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Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice
Neuroscience Letters 513 (2012) 72–77
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Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice Nobuaki Matsui ∗ , Yuki Kido, Hideki Okada, Miwa Kubo, Megumi Nakai, Nobuyuki Fukuishi, Yoshiyasu Fukuyama, Masaaki Akagi Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-bouji, Yamashiro-cho, Tokushima 770-8514, Japan
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Article history: Received 4 November 2011 Received in revised form 31 January 2012 Accepted 3 February 2012 Keywords: Olfactory bulbectomy Neurogenesis Neurotrophin Antidepressant Phenylbutenoid dimer Zingiber purpureum
a b s t r a c t Trans-3-(3 4 -dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3 4 -dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 (10–30 M) or Comp.2 (30 M) significantly induced neurite sprouting in PC12 cells. Comp.1 (0.03–3 M) or Comp.2 (0.3–3 M) significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 (30 M) and Comp.2 (3–30 M) also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1 (50 mg/kg p.o.), Comp.2 (50 mg/kg p.o.), or fluoxetine (10 mg/kg i.p.), an antidepressant, were administrated once a day on days 15–28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2 -deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia. © 2012 Elsevier Ireland Ltd. All rights reserved.
Neurotrophic factors, such as nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF), promote neurogenesis, neurodifferentiation, neuroprotection, and neuroplasticity [16]. Therefore, the therapeutic use of neurotrophic factors has been suggested for modification of neurodegenerative diseases [4,14,22]. More recently, it has been
Abbreviations: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; AD, Alzheimer’s disease; FGF-2, fibroblast growth factor-2; OBX, olfactory bulbectomy; NT-3, neurotrophin-3; HPLC, highperformance liquid chromatography; NMR, nuclear magnetic resonance; DMEM, Dulbecco’s modified Eagle’s medium; HS, horse serum; FBS, fetal bovine serum; PFA, paraformaldehyde; PBS, phosphate buffered saline; TBS, Tris-buffered saline; MAP2, microtubule-associated protein 2; HPLC/MS, high-performance liquid chromatography/mass spectrometry; BrdU, 5-bromo-2 -deoxyuridine; DG, dentate gyrus; SGZ, subgranular zone; SD, Sprague-Dawley; S.E.M., standard error of the mean; ANOVA, analysis of variance; SAM, senescence-accelerated mouse; A, amyloid-; CMC, carboxymethylcellulose. ∗ Corresponding author. Tel.: +81 88 602 8470; fax: +81 88 655 3051. E-mail address: [email protected] (N. Matsui). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.010
found that endogenous upregulation of BDNF contributes to the behavioral effect of antidepressants through enhancement of adult hippocampal neurogenesis, and it is thought that exogenous neurotrophins may ameliorate depression [2], as has been shown in many other studies [11,21]. Intracerebroventricular application of fibroblast growth factor-2 (FGF-2) ameliorates the reduction in hippocampal neurogenesis and reverses depressive behavior induced by olfactory bulbectomy (OBX) in mice, an experimental depression model [11]. Intrahippocampal infusion of neuropeptide VGF (non-acronymic) increases hippocampal neurogenesis and shows antidepressant-like effects on behavior in rats. In addition, intrahippocampal infusion of BDNF and neurotrophin-3 (NT-3) produces an antidepressant-like behavioral effect in rats [21]. However, since these neurotrophic factors are high molecular weight polypeptides, they cannot cross the blood–brain barrier. Therefore, these factors must be applied intracerebrally, and cannot be administered orally because they are disintegrated by gastric acid and proteases. Small molecular weight compounds with neurotrophic effects may solve this problem. Thus, efforts have been made to discover
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and synthesize small molecular weight compounds that can directly maintain neuronal function or upregulate neurotrophic factors [5,13,17,23]. For example, magnolol and honokiol, which are phenylpropanoid dimers from the stem bark of Magnolia obovata, promote neurite outgrowth and enhance neuronal survival in rat cortical neurons, and prevent age-related cholinergic deficits in the forebrain, and learning and memory impairment in senescence-accelerated mice (SAM) P8, an AD mouse model [5,15]. Nobiletin, a flavonoid found in Citrus depressa, induces neurite outgrowth in PC12D cells, and improves memory impairment in a transgenic mouse model of AD and OBX mice [17–19]. Moreover, we have found two phenylbutenoid dimers, trans-3-(3 4 dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene and cis-3-(3 4 -dimethoxyphenyl)-4-[(E)-3 ,4 (Comp.1) dimethoxystyryl]cyclohex-1-ene (Comp.2), from BANGLE (Zingiber purpureum), an Indonesian medicinal plant, which may have effects similar to other small molecular weight compounds with neurotrophic activity. Comp.1 has been reported to inhibit cell proliferation in various human cancer cells [6], but its neurotrophic activity has not been reported. In the present study, we describe the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. To assess the in vitro effects of both compounds, we examined neuritogenesis, neurite outgrowth, and neuroprotective activity in PC12 cells and primary cultured rat cortical neurons. The in vivo effects of both compounds were evaluated on hippocampal neurogenesis in OBX mice, an experimental depression and dementia animal model [7,8,10,11,18]. Comp.1 and Comp.2 (Fig. 1A) were isolated from methanol extracts of the root of BANGLE (Z. purpureum) using a combination of silica gel chromatography. Comp.1 and Comp.2 were purified by reverse-phase high-performance liquid chromatography (HPLC) using methanol–water (4:1) (Fig. 1B). The purity of Comp.1 (>99%) or Comp.2 (>99%) was determined by nuclear magnetic resonance (NMR) and HPLC. Fluoxetine hydrochloride was purchased from LKT Laboratories (St. Paul, MN, USA). Assays for the neuritogenic effects in PC12 cells were performed according to previously described methods [9]. The PC12 cell line (RCB0009) was obtained from Cell Bank, RIKEN BioResource Center (Tokyo, Japan). PC12 cells were cultured at a density of 2000 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM)/10% horse serum (HS) and 5% fetal bovine serum (FBS) for 24 h, and then the medium was changed to DMEM/2% HS, 1% FBS containing test samples. After being cultured for a further 96 h, neuritogenesis of PC12 cells were quantified. The cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 20 min, and washed with 0.1 M borate buffer and stained with 0.1% methylene blue. Ten photographs per well were obtained from three wells for each group. Neurites were defined as processes with a length longer than the cell diameter. The percentage of cells forming neurites was calculated. Assays for neurite outgrowth and neuroprotective activity in primary cultured neurons were performed according to previously described methods [5,23]. The neuronal cells were separated from the cerebral hemispheres of 18-day fetal Sprague-Dawley (SD) rats obtained from Japan SLC (Hamamatsu, Japan). After isolated cortical neurons had been cultured in poly-l-lysine-coated plates at a density of 5000 cells/cm2 for 24 h, the culture medium was changed from DMEM/10% FBS to NeurobasalTM medium/2% B27 supplement with test samples. After further culture for 6 days, the neurons were fixed with 4% PFA in PBS for 20 min, and soaked in 0.3% H2 O2 for 20 min. The cell membranes were permeabilized with 0.1% Triton X-100 in PBS for 20 min. For anti-microtubuleassociated protein 2 (MAP2) staining, the neurons were incubated with an anti-MAP2 antibody (Sternberger Monoclonals, Inc., Baltimore, MD, USA) overnight at 4 ◦ C followed by incubation with
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SimpleStain PO, a peroxidase-conjugated secondary antibody (Nichirei, Tokyo, Japan), for 1 h, and then peroxidase was developed with dimethylaminoazobenzene solution. Ten photographs per well were obtained from three wells for each group. At most five well-stained neurons that made connections to no more than two cells were selected for measurements of their primary neurite length and neurite number in each photograph. Neuronal viability was determined with the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay as previously described [23]. Briefly, isolated cortical neurons were cultured in poly-l-lysine-coated 96-well plates at a density of 2 × 105 cells/cm2 for 24 h, then the culture medium was changed to DMEM/1% N-2 supplement with various concentrations of test samples. After 48 h of further culture, MTT (0.5 mg/mL in PBS) was added to the culture medium (MTT:medium at 1:10). After a 2-h incubation, the medium was pipetted off, and 50% ethanol/50% dimethyl sulfoxide was used to dissolve the MTT formazan. Then, absorbance was measured at 570 nm and translated into neuronal viability. A mean value of six wells was used for each group. For experiments in vivo, male ddY mice (6 weeks old, n = 71, Japan SLC, Hamamatsu, Japan) were used. Mice were housed in a conventional animal facility with a 12/12 light/dark cycle (light from 08:00 to 20:00) and were kept in an air-conditioned room maintained at 23 ± 1◦ C with humidity at 55 ± 5%. Food and water were given ad libitum. All animal experiments were carried out under approved guidelines provided by the animal use committee at Tokushima Bunri University in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. For determination of pharmacokinetics, Comp.1 (50 mg/kg) or Comp.2 (50 mg/kg) was orally administered to the mice. Comp.1 and Comp.2 were dispersed in a solution of 0.5% carboxymethylcellulose (CMC). The mice were sacrificed after being anesthetized with sodium pentobarbital (90 mg/kg i.p.), and blood and brain tissues were collected at 0.5, 1, 2, and 24 h after administration. Blood was centrifuged at 2000 × g for 10 min at 4 ◦ C, and the plasma was collected. Plasma was diluted four-fold with methanol and was centrifuged at 20,000 × g for 20 min at 4 ◦ C, and the supernatant was collected. Brain tissue was homogenized with a nine-fold volume of methanol and centrifuged at 20,000 × g for 20 min at 4 ◦ C, and the supernatant was collected. The contents of compounds were analyzed with a HPLC/mass spectrometry (HPLC/MS) system (pump and detector, Agilent 1100 LC/MSD (Santa Clara, CA, USA); column, TSK gel ODS-80TS 4.6 × 150 mm (TOSOH, Tokyo, Japan); mobile phase, water:methanol = 20:80, flow rate; 0.5 mL/min). The OBX procedure was performed according to the procedure of Hozumi et al. [8]. At day 0, mice were anesthetized with sodium pentobarbital (90 mg/kg i.p.; Kyoritsu Seiyaku Corp., Tokyo, Japan). After exposure of the skull, 1-mm-diameter holes were drilled on both sides of the olfactory bulbs. Olfactory bulbs were removed through the hole by gentle aspiration with a suction pump, and care was taken not to damage the frontal cortex. Holes were filled with gelatin, and the skin was closed. Sham-operated mice were treated similarly but bulbs were left intact. On days 15–28, in treatment groups, vehicle (0.5% CMC p.o.), vehicle (saline i.p.), Comp.1 (50 mg/kg p.o.), Comp.2 (50 mg/kg p.o.), or fluoxetine (10 mg/kg i.p.) were administered to the mice once a day for 14 days. Comp.1 and Comp.2 were dispersed in a 0.5% CMC. Fluoxetine was dissolved in saline. On days 22–28, 5-bromo-2 -deoxyuridine (BrdU) (Sigma–Aldrich, St. Louis, MO, USA; 50 mg/kg i.p.) was intraperitoneally injected once daily for 7 days. On day 30, animals were sacrificed. For immunohistochemistry, animals were anesthetized with sodium pentobarbital and transcardially perfused with 4 ◦ C saline, followed by 4% PFA in 0.1 M PBS. The brain was removed from the skull and placed in the same fixative at 4 ◦ C overnight. Then
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A
MeO
MeO OMe
OMe
OMe
OMe
OMe
OMe
Comp.1
Comp.2
trans-3-(3',4'-dimethoxyphenyl)-4-[(E)3'',4''-dimethoxystyryl]cyclohex-1-ene
cis-3-(3',4'-dimethoxyphenyl)-4-[(E)3'',4''-dimethoxystyryl]cyclohex-1-ene
700 600 500 400 300 200 100 -0 0
Comp.1 17.681 mV
mV
B
2 4 6 8 10 12 14 16 18 20 22 24 min
700 600 500 400 300 200 100 -0 0
Comp.2 19.506
5
10
15 20 min
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Fig. 1. Compounds: (A) chemical structures of Comp.1 and Comp.2 and (B) HPLC chromatogram of Comp.1 and Comp.2.
tissues were washed with 0.1 M PBS and were equilibrated in 0.1 M PBS containing 30% sucrose at 4 ◦ C overnight. Tissues were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan) and snap-frozen in liquid nitrogen. The tissue was cut in coronal sections 20 m thick on a cryostat (CM1850; Leica, Nussloch, Germany). Frozen sections (from bregma −2.20 mm to −2.80 mm) were mounted on glass slides (MAS-coated; Matsunami Glass, Osaka, Japan). Sections were washed in Tris-buffered saline (TBS) and heated in a microwave for 5 min in distilled water. Following blocking with normal goat serum, sections were incubated overnight at room temperature with rat anti-BrdU monoclonal antibody (1:200; AbD Serotec, Oxford, UK) and mouse anti-NeuN monoclonal antibody (1:500; Millipore, Temecula, CA, USA). Sections were washed and incubated for 1 h at room temperature with goat anti-rat IgG Alexa Fluor 488 (1:500; Invitrogen, Carlsbad, CA, USA) and goat anti-mouse IgG Alexa Fluor 568 (1:500; Invitrogen, Carlsbad, CA, USA) with TBS containing Tween 20. Finally, sections were washed and coverslipped with VECTASHIELD Hard Set (Vector Laboratories, Burlingame, CA, USA). Immunofluorescent images were analyzed using a confocal laser-scanning microscope (A1Rsi: Nikon, Tokyo, Japan). Four sections per mouse were used, and two fluorescent images (640 × 640 m) of the dentate gyrus (DG) region of the hippocampus were obtained from each section. The number of BrdU/NeuN double-positive cells was counted in each image. In addition, the non-specific binding of the secondary antibody was barely detectable in the experiment in the absence of primary antibody. A mean value of eight images was used for each mouse, and there were 5–7 mice in each group. Each value was expressed as the mean ± standard error of the mean (S.E.M.). Statistical differences between each group were evaluated by Chi-square test, Dunnett’s t-test, and one-way analysis of variance (ANOVA) with Bonferroni correction. Differences were considered to be significant at p < 0.05. We first examined the neuritogenic effects of Comp.1 and Comp.2 in PC12 cells (Fig. 2A and B). In the control group (0.5% EtOH), only a few cells had neurites. NGF as a positive control induced significant neurite sprouting and outgrowth in PC12 cells. Similarly, Comp.1 or Comp.2 significantly and dose-dependently induced neurite sprouting in PC12 cells.
Next, we examined the effects of Comp.1 and Comp.2 on neurite outgrowth and neuronal survival in primary cultured rat cortical neurons (Fig. 2C–F). In the neurite outgrowth assay, Comp.1, Comp.2, or bFGF as a positive control significantly and dosedependently increased the neurite length and number of neurites in the cultured cortical neurons (Fig. 2C–E). To determine the neuroprotective effects of Comp.1 and Comp.2, we used a trophic withdrawal model. After the culture medium was changed to serum-free DMEM/N-2 supplement medium, the number of neurons decreased gradually. In the presence of Comp.1, Comp.2, or bFGF as a positive control, a large number of surviving cells was observed compared with the control group (0.5% EtOH) 48 h after exchanging the medium (Fig. 2F). Next, we investigated whether Comp.1 and Comp.2 showed neurotrophic activity on the central nervous system in vivo. We first determined the concentration of Comp.1 and Comp.2 in plasma and brain after oral administration (Fig. 3A). Concentrations of Comp.1 and Comp.2 increased in the brain within 30 min and peaked at 1 h after oral administration, and peak concentrations in brain were more than in the plasma for each. To determine the rate of hippocampal neurogenesis, animals were injected with BrdU after OBX. BrdU incorporation into a cell indicated that the cell was newly generated during the BrdU administration period. Anti-neuronal nuclear antigen, NeuN, was used as a neuron marker. Therefore, cells double-labeled for BrdU and NeuN were identifiable as newly generated neurons. Confocal microscopy analysis demonstrated that BrdU/NeuN double-positive cells (yellow) in the granular cell layer, and SGZ of DG, were observed in non-operated and sham-operated mice, but the number was significantly decreased 30 days after OBX (Fig. 3B and C). Chronic treatment with Comp.1 or Comp.2 increased the number of BrdU/NeuN double-positive cells compared with vehicle. Continuous administration of fluoxetine, an antidepressant, also increased the number of BrdU/NeuN double-positive cells compared with vehicle (Fig. 3B and C). The aim of the present study was to evaluate the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. We have shown that Comp.1 and Comp.2, phenylbutenoid dimers from Z. purpureum, have neurotrophic effects characterized by neuritogenesis, neurite outgrowth promotion, and neuronal survival enhancement
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Fig. 2. Neurotrophic effects of Comp.1 and Comp.2 in cultured cells. (A and B) Neuritogenic activity of Comp.1 and Comp.2 in PC12 cells. To evaluate neuritogenic activity, PC12 cells were cultured in DMEM/2% HS + 1% FBS containing test samples for 96 h. (A) Morphology of PC12 cells. (B) Percentage of PC12 cells with neurites (n = 3). (C–E) Neurite outgrowth activities of Comp.1 and Comp.2 in primary cultured rat cortical neurons. To evaluate neurite outgrowth activity, primary cultured rat cortical neurons were cultured in NB/2% B-27 medium containing test samples for 6 days. (C) Morphology of primary cultured rat cortical neurons. (D) Neurite length of primary cultured rat cortical neurons (n = 138–150). (E) Neurite number of primary cultured rat cortical neurons (n = 60). (F) Neuroprotective activities of Comp.1 and Comp.2 in primary cultured rat cortical neurons. To evaluate the neuroprotective activity, the primary cultured rat cortical neurons were cultured in DMEM/1% N-2 medium containing test samples for 48 h. (F) Numbers of surviving cells (% of control) (n = 6). Values are expressed as means ± S.E.M. *p < 0.05; **p < 0.01 compared with C by Chi-square test (B) or Dunnett’s t-test (D–F). C, control (0.5% EtOH).
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Fig. 3. Effects of compounds on hippocampal neurogenesis in OBX mice. (A) The concentration of Comp.1 and Comp.2 in plasma and brains of mice after oral administration (n = 3–5). Comp.1 (50 mg/kg p.o.) and Comp.2 (50 mg/kg p.o.) were administrated orally to the mice. The mice were sacrificed at various time points, and blood and brain tissues were collected. The concentrations of Comp.1 and Comp.2 in plasma and brain tissue were analyzed by HPLC/MS. (B) Confocal microscopy images of double staining for BrdU (green), and NeuN (red), and merged (yellow) in the DG region of the hippocampus. (C) Quantitative analysis of the number of BrdU and NeuN co-expressing cells in DG regions of the hippocampus (n = 5–7). Values are expressed as mean values ± S.E.M. *p < 0.05; **p < 0.01. Unrelated ANOVA with Bonferroni correction. Non, non-operated age-matched mice; Veh., vehicle; FLU, fluoxetine 10 mg/kg/day; Comp.1, Comp.1 50 mg/kg/day; Comp.2, Comp.2 50 mg/kg/day.
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in PC12 cells and primary cultured rat cortical neurons. We then showed that chronic treatment with neurotrophic small molecular weight compounds, including Comp.1 and Comp.2, enhanced hippocampal neurogenesis in OBX mice. These results suggest that Comp.1 and Comp.2 enhance hippocampal neurogenesis by their neurotrophic activity. Our results are consistent with the reports that exogenous treatment with neurotrophins enhances adult neurogenesis in various animal depression models [11,21]. In addition, although neurotrophins are not available to the central nervous system when administered orally, Comp.1 and Comp.2 accumulated in the brain by crossing the blood–brain barrier after oral administration. Chronic treatment with antidepressants enhances hippocampal neurogenesis in various animal models of depression, including the rodent OBX model [10,11,20]. Conversely, X-irradiation of mouse brains prevents hippocampal neurogenesis and behavioral improvement caused by antidepressant treatment [20]. This suggests that agents that enhance hippocampal neurogenesis probably have antidepressant-like effects. In the present study, Comp.1 and Comp.2 showed a similar effect as fluoxetine, a commonly used antidepressant, on hippocampal neurogenesis in OBX mice. This suggests that Comp.1 and Comp.2 may be able to induce antidepressant-like behavioral changes. Since most antidepressants act by increasing extracellular concentrations of serotonin [2], the risk of adverse side effects caused by high levels of serotonin, called serotonin syndrome [3] is difficult to avoid. In addition, tricyclic antidepressants also have antimuscarinic adverse side effects. Therefore, compounds with neurotrophic effects such as Comp.1 and Comp.2 may be more useful and have fewer adverse side effects than existing antidepressants. Hippocampal neurogenesis is implicated in learning and memory. Moreover, recent studies have revealed dysregulation of adult neurogenesis in AD patients and in various AD mouse models [1,12,13]. Therefore, neurotrophic compounds that have neurogenetic as well as neuroprotective effects may have great therapeutic potential for treatment of AD. For example, T-817MA, a neurotrophic agent, improves learning deficits while preventing neuron loss and increasing neurogenesis in rats infused intracerebroventricularly with amyloid- [13]. The OBX model mice exhibit not only characteristics of depression but also characteristics of AD, such as amyloid- (A) accumulation in the neocortex and hippocampus, cholinergic neuron loss in the basal forebrain, and spatial memory impairment. Therefore, Comp.1 and Comp.2 may have the potential to improve cognitive disorders such as AD. However, the effects of Comp.1 and Comp.2 on depressive behavior and cognitive impairment have not been examined. Further studies are needed to determine the behavioral effects of these compounds. Indeed, because the site of action and intracellular mechanism of neurotrophic activity of Comp.1 and Comp.2 are still unclear, further studies are needed to determine the mechanism of action of these compounds. The present study has demonstrated that cis- and trans-3-(3 4 dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene, phenylbutenoid dimers from Z. purpureum (BANGLE) have neurotrophic effects characterized by neuritogenesis, neurite outgrowth promotion, and neuronal survival enhancement in cultured neurons. Furthermore, chronic treatment of these compounds enhanced hippocampal neurogenesis in OBX mice. These results indicate that these compounds may have valuable therapeutic potential for depression and neurodegenerative diseases such as AD.
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Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice Nobuaki Matsui ∗ , Yuki Kido, Hideki Okada, Miwa Kubo, Megumi Nakai, Nobuyuki Fukuishi, Yoshiyasu Fukuyama, Masaaki Akagi Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-bouji, Yamashiro-cho, Tokushima 770-8514, Japan
a r t i c l e
i n f o
Article history: Received 4 November 2011 Received in revised form 31 January 2012 Accepted 3 February 2012 Keywords: Olfactory bulbectomy Neurogenesis Neurotrophin Antidepressant Phenylbutenoid dimer Zingiber purpureum
a b s t r a c t Trans-3-(3 4 -dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3 4 -dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 (10–30 M) or Comp.2 (30 M) significantly induced neurite sprouting in PC12 cells. Comp.1 (0.03–3 M) or Comp.2 (0.3–3 M) significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 (30 M) and Comp.2 (3–30 M) also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1 (50 mg/kg p.o.), Comp.2 (50 mg/kg p.o.), or fluoxetine (10 mg/kg i.p.), an antidepressant, were administrated once a day on days 15–28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2 -deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia. © 2012 Elsevier Ireland Ltd. All rights reserved.
Neurotrophic factors, such as nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF), promote neurogenesis, neurodifferentiation, neuroprotection, and neuroplasticity [16]. Therefore, the therapeutic use of neurotrophic factors has been suggested for modification of neurodegenerative diseases [4,14,22]. More recently, it has been
Abbreviations: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; AD, Alzheimer’s disease; FGF-2, fibroblast growth factor-2; OBX, olfactory bulbectomy; NT-3, neurotrophin-3; HPLC, highperformance liquid chromatography; NMR, nuclear magnetic resonance; DMEM, Dulbecco’s modified Eagle’s medium; HS, horse serum; FBS, fetal bovine serum; PFA, paraformaldehyde; PBS, phosphate buffered saline; TBS, Tris-buffered saline; MAP2, microtubule-associated protein 2; HPLC/MS, high-performance liquid chromatography/mass spectrometry; BrdU, 5-bromo-2 -deoxyuridine; DG, dentate gyrus; SGZ, subgranular zone; SD, Sprague-Dawley; S.E.M., standard error of the mean; ANOVA, analysis of variance; SAM, senescence-accelerated mouse; A, amyloid-; CMC, carboxymethylcellulose. ∗ Corresponding author. Tel.: +81 88 602 8470; fax: +81 88 655 3051. E-mail address: [email protected] (N. Matsui). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.010
found that endogenous upregulation of BDNF contributes to the behavioral effect of antidepressants through enhancement of adult hippocampal neurogenesis, and it is thought that exogenous neurotrophins may ameliorate depression [2], as has been shown in many other studies [11,21]. Intracerebroventricular application of fibroblast growth factor-2 (FGF-2) ameliorates the reduction in hippocampal neurogenesis and reverses depressive behavior induced by olfactory bulbectomy (OBX) in mice, an experimental depression model [11]. Intrahippocampal infusion of neuropeptide VGF (non-acronymic) increases hippocampal neurogenesis and shows antidepressant-like effects on behavior in rats. In addition, intrahippocampal infusion of BDNF and neurotrophin-3 (NT-3) produces an antidepressant-like behavioral effect in rats [21]. However, since these neurotrophic factors are high molecular weight polypeptides, they cannot cross the blood–brain barrier. Therefore, these factors must be applied intracerebrally, and cannot be administered orally because they are disintegrated by gastric acid and proteases. Small molecular weight compounds with neurotrophic effects may solve this problem. Thus, efforts have been made to discover
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and synthesize small molecular weight compounds that can directly maintain neuronal function or upregulate neurotrophic factors [5,13,17,23]. For example, magnolol and honokiol, which are phenylpropanoid dimers from the stem bark of Magnolia obovata, promote neurite outgrowth and enhance neuronal survival in rat cortical neurons, and prevent age-related cholinergic deficits in the forebrain, and learning and memory impairment in senescence-accelerated mice (SAM) P8, an AD mouse model [5,15]. Nobiletin, a flavonoid found in Citrus depressa, induces neurite outgrowth in PC12D cells, and improves memory impairment in a transgenic mouse model of AD and OBX mice [17–19]. Moreover, we have found two phenylbutenoid dimers, trans-3-(3 4 dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene and cis-3-(3 4 -dimethoxyphenyl)-4-[(E)-3 ,4 (Comp.1) dimethoxystyryl]cyclohex-1-ene (Comp.2), from BANGLE (Zingiber purpureum), an Indonesian medicinal plant, which may have effects similar to other small molecular weight compounds with neurotrophic activity. Comp.1 has been reported to inhibit cell proliferation in various human cancer cells [6], but its neurotrophic activity has not been reported. In the present study, we describe the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. To assess the in vitro effects of both compounds, we examined neuritogenesis, neurite outgrowth, and neuroprotective activity in PC12 cells and primary cultured rat cortical neurons. The in vivo effects of both compounds were evaluated on hippocampal neurogenesis in OBX mice, an experimental depression and dementia animal model [7,8,10,11,18]. Comp.1 and Comp.2 (Fig. 1A) were isolated from methanol extracts of the root of BANGLE (Z. purpureum) using a combination of silica gel chromatography. Comp.1 and Comp.2 were purified by reverse-phase high-performance liquid chromatography (HPLC) using methanol–water (4:1) (Fig. 1B). The purity of Comp.1 (>99%) or Comp.2 (>99%) was determined by nuclear magnetic resonance (NMR) and HPLC. Fluoxetine hydrochloride was purchased from LKT Laboratories (St. Paul, MN, USA). Assays for the neuritogenic effects in PC12 cells were performed according to previously described methods [9]. The PC12 cell line (RCB0009) was obtained from Cell Bank, RIKEN BioResource Center (Tokyo, Japan). PC12 cells were cultured at a density of 2000 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM)/10% horse serum (HS) and 5% fetal bovine serum (FBS) for 24 h, and then the medium was changed to DMEM/2% HS, 1% FBS containing test samples. After being cultured for a further 96 h, neuritogenesis of PC12 cells were quantified. The cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 20 min, and washed with 0.1 M borate buffer and stained with 0.1% methylene blue. Ten photographs per well were obtained from three wells for each group. Neurites were defined as processes with a length longer than the cell diameter. The percentage of cells forming neurites was calculated. Assays for neurite outgrowth and neuroprotective activity in primary cultured neurons were performed according to previously described methods [5,23]. The neuronal cells were separated from the cerebral hemispheres of 18-day fetal Sprague-Dawley (SD) rats obtained from Japan SLC (Hamamatsu, Japan). After isolated cortical neurons had been cultured in poly-l-lysine-coated plates at a density of 5000 cells/cm2 for 24 h, the culture medium was changed from DMEM/10% FBS to NeurobasalTM medium/2% B27 supplement with test samples. After further culture for 6 days, the neurons were fixed with 4% PFA in PBS for 20 min, and soaked in 0.3% H2 O2 for 20 min. The cell membranes were permeabilized with 0.1% Triton X-100 in PBS for 20 min. For anti-microtubuleassociated protein 2 (MAP2) staining, the neurons were incubated with an anti-MAP2 antibody (Sternberger Monoclonals, Inc., Baltimore, MD, USA) overnight at 4 ◦ C followed by incubation with
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SimpleStain PO, a peroxidase-conjugated secondary antibody (Nichirei, Tokyo, Japan), for 1 h, and then peroxidase was developed with dimethylaminoazobenzene solution. Ten photographs per well were obtained from three wells for each group. At most five well-stained neurons that made connections to no more than two cells were selected for measurements of their primary neurite length and neurite number in each photograph. Neuronal viability was determined with the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay as previously described [23]. Briefly, isolated cortical neurons were cultured in poly-l-lysine-coated 96-well plates at a density of 2 × 105 cells/cm2 for 24 h, then the culture medium was changed to DMEM/1% N-2 supplement with various concentrations of test samples. After 48 h of further culture, MTT (0.5 mg/mL in PBS) was added to the culture medium (MTT:medium at 1:10). After a 2-h incubation, the medium was pipetted off, and 50% ethanol/50% dimethyl sulfoxide was used to dissolve the MTT formazan. Then, absorbance was measured at 570 nm and translated into neuronal viability. A mean value of six wells was used for each group. For experiments in vivo, male ddY mice (6 weeks old, n = 71, Japan SLC, Hamamatsu, Japan) were used. Mice were housed in a conventional animal facility with a 12/12 light/dark cycle (light from 08:00 to 20:00) and were kept in an air-conditioned room maintained at 23 ± 1◦ C with humidity at 55 ± 5%. Food and water were given ad libitum. All animal experiments were carried out under approved guidelines provided by the animal use committee at Tokushima Bunri University in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. For determination of pharmacokinetics, Comp.1 (50 mg/kg) or Comp.2 (50 mg/kg) was orally administered to the mice. Comp.1 and Comp.2 were dispersed in a solution of 0.5% carboxymethylcellulose (CMC). The mice were sacrificed after being anesthetized with sodium pentobarbital (90 mg/kg i.p.), and blood and brain tissues were collected at 0.5, 1, 2, and 24 h after administration. Blood was centrifuged at 2000 × g for 10 min at 4 ◦ C, and the plasma was collected. Plasma was diluted four-fold with methanol and was centrifuged at 20,000 × g for 20 min at 4 ◦ C, and the supernatant was collected. Brain tissue was homogenized with a nine-fold volume of methanol and centrifuged at 20,000 × g for 20 min at 4 ◦ C, and the supernatant was collected. The contents of compounds were analyzed with a HPLC/mass spectrometry (HPLC/MS) system (pump and detector, Agilent 1100 LC/MSD (Santa Clara, CA, USA); column, TSK gel ODS-80TS 4.6 × 150 mm (TOSOH, Tokyo, Japan); mobile phase, water:methanol = 20:80, flow rate; 0.5 mL/min). The OBX procedure was performed according to the procedure of Hozumi et al. [8]. At day 0, mice were anesthetized with sodium pentobarbital (90 mg/kg i.p.; Kyoritsu Seiyaku Corp., Tokyo, Japan). After exposure of the skull, 1-mm-diameter holes were drilled on both sides of the olfactory bulbs. Olfactory bulbs were removed through the hole by gentle aspiration with a suction pump, and care was taken not to damage the frontal cortex. Holes were filled with gelatin, and the skin was closed. Sham-operated mice were treated similarly but bulbs were left intact. On days 15–28, in treatment groups, vehicle (0.5% CMC p.o.), vehicle (saline i.p.), Comp.1 (50 mg/kg p.o.), Comp.2 (50 mg/kg p.o.), or fluoxetine (10 mg/kg i.p.) were administered to the mice once a day for 14 days. Comp.1 and Comp.2 were dispersed in a 0.5% CMC. Fluoxetine was dissolved in saline. On days 22–28, 5-bromo-2 -deoxyuridine (BrdU) (Sigma–Aldrich, St. Louis, MO, USA; 50 mg/kg i.p.) was intraperitoneally injected once daily for 7 days. On day 30, animals were sacrificed. For immunohistochemistry, animals were anesthetized with sodium pentobarbital and transcardially perfused with 4 ◦ C saline, followed by 4% PFA in 0.1 M PBS. The brain was removed from the skull and placed in the same fixative at 4 ◦ C overnight. Then
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A
MeO
MeO OMe
OMe
OMe
OMe
OMe
OMe
Comp.1
Comp.2
trans-3-(3',4'-dimethoxyphenyl)-4-[(E)3'',4''-dimethoxystyryl]cyclohex-1-ene
cis-3-(3',4'-dimethoxyphenyl)-4-[(E)3'',4''-dimethoxystyryl]cyclohex-1-ene
700 600 500 400 300 200 100 -0 0
Comp.1 17.681 mV
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Comp.2 19.506
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Fig. 1. Compounds: (A) chemical structures of Comp.1 and Comp.2 and (B) HPLC chromatogram of Comp.1 and Comp.2.
tissues were washed with 0.1 M PBS and were equilibrated in 0.1 M PBS containing 30% sucrose at 4 ◦ C overnight. Tissues were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan) and snap-frozen in liquid nitrogen. The tissue was cut in coronal sections 20 m thick on a cryostat (CM1850; Leica, Nussloch, Germany). Frozen sections (from bregma −2.20 mm to −2.80 mm) were mounted on glass slides (MAS-coated; Matsunami Glass, Osaka, Japan). Sections were washed in Tris-buffered saline (TBS) and heated in a microwave for 5 min in distilled water. Following blocking with normal goat serum, sections were incubated overnight at room temperature with rat anti-BrdU monoclonal antibody (1:200; AbD Serotec, Oxford, UK) and mouse anti-NeuN monoclonal antibody (1:500; Millipore, Temecula, CA, USA). Sections were washed and incubated for 1 h at room temperature with goat anti-rat IgG Alexa Fluor 488 (1:500; Invitrogen, Carlsbad, CA, USA) and goat anti-mouse IgG Alexa Fluor 568 (1:500; Invitrogen, Carlsbad, CA, USA) with TBS containing Tween 20. Finally, sections were washed and coverslipped with VECTASHIELD Hard Set (Vector Laboratories, Burlingame, CA, USA). Immunofluorescent images were analyzed using a confocal laser-scanning microscope (A1Rsi: Nikon, Tokyo, Japan). Four sections per mouse were used, and two fluorescent images (640 × 640 m) of the dentate gyrus (DG) region of the hippocampus were obtained from each section. The number of BrdU/NeuN double-positive cells was counted in each image. In addition, the non-specific binding of the secondary antibody was barely detectable in the experiment in the absence of primary antibody. A mean value of eight images was used for each mouse, and there were 5–7 mice in each group. Each value was expressed as the mean ± standard error of the mean (S.E.M.). Statistical differences between each group were evaluated by Chi-square test, Dunnett’s t-test, and one-way analysis of variance (ANOVA) with Bonferroni correction. Differences were considered to be significant at p < 0.05. We first examined the neuritogenic effects of Comp.1 and Comp.2 in PC12 cells (Fig. 2A and B). In the control group (0.5% EtOH), only a few cells had neurites. NGF as a positive control induced significant neurite sprouting and outgrowth in PC12 cells. Similarly, Comp.1 or Comp.2 significantly and dose-dependently induced neurite sprouting in PC12 cells.
Next, we examined the effects of Comp.1 and Comp.2 on neurite outgrowth and neuronal survival in primary cultured rat cortical neurons (Fig. 2C–F). In the neurite outgrowth assay, Comp.1, Comp.2, or bFGF as a positive control significantly and dosedependently increased the neurite length and number of neurites in the cultured cortical neurons (Fig. 2C–E). To determine the neuroprotective effects of Comp.1 and Comp.2, we used a trophic withdrawal model. After the culture medium was changed to serum-free DMEM/N-2 supplement medium, the number of neurons decreased gradually. In the presence of Comp.1, Comp.2, or bFGF as a positive control, a large number of surviving cells was observed compared with the control group (0.5% EtOH) 48 h after exchanging the medium (Fig. 2F). Next, we investigated whether Comp.1 and Comp.2 showed neurotrophic activity on the central nervous system in vivo. We first determined the concentration of Comp.1 and Comp.2 in plasma and brain after oral administration (Fig. 3A). Concentrations of Comp.1 and Comp.2 increased in the brain within 30 min and peaked at 1 h after oral administration, and peak concentrations in brain were more than in the plasma for each. To determine the rate of hippocampal neurogenesis, animals were injected with BrdU after OBX. BrdU incorporation into a cell indicated that the cell was newly generated during the BrdU administration period. Anti-neuronal nuclear antigen, NeuN, was used as a neuron marker. Therefore, cells double-labeled for BrdU and NeuN were identifiable as newly generated neurons. Confocal microscopy analysis demonstrated that BrdU/NeuN double-positive cells (yellow) in the granular cell layer, and SGZ of DG, were observed in non-operated and sham-operated mice, but the number was significantly decreased 30 days after OBX (Fig. 3B and C). Chronic treatment with Comp.1 or Comp.2 increased the number of BrdU/NeuN double-positive cells compared with vehicle. Continuous administration of fluoxetine, an antidepressant, also increased the number of BrdU/NeuN double-positive cells compared with vehicle (Fig. 3B and C). The aim of the present study was to evaluate the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. We have shown that Comp.1 and Comp.2, phenylbutenoid dimers from Z. purpureum, have neurotrophic effects characterized by neuritogenesis, neurite outgrowth promotion, and neuronal survival enhancement
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Fig. 2. Neurotrophic effects of Comp.1 and Comp.2 in cultured cells. (A and B) Neuritogenic activity of Comp.1 and Comp.2 in PC12 cells. To evaluate neuritogenic activity, PC12 cells were cultured in DMEM/2% HS + 1% FBS containing test samples for 96 h. (A) Morphology of PC12 cells. (B) Percentage of PC12 cells with neurites (n = 3). (C–E) Neurite outgrowth activities of Comp.1 and Comp.2 in primary cultured rat cortical neurons. To evaluate neurite outgrowth activity, primary cultured rat cortical neurons were cultured in NB/2% B-27 medium containing test samples for 6 days. (C) Morphology of primary cultured rat cortical neurons. (D) Neurite length of primary cultured rat cortical neurons (n = 138–150). (E) Neurite number of primary cultured rat cortical neurons (n = 60). (F) Neuroprotective activities of Comp.1 and Comp.2 in primary cultured rat cortical neurons. To evaluate the neuroprotective activity, the primary cultured rat cortical neurons were cultured in DMEM/1% N-2 medium containing test samples for 48 h. (F) Numbers of surviving cells (% of control) (n = 6). Values are expressed as means ± S.E.M. *p < 0.05; **p < 0.01 compared with C by Chi-square test (B) or Dunnett’s t-test (D–F). C, control (0.5% EtOH).
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Fig. 3. Effects of compounds on hippocampal neurogenesis in OBX mice. (A) The concentration of Comp.1 and Comp.2 in plasma and brains of mice after oral administration (n = 3–5). Comp.1 (50 mg/kg p.o.) and Comp.2 (50 mg/kg p.o.) were administrated orally to the mice. The mice were sacrificed at various time points, and blood and brain tissues were collected. The concentrations of Comp.1 and Comp.2 in plasma and brain tissue were analyzed by HPLC/MS. (B) Confocal microscopy images of double staining for BrdU (green), and NeuN (red), and merged (yellow) in the DG region of the hippocampus. (C) Quantitative analysis of the number of BrdU and NeuN co-expressing cells in DG regions of the hippocampus (n = 5–7). Values are expressed as mean values ± S.E.M. *p < 0.05; **p < 0.01. Unrelated ANOVA with Bonferroni correction. Non, non-operated age-matched mice; Veh., vehicle; FLU, fluoxetine 10 mg/kg/day; Comp.1, Comp.1 50 mg/kg/day; Comp.2, Comp.2 50 mg/kg/day.
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in PC12 cells and primary cultured rat cortical neurons. We then showed that chronic treatment with neurotrophic small molecular weight compounds, including Comp.1 and Comp.2, enhanced hippocampal neurogenesis in OBX mice. These results suggest that Comp.1 and Comp.2 enhance hippocampal neurogenesis by their neurotrophic activity. Our results are consistent with the reports that exogenous treatment with neurotrophins enhances adult neurogenesis in various animal depression models [11,21]. In addition, although neurotrophins are not available to the central nervous system when administered orally, Comp.1 and Comp.2 accumulated in the brain by crossing the blood–brain barrier after oral administration. Chronic treatment with antidepressants enhances hippocampal neurogenesis in various animal models of depression, including the rodent OBX model [10,11,20]. Conversely, X-irradiation of mouse brains prevents hippocampal neurogenesis and behavioral improvement caused by antidepressant treatment [20]. This suggests that agents that enhance hippocampal neurogenesis probably have antidepressant-like effects. In the present study, Comp.1 and Comp.2 showed a similar effect as fluoxetine, a commonly used antidepressant, on hippocampal neurogenesis in OBX mice. This suggests that Comp.1 and Comp.2 may be able to induce antidepressant-like behavioral changes. Since most antidepressants act by increasing extracellular concentrations of serotonin [2], the risk of adverse side effects caused by high levels of serotonin, called serotonin syndrome [3] is difficult to avoid. In addition, tricyclic antidepressants also have antimuscarinic adverse side effects. Therefore, compounds with neurotrophic effects such as Comp.1 and Comp.2 may be more useful and have fewer adverse side effects than existing antidepressants. Hippocampal neurogenesis is implicated in learning and memory. Moreover, recent studies have revealed dysregulation of adult neurogenesis in AD patients and in various AD mouse models [1,12,13]. Therefore, neurotrophic compounds that have neurogenetic as well as neuroprotective effects may have great therapeutic potential for treatment of AD. For example, T-817MA, a neurotrophic agent, improves learning deficits while preventing neuron loss and increasing neurogenesis in rats infused intracerebroventricularly with amyloid- [13]. The OBX model mice exhibit not only characteristics of depression but also characteristics of AD, such as amyloid- (A) accumulation in the neocortex and hippocampus, cholinergic neuron loss in the basal forebrain, and spatial memory impairment. Therefore, Comp.1 and Comp.2 may have the potential to improve cognitive disorders such as AD. However, the effects of Comp.1 and Comp.2 on depressive behavior and cognitive impairment have not been examined. Further studies are needed to determine the behavioral effects of these compounds. Indeed, because the site of action and intracellular mechanism of neurotrophic activity of Comp.1 and Comp.2 are still unclear, further studies are needed to determine the mechanism of action of these compounds. The present study has demonstrated that cis- and trans-3-(3 4 dimethoxyphenyl)-4-[(E)-3 ,4 -dimethoxystyryl]cyclohex-1-ene, phenylbutenoid dimers from Z. purpureum (BANGLE) have neurotrophic effects characterized by neuritogenesis, neurite outgrowth promotion, and neuronal survival enhancement in cultured neurons. Furthermore, chronic treatment of these compounds enhanced hippocampal neurogenesis in OBX mice. These results indicate that these compounds may have valuable therapeutic potential for depression and neurodegenerative diseases such as AD.
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