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. Author manuscript; available in PMC: 2013 Mar 14.
Published in final edited form as: Neurotox Res. 2012 May 10;23(2):145–153. doi: 10.1007/s12640-012-9328-5

Anti-Inflammatory Role of the Isoflavone Diadzein in Lipopolysaccharide-Stimulated Microglia: Implications for Parkinson’s Disease

Shankar J Chinta 1, Abirami Ganesan 1, Pedro Reis-Rodrigues 1, Gordon J Lithgow 1, Julie K Andersen 1,✉
PMCID: PMC3597389  NIHMSID: NIHMS447655  PMID: 22573480

Abstract

Microglial activation and subsequent release of toxic pro-inflammatory factors are believed to play an important role in neuronal cell death associated with Parkinson’s disease (PD). Compounds that inhibit microglia activation and suppress pro-inflammatory factor release have been reported to have neuroprotective effects in animal models of PD. In this study, we tested whether diadzein, a natural isoflavone found in soybean, attenuated lipopolysaccharide (LPS)-induced release of inflammatory mediators in BV-2, a murine microglial cell line. Diadzein pretreatment was found to significantly suppress the production of the pro-inflammatory factors nitric oxide and IL-6 as well as their mRNA expression in conjunction with reductions in ROS production, p38 MAPK phosphorylation, and NF-κB activation. Furthermore, transfer of conditioned media (CM) from BV-2 cells pretreated with diadzein resulted in a significantly reduction in dopaminergic neurotoxicity compared with CM from microglia stimulated with LPS alone. Together, our results suggest that diadzein’s neuroprotective properties may be due to its ability to dampen induction of microglial activation and the subsequent release of soluble pro-inflammatory factors. This appears to be via inhibition of oxidative induction of the p38 MAP kinase-NFκB pathway, resulting in reduced expression of pro-inflammatory genes and release of their corresponding gene products.

Keywords: Parkinson’s disease (PD), Isoflavones, Microglia, Inflammation, Lipopolysaccharide (LPS), Diadzein, Neuroprotection

Introduction

Parkinson’s disease (PD) is a progressive age-related neurodegenerative disorder characterized by preferential loss of dopaminergic neurons in the substantia nigra. Although PD has been heavily researched over the last several decades, the exact cause and underlying mechanisms responsible for this dopaminergic cell death is still not completely clear (Moore et al. 2005; Thomas and Beal 2011). Microglia-mediated neuroinflammation has, however, been widely hypothesized to contribute to the cascade of events leading to neuronal cell death (Hirsch and Hunot 2009).

Microglia are the resident macrophages of the central nervous system (CNS) and as such play a major role in host defense and tissue repair in the CNS. In their resting state, microglial cells display a ramified morphology and express low levels of surface receptors that mediate their normal macrophage functions (Kreutzberg 1996). Upon exposure to immunological challenges, including invading pathogens and neuronal injuries, microglia become activated undergoing alterations in morphology, number, and function. Activated microglia produced large numbers of pro-inflammatory factors including cytokines and reactive oxygen and nitrogen species which can have deleterious effects on neighboring neurons (Neumann et al. 2008). Elevated levels of these pro-inflammatory compounds have been reported in the brain, cerebrospinal fluid, and sera of PD patients (Blum-Degen et al. 1995; Dobbs et al. 1999; Hunot et al. 1999; Imamura et al. 2003). Furthermore, inhibition of microglia activation and suppression of pro-inflammatory mediators has been reported to attenuate or delay the disease progression in both in vitro and in vivo models of PD (Carta et al. 2011; Qian et al. 2010). So, efforts to identify the compounds or molecules that inhibit microglial activation and release of pro-inflammatory mediators may offer a potential therapeutic strategy for the treatment of PD.

One group of compounds which appear to have anti-inflammatory effects that may attenuate or delay neuronal cell loss is the phytoestrogens. Phytoestrogens containing an isoflavone core are commonly derived from a variety of plant tissues and structurally or functionally mimic mammalian estrogens. Isoflavones are the most widespread type of phytoestrogen found in legume plants. Daidzein, genistein, and their metabolites are the most prevalent isoflavones found in soybean (Axelson et al. 1984). Diadzein is the most commonly ingested and studied type of phytoestrogen, often found in nuts, fruits, soybeans, and soy-based products (Liggins et al. 2000, 2002). Diadzein and its derivatives, like other isoflavones, have both estrogenic and anti-estrogenic effects and has been shown to be beneficial for human health including menopausal symptoms and osteoporosis (Jiang et al. 2010; Levis et al. 2011; Silvina et al. 2011). They have been shown to protect against neurotoxicity in various paradigms, possibly in part via inhibition of neuroinflammation (Occhiuto et al. 2008, 2009; Chen et al. 2007; Schreihofer and Redmond 2009).

The focus of this current study was to test whether the phytoestrogen diadzein may act as a neuroprotectant via anti-inflammatory mechanisms in an in vitro model of microglial activation. In this current study, we used BV-2, a murine microglial cell line, to investigate the potential anti-inflammatory effects of diadzein as well as the possible mechanisms involved. Diadzein was found to significantly inhibit the induction and release of inflammatory mediators in BV-2 cells stimulated with the microglial activator lipopolysaccharide (LPS); this inhibition was found to be at the transcript level. This occurred in conjunction with reduced ROS production, p38 MAP kinase (MAPK) phosphorylation, and NFκB pathway activation. Diadzein was also found to protect against the death of dopaminergic neurons elicited by transfer of CM from LPS-activated BV-2 cells. Our results suggest that diadzein’s neuroprotective properties may be due to its ability to dampen the induction of microglial activation and the subsequent release of soluble pro-inflammatory factors.

Materials and Methods

Reagents

All reagents used for cell culture were purchased from Mediatech, Inc. (Manassas, VA, USA). LPS (Escherichia coli O111:B4) and diadzein were purchased from Sigma Chemical Co. (St Louis, MO, USA). Sigma LPS contains endotoxin levels of not less than 500,000 EU (endotoxin units)/mg unless otherwise noted. Antibodies against phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, actin, iNOS, and p65NFκB were purchased form Cell Signaling Technology (Beverley, MA, USA). All the reagents for RT-PCR were purchased from Promega (Madison, WI, USA).

Microglial and Neuronal Cultures

The BV-2 cell line was obtained from Dr. Luc Vallieres, Quebec City, Canada. BV-2 cells were immortalized by infecting murine primary microglial cell cultures with a v-raf/v-myc oncogene carrying retrovirus (J2) as previously described (Blasi et al. 1990). BV-2 cells were maintained in Dulbecco’s modified essential medium supplemented with 10 % heat-inactivated fetal bovine serum, streptomycin (10 μg/mL), and penicillin (10 U/mL) at 37 °C. The dopaminergic N27 neuronal cell line used in in vitro neuronal viability studies was derived from embryonic rat dopaminergic mesencephalic neurons via SV40 large T antigen immortalization. The cells were grown in RPMI medium 1640 containing 10 % fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL), and were differentiated with 2.0 mM dibutyryl adenosine 3′,5′-cyclic monophosphate and 60 μg/mL dehydroepiandrosterone for 48 h (Chinta and Andersen 2006) before the addition of microglial CM.

Measurement of Pro-Inflammatory Cytokine IL-6 Levels in BV-2 CM

Microglial cells (1 × 105 cells per well, 24-well plates) were pretreated with diadzein for 2 h followed by stimulation with LPS (100 ng/mL). The 2 h pretreatment was performed to assess the potential neuroprotective capabilities of the compound. CM was collected after 24 h of LPS stimulation and the concentrations of IL-6 was measured by ELISA.

Determination of Nitrite (NO2−) Levels in BV-2 CM

Levels of NO2−, a stable downstream product of NO, were determined by the Griess assay. Accumulated nitrite was measured in collected BV-2 CM using Griess reagent (Sigma) as described previously (Bi et al. 2011).

iNOS Protein Levels by Western Blot Analysis

Whole cell protein lysates from BV-2 cells were prepared in lysis buffer; protein samples were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Invitrogen). Membranes were blocked with 5 % skim milk in 10 mM Tris–HCl containing 150 mM NaCl and 0.5 % Tween 20 (TBST) and incubated with primary antibodies (1:1,000) against iNOS versus actin as loading control. After thorough washing with TBST, horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution in TBST; Millipore, CA, USA) were applied and blots were developed using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA). Optical density was assessed by the NIH ImageJ program.

RT-PCR

BV-2 cells (7.5 × 105 cells per 6-cm dish) were treated with LPS in the presence or absence of diadzein and total RNA was extracted using TRIZOL reagent (Sigma). For RT-PCR, total RNA (1 μg) was reverse transcribed in a reaction mixture containing 1 U RNase inhibitor, 500 ng random primers, 3 mM MgCl2, 0.5 mM dNTP, and 10 U reverse transcriptase in RT buffer (Promega). The synthesized cDNA was used as a template for PCR reactions using GoTaq polymerase (Promega) and appropriate primers. qPCR analysis of GAPDH, TNF-α, IL-6, iNOS, and Cox-2 was performed using the Roche universal probe library detection system. Relative quantification of gene expression was performed by the comparative threshold (CT) method. Changes in mRNA expression levels were calculated following normalization to GAPDH. The ratios obtained after normalization are expressed as fold change versus corresponding controls (Sriram et al. 2006).

Measurement of Intracellular Reactive Oxygen Species (ROS) Levels

Intracellular accumulation of ROS was measured using H2DCF-DA (Sigma) as previously described (Jung et al. 2010). In brief, microglial cells were stimulated with LPS for 16 h in the absence or presence of diadzein then stained with 20 μM H2DCF-DA in Hank’s balanced salt solution buffer for 1 h at 37 °C. DCF fluorescence intensity was measured on a fluorescence plate reader at 485-nm excitation and 535-nm emission (Molecular Devices, CA).

Measurement of p38 Phosphorylation

BV-2 microglia cultured in six-well dishes were pretreated for 2 h with diadzein (75 μM) and then stimulated with LPS (100 ng/mL) for 1 h. The phosphorylation status of p38 MAPK was assessed by western blot analysis, as described above, following overnight incubation at 4 °C in primary antibodies (1:1,000) against p38 versus phosphorylated p38 MAPK (Peng et al. 2004).

Transient Transfection of NFkappaB Reporter Construct and Assay by Luciferase

Transfection of the NFkappaB binding reporter gene into BV-2 cells was performed using lipofectamine 2000 (Invitrogen, USA). The NFkappaB binding reporter plasmid contains three copies of the κB-binding sequence fused to the firefly luciferase gene (Clontech, Mountain View, CA, USA). BV-2 cells (2 × 105 cells per well, 12-well plates) were transfected with 1 μg of the reporter construct mixed with lipofectamine 2000. After 48 h, cells were harvested and luciferase activity was assayed as previously described (Kim et al. 2005). To determine the effect of diadzein on reporter gene activity, cells were pretreated for 1 h with the agent before treating with LPS (100 ng/mL) for 6 h before cell harvest.

Immunocytofluorescent Analysis of Nuclear NFkappaB Translocation

BV-2 cells were seeded onto glass cover slips and stimulated with LPS (100 ng/mL) following pretreatment with diadzein or media for 1 h. Then, cells were fixed in 4 % paraformaldehyde, permeabilized in 0.5 % Trition X-100 for 30 min. After blocking with 5 % nonfat milk in PBS buffer, cells were incubated with rabbit anti-p65 antibodies for 1 h at room temperature. After a brief wash, cells were incubated with Alexa fluor-conjugated secondary antibody (1:500, Molecular Probes). Finally, the cells were washed again, mounted using Vectashield hard mount with DAPI, and visualized using a Zeiss LSM 510 confocal microscope.

Assay of Affects of CM from BV-2 on Cell Viability of Dopaminergic N27 Cells

BV-2 cells were seeded in 24-well tissue culture plates (6 × 104 cells/well) and incubated at 37 °C. After 24 h, the cells were stimulated with LPS (100 ng/mL) in the absence or presence or diadzein (75 μM) for 24 h. The condition media was collected from all the groups and centrifuged at 2,000× g for 10 min to remove any cell debris. The CM from control, diadzein, LPS, versus LPS + diadzein-treated cells was diluted (1:3) in neuronal culture media before adding to differentiated dopaminergic N27 cells plated earlier at 1 × 104 in 96-well plates. After 48 h, N27 cell viability was assessed via the MTT assay as previously described (Chinta and Andersen 2006).

Statistical Analysis

Unless otherwise stated, all experiments were performed in triplicate and repeated at least three times. The data are presented as mean ± SEM and statistical comparisons between groups were performed by one-way ANOVA followed by Student’s t test. Multiple comparisons of data from in vitro experiments were evaluated by two way ANOVA followed by Bonferroni post hoc testing. Statistical significance was set at p < 0.05 for all analyses.

Results

Diadzein Inhibits LPS-Stimulated Induction of the Pro-Inflammatory Mediators IL-6 and NO in Microglial BV-2 Cells

Activated microglia are known to be a major source of cytotoxic pro-inflammatory factors that can in turn contribute to subsequent neurotoxicity (Gao et al. 2002; Wang et al. 2002). To investigate the possible anti-inflammatory effects of diadzein, we first examined the effects of the drug on levels of two important pro-inflammatory mediators, NO and IL-6, in the media of BV-2 microglia challenged with LPS. Pretreatment of cells with diadzein was found to reduce the release of both NO and IL-6 into the media in a dose-dependent manner (Fig. 1a, c) as well as protein levels of the NO-producing enzyme iNOS (Fig. 1b). Diadzein at the concentrations used in this study (25–75 μM) did not affect cell viability of BV-2 cells as measured by MTT assay (Fig. 1d).

Fig. 1.

Fig. 1

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Diadzein suppresses LPS-induced production and release of the pro-inflammatory factors IL-6 and NO in microglial BV-2 cells. BV-2 cells were pretreated with diadzein (D) for 2 h before application of LPS (100 ng/mL). a Conditioned media (CM) was collected after 24 h and IL-6 levels determined using an ELISA kit according to the manufacturer’s recommendations. Data are presented as mean ± SD of at least four independent experiments; *p < 0.05 compared with the control group, **p < 0.01 compared with the LPS treated group. b Nitrite levels were determined in CM using the Griess reagent; *p < 0.05 compared with control group, **p < 0.05 compared with LPS-treated group. c Protein was extracted from whole cell lysates and subjected to western blot analysis for iNOS protein levels; actin was used as a loading control. The band density (integrated density value) is expressed graphically as a percentage ratio of densitometric optical density of iNOS to that of actin with denotations of significance obtained from statistical analyses of pooled raw data; *p < 0.05 relative to the band density of the untreated control sample. d Cytotoxicity of diadzein was assessed calorimetrically by the MTT assay as described in the “Materials and Methods” section; *p < 0.05 compared with control group

Diadzein Suppresses LPS-Induced Pro-Inflammatory Production at the Transcript Level

To examine whether suppression of NO and IL-6 production and release by diadzein was due to reduced mRNA expression, real-time PCR analyses from LPS-stimulated BV-2 microglia cells were conducted (Fig. 2). As previously reported, LPS treatment led to marked increases in mRNA expression of TNF-α, iNOS, Cox-2, and IL-6 in BV-2 microglia cultures (Jung et al. 2010). However, pretreatment of cells with diadzein was found to inhibit LPS-induced mRNA expression of these pro-inflammatory factors in a dose-dependent fashion.

Fig. 2.

Fig. 2

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Diadzein suppresses LPS-induced increases in transcript levels of pro-inflammatory genes. BV-2 microglial cells were pretreated with diadzein (50 and 75 μM) for 2 h followed by co-treatment with LPS (100 ng/mL) for 6 h. Total RNA was isolated and real-time PCR analysis was performed. Relative mRNA levels for Cox-2, iNOS, TNF-α, and IL-6 were normalized for GAPDH. Data are presented as mean ± SEM; *p < 0.05 compared with control group, **p < 0.05 compared with LPS-treated group

Diadzein Attenuates LPS-Induced Intracellular ROS Production, MAP Kinase Phosphorylation, and Activation of the NFkappa B Pathway

Intracellular ROS can trigger a cascade of deleterious events in the inflammatory process (Qin et al. 2004) via the phosphorylation of p38 MAPK resulting in subsequent activation of the NFκB pathway (Guyton et al. 1996; Schreck et al. 1991). NFκB is known to be a major regulator of pro-inflammatory gene expression in LPS-stimulated microglia. LPS exposure was found to not only induce increased intracellular ROS production in microglial BV-2 cells (Fig. 3) but also phosphorylation of p38MAPK (Fig. 4). Pretreatment with diadzein significantly blocked LPS-induced ROS production in a dose-dependent manner (Fig. 3) as well as suppressed LPS-induced p38 phosphorylation in activated microglial cells (Fig. 4). Moreover, LPS exposure was found to result in significant NFκB p65 nuclear translocation and upregulation of NFκB-dependent transcriptional activity, and both were significantly attenuated by diadzein pretreatment (Fig. 5). These data suggest that diadzein could elicit neuroprotective affects by preventing oxidative stress-mediated induction of these inflammatory pathways which can lead in turn to the induction of pro-inflammatory genes and subsequent production of their corresponding soluble cytotoxic gene products.

Fig. 3.

Fig. 3

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Effect of diadzein on LPS-induced intracellular ROS production. BV-2 cells were pre-incubated with diadzein for 2 h followed by replacement with medium containing LPS (100 ng/mL) for 4 h. Cells were then exposed to HBSS containing DCFH-DA (10 μM) for 30 min. After cells were washed with PBS, ROS was measured at the 488-nm excitation and 535-nm emission on a fluorescence plate reader. The data are expressed as mean ± SD, n = 4; *p < 0.05 compared with control group, **p < 0.05 compared with LPS-treated group

Fig. 4.

Fig. 4

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Diadzein inhibits LPS-induced phosphorylation of p38 MAPK. BV-2 cells were pretreated with diadzein for 2 h (75 μM) and then stimulated with LPS (100 ng/mL) for a 1-h incubation period. Cells were lysed, run on a SDS-PAGE gel, transferred to PVDF membranes, and blotted with phospho-p38 MAPK (Thr180/Tyr182) and p38 MAPK antibodies. The band density (integrated density value) is expressed graphically as a percentage ratio of densitometric optical density of phospho-p38 MAPK to that of p38 MAPK. Data (mean ± SD) are from four independent experiments; *p < 0.05 relative to the band density of the untreated control sample

Fig. 5.

Fig. 5

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Diadzein inhibits LPS-induced NFκB nuclear translocation and DNA binding activity. a After pretreatment with diadzein (75 μM) for 2 h, BV-2 cells were stimulated with 100 ng/mL LPS for 1 h. Nuclear translocation of NFκB subunit p65 was assessed by confocal fluorescence microscopy using anti-p65 antibody. Representative laser confocal microcopy images demonstrate localization of p65 (red) with nuclei stained with DAPI (blue) in cells exposed to LPS with or without diadzein pretreatment; pink, merged. Arrows indicate the presence of p65 within the cell nucleus. Bar = 20 μm. b BV-2 cells were transiently transfected with NFκB-Luc for 24 h then treated with 100 ng/mL LPS for 4 h ± diadzein. Cell lysates were then assayed for luciferase activity (mean ± SE, n = 4); *p < 0.05 versus control, **p < 0.05 versus LPS. (Color figure online)

Diadzein Protects Neurons Against Neurotoxicity Elicited via the Presence of Soluble Factors in the Media of LPS-Stimulated BV-2 Microglial Cells

A number of studies have demonstrated that activated microglia can induce neuronal toxicity (Wang et al. 2011). To investigate whether diadzein’s ability to suppress microglial activation and the subsequent release of pro-inflammatory cytotoxic factors has a neuroprotective impact, we evaluated the affects of CM from LPS-treated BV-2 cells grown in the absence and presence of diadzein on the viability of dopaminergic N27 cells. CM from LPS-stimulated microglia (LPS-CM) was found to produce significant toxicity in N27 cells that was attenuated in CM from BV-2 cells grown in the presence of diadzein pretreatment (Fig. 6). This suggests that the toxicity of the CM derived from LPS-treated activated microglia are dependent on elevated levels of soluble neurotoxic factors and that this is prevented by growth of microglial cells in the presence of diadzein.

Fig. 6.

Fig. 6

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Diadzein protects against dopaminergic cell loss induced by CM from LPS-activated BV-2 cells. BV-2 cells were stimulated with LPS (100 ng/mL) ± diadzein (75 μM) for 24 h. CM from control (BV-2-CM), diadzein-treated (Dia-CM), LPS-treated (LPS-CM), and LPS/diadzein-treated (50 and 75 μM; LPD50-CM, LDP100-CM) BV-2 cells was added to dopaminergic N27 cells plated in 96-well plates. After 48 h, N27 cell viability was assessed via the MTT assay. Data are expressed as mean ± SD, n = 4; *p < 0.01, compared with control-CM group, **p < 0.05, compared with LPS-CM group

Discussion

In this study, we report that the isoflavone compound diadzein can elicit neuroprotective affects in part via its ability to dampen microglial activation and the subsequent production and release of soluble pro-inflammatory factors. This is consistent with previous studies in which related isoflavones including genistein and Biochanin A were reported to protect dopaminergic neurons against LPS-induced neurotoxicity via inhibition of neuroinflammatory events (Wang et al. 2005; Chen et al. 2007). In these previous studies, however, the possible molecular mechanisms responsible for these affects were not addressed. Based on our current studies, these affects mechanistically appear to involve the capacity of diadzein and perhaps other isoflavones to inhibit oxidative induction of the MAP kinase-NFκB signaling pathway, preventing increased synthesis and release of these neurotoxic factors. LPS is known to induce oxidative phosphorylation of p38 MAPK within microglia (Bhat et al. 1998; Xie et al. 2004). This in turn can stimulate the activation of NFκB via phosphorylation of its p65 subunit. Subsequent nuclear translocation of p65 results in increased expression of several pro-inflammatory genes (Moon et al. 2007).

Microglia are the primary immune cells of the brain. In response to injury, infection, stress, or exposure to environmental factors, microglia become metabolically active and assumes an ameboid profile. Activated microglia secretes various pro-inflammatory mediators including cytokines such as IL-6 and TNF-α, reactive oxygen species, and reactive nitrative species such as NO. These factors are believed to contribute to microglia-mediated neurotoxicity (Jeohn et al. 1998). In this study, we have demonstrated that production of NO, IL-6, TNF-α, and ROS by LPS-activated microglia is significantly inhibited in a dose-dependent manner by diadzein pretreatment. Furthermore, inhibition of this pro-inflammatory mediator secretion provides significant protection to N27 dopaminergic neurons against inflammation-mediated degeneration. The inhibitory and neuroprotective profile of diadzein seem to be similar to that of other isoflavones. Previous in vitro studies using primary mesencephalic cultures have demonstrated that both genistein and Biochanin A protect dopaminergic neurons against LPS-induced damage through inhibition of microglia activation and pro-inflammatory secretion (Wang et al. 2005; Chen et al. 2007). Our present results demonstrated that diadzein inhibits LPS-induced increases in microglial ROS levels along with phosphorylation of p38 MAPK, subsequent p65 translocation, and mediation of transcriptional activation. This was associated with reduced expression of pro-inflammatory genes in LPS-induced microglial cells along with reduced release of their corresponding neurotoxic gene products. It will be important to determine whether the neuroprotective effect of these isoflavones can be observed in animal models of inflammation-mediated neurodegenerative diseases including PD. Ongoing studies are toward assessing the neuroprotective ability of diadzein in in vivo models of PD.

In summary, our results demonstrate that diadzein’s neuroprotective properties may be associated with inhibition of microglia activation and the generation of pro-inflammatory factors. Mechanistically, this involves inhibition of oxidative induction of the p38 MAP kinase-NFκB pathway resulting in reduced expression of pro-inflammatory genes and release of their corresponding gene products.

Acknowledgments

These studies were funded by R01 NS045615 (JKA) and R01 AG029631 (GJL). We thank Mr. Anand Rane for help with immunocytochemistry.

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