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Protective effect of isoflavones from Trifolium pratense on dopaminergic neurons
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Neuroscience Research 62 (2008) 123–130 www.elsevier.com/locate/neures
Protective effect of isoflavones from Trifolium pratense on dopaminergic neurons Han-Qing Chen a,b, Xi-Jin Wang c, Zheng-Yu Jin a,b,*, Xue-Ming Xu a,b, Jian-Wei Zhao a,b, Zheng-Jun Xie a,b a
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China b School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China c Department of Neurology & Institute of Neurology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, PR China Received 13 April 2008; accepted 7 July 2008 Available online 16 July 2008
Abstract In the present study, protective effect of five isoflavones (formononetin, daidzein, pratensein, calycosin and irilone) from Trifolium pratense on lipopolysaccharide-induced dopaminergic neurodegeneration was studied for the first time. The results showed that all five isoflavones attenuated LPS-induced decrease in dopamine uptake and the number of dopaminergic neurons in a dose-dependent manner in rat mesencephalic neuron-glia cultures. Moreover, they also significantly inhibited LPS-induced activation of microglia and production of tumor necrosis factor-a, nitric oxide and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures. In addition, the rank order of protective potency of five isoflavones was: pratensein > daidzein > calycosin > formononetin > irilone. This study suggested that all five isoflavones protected dopaminergic neurons against LPS-induced injury through inhibition of microglia activation and proinflammatory factors generation. # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Isoflavone; Inflammation; Microglia; Dopaminergic neurodegeneration; Parkinson’s disease
1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by cardinal features, including resting tremor, slowing of movement, rigidity and postural instability. The major pathological change of PD is the progressive and selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Olanow and Tatton, 1999). The exact cause and underlying mechanism responsible for the progressive neurodegeneration of PD remains largely unknown. Increasing evidence has suggested that inflammation in the brain, in particular, activation of microglia, participates in the pathogenesis of PD, as well as several other neurodegenerative disorders, including Alzheimer’s disease (Giulian, 1999; Liu and Hong, 2003; Wang et al., 2005b, 2007). Microglia, the resident immune cells in the brain, serve the role of immune surveillance * Corresponding author at: School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China. Tel.: +86 510 85913299; fax: +86 510 85913299. E-mail address: [email protected] (Z.-Y. Jin).
and host defense under normal conditions (Kreutzberg, 1996). However, microglia become readily activated in response to injury, infection, or inflammation. Activated microglia secrete a variety of proinflammatory factors, including cytokines such as tumor necrosis factor-a (TNF-a) and the free radicals nitric oxide (NO) and superoxide. Many studies have demonstrated that the accumulation of these proinflammatory factors contributes to the degeneration of dopaminergic neurons (Gao et al., 2002a; Gayle et al., 2002; Kim et al., 2000; Liu et al., 2000a, 2002a). Because the midbrain region that encompasses the substantia nigra is particularly rich in microglia (Kim et al., 2000), activation of microglia and release of proinflammatory factors may be a crucial component of the degenerative process of dopaminergic neurons in PD. Hence, identification of compounds that prevent microglial activation is highly desirable in the search for therapeutic agents for inflammation-mediated neurodegenerative diseases including PD. Phytoestrogens are plant-derived compounds that structurally or functionally mimic mammalian estrogens and therefore are considered to play an important role in the prevention of cancers, heart disease, menopausal symptoms and osteoporosis
0168-0102/$ – see front matter # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2008.07.001
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(Ososki and Kennelly, 2003). Phytoestrogens include isoflavones, coumestans, and lignans. Isoflavones are the most important type of phytoestrogens found in legume plants such as Trifolium pratense and Glycine max. Preliminary clinical reports suggest a potential protective effect of estrogen replacement therapy (ERT) on the onset and progression of both PD and PD-related dementia (Marder et al., 1998; Blanchet et al., 1999). Furthermore, in vitro studies have demonstrated that estrogens protect dopaminergic neurons against inflammation-induced neurotoxicity (Sawada et al., 1998, 2002; Liu et al., 2005). Despite the beneficial effect of ERT on PD, estrogen use in postmenopausal women is associated with increased risks of uterine cancer and potentially an increase in neoplasms of the breast (Hammond, 1994), especially after long-term use (Grady et al., 1995; Colditz et al., 1995). In contrast, several reports indicate that phytoestrogens do not promote neoplasms of the breast and uterus, but instead reduce the risk of developing several types of cancer, most notably breast cancer (Peterson and Barnes, 1991; Hsu et al., 1999; Messina et al., 1997). Therefore, phytoestrogens receive more attention in recent years and have been shown to provide neuroprotection in vitro. Zhao et al. (2002) report that phytoestrogens can exert neuroprotective effect in cultured hippocampal neurons. Similarly, Ge´linas and Martinoli (2002) also demonstrate that phytoestrogens have neuroprotective effect on MPP+-induced cytotoxicity in neuronal PC12 cells. However, very limited investigations have explored the effect of isoflavones from T. pratense on inflammation-mediated dopaminergic neurodegeneration. Recently, Wang et al. (2005a) report that genistein, the primary soybean isoflavone, protects dopaminergic neurons by inhibiting microglial activation. Our previous study also demonstrates that biochanin A, one of the predominant isoflavones in T. pratense, protects dopaminergic neurons against lipopolysaccharide (LPS)-induced damage (Chen et al., 2007). However, except for biochanin A and genistein in T. pratense, there are other isoflavones such as pratensein, calycosin, daidzein, irilone, and formononetin, the effects of them on inflammation-induced dopaminergic neurodegeneration remain unknown. In this study, using rat primary mesencephalic neuron-glia cultures and microglia-enriched cultures, we further investigate the effects of five isoflavones (formononetin, pratensein, daidzein, calycosin, and irilone) from T. pratense on LPSinduced dopaminergic neurodegeneration. We find that all these isoflavones from T. pratense protect dopaminergic neurons from LPS-induced damage to a certain extent. Moreover, the rank order of protective capacities of them on dopaminergic neurons is: pratensein > daidzein > calycosin > formononetin > irilone.
O111:B4) and the monoclonal antibody against tyrosine hydroxylase (TH) were purchased from Sigma (St. Louis, MO). [3H]dopamine (DA) (30 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). The monoclonal antibodies against the CR3 complement receptor (OX-42) and glial fibrillary acidic protein (GFAP) were purchased from chemicon (Temecula, CA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
2.2. Primary rat ventral mesencephalic neuron-glia cultures Primary rat ventral mesencephalic neuron-glia cultures were prepared following previously described protocol (Gao et al., 2002b; Liu et al., 2000a) with some modifications. Briefly, ventral mesencephalic tissues were dissected from embryonic day 14 Sprague–Dawley rats under pentobarbital anaesthesia and dissociated by a mild mechanical trituration. According to the experimental arrangement, dissociated cells were seeded to 24-well (5 105/ well) culture plates precoated with poly-D-lysine (20 mg/ml). The cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% heat-inactivated horse serum (HS), 1 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM non-essential amino acids, 50 U/ml penicillin, and 50 mg/ml streptomycin. After the initial seeding for 3 days, the cultures were replenished with 0.5 ml of fresh medium. Seven-day-old cultures were used for treatment. During the course of the experiment, all animals were treated in strict accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals, and all efforts were made to minimize the number of animals and their suffering.
2.3. Primary microglia-enriched cultures Rat microglia-enriched cultures were prepared according to previously described protocol (Liu et al., 2000a). Briefly, whole brains of 1-day-old Sprague–Dawley rats, with the blood vessels and meninges removed, were triturated. Cells (5 107) were seeded in 150 cm2 culture flasks in 30 ml of a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM non-essential amino acids, 50 U/ml penicillin, and 50 mg/ml streptomycin. The cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. Medium (30 ml/flask) was replenished 1 and 4 days after the initial seeding and changed thereafter every third day. On reaching confluence (day 14), microglia were shaken off (180 rpm for 5 h on an orbital shaker), pelleted at 800 g for 10 min, resuspended in fresh medium, and plated (105 cells/well) into 24-well culture plates. Twenty-four hours later, cells were ready for treatment. The enriched microglia were found to be >98% pure as determined by OX-42- and GFAPimmunoreactive (IR).
2.4. Immunocytochemical staining Dopaminergic neurons were recognized with anti-TH antibody and microglia were detected with OX-42 antibody, which recognizes the CR3 complement receptor as described previously (Gao et al., 2002b; Liu et al., 2000a). Briefly, cells were fixed with 3.7% paraformaldehyde in PBS at room temperature for 20 min and then treated with 1% hydrogen peroxide for 10 min. After incubation with blocking solution for 40 min, the cells were incubated with primary antibodies at appropriate concentrations at 4 8C overnight. Afterward, the cells were incubated with appropriate biotinylated secondary antibody for 2 h followed by the Vectastain ABC reagents for 40 min. Color was developed with 3,30 -diaminobenzidine. For visual counting of TH-, OX-42-, or GFAP-IR neurons, 10 representative areas per well of the 24-well plates were counted under the microscope at 100 magnification. Counting was always performed by two to three individuals in a blind manner.
2.5. Uptake assay for [3H]dopamine (DA) 2. Materials and methods 2.1. Reagents Formononetin, pratensein, daidzein, calycosin and irilone were isolated from T. pratense and the purities of them were no less than 95%. LPS (Escherichia coli
Uptake assay was performed using previously described methods (Gao et al., 2002b; Liu et al., 2000a). Briefly, cells in each well were washed with Krebs-Ringer buffer (KRB, 16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4) twice. The cells were then incubated with 1 mM [3H]DA in KRB at 37 8C
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for 15 min. Non-specific uptake for dopamine was determined in parallel wells receiving both tritiated dopamine and 10 mM mazindol, an inhibitor of neuronal high-affinity dopamine uptake. Afterward, the cells were washed with ice-cold KRB three times and solublized in 1 N NaOH and radioactivity was determined by a liquid scintillation counter. The specific uptake for dopamine was calculated by subtracting the amount of radioactivity observed in the presence of mazindol from that observed in the absence of mazindol.
2.6. Nitrite and TNF-a assays The accumulation of nitrite in the culture supernatant, an indicator of the production of NO, was determined with a colorimetric assay with the Griess reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4) (Green et al., 1982). Equal volumes of culture supernatant and Griess reagent were mixed, the mixture was incubated at room temperature for 10 min, and absorbance at 540 nm was determined with a microplate reader (BIORAD Laboratories, CA). The concentrations of nitrite in the samples were determined from a sodium nitrite standard curve (Liu et al., 2000a). The level of tumor necrosis factor-a (TNF-a) in the culture medium was measured with a rat TNF-a enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) (Liu et al., 2000a).
2.7. Superoxide assay The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c as previously described (Gao et al., 2002b; Liu et al., 2000a). Briefly, mesencephalic neuron-glia or microglia-enriched cultures grown in 96-well culture plates were treated with vehicle or LPS in treatment medium containing phenol red-free MEM (150 ml/ well), with or without 600 U/ml SOD. To each well, 50 ml of ferricytochrome C (100 mM) in treatment medium was added. The cultures were then incubated at 37 8C for 30 min. Afterwards, the absorbance at 550 nm was recorded with a SpectraMax Plus microplate spectrophotometer. To determine the effect of isoflavones on superoxide release, cultures were preincubated with isoflavones at 37 8C for 30 min prior to the addition of LPS.
2.8. Statistical analysis The data were expressed as the mean S.E.M. Statistical significance was assessed with one-way analysis of variance (ANOVA), followed by the Newman–keuls test (SAS software, Version 8.0). A value of P < 0.05 was considered statistically significant.
3. Results 3.1. Different isoflavones from T. pratense protected dopaminergic neurons against LPS-induced neurotoxicity in a dose-dependent manner in mesencephalic neuron-glia cultures In preliminary experiments, through MTT assay, we found that different isoflavones from T. pratense (24 h) induced cell death at a concentration of 50 mM in rat mesencephalic neuronglia cultures, but at 10 mM they showed no obvious toxicity to primary cells (data not shown). Therefore, in order to avoid the cytotoxicity of different isoflavones, their concentrations used in this study were 0.25–2.5 mM. Mesencephalic neuron-glia cultures were pretreated with vehicle or 0.25–2.5 mM different isoflavones for 30 min before treatment with 10 ng/ml LPS. Seven days later, the degeneration of dopaminergic neurons was assessed by [3H]DA uptake or TH immunostaining. [3H]DA uptake assay indicated that LPS treatment decreased the uptake capacity to 37.2% of that of vehicle-treated control
Fig. 1. Effect of different isoflavones from Trifolium pratense on DA uptake in primary mesencephalic neuron-glia cultures. (A) Isoflavones (0.25 mM, 1.0 mM, and 2.5 mM) from Trifolium pratense attenuated LPS-induced decrease in DA uptake in primary mesencephalic neuron-glia cultures; (B) Isoflavones (2.5 mM) from Trifolium pratense alone did not affect DA uptake in primary mesencephalic neuron-glia cultures. Rat ventral mesencephalic neuron-glia cultures were pretreated with vehicle (Veh) or indicated concentrations of isoflavones for 30 min before treatment with 10 ng/ml LPS for 7 days. Results are mean S.E.M. of five experiments performed triplicate and are expressed as a percentage of vehicletreated control culture. *P < 0.05 compared with LPS-treated culture. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
culture (Fig. 1A). However, all tested isoflavones significantly attenuated the LPS-induced decrease in DA uptake in a dosedependent manner. The most effective concentration was found at 2.5 mM. At the concentration of 2.5 mM, DA uptake of cultures pretreatment with pratensein, daidzein, calycosin, formononetin, and irilone before treatment with LPS were 80.2%, 70.1%, 65.7%, 60.7%, and 56.1% of that of control cultures, respectively (Fig. 1A). However, DA uptake of cultures treated with any isoflavone alone did not differ significantly from that of control cultures (Fig. 1B). In addition to DA uptake, counting the number of TH-IR neurons in the cultures revealed that LPS treatment reduced the number of TH-IR neurons by 53.3% compared with vehicletreated control cultures (Fig. 2A). However, all tested isoflavones significantly attenuated LPS-induced reduction in the number of TH-IR neurons in a dose-dependent manner. The number of TH-IR neurons of cultures pretreated with pratensein, daidzein, calycosin, formononetin and irilone at the concentration of 2.5 mM before treatment with LPS were 81.1%, 71.3%, 66.8%, 61.8%, and 57.1% of that of control cultures, respectively (Fig. 2A). But treatment of cultures with 2.5 mM any isoflavone alone did not have significant effect on the number of the TH-IR neurons (Fig. 2B). 3.2. Different isoflavones from T. pratense inhibited LPSinduced activation of microglia and release of proinflammatory factors in mesencephalic neuron-glia cultures and microglia-enriched cultures To elucidate the underlying mechanism of the neuroprotective activity of different isoflavones from T. pratense, we
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Fig. 2. Effect of different isoflavones on the number of DA neurons in primary mesencephalic neuron-glia cultures. (A) Isoflavones (0.25 mM, 1.0 mM, and 2.5 mM) from Trifolium pratense attenuated LPS-induced decrease in the number of DA neurons in primary mesencephalic neuron-glia cultures; (B) Isoflavones (2.5 mM) from Trifolium pratense alone did not affect the number of DA neurons in primary mesencephalic neuron-glia cultures. Rat ventral mesencephalic neuron-glia cultures were pretreated with vehicle (Veh) or indicated concentrations of isoflavones for 30 min before treatment with 10 ng/ml LPS for 7 days. Results are mean S.E.M. of five experiments performed triplicate and are expressed as a percentage of vehicle-treated control culture. *P < 0.05 compared with LPS-treated cultures. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
investigated the effect of different isoflavones on the LPSinduced microglial activation. Culture supernatants were taken at optimal time points for determining the levels of some key proinflammatory factors (TNF-a, NO, and superoxide) and cells were fixed and immunostained for specific markers of microglia. As shown in Fig. 3A, after 24 h exposure to 10 ng/ml LPS, the number of OX-42-IR microglia in the cultures significantly increased compared with vehicle-treated control cultures. All tested isoflavones effectively prevented the increase of OX-42-IR microglia in LPS-exposed cultures. Pretreatment with pratensein, daidzein, calycosin, formononetin, and irilone at the concentration of 2.5 mM before treatment with LPS reduced the number of OX-42-IR microglia in the cultures to 48.7%, 54.7%, 59.5%, 63.6%, and 67.9% of that of LPS-treated culture, respectively. But different isoflavone (2.5 mM) alone had no effect on their number. In addition to preventing the activation of microglia, all tested isoflavones from T. pratense significantly inhibited the production of proinflammatory factors in neuron-glia cultures. As shown in Fig. 3B–D, significant levels of TNF-a, NO and superoxide were observed in neuron-glia cultures exposed to LPS treatment. The release of TNF-a, NO and superoxide was inhibited by pretreatment with any isoflavone in a dosedependent manner, and rank order of action was: pratensein > daidzein > calycosin > formononetin > irilone. In microglia-enriched cultures, we also determined the effect of different isoflavones from T. pratense on the LPSinduced production of TNF-a, NO and superoxide. As shown in Fig. 4A–C, consistent with the results from neuron-glia
Fig. 3. Effect of different isoflavones from Trifolium pratense on microglia activation and the production of proinflammatory factors in primary mesencephalic neuron-glia cultures. (A) Isoflavones (2.5 mM) from Trifolium pratense inhibited LPS-induced microglia activation in primary mesencephalic neuron-glia cultures; (B–D) Isoflavones (0.25 mM, 1.0 mM, and 2.5 mM) from Trifolium pratense inhibited LPS-induced proinflammatory factor (B: TNF-a; C: NO; D: Superoxide) generation in primary mesencephalic neuron-glia cultures. Rat ventral mesencephalic neuron-glia cultures were pretreated with vehicle (Veh) or isoflavones (2.5 mM) for 30 min before treatment with 10 ng/ml LPS for 24 h. Microglial activation was evaluated by quantification of OX-42-IR microglia. Neuron-glia cultures were pretreated with vehicle or indicated concentrations of isoflavones for 30 min before treatment with 10 ng/ml LPS. Supernatants were removed for the measurement of TNF-a at 6 h, and for NO at 24 h. Superoxide assay was performed as described in Section 2. Results are expressed as a percentage of LPS-treated cultures and are mean S.E.M. of five experiments. *P < 0.05 compared with LPS-treated cultures. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
cultures, pretreatment with any isoflavone (2.5 mM) significantly inhibited LPS-induced production of TNF-a, NO and superoxide, and rank order of action was: pratensein > daidzein > calycosin > formononetin > irilone. But different isoflavone (2.5 mM) alone had no significant effect on production of these proinflammatory factors (data not shown).
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Fig. 4. Effect of different isoflavones from Trifolium pratense on the production of proinflammatory factors in primary microglia-enriched cultures. (A–C) Isoflavones (2.5 mM) inhibited the production of proinflammatory factors (A: TNF-a; B: NO; C: Superoxide) in primary microglia-enriched cultures. Microglia-enriched cultures were pretreated with vehicle or different isoflavones (2.5 mM) for 30 min before treatment with 10 ng/ml LPS. Supernatants were removed for the measurement of TNF-a at 6 h, and for NO at 24 h. Superoxide assay was performed as described in Section 2. Results are expressed as a percentage of LPS-treated cultures and are mean S.E.M. of five experiments. *P < 0.05 compared with LPS-treated culture. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
4. Discussion T. pratense is an important kind of legume plant which is widely grown in China and other countries in the world. T. pratense contains isoflavones, proteins, amino acids, sugars, vitamins and so on. The content of isoflavone in T. pratense is as high as 1–2% on the basis of dry weight. Biochanin A, formononetin, pratensein, genistein, daidzein, calycosin and irilone are seven isoflavones in T. pratense. Previous studies have showed that genistein and biochanin A protect dopaminergic neurons against LPS-induced injury (Wang et al., 2005a; Chen et al., 2007). However, except for genistein and biochanin A, there are other five isoflavones in T. pratense. To the best of our knowledge, the effects of those five isoflavones on dopaminergic neurons have not been reported. In this study, using rat mesencephalic neuron-glia cultures as a model and LPS as a tool, we explored for the first time the effects of five isoflavones from T. pratense on LPS-induced dopaminergic neurodegeneration. Our results indicated that pratensein, daidzein, calycosin, formononetin and irilone could
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effectively protect dopaminergic neurons against LPS-induced neurotoxicity by measuring the [3H]DA uptake and counting TH-immunoreactive cells. Furthermore, these five isoflavones significantly inhibited the activation of microglia and release of proinflammatory factors, including NO, TNF-a, and superoxide, in neuron-glia cultures and in microglia-enriched cultures exposed to LPS treatment. Moreover, the rank order of neuroprotective efficacy of these five isoflavones was: pratensein > daidzein > calycosin > formononetin > irilone. Microglia, the resident immune cells in the central nervous system, play a role in immune surveillance under normal condition (Kreutzberg, 1996). However, in response to injury or infection, microglia become readily activated and consequently release a variety of factors, including cytokines such as TNF-a and IL-b, free radicals such as NO and superoxide. These factors are believed to contribute to microglia-mediated neurodegeneration (Chao et al., 1992; Jeohn et al., 1998; Le et al., 2001). Many studies indicate that neuroprotection can be obtained by inhibiting microglia activation in vivo and in vitro. Liu et al. (2000a) have shown that naloxone protects dopaminergic neurons against inflammatory damage through inhibition of microglia activation. Similarly, Wang et al. (2002) have demonstrated that silymarin, a polyphenol flavonoid derived from milk thistle, protects dopaminergic neurons against LPS-induced neurotoxicity by inhibiting microglia activation in mesencephalic neuron-glia cultures. Moreover, Liu et al. (2003) have reported that dextromethorphan protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation. Li et al. (2004) and Zhou et al. (2005) have shown that triptolide, a Chinese herbal extract, protects dopaminergic neurons from inflammation-mediated damage through inhibition of microglial activation in vitro and in vivo. Recently, Wang et al. (2005a) reported that genistein, the primary soybean isoflavone, protected dopaminergic neurons by inhibiting microglial activation. Our previous study also indicated that biochanin A, one of the predominant isoflavones in T. pratense, protected dopaminergic neurons against LPS-induced damage through inhibition of microglia activation (Chen et al., 2007). These observations suggest that the agents which inhibit microglia activation will provide neuroprotective effects. In agreement with the above reports, in the present study, we first found that pratensein, daidzein, calycosin, formononetin and irilone from T. pratense also effectively inhibited microglia activation and reduced the production of TNF-a, NO, and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures exposed to LPS treatment. These findings indicated that the mechanism of action underlying the neuroprotective role of these five isoflavones from T. pratense, at least partially, is attributed to the inhibition of microglia activation. Among the neurotoxic factors secreted by activated microglia, the consequences of overproduction of TNF-a, NO, and superoxide free radicals have been relatively well studied. McGuire et al. (2002) demonstrated that TNF-a was capable of inducing the death of cultured dopaminergic neurons. Excessive accumulation of NO has long been known
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to be toxic to neurons (Chao et al., 1992; Jeohn et al., 2000; Liu et al., 2002a). The overproduction of free radicals is especially deleterious to neurons (Cadet and Brannock, 1998; Floyd, 1999). Moreover, when NO meets with superoxide, a more deadly nitrite, peroxynitrite, is formed, which is a potent oxidant and nitrating agent capable of attacking and modifying proteins, lipids and DNA as well as depleting antioxidant defenses (Torreilles et al., 1999). In fact, a recent study has identified peroxynitrite as a key mediator of neurotoxicity induced by LPS-activated microglia (Xie et al., 2002). In addition, several studies have shown that factors produced by activated microglia may work in concert to induce neurodegeneration. For example, Chao et al. (1995) reported that the combination of TNF-a and IL-1b, but not either cytokine alone, induced the degeneration of cortical neurons. Similarly, Jeohn et al. (1998) also demonstrated that the combination of TNF-a, IL-1b, and interferon-g work in synergy to induce degeneration of cortical neurons. Therefore, neurodegeneration in such diseases as PD is, at least in part, induced by the combined impact of multiple factors generated from activated microglia. Hence, the agents that are capable of inhibiting the production of multiple factors such as NO, TNF-a, and superoxide in activated microglia may be highly relevant to the development of potential therapeutic agents. In this study, we have demonstrated that the production of NO, TNF-a, and superoxide by LPS-activated microglia is significantly inhibited by pratensein, daidzein, calycosin, formononetin and irilone and the inhibition of the production of these factors confers significant protection to dopaminergic neurons against inflammation-mediated degeneration. Consistent with our previous study on biochanin A (Chen et al., 2007), in this study, we also found that pratensein, daidzein, calycosin, formononetin and irilone from T. pratense were significantly more potent in inhibiting LPS-induced superoxide production than the production of NO and TNF-a. This result implied that LPS-induced superoxide generation may play a more critical role than other microglia-originated factors in the induction of dopaminergic neurodegeneration. In fact, in mesencephalic neuron-glia cultures stimulated with very low concentrations of LPS (<1 ng/ml), production of superoxide, but not NO and TNF-a, seemed to mediate LPSinduced dopaminergic neurotoxicity (Gao et al., 2002a). It is possible that agents having a preferential inhibitory activity toward free radical generation may prove to confer potent neuroprotection against inflammation-mediated degeneration. The inhibitory and neuroprotective profiles of pratensein, daidzein, calycosin, formononetin and irilone seem to be similar to that of naloxone stereoisomers. Naloxone is more effective in the inhibition of superoxide generation than in that of TNF-a, NO, or IL-1b (Chang et al., 2000; Liu et al., 2000a, 2002b). The neuroprotective effect of naloxone has been observed in both in vitro and in vivo models of inflammationmediated neurodegeneration (Liu et al., 2000a,b, 2002b). Hence, it is important to determine whether the neuroprotective effects of pratensein, daidzein, calycosin, formononetin and irilone can be observed in animal models of inflammationmediated neurodegenerative diseases including PD.
In this study, we also found that neuroprotective capacity of five isoflavones was different, and the rank order of protective potency was: pratensein > daidzein > calycosin > formononetin > irilone. The reason for differences in neuroprotective potency of five isoflavones is not yet clear, but it may be related to their estrogenic activity. Previous study has indicated that estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor (ER)-a and estrogen receptor-b in microglia (Liu et al., 2005). Moreover, Baker et al. (2004) have reported that estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor b. Due to the similarities of isoflavones and estrogen in structure, isoflavones may exhibit neuroprotective effect possibly by interactions with estrogen receptors in microglia. Phytoestrogens exert estrogenic activity by binding to estrogen receptors. The binding affinity of isoflavones to estrogen receptor may be used to evaluate the estrogenic activity of different isoflavones. Kuiper et al. (1998) compared the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with estrogen receptor a and estrogen receptor b, and found that some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol competed stronger with 17b-estradiol for binding to ERb than to ERa. The ranking to the estrogenic potency of phytoestrogens for ERb in the transactivation assays was different, that was: genistein = coumestrol > zearalenone > daidzein > apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin, which was in agreement with the rank order of neuroprotective potency of isoflavones. Our study indicated that the neuroprotective capacity of daidzein was stronger than that of formononetin. Therefore, the difference in estrogenic activity of five isoflavones may contribute to the difference of neuroprotective potency. However, except difference in estrogenic activity, it might not rule out the other possibility. Therefore, further study is needed to elucidate the difference in neuroprotective potency of five isoflavones. Microglia are the major cells in the CNS that express Tolllike receptor 4 (TLR4). TLR4 functions as the signaltransducing receptor for LPS, in this study, we did not examine the effect of those five isoflavones on TLR4. However, previous studies indicate that activation of innate immunity in the CNS triggers neurodegeneration through a TLR4-dependent pathway (Lehnardt et al., 2003) and TLR4 plays a pivotal role in ischemic brain injury and functional deficits (Tang et al., 2007). Therefore, further study is needed to elucidate the effect of those isoflavones on TLR4 in the future. In conclusion, our results show for the first time that pratensein, daidzein, calycosin, formononetin and irilone from T. pratense may protect dopaminergic neurons against LPSinduced neurotoxicity and that the neuroprotective effect of them may be associated with inhibition of microglia activation and proinflammatory factors generation. Moreover, we also find that the rank order of neuroprotective potency of five isoflavones from T. pratense was: pratensein > daidzein >
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calycosin > formononetin > irilone. However, the mechanisms underlying the neuroprotective effect of pratensein, daidzein, calycosin, formononetin and irilone, except inhibition of microglia activation, might not rule out the possibility of other mechanisms. Therefore, further investigation is required to delineate the precise mechanism of action responsible for the inhibitory effect of pratensein, daidzein, calycosin, formononetin and irilone from T. pratense on microglia activation. Nevertheless, our study may provide insight into the therapeutic value of pratensein, daidzein, calycosin, formononetin and irilone in the treatment of neurodegenerative diseases including PD. References Baker, A.E., Brautigam, V.M., Watters, J.J., 2004. Estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor b. Endocrinology 145, 5021–5032. Blanchet, P.J., Fang, J., Hyland, K., Arnold, L.A., Mouradian, M.M., Chase, T.N., 1999. Short-term effects of high-dose 17b-estradiol in postmenopausal PD patients. Neurology 53, 91–95. Cadet, J.L., Brannock, C., 1998. Free radicals and the pathobiology of brain dopamine systems. Neurochem. Int. 32, 117–131. Chang, R.C., Rota, C., Glover, R.E., Mason, R.P., Hong, J.S., 2000. A novel effect of an opioid receptor antagonist, naloxone, on the production of reactive oxygen species by microglia: a study by electron paramagnetic resonance spectroscopy. Brain Res. 854, 224–229. Chao, C.C., Hu, S., Ehrlich, L., Peterson, P.K., 1995. Interleukin-1 and tumor necrosis factor-a synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors. Brain Behav. Immun. 9, 355–365. Chao, C.C., Hu, S., Moliter, T.W., Shaskan, E.G., Peterson, P.K., 1992. Activated microglia mediate cell injury via a nitric oxide mechanism. J. Immunol. 149, 2736–2741. Chen, H.Q., Jin, Z.Y., Li, G.H., 2007. Biochanin A protects dopaminergic neurons against lipopolysaccharide-induced damage through inhibition of microglia activation and proinflammatory factors generation. Neurosci. Lett. 417, 112–117. Colditz, G.A., Hankinson, S.E., Hunter, D.J., Willett, W.C., Manson, J.E., Stampfer, M.J., Hennekens, C., Rosner, B., Speizer, F.E., 1995. The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N. Engl. J. Med. 332, 1589–1593. Floyd, R.A., 1999. Neuroinflammatory processes are important in neurodegenerative diseases: a hypothesis to explain the increased formation of reactive oxygen and nitrogen species as major factors involved in neurodegenerative disease development. Free Radic. Biol. Med. 26, 1346–1355. Gao, H.M., Hong, J.S., Zhang, W.Q., Liu, B., 2002a. Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J. Neurosci. 22, 782–790. Gao, H.M., Jiang, J., Wilson, B., Zhang, W., Hong, J.S., Liu, B., 2002b. Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson’s disease. J. Neurochem. 81, 1285–1297. Gayle, D.A., Ling, Z., Tong, C., Landers, T., Lipton, J.W., Carvey, P.M., 2002. Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-a, interleukin-1b and nitric oxide. Brain Res. Dev. Brain Res. 133, 27–35. Ge´linas, S., Martinoli, M.G., 2002. Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells. J. Neurosci. Res. 70, 90–96. Giulian, D., 1999. Microglia and the immune pathology of Alzheimer disease. Am. J. Hum. Genet. 65, 13–18. Grady, D., Gebretsadik, T., Kerlikowske, K., Ernster, V., Petitti, D., 1995. Hormone replacement therapy and endometrial cancer risk: a metaanalysis. Obstet. Glynecol. 85, 304–313.
129
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131–138. Hammond, C.B., 1994. Women’s concerns with hormone replacement therapycompliance issues. Fertil. Steril. 62, 157S–160S. Hsu, J.T., Hung, H.C., Chen, C.J., Hsu, W.L., Ying, C., 1999. Effects of the dietary phytoestrogen biochanin A on cell growth in the mammary carcinoma cell line MCF-7. J. Nutr. Biochem. 10, 510–517. Jeohn, G.H., Kim, W.G., Hong, J.S., 2000. Time dependency of the action of nitric oxide in lipopolysaccharide-interferon-g-induced neuronal cell death in murine primary neuron-glia co-cultures. Brain Res. 880, 173–177. Jeohn, G.H., Kong, L.Y., Wilson, B., Hudson, P., Hong, J.S., 1998. Synergistic neurotoxic effects of combined treatments with cytokines in murine primary mixed neuron/glia cultures. J. Neuroimmunol. 85, 1–10. Kim, W.G., Mohney, R.P., Wilson, B., Jeohn, G.H., Liu, B., Hong, J.S., 2000. Regional difference in the susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J. Neurosci. 20, 6309– 6316. Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der ˚ ., 1998. Interaction of Saag, P.T., van der Burg, B., Gustafsson, J.-A estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 139, 4252–4263. Le, W., Rowe, D., Xie, W., Ortiz, I., He, Y., Appel, S.H., 2001. Microglial activation and dopaminergic cell injury: an in vitro model relevant to Parkinson’s disease. J. Neurosci. 21, 8447–8455. Lehnardt, S., Massillon, L., Follett, P., Jensen, F.E., Ratan, R., Rosenberg, P.A., Volpe, J.J., Vartanian, T., 2003. Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc. Natl. Acad. Sci. U.S.A. 100, 8514–8519. Li, F.Q., Lu, X.Z., Liang, X.B., Zhou, H.F., Xue, B., Liu, X.Y., Niu, D.B., Han, J.S., Wang, X.M., 2004. Triptolide, a Chinese herbal extract, protects dopaminergic neurons from inflammation-mediated damage through inhibition of microglial activation. J. Neuroimmunol. 148, 24–31. Liu, B., Du, L., Hong, J.S., 2000a. Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J. Pharmacol. Exp. Ther. 293, 607–617. Liu, B., Gao, H.M., Wang, J.Y., Jeohn, G.H., Cooper, C.L., Hong, J.S., 2002a. Role of nitric oxide in inflammation-mediated neurodegeneration. Ann. N.Y. Acad. Sci. 962, 256–263. Liu, B., Hong, J.S., 2003. Role of microglia in inflammation-mediated neurodegenerative diseases: mechanism and strategies for therapeutic intervention. J. Pharmacol. Exp. Ther. 304, 1–7. Liu, B., Jiang, J.W., Wilson, B.C., Du, L., Yang, S.N., Wang, J.Y., Wu, G.C., Cao, X.D., Hong, J.S., 2000b. Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide. J. Pharmacol. Exp. Ther. 295, 125–132. Liu, X., Fan, X.L., Zhao, Y., Luo, G.R., Li, X.P., Li, R., Le, W.D., 2005. Estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor-a and estrogen receptor-b in microglia. J. Neurosci. Res. 81, 653–665. Liu, Y.X., Qin, L., Wilson, B.C., An, L., Hong, J.S., Liu, B., 2002b. Inhibition by naloxone stereoisomers of b-amyloid peptide (1–42)-induced superoxide production in microglia and degeneration of cortical and mesencephalic neurons. J. Pharmacol. Exp. Ther. 302, 1212–1219. Liu, Y.X., Qin, L.Y., Li, G.R., Zhang, W., An, L.J., Liu, B., Hong, J.S., 2003. Dextromethorphan protects dopaminergic neurons against inflammationmediated degeneration through inhibition of microglial activation. J. Pharmacol. Exp. Ther. 305, 212–218. Marder, K., Tang, M.X., Alfaro, B., Mejia, H., Cote, L., Jacobs, D., Stern, Y., Sano, M., Mayeux, R., 1998. Postmenopausal estrogen use and Parkinson’s disease with and without dementia. Neurology 50, 1141–1143. McGuire, S.O., Ling, Z.D., Lipton, J.W., Sortwell, C.E., Collier, T.J., Carvey, P.M., 2002. Tumor necrosis factor-a is toxic to embryonic mesencephalic dopamine neurons. Exp. Neurol. 169, 219–230.
130
H.-Q. Chen et al. / Neuroscience Research 62 (2008) 123–130
Messina, M., Barnes, S., Setchell, K.D., 1997. Phyto-oestrogens and breast cancer [comment]. Lancet 350, 971–972. Olanow, C.W., Tatton, W.G., 1999. Etiology and pathogenesis of Parkinson’s disease. Annu. Rev. Neurosci. 22, 123–144. Ososki, A.L., Kennelly, E.J., 2003. Phytoestrogens: a review of the present state of research. Phytother. Res. 17, 845–869. Peterson, G., Barnes, S., 1991. Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multidrug resistance gene. Biochem. Biophys. Res. Commun. 179, 661–667. Sawada, H., Ibi, M., Kihara, T., Honda, K., Nakamizo, T., Kanki, R., Nakanishi, M., Sakka, N., Akaike, A., Shimohama, S., 2002. Estradiol protects dopaminergic neurons in a MPP+ Parkinson’s disease model. Neuropharmacology 42, 1056–1064. Sawada, H., Ibi, M., Kihara, T., Urushitani, M., Akaike, A., Shimohama, S., 1998. Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death. J. Neurosci. Res. 54, 707–719. Tang, S.-C., Arumugam, T.V., Xu, X., Cheng, A., Mughal, M.R., Jo, D.G., Lathia, J.D., Siler, D.A., Chigurupati, S., Ouyang, X., Magnus, T., Camandola, S., Mattson, M.P., 2007. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc. Natl. Acad. Sci. U.S.A. 104, 13798–13803. Torreilles, F., Salman-Tabcheh, S., Guerin, M., Torreilles, J., 1999. Neurodegenerative disorders: the role of peroxynitrite. Brain Res. Brain Res. Rev. 30, 153–163.
Wang, M.J., Lin, W.W., Chen, H.L., Chang, Y.H., Ou, H.C., Kuo, J.S., Hong, J.S., Jeng, K.-C.G., 2002. Silymarin protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity by inhibiting microglia activation. Eur. J. Neurosci. 16, 2103–2112. Wang, X., Chen, S., Ma, G., Ye, M., Lu, G., 2005a. Genistein protects dopaminergic neurons by inhibiting microglial activation. NeuroReport 16, 267–270. Wang, X.J., Chen, S.D., Ma, G.Z., Ye, M., Lu, G.Q., 2005b. Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration. Mech. Ageing Dev. 126, 1241–1254. Wang, X.J., Yan, Z.Q., Lu, G.Q., Stuart, S., Chen, S.D., 2007. Parkinson disease IgG and C5a-induced synergistic dopaminergic neurotoxicity: role of microglia. Neurochem. Int. 50, 39–50. Xie, Z., Wei, M., Morgan, T.E., Fabrizio, P., Han, D., Finch, C.E., Longo, V.D., 2002. Peroxynitrite mediates neurotoxicity of amyloid b-peptide 1–42- and lipopolysaccharide-activated microglia. J. Neurosci. 22, 3484–3492. Zhao, L., Chen, Q., Brinton, R.D., 2002. Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons. Exp. Biol. Med. 227, 509–519. Zhou, H.F., Liu, X.Y., Niu, D.B., Li, F.Q., He, Q.H., Wang, X.M., 2005. Triptolide protects dopaminergic neurons from inflammation-mediated damage induced by lipopolysaccharide intranigral injection. Neurobiol. Dis. 18, 441–449.
Neuroscience Research 62 (2008) 123–130 www.elsevier.com/locate/neures
Protective effect of isoflavones from Trifolium pratense on dopaminergic neurons Han-Qing Chen a,b, Xi-Jin Wang c, Zheng-Yu Jin a,b,*, Xue-Ming Xu a,b, Jian-Wei Zhao a,b, Zheng-Jun Xie a,b a
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China b School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China c Department of Neurology & Institute of Neurology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, PR China Received 13 April 2008; accepted 7 July 2008 Available online 16 July 2008
Abstract In the present study, protective effect of five isoflavones (formononetin, daidzein, pratensein, calycosin and irilone) from Trifolium pratense on lipopolysaccharide-induced dopaminergic neurodegeneration was studied for the first time. The results showed that all five isoflavones attenuated LPS-induced decrease in dopamine uptake and the number of dopaminergic neurons in a dose-dependent manner in rat mesencephalic neuron-glia cultures. Moreover, they also significantly inhibited LPS-induced activation of microglia and production of tumor necrosis factor-a, nitric oxide and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures. In addition, the rank order of protective potency of five isoflavones was: pratensein > daidzein > calycosin > formononetin > irilone. This study suggested that all five isoflavones protected dopaminergic neurons against LPS-induced injury through inhibition of microglia activation and proinflammatory factors generation. # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Isoflavone; Inflammation; Microglia; Dopaminergic neurodegeneration; Parkinson’s disease
1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by cardinal features, including resting tremor, slowing of movement, rigidity and postural instability. The major pathological change of PD is the progressive and selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Olanow and Tatton, 1999). The exact cause and underlying mechanism responsible for the progressive neurodegeneration of PD remains largely unknown. Increasing evidence has suggested that inflammation in the brain, in particular, activation of microglia, participates in the pathogenesis of PD, as well as several other neurodegenerative disorders, including Alzheimer’s disease (Giulian, 1999; Liu and Hong, 2003; Wang et al., 2005b, 2007). Microglia, the resident immune cells in the brain, serve the role of immune surveillance * Corresponding author at: School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China. Tel.: +86 510 85913299; fax: +86 510 85913299. E-mail address: [email protected] (Z.-Y. Jin).
and host defense under normal conditions (Kreutzberg, 1996). However, microglia become readily activated in response to injury, infection, or inflammation. Activated microglia secrete a variety of proinflammatory factors, including cytokines such as tumor necrosis factor-a (TNF-a) and the free radicals nitric oxide (NO) and superoxide. Many studies have demonstrated that the accumulation of these proinflammatory factors contributes to the degeneration of dopaminergic neurons (Gao et al., 2002a; Gayle et al., 2002; Kim et al., 2000; Liu et al., 2000a, 2002a). Because the midbrain region that encompasses the substantia nigra is particularly rich in microglia (Kim et al., 2000), activation of microglia and release of proinflammatory factors may be a crucial component of the degenerative process of dopaminergic neurons in PD. Hence, identification of compounds that prevent microglial activation is highly desirable in the search for therapeutic agents for inflammation-mediated neurodegenerative diseases including PD. Phytoestrogens are plant-derived compounds that structurally or functionally mimic mammalian estrogens and therefore are considered to play an important role in the prevention of cancers, heart disease, menopausal symptoms and osteoporosis
0168-0102/$ – see front matter # 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2008.07.001
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(Ososki and Kennelly, 2003). Phytoestrogens include isoflavones, coumestans, and lignans. Isoflavones are the most important type of phytoestrogens found in legume plants such as Trifolium pratense and Glycine max. Preliminary clinical reports suggest a potential protective effect of estrogen replacement therapy (ERT) on the onset and progression of both PD and PD-related dementia (Marder et al., 1998; Blanchet et al., 1999). Furthermore, in vitro studies have demonstrated that estrogens protect dopaminergic neurons against inflammation-induced neurotoxicity (Sawada et al., 1998, 2002; Liu et al., 2005). Despite the beneficial effect of ERT on PD, estrogen use in postmenopausal women is associated with increased risks of uterine cancer and potentially an increase in neoplasms of the breast (Hammond, 1994), especially after long-term use (Grady et al., 1995; Colditz et al., 1995). In contrast, several reports indicate that phytoestrogens do not promote neoplasms of the breast and uterus, but instead reduce the risk of developing several types of cancer, most notably breast cancer (Peterson and Barnes, 1991; Hsu et al., 1999; Messina et al., 1997). Therefore, phytoestrogens receive more attention in recent years and have been shown to provide neuroprotection in vitro. Zhao et al. (2002) report that phytoestrogens can exert neuroprotective effect in cultured hippocampal neurons. Similarly, Ge´linas and Martinoli (2002) also demonstrate that phytoestrogens have neuroprotective effect on MPP+-induced cytotoxicity in neuronal PC12 cells. However, very limited investigations have explored the effect of isoflavones from T. pratense on inflammation-mediated dopaminergic neurodegeneration. Recently, Wang et al. (2005a) report that genistein, the primary soybean isoflavone, protects dopaminergic neurons by inhibiting microglial activation. Our previous study also demonstrates that biochanin A, one of the predominant isoflavones in T. pratense, protects dopaminergic neurons against lipopolysaccharide (LPS)-induced damage (Chen et al., 2007). However, except for biochanin A and genistein in T. pratense, there are other isoflavones such as pratensein, calycosin, daidzein, irilone, and formononetin, the effects of them on inflammation-induced dopaminergic neurodegeneration remain unknown. In this study, using rat primary mesencephalic neuron-glia cultures and microglia-enriched cultures, we further investigate the effects of five isoflavones (formononetin, pratensein, daidzein, calycosin, and irilone) from T. pratense on LPSinduced dopaminergic neurodegeneration. We find that all these isoflavones from T. pratense protect dopaminergic neurons from LPS-induced damage to a certain extent. Moreover, the rank order of protective capacities of them on dopaminergic neurons is: pratensein > daidzein > calycosin > formononetin > irilone.
O111:B4) and the monoclonal antibody against tyrosine hydroxylase (TH) were purchased from Sigma (St. Louis, MO). [3H]dopamine (DA) (30 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). The monoclonal antibodies against the CR3 complement receptor (OX-42) and glial fibrillary acidic protein (GFAP) were purchased from chemicon (Temecula, CA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
2.2. Primary rat ventral mesencephalic neuron-glia cultures Primary rat ventral mesencephalic neuron-glia cultures were prepared following previously described protocol (Gao et al., 2002b; Liu et al., 2000a) with some modifications. Briefly, ventral mesencephalic tissues were dissected from embryonic day 14 Sprague–Dawley rats under pentobarbital anaesthesia and dissociated by a mild mechanical trituration. According to the experimental arrangement, dissociated cells were seeded to 24-well (5 105/ well) culture plates precoated with poly-D-lysine (20 mg/ml). The cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% heat-inactivated horse serum (HS), 1 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM non-essential amino acids, 50 U/ml penicillin, and 50 mg/ml streptomycin. After the initial seeding for 3 days, the cultures were replenished with 0.5 ml of fresh medium. Seven-day-old cultures were used for treatment. During the course of the experiment, all animals were treated in strict accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals, and all efforts were made to minimize the number of animals and their suffering.
2.3. Primary microglia-enriched cultures Rat microglia-enriched cultures were prepared according to previously described protocol (Liu et al., 2000a). Briefly, whole brains of 1-day-old Sprague–Dawley rats, with the blood vessels and meninges removed, were triturated. Cells (5 107) were seeded in 150 cm2 culture flasks in 30 ml of a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM non-essential amino acids, 50 U/ml penicillin, and 50 mg/ml streptomycin. The cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. Medium (30 ml/flask) was replenished 1 and 4 days after the initial seeding and changed thereafter every third day. On reaching confluence (day 14), microglia were shaken off (180 rpm for 5 h on an orbital shaker), pelleted at 800 g for 10 min, resuspended in fresh medium, and plated (105 cells/well) into 24-well culture plates. Twenty-four hours later, cells were ready for treatment. The enriched microglia were found to be >98% pure as determined by OX-42- and GFAPimmunoreactive (IR).
2.4. Immunocytochemical staining Dopaminergic neurons were recognized with anti-TH antibody and microglia were detected with OX-42 antibody, which recognizes the CR3 complement receptor as described previously (Gao et al., 2002b; Liu et al., 2000a). Briefly, cells were fixed with 3.7% paraformaldehyde in PBS at room temperature for 20 min and then treated with 1% hydrogen peroxide for 10 min. After incubation with blocking solution for 40 min, the cells were incubated with primary antibodies at appropriate concentrations at 4 8C overnight. Afterward, the cells were incubated with appropriate biotinylated secondary antibody for 2 h followed by the Vectastain ABC reagents for 40 min. Color was developed with 3,30 -diaminobenzidine. For visual counting of TH-, OX-42-, or GFAP-IR neurons, 10 representative areas per well of the 24-well plates were counted under the microscope at 100 magnification. Counting was always performed by two to three individuals in a blind manner.
2.5. Uptake assay for [3H]dopamine (DA) 2. Materials and methods 2.1. Reagents Formononetin, pratensein, daidzein, calycosin and irilone were isolated from T. pratense and the purities of them were no less than 95%. LPS (Escherichia coli
Uptake assay was performed using previously described methods (Gao et al., 2002b; Liu et al., 2000a). Briefly, cells in each well were washed with Krebs-Ringer buffer (KRB, 16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4) twice. The cells were then incubated with 1 mM [3H]DA in KRB at 37 8C
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for 15 min. Non-specific uptake for dopamine was determined in parallel wells receiving both tritiated dopamine and 10 mM mazindol, an inhibitor of neuronal high-affinity dopamine uptake. Afterward, the cells were washed with ice-cold KRB three times and solublized in 1 N NaOH and radioactivity was determined by a liquid scintillation counter. The specific uptake for dopamine was calculated by subtracting the amount of radioactivity observed in the presence of mazindol from that observed in the absence of mazindol.
2.6. Nitrite and TNF-a assays The accumulation of nitrite in the culture supernatant, an indicator of the production of NO, was determined with a colorimetric assay with the Griess reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4) (Green et al., 1982). Equal volumes of culture supernatant and Griess reagent were mixed, the mixture was incubated at room temperature for 10 min, and absorbance at 540 nm was determined with a microplate reader (BIORAD Laboratories, CA). The concentrations of nitrite in the samples were determined from a sodium nitrite standard curve (Liu et al., 2000a). The level of tumor necrosis factor-a (TNF-a) in the culture medium was measured with a rat TNF-a enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) (Liu et al., 2000a).
2.7. Superoxide assay The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c as previously described (Gao et al., 2002b; Liu et al., 2000a). Briefly, mesencephalic neuron-glia or microglia-enriched cultures grown in 96-well culture plates were treated with vehicle or LPS in treatment medium containing phenol red-free MEM (150 ml/ well), with or without 600 U/ml SOD. To each well, 50 ml of ferricytochrome C (100 mM) in treatment medium was added. The cultures were then incubated at 37 8C for 30 min. Afterwards, the absorbance at 550 nm was recorded with a SpectraMax Plus microplate spectrophotometer. To determine the effect of isoflavones on superoxide release, cultures were preincubated with isoflavones at 37 8C for 30 min prior to the addition of LPS.
2.8. Statistical analysis The data were expressed as the mean S.E.M. Statistical significance was assessed with one-way analysis of variance (ANOVA), followed by the Newman–keuls test (SAS software, Version 8.0). A value of P < 0.05 was considered statistically significant.
3. Results 3.1. Different isoflavones from T. pratense protected dopaminergic neurons against LPS-induced neurotoxicity in a dose-dependent manner in mesencephalic neuron-glia cultures In preliminary experiments, through MTT assay, we found that different isoflavones from T. pratense (24 h) induced cell death at a concentration of 50 mM in rat mesencephalic neuronglia cultures, but at 10 mM they showed no obvious toxicity to primary cells (data not shown). Therefore, in order to avoid the cytotoxicity of different isoflavones, their concentrations used in this study were 0.25–2.5 mM. Mesencephalic neuron-glia cultures were pretreated with vehicle or 0.25–2.5 mM different isoflavones for 30 min before treatment with 10 ng/ml LPS. Seven days later, the degeneration of dopaminergic neurons was assessed by [3H]DA uptake or TH immunostaining. [3H]DA uptake assay indicated that LPS treatment decreased the uptake capacity to 37.2% of that of vehicle-treated control
Fig. 1. Effect of different isoflavones from Trifolium pratense on DA uptake in primary mesencephalic neuron-glia cultures. (A) Isoflavones (0.25 mM, 1.0 mM, and 2.5 mM) from Trifolium pratense attenuated LPS-induced decrease in DA uptake in primary mesencephalic neuron-glia cultures; (B) Isoflavones (2.5 mM) from Trifolium pratense alone did not affect DA uptake in primary mesencephalic neuron-glia cultures. Rat ventral mesencephalic neuron-glia cultures were pretreated with vehicle (Veh) or indicated concentrations of isoflavones for 30 min before treatment with 10 ng/ml LPS for 7 days. Results are mean S.E.M. of five experiments performed triplicate and are expressed as a percentage of vehicletreated control culture. *P < 0.05 compared with LPS-treated culture. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
culture (Fig. 1A). However, all tested isoflavones significantly attenuated the LPS-induced decrease in DA uptake in a dosedependent manner. The most effective concentration was found at 2.5 mM. At the concentration of 2.5 mM, DA uptake of cultures pretreatment with pratensein, daidzein, calycosin, formononetin, and irilone before treatment with LPS were 80.2%, 70.1%, 65.7%, 60.7%, and 56.1% of that of control cultures, respectively (Fig. 1A). However, DA uptake of cultures treated with any isoflavone alone did not differ significantly from that of control cultures (Fig. 1B). In addition to DA uptake, counting the number of TH-IR neurons in the cultures revealed that LPS treatment reduced the number of TH-IR neurons by 53.3% compared with vehicletreated control cultures (Fig. 2A). However, all tested isoflavones significantly attenuated LPS-induced reduction in the number of TH-IR neurons in a dose-dependent manner. The number of TH-IR neurons of cultures pretreated with pratensein, daidzein, calycosin, formononetin and irilone at the concentration of 2.5 mM before treatment with LPS were 81.1%, 71.3%, 66.8%, 61.8%, and 57.1% of that of control cultures, respectively (Fig. 2A). But treatment of cultures with 2.5 mM any isoflavone alone did not have significant effect on the number of the TH-IR neurons (Fig. 2B). 3.2. Different isoflavones from T. pratense inhibited LPSinduced activation of microglia and release of proinflammatory factors in mesencephalic neuron-glia cultures and microglia-enriched cultures To elucidate the underlying mechanism of the neuroprotective activity of different isoflavones from T. pratense, we
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Fig. 2. Effect of different isoflavones on the number of DA neurons in primary mesencephalic neuron-glia cultures. (A) Isoflavones (0.25 mM, 1.0 mM, and 2.5 mM) from Trifolium pratense attenuated LPS-induced decrease in the number of DA neurons in primary mesencephalic neuron-glia cultures; (B) Isoflavones (2.5 mM) from Trifolium pratense alone did not affect the number of DA neurons in primary mesencephalic neuron-glia cultures. Rat ventral mesencephalic neuron-glia cultures were pretreated with vehicle (Veh) or indicated concentrations of isoflavones for 30 min before treatment with 10 ng/ml LPS for 7 days. Results are mean S.E.M. of five experiments performed triplicate and are expressed as a percentage of vehicle-treated control culture. *P < 0.05 compared with LPS-treated cultures. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
investigated the effect of different isoflavones on the LPSinduced microglial activation. Culture supernatants were taken at optimal time points for determining the levels of some key proinflammatory factors (TNF-a, NO, and superoxide) and cells were fixed and immunostained for specific markers of microglia. As shown in Fig. 3A, after 24 h exposure to 10 ng/ml LPS, the number of OX-42-IR microglia in the cultures significantly increased compared with vehicle-treated control cultures. All tested isoflavones effectively prevented the increase of OX-42-IR microglia in LPS-exposed cultures. Pretreatment with pratensein, daidzein, calycosin, formononetin, and irilone at the concentration of 2.5 mM before treatment with LPS reduced the number of OX-42-IR microglia in the cultures to 48.7%, 54.7%, 59.5%, 63.6%, and 67.9% of that of LPS-treated culture, respectively. But different isoflavone (2.5 mM) alone had no effect on their number. In addition to preventing the activation of microglia, all tested isoflavones from T. pratense significantly inhibited the production of proinflammatory factors in neuron-glia cultures. As shown in Fig. 3B–D, significant levels of TNF-a, NO and superoxide were observed in neuron-glia cultures exposed to LPS treatment. The release of TNF-a, NO and superoxide was inhibited by pretreatment with any isoflavone in a dosedependent manner, and rank order of action was: pratensein > daidzein > calycosin > formononetin > irilone. In microglia-enriched cultures, we also determined the effect of different isoflavones from T. pratense on the LPSinduced production of TNF-a, NO and superoxide. As shown in Fig. 4A–C, consistent with the results from neuron-glia
Fig. 3. Effect of different isoflavones from Trifolium pratense on microglia activation and the production of proinflammatory factors in primary mesencephalic neuron-glia cultures. (A) Isoflavones (2.5 mM) from Trifolium pratense inhibited LPS-induced microglia activation in primary mesencephalic neuron-glia cultures; (B–D) Isoflavones (0.25 mM, 1.0 mM, and 2.5 mM) from Trifolium pratense inhibited LPS-induced proinflammatory factor (B: TNF-a; C: NO; D: Superoxide) generation in primary mesencephalic neuron-glia cultures. Rat ventral mesencephalic neuron-glia cultures were pretreated with vehicle (Veh) or isoflavones (2.5 mM) for 30 min before treatment with 10 ng/ml LPS for 24 h. Microglial activation was evaluated by quantification of OX-42-IR microglia. Neuron-glia cultures were pretreated with vehicle or indicated concentrations of isoflavones for 30 min before treatment with 10 ng/ml LPS. Supernatants were removed for the measurement of TNF-a at 6 h, and for NO at 24 h. Superoxide assay was performed as described in Section 2. Results are expressed as a percentage of LPS-treated cultures and are mean S.E.M. of five experiments. *P < 0.05 compared with LPS-treated cultures. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
cultures, pretreatment with any isoflavone (2.5 mM) significantly inhibited LPS-induced production of TNF-a, NO and superoxide, and rank order of action was: pratensein > daidzein > calycosin > formononetin > irilone. But different isoflavone (2.5 mM) alone had no significant effect on production of these proinflammatory factors (data not shown).
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Fig. 4. Effect of different isoflavones from Trifolium pratense on the production of proinflammatory factors in primary microglia-enriched cultures. (A–C) Isoflavones (2.5 mM) inhibited the production of proinflammatory factors (A: TNF-a; B: NO; C: Superoxide) in primary microglia-enriched cultures. Microglia-enriched cultures were pretreated with vehicle or different isoflavones (2.5 mM) for 30 min before treatment with 10 ng/ml LPS. Supernatants were removed for the measurement of TNF-a at 6 h, and for NO at 24 h. Superoxide assay was performed as described in Section 2. Results are expressed as a percentage of LPS-treated cultures and are mean S.E.M. of five experiments. *P < 0.05 compared with LPS-treated culture. Pratensein, Pra; Daidzein, Daid; Calycosin, Cal; Formononetin, For; Irilone, Iri.
4. Discussion T. pratense is an important kind of legume plant which is widely grown in China and other countries in the world. T. pratense contains isoflavones, proteins, amino acids, sugars, vitamins and so on. The content of isoflavone in T. pratense is as high as 1–2% on the basis of dry weight. Biochanin A, formononetin, pratensein, genistein, daidzein, calycosin and irilone are seven isoflavones in T. pratense. Previous studies have showed that genistein and biochanin A protect dopaminergic neurons against LPS-induced injury (Wang et al., 2005a; Chen et al., 2007). However, except for genistein and biochanin A, there are other five isoflavones in T. pratense. To the best of our knowledge, the effects of those five isoflavones on dopaminergic neurons have not been reported. In this study, using rat mesencephalic neuron-glia cultures as a model and LPS as a tool, we explored for the first time the effects of five isoflavones from T. pratense on LPS-induced dopaminergic neurodegeneration. Our results indicated that pratensein, daidzein, calycosin, formononetin and irilone could
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effectively protect dopaminergic neurons against LPS-induced neurotoxicity by measuring the [3H]DA uptake and counting TH-immunoreactive cells. Furthermore, these five isoflavones significantly inhibited the activation of microglia and release of proinflammatory factors, including NO, TNF-a, and superoxide, in neuron-glia cultures and in microglia-enriched cultures exposed to LPS treatment. Moreover, the rank order of neuroprotective efficacy of these five isoflavones was: pratensein > daidzein > calycosin > formononetin > irilone. Microglia, the resident immune cells in the central nervous system, play a role in immune surveillance under normal condition (Kreutzberg, 1996). However, in response to injury or infection, microglia become readily activated and consequently release a variety of factors, including cytokines such as TNF-a and IL-b, free radicals such as NO and superoxide. These factors are believed to contribute to microglia-mediated neurodegeneration (Chao et al., 1992; Jeohn et al., 1998; Le et al., 2001). Many studies indicate that neuroprotection can be obtained by inhibiting microglia activation in vivo and in vitro. Liu et al. (2000a) have shown that naloxone protects dopaminergic neurons against inflammatory damage through inhibition of microglia activation. Similarly, Wang et al. (2002) have demonstrated that silymarin, a polyphenol flavonoid derived from milk thistle, protects dopaminergic neurons against LPS-induced neurotoxicity by inhibiting microglia activation in mesencephalic neuron-glia cultures. Moreover, Liu et al. (2003) have reported that dextromethorphan protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation. Li et al. (2004) and Zhou et al. (2005) have shown that triptolide, a Chinese herbal extract, protects dopaminergic neurons from inflammation-mediated damage through inhibition of microglial activation in vitro and in vivo. Recently, Wang et al. (2005a) reported that genistein, the primary soybean isoflavone, protected dopaminergic neurons by inhibiting microglial activation. Our previous study also indicated that biochanin A, one of the predominant isoflavones in T. pratense, protected dopaminergic neurons against LPS-induced damage through inhibition of microglia activation (Chen et al., 2007). These observations suggest that the agents which inhibit microglia activation will provide neuroprotective effects. In agreement with the above reports, in the present study, we first found that pratensein, daidzein, calycosin, formononetin and irilone from T. pratense also effectively inhibited microglia activation and reduced the production of TNF-a, NO, and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures exposed to LPS treatment. These findings indicated that the mechanism of action underlying the neuroprotective role of these five isoflavones from T. pratense, at least partially, is attributed to the inhibition of microglia activation. Among the neurotoxic factors secreted by activated microglia, the consequences of overproduction of TNF-a, NO, and superoxide free radicals have been relatively well studied. McGuire et al. (2002) demonstrated that TNF-a was capable of inducing the death of cultured dopaminergic neurons. Excessive accumulation of NO has long been known
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to be toxic to neurons (Chao et al., 1992; Jeohn et al., 2000; Liu et al., 2002a). The overproduction of free radicals is especially deleterious to neurons (Cadet and Brannock, 1998; Floyd, 1999). Moreover, when NO meets with superoxide, a more deadly nitrite, peroxynitrite, is formed, which is a potent oxidant and nitrating agent capable of attacking and modifying proteins, lipids and DNA as well as depleting antioxidant defenses (Torreilles et al., 1999). In fact, a recent study has identified peroxynitrite as a key mediator of neurotoxicity induced by LPS-activated microglia (Xie et al., 2002). In addition, several studies have shown that factors produced by activated microglia may work in concert to induce neurodegeneration. For example, Chao et al. (1995) reported that the combination of TNF-a and IL-1b, but not either cytokine alone, induced the degeneration of cortical neurons. Similarly, Jeohn et al. (1998) also demonstrated that the combination of TNF-a, IL-1b, and interferon-g work in synergy to induce degeneration of cortical neurons. Therefore, neurodegeneration in such diseases as PD is, at least in part, induced by the combined impact of multiple factors generated from activated microglia. Hence, the agents that are capable of inhibiting the production of multiple factors such as NO, TNF-a, and superoxide in activated microglia may be highly relevant to the development of potential therapeutic agents. In this study, we have demonstrated that the production of NO, TNF-a, and superoxide by LPS-activated microglia is significantly inhibited by pratensein, daidzein, calycosin, formononetin and irilone and the inhibition of the production of these factors confers significant protection to dopaminergic neurons against inflammation-mediated degeneration. Consistent with our previous study on biochanin A (Chen et al., 2007), in this study, we also found that pratensein, daidzein, calycosin, formononetin and irilone from T. pratense were significantly more potent in inhibiting LPS-induced superoxide production than the production of NO and TNF-a. This result implied that LPS-induced superoxide generation may play a more critical role than other microglia-originated factors in the induction of dopaminergic neurodegeneration. In fact, in mesencephalic neuron-glia cultures stimulated with very low concentrations of LPS (<1 ng/ml), production of superoxide, but not NO and TNF-a, seemed to mediate LPSinduced dopaminergic neurotoxicity (Gao et al., 2002a). It is possible that agents having a preferential inhibitory activity toward free radical generation may prove to confer potent neuroprotection against inflammation-mediated degeneration. The inhibitory and neuroprotective profiles of pratensein, daidzein, calycosin, formononetin and irilone seem to be similar to that of naloxone stereoisomers. Naloxone is more effective in the inhibition of superoxide generation than in that of TNF-a, NO, or IL-1b (Chang et al., 2000; Liu et al., 2000a, 2002b). The neuroprotective effect of naloxone has been observed in both in vitro and in vivo models of inflammationmediated neurodegeneration (Liu et al., 2000a,b, 2002b). Hence, it is important to determine whether the neuroprotective effects of pratensein, daidzein, calycosin, formononetin and irilone can be observed in animal models of inflammationmediated neurodegenerative diseases including PD.
In this study, we also found that neuroprotective capacity of five isoflavones was different, and the rank order of protective potency was: pratensein > daidzein > calycosin > formononetin > irilone. The reason for differences in neuroprotective potency of five isoflavones is not yet clear, but it may be related to their estrogenic activity. Previous study has indicated that estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor (ER)-a and estrogen receptor-b in microglia (Liu et al., 2005). Moreover, Baker et al. (2004) have reported that estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor b. Due to the similarities of isoflavones and estrogen in structure, isoflavones may exhibit neuroprotective effect possibly by interactions with estrogen receptors in microglia. Phytoestrogens exert estrogenic activity by binding to estrogen receptors. The binding affinity of isoflavones to estrogen receptor may be used to evaluate the estrogenic activity of different isoflavones. Kuiper et al. (1998) compared the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with estrogen receptor a and estrogen receptor b, and found that some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol competed stronger with 17b-estradiol for binding to ERb than to ERa. The ranking to the estrogenic potency of phytoestrogens for ERb in the transactivation assays was different, that was: genistein = coumestrol > zearalenone > daidzein > apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin, which was in agreement with the rank order of neuroprotective potency of isoflavones. Our study indicated that the neuroprotective capacity of daidzein was stronger than that of formononetin. Therefore, the difference in estrogenic activity of five isoflavones may contribute to the difference of neuroprotective potency. However, except difference in estrogenic activity, it might not rule out the other possibility. Therefore, further study is needed to elucidate the difference in neuroprotective potency of five isoflavones. Microglia are the major cells in the CNS that express Tolllike receptor 4 (TLR4). TLR4 functions as the signaltransducing receptor for LPS, in this study, we did not examine the effect of those five isoflavones on TLR4. However, previous studies indicate that activation of innate immunity in the CNS triggers neurodegeneration through a TLR4-dependent pathway (Lehnardt et al., 2003) and TLR4 plays a pivotal role in ischemic brain injury and functional deficits (Tang et al., 2007). Therefore, further study is needed to elucidate the effect of those isoflavones on TLR4 in the future. In conclusion, our results show for the first time that pratensein, daidzein, calycosin, formononetin and irilone from T. pratense may protect dopaminergic neurons against LPSinduced neurotoxicity and that the neuroprotective effect of them may be associated with inhibition of microglia activation and proinflammatory factors generation. Moreover, we also find that the rank order of neuroprotective potency of five isoflavones from T. pratense was: pratensein > daidzein >
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calycosin > formononetin > irilone. However, the mechanisms underlying the neuroprotective effect of pratensein, daidzein, calycosin, formononetin and irilone, except inhibition of microglia activation, might not rule out the possibility of other mechanisms. Therefore, further investigation is required to delineate the precise mechanism of action responsible for the inhibitory effect of pratensein, daidzein, calycosin, formononetin and irilone from T. pratense on microglia activation. Nevertheless, our study may provide insight into the therapeutic value of pratensein, daidzein, calycosin, formononetin and irilone in the treatment of neurodegenerative diseases including PD. References Baker, A.E., Brautigam, V.M., Watters, J.J., 2004. Estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor b. Endocrinology 145, 5021–5032. Blanchet, P.J., Fang, J., Hyland, K., Arnold, L.A., Mouradian, M.M., Chase, T.N., 1999. Short-term effects of high-dose 17b-estradiol in postmenopausal PD patients. Neurology 53, 91–95. Cadet, J.L., Brannock, C., 1998. Free radicals and the pathobiology of brain dopamine systems. Neurochem. Int. 32, 117–131. Chang, R.C., Rota, C., Glover, R.E., Mason, R.P., Hong, J.S., 2000. A novel effect of an opioid receptor antagonist, naloxone, on the production of reactive oxygen species by microglia: a study by electron paramagnetic resonance spectroscopy. Brain Res. 854, 224–229. Chao, C.C., Hu, S., Ehrlich, L., Peterson, P.K., 1995. Interleukin-1 and tumor necrosis factor-a synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors. Brain Behav. Immun. 9, 355–365. Chao, C.C., Hu, S., Moliter, T.W., Shaskan, E.G., Peterson, P.K., 1992. Activated microglia mediate cell injury via a nitric oxide mechanism. J. Immunol. 149, 2736–2741. Chen, H.Q., Jin, Z.Y., Li, G.H., 2007. Biochanin A protects dopaminergic neurons against lipopolysaccharide-induced damage through inhibition of microglia activation and proinflammatory factors generation. Neurosci. Lett. 417, 112–117. Colditz, G.A., Hankinson, S.E., Hunter, D.J., Willett, W.C., Manson, J.E., Stampfer, M.J., Hennekens, C., Rosner, B., Speizer, F.E., 1995. The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N. Engl. J. Med. 332, 1589–1593. Floyd, R.A., 1999. Neuroinflammatory processes are important in neurodegenerative diseases: a hypothesis to explain the increased formation of reactive oxygen and nitrogen species as major factors involved in neurodegenerative disease development. Free Radic. Biol. Med. 26, 1346–1355. Gao, H.M., Hong, J.S., Zhang, W.Q., Liu, B., 2002a. Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J. Neurosci. 22, 782–790. Gao, H.M., Jiang, J., Wilson, B., Zhang, W., Hong, J.S., Liu, B., 2002b. Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson’s disease. J. Neurochem. 81, 1285–1297. Gayle, D.A., Ling, Z., Tong, C., Landers, T., Lipton, J.W., Carvey, P.M., 2002. Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-a, interleukin-1b and nitric oxide. Brain Res. Dev. Brain Res. 133, 27–35. Ge´linas, S., Martinoli, M.G., 2002. Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells. J. Neurosci. Res. 70, 90–96. Giulian, D., 1999. Microglia and the immune pathology of Alzheimer disease. Am. J. Hum. Genet. 65, 13–18. Grady, D., Gebretsadik, T., Kerlikowske, K., Ernster, V., Petitti, D., 1995. Hormone replacement therapy and endometrial cancer risk: a metaanalysis. Obstet. Glynecol. 85, 304–313.
129
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131–138. Hammond, C.B., 1994. Women’s concerns with hormone replacement therapycompliance issues. Fertil. Steril. 62, 157S–160S. Hsu, J.T., Hung, H.C., Chen, C.J., Hsu, W.L., Ying, C., 1999. Effects of the dietary phytoestrogen biochanin A on cell growth in the mammary carcinoma cell line MCF-7. J. Nutr. Biochem. 10, 510–517. Jeohn, G.H., Kim, W.G., Hong, J.S., 2000. Time dependency of the action of nitric oxide in lipopolysaccharide-interferon-g-induced neuronal cell death in murine primary neuron-glia co-cultures. Brain Res. 880, 173–177. Jeohn, G.H., Kong, L.Y., Wilson, B., Hudson, P., Hong, J.S., 1998. Synergistic neurotoxic effects of combined treatments with cytokines in murine primary mixed neuron/glia cultures. J. Neuroimmunol. 85, 1–10. Kim, W.G., Mohney, R.P., Wilson, B., Jeohn, G.H., Liu, B., Hong, J.S., 2000. Regional difference in the susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J. Neurosci. 20, 6309– 6316. Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Kuiper, G.G.J.M., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der ˚ ., 1998. Interaction of Saag, P.T., van der Burg, B., Gustafsson, J.-A estrogenic chemicals and phytoestrogens with estrogen receptor b. Endocrinology 139, 4252–4263. Le, W., Rowe, D., Xie, W., Ortiz, I., He, Y., Appel, S.H., 2001. Microglial activation and dopaminergic cell injury: an in vitro model relevant to Parkinson’s disease. J. Neurosci. 21, 8447–8455. Lehnardt, S., Massillon, L., Follett, P., Jensen, F.E., Ratan, R., Rosenberg, P.A., Volpe, J.J., Vartanian, T., 2003. Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc. Natl. Acad. Sci. U.S.A. 100, 8514–8519. Li, F.Q., Lu, X.Z., Liang, X.B., Zhou, H.F., Xue, B., Liu, X.Y., Niu, D.B., Han, J.S., Wang, X.M., 2004. Triptolide, a Chinese herbal extract, protects dopaminergic neurons from inflammation-mediated damage through inhibition of microglial activation. J. Neuroimmunol. 148, 24–31. Liu, B., Du, L., Hong, J.S., 2000a. Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J. Pharmacol. Exp. Ther. 293, 607–617. Liu, B., Gao, H.M., Wang, J.Y., Jeohn, G.H., Cooper, C.L., Hong, J.S., 2002a. Role of nitric oxide in inflammation-mediated neurodegeneration. Ann. N.Y. Acad. Sci. 962, 256–263. Liu, B., Hong, J.S., 2003. Role of microglia in inflammation-mediated neurodegenerative diseases: mechanism and strategies for therapeutic intervention. J. Pharmacol. Exp. Ther. 304, 1–7. Liu, B., Jiang, J.W., Wilson, B.C., Du, L., Yang, S.N., Wang, J.Y., Wu, G.C., Cao, X.D., Hong, J.S., 2000b. Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide. J. Pharmacol. Exp. Ther. 295, 125–132. Liu, X., Fan, X.L., Zhao, Y., Luo, G.R., Li, X.P., Li, R., Le, W.D., 2005. Estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor-a and estrogen receptor-b in microglia. J. Neurosci. Res. 81, 653–665. Liu, Y.X., Qin, L., Wilson, B.C., An, L., Hong, J.S., Liu, B., 2002b. Inhibition by naloxone stereoisomers of b-amyloid peptide (1–42)-induced superoxide production in microglia and degeneration of cortical and mesencephalic neurons. J. Pharmacol. Exp. Ther. 302, 1212–1219. Liu, Y.X., Qin, L.Y., Li, G.R., Zhang, W., An, L.J., Liu, B., Hong, J.S., 2003. Dextromethorphan protects dopaminergic neurons against inflammationmediated degeneration through inhibition of microglial activation. J. Pharmacol. Exp. Ther. 305, 212–218. Marder, K., Tang, M.X., Alfaro, B., Mejia, H., Cote, L., Jacobs, D., Stern, Y., Sano, M., Mayeux, R., 1998. Postmenopausal estrogen use and Parkinson’s disease with and without dementia. Neurology 50, 1141–1143. McGuire, S.O., Ling, Z.D., Lipton, J.W., Sortwell, C.E., Collier, T.J., Carvey, P.M., 2002. Tumor necrosis factor-a is toxic to embryonic mesencephalic dopamine neurons. Exp. Neurol. 169, 219–230.
130
H.-Q. Chen et al. / Neuroscience Research 62 (2008) 123–130
Messina, M., Barnes, S., Setchell, K.D., 1997. Phyto-oestrogens and breast cancer [comment]. Lancet 350, 971–972. Olanow, C.W., Tatton, W.G., 1999. Etiology and pathogenesis of Parkinson’s disease. Annu. Rev. Neurosci. 22, 123–144. Ososki, A.L., Kennelly, E.J., 2003. Phytoestrogens: a review of the present state of research. Phytother. Res. 17, 845–869. Peterson, G., Barnes, S., 1991. Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multidrug resistance gene. Biochem. Biophys. Res. Commun. 179, 661–667. Sawada, H., Ibi, M., Kihara, T., Honda, K., Nakamizo, T., Kanki, R., Nakanishi, M., Sakka, N., Akaike, A., Shimohama, S., 2002. Estradiol protects dopaminergic neurons in a MPP+ Parkinson’s disease model. Neuropharmacology 42, 1056–1064. Sawada, H., Ibi, M., Kihara, T., Urushitani, M., Akaike, A., Shimohama, S., 1998. Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death. J. Neurosci. Res. 54, 707–719. Tang, S.-C., Arumugam, T.V., Xu, X., Cheng, A., Mughal, M.R., Jo, D.G., Lathia, J.D., Siler, D.A., Chigurupati, S., Ouyang, X., Magnus, T., Camandola, S., Mattson, M.P., 2007. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc. Natl. Acad. Sci. U.S.A. 104, 13798–13803. Torreilles, F., Salman-Tabcheh, S., Guerin, M., Torreilles, J., 1999. Neurodegenerative disorders: the role of peroxynitrite. Brain Res. Brain Res. Rev. 30, 153–163.
Wang, M.J., Lin, W.W., Chen, H.L., Chang, Y.H., Ou, H.C., Kuo, J.S., Hong, J.S., Jeng, K.-C.G., 2002. Silymarin protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity by inhibiting microglia activation. Eur. J. Neurosci. 16, 2103–2112. Wang, X., Chen, S., Ma, G., Ye, M., Lu, G., 2005a. Genistein protects dopaminergic neurons by inhibiting microglial activation. NeuroReport 16, 267–270. Wang, X.J., Chen, S.D., Ma, G.Z., Ye, M., Lu, G.Q., 2005b. Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration. Mech. Ageing Dev. 126, 1241–1254. Wang, X.J., Yan, Z.Q., Lu, G.Q., Stuart, S., Chen, S.D., 2007. Parkinson disease IgG and C5a-induced synergistic dopaminergic neurotoxicity: role of microglia. Neurochem. Int. 50, 39–50. Xie, Z., Wei, M., Morgan, T.E., Fabrizio, P., Han, D., Finch, C.E., Longo, V.D., 2002. Peroxynitrite mediates neurotoxicity of amyloid b-peptide 1–42- and lipopolysaccharide-activated microglia. J. Neurosci. 22, 3484–3492. Zhao, L., Chen, Q., Brinton, R.D., 2002. Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons. Exp. Biol. Med. 227, 509–519. Zhou, H.F., Liu, X.Y., Niu, D.B., Li, F.Q., He, Q.H., Wang, X.M., 2005. Triptolide protects dopaminergic neurons from inflammation-mediated damage induced by lipopolysaccharide intranigral injection. Neurobiol. Dis. 18, 441–449.