that ginsenoside Re promoted PPARγ protein and mRNA expression in the N2a/APP695 cells. 3.7. Ginsenoside Re Modulates BACE1 Activity via PPARγ To determine whether PPARγ was involved in the anti-BACE1 effects of ginsenoside Re, the cells were exposed to the PPARγ agonist pioglitazone and antagonist GW9662. We compared different concentrations of ginsenoside Re and the PPARγ agonist pioglitazone (10 μM) affected BACE1 protein and mRNA 10
expression and activity. Ginsenoside Re (100 μM) and pioglitazone (10 μM) significantly decreased the BACE1 protein level (P < 0.01) (Fig. 6A). Ginsenoside Re (50-100 μM) and pioglitazone (10 μM) significantly decreased BACE1 mRNA expression and activity (P < 0.001) (Fig. 7B and C). Furthermore, we determined the involvement of PPARγ in the inhibition of BACE1 in the N2a/APP695 cells. The cells were treated with 30 μM GW9662 for 1 h prior to incubation with ginsenoside Re. As shown in Fig. D and E, ginsenoside Re did not decrease the BACE1 protein and mRNA levels. Pretreatment with GW9662 significantly blocked the effect of ginsenoside Re on the BACE1 activity and the sAPPβ protein level in the N2a/APP695 cells (Fig. 7F and G). These data demonstrated that ginsenoside Re inhibited BACE1 transcription, translation and activity through PPARγ activation. 4. Discussion Alzheimer’s disease (AD) is an irreversible neurodegenerative brain disorder and is the most common cause of dementia that affects the elderly (Tramutola et al., 2016). Aβ deposits are involved in Alzheimer's disease like memory impairment; thus, reducing the amount of Aβ might be a potential therapeutic strategy for AD (Song et al., 2013). Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP). APP is processed through two pathways (non-amyloidogenic and amyloidogenic). In the non-amyloidogenic pathway, APP is cleaved to generate sAPPα and p3, thereby preventing the formation of Aβ. In the amyloidogenic pathway, APP cleavage generates sAPPβ and the Aβ peptide (Sathya et al., 2012). Thus, BACE1 gene expression plays an essential role in regulating the APP processing pathways. PPARγ is a transcription factor that regulates BACE1 gene expression (Sastre et al., 2006). The thiazolidinedione (TZD) PPARγ agonists, including rosiglitazone and pioglitazone, are widely used for the treatment of type 2 diabetes and have been tested as potential treatments for AD (Escribano et al., 11
2010; Gupta and Gupta, 2012; Pedersen et al., 2006; Toba et al., 2016). However, the thiazolidinediones have severe adverse effects, which has led to their withdrawal from the market or restricted their clinical applications. Thus, TZDs may not be suitable for the treatment of AD. Natural products have been recognized as sources of new lead compounds for the treatment of various diseases, including AD (Williams et al., 2011). Panax notoginseng (Burk.) F.H. Chen (P. notoginseng), which is known as Sanqi or Tianqi in East Asian countries, is one of the primary herbs in traditional Chinese medicine (Wang et al., 2016b). This herb has been used to treat a wide array of diseases, such as cerebrovascular disorders (He et al., 2015), cancer (Hsieh et al., 2016), and neurodegenerative diseases (Cho, 2012). P. notoginseng saponins (PNS) are the most important active ingredients. Previous studies have shown the beneficial effect of PNS on AD (Huang et al., 2012; Zhong et al., 2005). Ginsenoside Re, which is an important component of PNS, also plays a protective role in AD in vivo and in vitro. Ginsenoside Re attenuates β-amyloid and serum-free induced neurotoxicity in PC12 cells (Ji et al., 2006) and decreases the Aβ1-40 and Aβl-42 levels in the brains of Tg2576 mice (Chen, 2006). Interestingly, the notoginsenoside R1 and the ginsenosides Rg1 and Re belong to the 20(S)-protopanaxatriol-type. The ppt-type ginsenosides have a hydroxyl group at C-3 and sugar moieties at C-6 and/or C-20. A previous study showed that ginsenoside Rg1 and notoginsenoside R1 significantly upregulated PPARγ mRNA and protein expression in N2a/APP695 cells (Chen et al., 2012; Li et al., 2015). Additionally, research confirmed that ginsenoside Re enhanced PPARγ mRNA expression in 3T3-L1 cells. Thus, we speculated that the decrease in Aβ levels in response to ginsenoside Re might be related to PPARγ. In the present study, we investigated the mechanism underlying the ginsenoside Re induced decrease in 12
Aβ levels in N2a/APP695 cells. Based on our results, we drew three main conclusions. First, evaluation of the Aβ1-40 and Aβ1-42 levels demonstrated that ginsenoside Re remarkably decreased the Aβ levels in N2a/APP695 cells (Fig. 2). Second, we confirmed that ginsenoside Re decreased Aβ via the amyloidogenic pathway. The APP, sAPPα, sAPPβ, C99 and BACE1 protein levels and the BACE1 activity and mRNA level were evaluated. Ginsenoside Re did not change the APP and sAPPα protein levels but significantly decreased the sAPPβ, C99 and BACE1 protein levels and BACE1 activity and mRNA level (Figs. 3-5). Third, we confirmed that PPARγ was involved in the decrease in Aβ induced by ginsenoside Re. Ginsenoside Re treatment increased PPARγ protein and mRNA expression in the N2a/APP695 cells. Furthermore, we found that blocking PPARγ using GW9662 reversed the effects of ginsenoside Re decreased the BACE1 protein level, activity and mRNA level. The above evidence demonstrates that ginsenoside Re decreases Aβ through a mechanism that is dependent on PPARγ activation.
However, our study has two limitations. First, we use western blot method to detect the protein level. This method is a semi-quantitative method. The experimental results have certain error. So in Fig 5 A, N2a/WT has around 60% of the expression of BACE1 comparing to N2a/APP695, while in Fig 7 A around 80%. The similar phenomenon was congruent with previous study (Lin et al., 2015). About the change range of BACE1 protein and activity in N2a/APP695 cells and N2a/WT cells, similar study results shown that the change range is different (Li et al., 2010). The phenomenon may be caused by the error of the test method. Second, PPARγ is a transcription factor that regulates BACE1 gene expression. However, other transcription factors are also involved in the regulation of BACE1 gene expression, including NF-κB and Sp1. Therefore, we cannot rule out the possibility that ginsenoside Re may regulate the activity of other transcription factors that affect BACE1 gene promoter activity. This possibility may represent another 13
mechanism through which ginsenoside Re suppresses Aβ generation. Furthermore, it is not clear how Re regulates PPARγ expression. These issues will be explored in future experiments. In summary, our results clearly demonstrated that ginsenoside Re significantly attenuated Aβ generation in N2a/APP695 cells. The effect of ginsenoside Re on Aβ generation was mediated by PPARγ activation and BACE1 inhibition. Our study suggests that ginsenoside Re may be a therapeutic agent for Alzheimer's disease.
Conflict of interests
The authors confirm that this article content has no conflict of interest.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No.81274118), the Key New Drug Creation and Development Program of China (No.2012ZX09103-201) and the Fundamental Research Funds for the Central Universities.
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Figure legends˖ Fig. 1. Effects of ginsenoside Re on the viability of N2a/WT and N2a/APP695 cells. (A) The molecular structure of ginsenoside Re. (B and C) Cell viability was assessed by the MTT reduction assay. As shown,
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ginsenoside Re is safe for cells in the 0-100 μM range. Values are expressed as the mean±S.E.M. of three independent experiments. *P < 0.05, **P < 0.01 compared with the control group.
Fig. 2. Ginsenoside Re reduces the Aβ1-40 and Aβ1-42 levels in N2a/APP695 cells. The results showed that ginsenoside Re attenuated the release of Aβ 1-40 and Aβ 1-42 in a dose-dependent manner. Ginsenoside Re at the 50-100 μM doses significantly reduced the Aβ 1-40 and Aβ 1-42 levels. The data are expressed as the mean±S.E.M. n=3. *P < 0.05 and ** P < 0.01 compared with the N2a/APP695 control (0 μM) group.
Fig. 3. Effect of ginsenoside Re on the APP protein level in N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. The APP protein level was measured by western blotting. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P<0.01 and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 4. Effect of ginsenoside Re on the sAPPα, sAPPβ and C99 protein levels in the N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. (A , B, C) The sAPPα, sAPPβ and C99 protein levels were measured by western blotting. The data are expressed as the mean±S.E.M. n=3. *P < 0.05 , **P < 0.01 and ***P < 0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 5. Effect of ginsenoside Re on the BACE1 mRNA and protein levels and activity in N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. (A) The 20
BACE1 protein level was measured by western blotting. (B) BACE1 mRNA expression was measured by real-time PCR. (C) β-secretase activity was measured using a microplate reader. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P <0.01, and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 6. Effect of ginsenoside Re on the PPARγ protein and mRNA expression levels in N2a/WT and N2a/APP695 cells. The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) for 24 h. (A) PPARγ protein expression was measured by western blotting. (B) PPARγ mRNA expression was measured by real-time PCR. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P <0.01, and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
Fig. 7. Effect of ginsenoside Re on BACE1 through PPARγ in the N2a/WT and N2a/APP695 cells. The cells were exposed to the PPARγ agonist pioglitazone (10 μM) and antagonist, GW9662 (30 μM). (A-C) The cells were treated with ginsenoside Re (0, 25, 50 and 100 μM) and pioglitazone (10 μM) for 24 h. (D-G) The cells were treated with 30 μM GW9662 for 1 h before incubation with ginsenoside Re. (A, D and G) BACE1 and sAPPβ protein expression was measured by western blotting. (B and E) BACE1 mRNA expression was measured by real-time PCR. (C and F) β-secretase activity was measured using a microplate reader. The data are expressed as the mean±S.E.M. n=3. *P<0.05, **P <0.01, and ***P <0.001 compared with the N2a/APP695 control (0 μM) group.
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