antioxidant enzymes in microglia

International Immunopharmacology 17 (2013) 415–426

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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Schizandrin C exerts anti-neuroinflammatory effects by upregulating phase II detoxifying/antioxidant enzymes in microglia Sun Young Park a, Se Jin Park b, Tae Gyeong Park b, Seetharaman Rajasekar b, Sang-Joon Lee c, Young-Whan Choi b,⁎ a b c

Bio-IT Fusion Technology Research Institute, Pusan National University, Busan 609-735, Republic of Korea Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Republic of Korea Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea

a r t i c l e

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Article history: Received 3 May 2013 Received in revised form 24 June 2013 Accepted 26 June 2013 Available online 13 July 2013 Keywords: Microglia Schizandrin C Anti-neuroinflammation Lipoteichoic acid Phase II detoxifying/antioxidant enzymes

a b s t r a c t We investigated the anti-neuroinflammatory properties of schizandrin C by focusing on its roles in the induction of phase II detoxifying/antioxidant enzymes and in the modulation of upstream signaling pathways. Schizandrin C induced expression of phase II detoxifying/antioxidant enzymes including heme oxygenase-1 (HO-1) and NADPH dehydrogenase quinone-1 (NQO-1). Activation of upstream signaling pathways, such as the cAMP/protein kinase A/cAMP response element-binding protein (cAMP/PKA/CREB) and erythroid-specific nuclear factor-regulated factor 2 (Nrf-2) pathways, significantly increased following treatment with schizandrin C. In addition, expressions of schizandrin C-mediated phase II detoxifying/antioxidant enzymes were completely attenuated by adenylyl cyclase inhibitor (ddAdo) and protein kinase A (PKA) inhibitor (H-89). In microglia, schizandrin C significantly inhibited lipoteichoic acid (LTA)-stimulated pro-inflammatory cytokines and chemokines, prostaglandin E2 (PGE2), nitric oxide (NO), and reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metallopeptidase-9 (MMP-9) protein expressions. Moreover, schizandrin C suppressed LTA-induced nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1), janus-kinase/ signal transducer and activator of transcription (JAK-STATs), and mitogen-activated protein kinase (MAPK) activation. Schizandrin C also effectively suppressed ROS generation and NO production, as well as iNOS promoter activity in LTA-stimulated microglia. This suppressive effect was reversed by transfection with Nrf-2 and HO-1 siRNA and co-treatment with inhibitors ddAdo and H-89. Our results indicate that schizandrin C isolated from Schisandra chinensis could be used as a natural anti-neuroinflammatory agent, inducing phase II detoxifying/antioxidant enzymes via cAMP/PKA/CREB and Nrf-2 signaling. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Expressions of phase II detoxifying/antioxidant enzymes are regulated through the activation of transcription factors, including the erythroid-specific nuclear factor-regulated factor 2 (Nrf-2), nuclear factor-kappa B (NF-κB), cAMP response element (CRE)-binding protein (CREB), and activator protein-1 (AP-1). The promoter regions of phase II detoxifying/antioxidant enzyme genes contain antioxidant response elements (AREs) that directly bind Nrf-2 and regulate the expression of many genes involved in adaptive responses and inflammation [1,2]. Several kinase signaling pathways, including protein kinase A (PKA), phosphatidylinositol-3 kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK), have been suggested to be involved in regulating the Nrf-2 activation that facilitates its accumulation in the nucleus to

⁎ Corresponding author. Tel.: +82 55 350 5522; fax: +82 55 350 5529. E-mail address: [email protected] (Y.-W. Choi). 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.06.032

promote ARE-related gene expressions [3]. Elevated cAMP levels induce the expression of phase II detoxifying/antioxidant enzymes through PKA-mediated phosphorylation of CREB, a component of the transcription complex at the CRE site on the ARE promoter [4]. Additionally, heme oxygenase-1 (HO-1), and NADPH dehydrogenase quinone-1 (NQO-1) have been intensively studied in the brain for their neuroprotective and anti-inflammatory effects, and results have suggested that they are potential therapeutic targets for many inflammatory diseases [5]. The HO-1 and NQO-1 genes contain an ARE consensus sequence, which enables responses to Nrf-2, as well as to oxidative and nitrosative stresses such as hypoxia, cytokines, nitric oxide (NO), heat shock, and hydrogen peroxide [2]. The major anti-inflammatory function of HO-1 is its rate-limiting catabolic activity during heme degradation. In this reaction, the oxidation of free heme engenders ferrous iron and biliverdin, which are subsequently converted into ferritin and bilirubin, respectively, as well as carbon monoxide, all of which are known to have anti-inflammatory and anti-oxidant properties [6].

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Microglia are macrophages of the central nervous system (CNS) that play a crucial role in immunological defense against virulent factors in the brain [7]. Under normal conditions, microglia display a ramified morphology in the resting state while continually monitoring the surrounding environment for any changes in the homeostasis of the CNS that may be harmful to neurons or induce damage in the brain [8]. However, in the presence of certain stimuli, microglia become activated and enable proper brain development or repair injured sites via secretion of various inflammatory cytokines and phagocytosis, which protects neuronal tissue from subsidiary damages in healthy brains [9]. Upon microglia stimulation, intracellular phosphorylation cascades, including NF-κB, AP-1, janus-kinase/signal transducer and activator of transcription (JAK-STATs), and mitogen-activated protein kinases (MAPKs) become activated. Excessive levels of microglia activation induce chronic inflammatory circumstances via constituent activation of pro-inflammatory signal cascades, such as the NF-κB, AP-1, and STAT pathways, ultimately leading to neuronal death and brain injury rather than neuronal survival [10]. In addition, recent studies have emphasized the neuro-inflammation triggered by microglia activation, which has potent effects on the pathogenesis of several neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease [11]. Bacterial lipoteichoic acid (LTA) is known as the major antigenic agent against innate immune cells such as microglia, monocytes, and macrophages. LTA is the smallest bioactive fragment of peptidoglycan (PGN) of gram-positive bacteria membranes. The surfaces of immune cells contain Toll-like receptors (TLRs), which are involved in recognition of various bacterial products such as LTA, PGN, and lipopolysaccharides (LPS) [12]. Recognition by Toll-like receptors leads to activation of cellular signaling pathways involved in the defense against external stimulations. Little information is available pertaining to LTA, but they are both known to trigger activation of immune cells and to promote a diverse array of inflammatory responses through the release of various pro-inflammatory cytokines and mediators, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and NO. Moreover, there is accumulating evidence that TLR2 is specifically associated with LTA [13]. In several Asian countries, dried fruit of Schisandra chinensis has long been used in traditional medicine as sedatives, analgesics, and antipyretics, and for treatment of hyperlipidemia and hypertension [14]. Researchers have recently found that S. chinensis is also useful for treatment of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Moreover, schizandrin C isolated from S. chinensis has been shown to have neuroprotective effects on amyloid beta-stimulated PC12 cells [15]. In addition, schizandrin C has been shown to have inhibitory effects on LPS-induced inducible nitric oxide synthase (iNOS) expression via MAPKs in RAW264.7 cells [16]. However, to the best of our knowledge, no studies have been conducted to date focusing on the inhibitory effects of schizandrin C on neuroinflammatory responses via up-regulation of phase II detoxifying/antioxidant enzymes. Therefore, in order to determine its potential for further development as an anti-neuroinflammatory agent, we investigated the mechanism by which schizandrin C exerts its neuroinflammatory responses. Specifically, experiments were carried out to investigate the effects of schizandrin C on neuroinflammatory responses of murine primary microglia and BV2 microglia cell lines. Here, we provide the first evidence that schizandrin C induces the expression of phase II detoxifying/ antioxidant enzymes via cAMP/PKA/CREB and nuclear factor-regulated factor 2 (Nrf-2) activation in microglia. The molecular mechanism underlying the observed anti-neuroinflammatory properties of schizandrin C was determined by studying its effects on activation of cAMP/PKA/CREB, Nrf-2, NF-κB, AP-1, STATs, and MAPKs. Overall, results presented herein reveal novel mechanisms by which schizandrin C exerts anti-neuroinflammatory effects, and will therefore be helpful for the development of therapeutic strategies for LTA-mediated neurodegeneration in CNS diseases.

2. Materials and methods 2.1. Materials The cell culture medium, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were all purchased from Gibco BRL (now Invitrogen Corporation, Carlsbad, CA). LTA and other reagents were obtained from Sigma (St. Louis, MO). Protoporphyrin IX (SnPP), siRNAs against Nrf-2 and HO-1, and antibodies for iNOS, cyclooxygenase-2 (COX-2), HO-1, Nrf-2, c-Jun, c-Fos, NF-κB, IκBα, TATA-binding protein (TBP), and α-tubulin were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphorylated p-p38, p-JNK, p-ERK, p-IκBα, p-STAT-1, STAT-1, P-STAT-3, STAT-3, p-CREB, CREB, and matrix metallopeptidase-9 (MMP-9) were purchased from Cell Signaling Technology (Beverly, MA). The FuGENE 6 transfection reagent and the X-treme GENE siRNA Transfection Reagent were obtained from Roche (Indianapolis, IN). 2.2. Plant material Fruits of S. chinensis (Turcz.) Baill were collected in September 2005 from Moonkyong, Korea. A voucher specimen (accession number SC-PDRL-1) was deposited in the herbarium of the Pusan National University (Miryang, Korea). The plant was identified by one of the authors (Y.W. Choi). 2.3. Purification of schizandrin C The isolation and purification of schizandrin C from dried fruit of Schisandra chinensis, and subsequent evaluation of its structure, were conducted as described previously by Choi et al. Briefly, dried fruits of S. chinensis (2.5 kg) were ground to a fine powder and successively extracted at room temperature with n-hexane, EtOAc, and MeOH. The hexane extract (308 g) was then evaporated in vacuo, and the remaining sample was chromatographed on a 40 μm silica gel (J.T. Baker, NJ) column (70 × 8.0 cm) with a step gradient of 0, 5, 10, 20, and 30% EtOAc in hexane and 5% MeOH in CHCl3 to obtain 38 fractions. Fraction 8 (1.579 mg) was separated on a silica gel column (100 × 3.0 cm) with CH2Cl2 to obtain schizandrin C (501 mg). 2.4. Isolation of mouse primary microglia and cell culture Isolated primary microglia cultures were prepared as previously described [17]. Briefly, primary mixed glia cell cultures from whole brains of imprinting control region (ICR) mice at postnatal days 2–5 were prepared in culture flasks and maintained in DMEM/F12 containing 10% FBS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 mg/ml penicillin/streptomycin at 37 °C under 5% CO2. After 2 weeks, the culture flasks were shaken in an orbital shaker at 180 rpm at 37 °C for 5 h and the medium was harvested. The attached cells were then removed by trypsinization and seeded onto new plates for subsequent experiments. To monitor purity, cells were immunostained with CD11b antibody, which resulted in more than 90% of cells being stained positively. Mouse BV2 microglial cells were cultured in DMEM supplemented with 5% heat-inactivated FBS and 0.1% penicillin–streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. 2.5. Determination of intracellular cAMP levels The cAMP concentration was measured using a cAMP EIA kit (Cayman Chemical Company, Ann Arbor, MI). Briefly, cells were lysed in 0.1 M HCl to inhibit phosphodiesterase activity. After neutralization and dilution, a fixed amount of cAMP conjugate was added to compete with cAMP in the cell lysate for sites on rabbit polyclonal antibody immobilized on a 96-well plate. The protein content in the cell lysate was determined

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using the Bradford reagent. For cAMP measurements, 50 μg of protein was used for sample analysis, according to manufacturer's instructions.

cleared by centrifugation, mixed with 2 × sodium dodecyl sulfate (SDS) sample buffer (Invitrogen Corporation), and electrophoresed in a polyacrylamide gel containing 0.1% (w/v) gelatin. Following electrophoresis, gels were incubated in renaturing buffer (2.5% Triton X-100) with gentle agitation to remove the SDS, and were then incubated in developing buffer (50 mM Tris–HCl buffer, pH 7.4, 10 mM CaCl2) overnight at 37 °C to digest the gelatin. Gels were then stained with SimplyBlue SafeStain (Invitrogen Corporation) until clear bands appeared indicating gelatin digestion.

2.6. HO-1 activity assay HO-1 activity was determined by measuring the amount of bilirubin, as described previously. To determine bilirubin levels, 500 μl of each culture supernatant was collected and mixed with 250 mg barium chloride dihydrate (BaCl2·2H2O) by vortexing (10–15 s). Benzene (750 μl) was added to each sample, and the samples were then vortexed briskly (50–60 s). After centrifugation for 30 min at 13,000 rpm, the upper benzene layer was isolated, and the amount of bilirubin was determined based on absorbance at 450 nm, with a reference value at 600 nm, measured by UV/visible spectrophotometry (Ultrospec 6300 Pro, Amersham Biosciences, Piscataway, NJ). All steps of the assay were executed under dark conditions, and the bilirubin quantity was calculated using an extinction coefficient of ε450 = 27.3 mM/cm.

2.10. Transient transfection with siRNA Transfection of cells with siRNA was performed using the X-treme GENE siRNA Transfection Reagent (Roche Applied Science) according to manufacturer's instructions. Commercially available human HO-1and Nrf-2-specific siRNAs (Santa Cruz, Heidelberg, Germany) and negative control siRNAs (Santa Cruz) were used for transfection. Briefly, X-treme GENE siRNA Transfection Reagent (10 μl) was added to 100 μl of serum-free medium containing 2 μg of each siRNA and incubated for 20 min at room temperature. Gene silencing was measured after 48 h by western blotting.

2.7. Measurement of reactive oxygen species (ROS) To evaluate intracellular ROS levels, cells were treated with CM-H2DCFDA, an indicator of general oxidative stress, for 1 h at 37 °C under 5% CO2. Cells were then harvested and washed three times with phosphate-buffered saline (PBS), and the fluorescence intensity was then measured by flow cytometry using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Data analyses were performed using CXP 2.0 (Beckman Coulter).

2.11. Transient transfection and dual luciferase assay Cells were transfected with the κB-luc reporter plasmid consisting of 3 kb concatemers from the immunoglobulin-gamma (Igγ) chain, the firefly luciferase gene, and either an AP-1-reporter plasmid, an ARE-reporter plasmid, or an HO-1 promoter reporter plasmid (Stratagene, Grand Island, NY) using FuGENE-HD reagent (Roche Applied Science) according to manufacturer's instructions. A Renilla luciferase control plasmid, pRL-CMV (Promega), was co-transfected as an internal control for transfection efficiency. Luciferase activity was assayed using a dualluciferase assay kit (Promega) according to manufacturer's instructions. Luminescence was measured using a microplate luminometer (Wallac 1420).

2.8. Measurement of TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 (MCP-1), and prostaglandin E2 (PGE2) Cells were first incubated with various concentrations of schizandrin C for 1 h, and were then incubated with LTA for 16 h. Following 24 h incubation, TNF-α, IL-1β, and IL-6 levels were quantified in the culture media using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions.

2.12. Western blot analysis

2.9. Gelatin zymography assay

Cells were harvested in ice-cold lysis buffer consisting of 1% Triton X-100, 1% deoxycholate, and 0.1% SDS. The protein content of cell lysates was then determined using Bradford reagent (Bio-Rad, Hercules, CA). Total proteins in each sample (50 μg) were resolved by 7.5% SDSpolyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with appropriate antibodies. Proteins were visualized using an enhanced chemiluminescence

The activity of MMP-9 in the conditioned medium was determined by gelatin zymography protease assays. Briefly, 2 × 105 cells were seeded in 6-well plates and allowed to grow to 80% confluence. Cells were then maintained in serum-free medium for 12 h prior to treatment with schizandrin C or LTA for 16 h. Conditioned media were collected,

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Fig. 2. Effect of schizandrin C on the activation of cAMP/PKA/CREB and Nrf-2-mediated HO-1 and NQO-1. (A) BV-2 microglial cells were cultured with increasing doses of schizandrin C for 8 h, or with 20 μM of schizandrin C for the indicated times. HO-1 and NQO-1 expressions were determined by western blot analysis. (B) Cells were incubated with increasing doses of schizandrin C for 2 h or with 20 μM of schizandrin C for the indicated times. Nuclear localization of Nrf-2 was determined by western blot analysis. Western blot detection of TBP was used as a protein loading control for each lane. (C) Cells were transfected with the ARE-luciferase or the HO-1 promoter-luciferase constructs and were then treated with increasing doses of schizandrin C. Equal amounts of cell extract were assayed for dual-luciferase activity. Expression of the Renilla luciferase control was used to normalize ARE-luciferase and HO-1 promoter-luciferase activities. (D) Cells were co-incubated with 50 μM hemin for 2 h, after which they were exposed to increasing doses of schizandrin C, 20 μM SnPP (HO-1 inhibitor), or 20 μM CoPP (HO-1 activator) for 12 h. The quantity of bilirubin produced in the culture media was measured on a spectrophotometer and was calculated using the molar extinction coefficient of bilirubin dissolved in benzene. (E) Cells were cultured with increasing doses of schizandrin C for 2 h or with 20 μM of schizandrin C for the indicated times. p-CREB and CREB expressions were determined by western blot analysis. (F) Cells were cultured with increasing doses of schizandrin C for 24 h and the intracellular cAMP concentration was measured. (G) H-89 is a selective PKA inhibitor. Cells were pre-treated with ddAdo (100 μM) or H-89 (5 μM) for 1 h and were then treated with schizandrin C (20 μM) for 4 h. HO-1 and NQO-1 expression was determined by western blot analysis. Each bar represents the mean ± S.E. from three independent experiments per group. *P b 0.05 and **P b 0.01 relative to the LTA-treated group.

detection system (Amersham Biosciences, Piscataway, NJ) with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. Images were acquired using an ImageQuant 350 analyzer (Amersham Biosciences).

2.13. Immunofluorescence confocal microscopy Cells were cultured directly on glass coverslips in a 35 mm dish. Cells were fixed with 3.5% paraformaldehyde in PBS for 10 min at

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room temperature and then permeabilized with 100% MeOH for 10 min. To investigate the cellular localization of NF-κB p65, cells were treated with polyclonal antibody (1:100) against NF-κB p65 for 16 h. After extensive washing with PBS, cells were incubated with a secondary fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG antibody diluted to 1:1000 in PBS for 4 h at room temperature. Nuclei were then stained with 1 μg/ml of 4′-6-diamidino-2-phenylindole (DAPI), and observed by confocal microscopy using a Zeiss LSM 510 Meta microscope.

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treating primary microglia with schizandrin C (20 μΜ) for 0–8 h. Schizandrin C treatment induced a remarkable increase in the nuclear accumulation of Nrf-2 in primary microglia, especially in cells treated for 2 h. When cells were treated with varying concentrations of schizandrin C for 2 h, Nrf-2 nuclear accumulation was induced in a

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2.15. Statistical analysis Data are expressed as the mean ± S.E. Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (version 18.0) to identify significant differences based on either one-way or two-way analysis of variance (ANOVA) followed by Dunn's post-hoc tests. P-values b 0.05 were considered statistically significant. Each experiment was repeated at least three times.

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3. Results

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3.1. Schizandrin C induces the expression of HO-1 and NQO-1 via cAMP/ PKA/CREB and Nrf-2 activation

Schizandrin C + LTA NAC + LTA 100

Relative ROS

To verify the effect of schizandrin C on cell viability, primary microglia and BV 2 microglial cells were treated with schizandrin C for 24 h. When compared with the untreated control cells, primary microglia and BV 2 microglial cells treated with schizandrin C at concentrations between 0 and 20 μM exhibited no cytotoxicity. Thus, this concentration range of schizandrin C was applied in all the subsequent experiments (Fig. 1B). In the CNS, modulation of phase II detoxifying/antioxidant enzymes (HO-1 and NQO-1) is crucial to the pathogenesis of neurodegenerative diseases [19]. To assess the effects of schizandrin C on HO-1 and NQO-1 protein expressions, primary microglia were treated with schizandrin C (20 μΜ) for varying lengths of time (0–24 h). Levels of both proteins increased after schizandrin C treatment, especially in cells treated for 8 h. Next, we measured the protein expression levels of HO-1 and NQO-1 in primary microglia treated with schizandrin C (1, 5, 10, and 20 μΜ) or 20 μΜ of cobalt protoporphyrin (CoPP; HO-1 activator) for 8 h. A concentration-dependent increase in expression levels of both proteins was observed; the greatest increase was observed in response to 20 μΜ of schizandrin C (Fig. 2A). Nrf-2 is a critical player in the up-regulation of phase II detoxifying/antioxidant enzymes such as HO-1 and NQO-1. We attempted to confirm the effects of schizandrin C on Nrf-2 nuclear accumulation in primary microglia by

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To detect the in vivo association of nuclear proteins with the mouse iNOS promoter, chromatin immunoprecipitation (ChIP) analysis was conducted as previously described [18], with some modifications. Briefly, cells were incubated in cultured medium containing 1% formaldehyde for 10 min at room temperature. The cross-linking reaction was then quenched by adding glycine to a final concentration of 0.125 M. Isolated nuclei were subsequently digested with 200 U of MNase at 37 °C for 15 min, followed by sonication to produce chromatin of primarily mononucleosome size. Antibodies were incubated with the fragmented chromatin for 3 h at 4 °C. Protein–DNA complexes were recovered with protein-A agarose beads, washed, and eluted as previously described. Crosslinks were reversed at 65 °C in 0.25 M NaCl overnight, and DNA was digested with proteinase K for 2 h at 50 °C. The DNA was then isolated using a BioRad kit, which was used as a template for polymerase chain reaction (PCR). Primers for amplification of the iNOS promoter (including the NF-κB binding sequence: −85 to −76, from −184 to +102, 287 bp) were as follows: sense: 5′-CATGAGGA TACACCACAGAG-3′ and antisense: 5′-AAGACCCAAGCGTGAGGAGC-3′.

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Fig. 3. Effect of schizandrin C on LTA-induced ROS production in microglia. Primary microglia cells were treated with schizandrin C (20 μM) or NAC (10 mM) for 1 h, and were then treated with LTA. After 24 h of stimulation, cells were incubated with CM-H2DCFDA for an additional 1 h. Intracellular ROS levels were then determined by confocal microscopy (A) and flow cytometry (B). Each bar represents the mean ± S.E. from three independent experiments per group. **P b 0.01 relative to LTA-treated group.

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concentration-dependent manner (Fig. 2B). To clarify the effects of schizandrin C on Nrf-2 transactivity, BV-2 microglia were transiently transfected with luciferase reporter genes driven by ARE,

which bind Nrf-2. Schizandrin C increased ARE promoter activity in a dose-dependent manner (Fig. 2C), as well as HO-1 promoter activity and enzymatic activity (Fig. 2D). We then investigated whether

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MMP-9,2 (Zym) MMP-9 MMP-2 iNOS COX-2 α-tubulin Fig. 4. Effect of schizandrin C on neuroinflammatory molecules in LTA-stimulated microglia. (A) Primary microglia were treated with increasing doses of schizandrin C for 1 h and were then treated with LTA for 24 h. Nitrite content was measured using the Griess reaction. The concentrations of PGE2, TNF-α, IL-1β, IL-6, and MCP-1 in the culture media were measured using a commercial ELISA kit. (B) MMP-9 enzymatic activity was analyzed by gelatin zymography (Zym). Protein expressions of MMP-9, iNOS, COX-2, and tubulin were detected by western blot analysis using specific antibodies. Each bar represents the mean ± S.E. of three independent experiments per group. *P b 0.05 and **P b 0.01 relative to the TLR2/4 agonist-treated group.

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cAMP/PKA/CREB activation by schizandrin C contributed to the regulation of HO-1 and NQO-1 expression using their inhibitors. Schizandrin C led to a dose dependent increase in CREB phosphorylation in primary microglia (Fig. 2E). Additionally, schizandrin C increased intracellular cAMP levels in a dose-dependent manner (Fig. 2F). Moreover, schizandrin C-mediated HO-1 and NQO-1 expression was completely attenuated by adenylyl cyclase inhibitor (ddAdo) and PKA inhibitor (H-89) (Fig. 2G). Taken together, these results suggested that schizandrin C-induced HO-1 and NQO-1 expression was associated with activation of cAMP/PKA/CREB and NRf-2.

C as a potential mechanism contributing to the observed inhibition of neuroinflammatory molecules. To accomplish this, we measured ROS generation using CM-H2DCFDA, a reagent that measures intracellular ROS, as well as confocal microscopy and flow cytometry. To clarify the effects that schizandrin C might have on LTA-induced ROS production, cells were pretreated with schizandrin C and NAC (positive anti-oxidant control) for 1 h, followed by incubation with LTA for 24 h. As shown in Fig. 3, LTA-induced ROS production was inhibited by schizandrin C and NAC. These results suggested that schizandrin C modulated ROS production in LTA-stimulated microglia.

3.2. Schizandrin C inhibited LTA-induced ROS production in microglia

3.3. Schizandrin C suppresses neuroinflammatory molecules in LTAstimulated microglia

Previous studies have revealed that excessive ROS generation by microglia is a significant portent of neurodegenerative disorders and that they act as secondary messengers in neuroinflammatory responses [20]. We evaluated the antioxidant capability of schizandrin

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LTA Fig. 5. Effect of schizandrin C on LTA-induced activation of NF-κB and AP-1. (A) BV-2 microglial cells were treated with increasing doses of schizandrin C for 1 h followed by LTA treatment for 0.5 h. Nuclear extracts were analyzed by western blot analysis with anti-NF-κB p65, anti-p-NF-κB p65, c-Jun, and c-Fos antibodies. Cytosolic extracts were analyzed by western blotting with anti-IκB-α and anti-p-IκB-α antibodies. For western blot detection of TBP, α-tubulin was used as a protein-loading control for each lane. (B) Cells were pre-treated with schizandrin C (20 μM) for 1 h and stimulated with LTA for 1 h. Fixed cells were stained with DAPI and anti-NF-κB p65 antibody followed by incubation with FITC-conjugated anti-rabbit IgG secondary antibody. Samples were then observed by confocal microscopy. (C) Cells were co-transfected with the κB-luciferase reporter or with the AP-1 luciferase reporter and the control Renilla luciferase plasmid, pRL-CMV. After 24 h, cells were incubated with the indicated concentrations of schizandrin C for 1 h and were then stimulated with LTA for 24 h. Equal amounts of cell extract were assayed for dual-luciferase activity. Expression of the Renilla luciferase control was used to normalize the κB-luciferase and AP-1 luciferase activities. (D) Cells were incubated with schizandrin C (20 μM) for 0.5 h and then with LTA for 4 h. DNA was immunoprecipitated by an anti-NF-κB p65 antibody and purified, after which the precipitated iNOS promoter region was amplified by PCR and real-time PCR. The input represents PCR products from chromatin pellets prior to immunoprecipitation. Each bar represents the mean ± S.E. of 3 independent experiments per group. *P b 0.05 and **P b 0.01 relative to the LTA-treated group.

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microglia [21]. To investigate the anti-neuroinflammatory effects of schizandrin C, microglia were stimulated with LTA in the presence or absence of schizandrin C. As shown in Fig. 4A, schizandrin C significantly inhibited NO, PGE2, TNF-α, IL-1β, IL-6, and MCP-1 production in LTA-stimulated microglia. Consistent with results of previous studies, schizandrin C suppressed LTA-induced iNOS and COX-2 protein expression. Subsequently, we investigated the effects of schizandrin C on MMP-9 expression and enzyme activity in microglia. LTA treatment resulted in an increase in MMP-9 expression and enzyme activity, whereas pretreatment with schizandrin C inhibited MMP-9 expression and enzyme activity in comparison with LTA agonist-treated control cells (Fig. 4B). 3.4. Schizandrin C inhibited NF-κB and AP-1 activation, which was associated with inhibition of neuroinflammatory molecules

BV2 cells, the degree of MAPK phosphorylation was determined by western blot assay. As shown in Fig. 6C, schizandrin C inhibited LTA-induced ERK, JNK, and p38 phosphorylations. These results suggested that STAT and MAPK pathways were involved in anti-neuroinflammatory effects of schizandrin C in LTA-stimulated microglia.

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The NF-κB and AP-1 signal pathway acts as a central factor in response to cellular damage, stress, and inflammatory processes [22]. The activation of NF-κB and AP-1 regulates more than 100 genes associated with immune and inflammatory responses including TNF-α, IL-1β, IL-6, and COX-2, and adhesion molecules. It has also been reported that the activation of NF-κB and AP-1 is triggered by MAPKs, including ERK, p38, and JNK. To elucidate whether schizandrin C could affect the nuclear translocation of NF-κB and AP-1, cells were treated with various concentrations of schizandrin C in the presence of LTA for 1 h, and nuclear extracts were prepared and examined by western blot analysis. LTA induced the nuclear translocation of NF-κB p65 and AP-1 (c-Jun and c-FOS), whereas schizandrin C inhibited their nuclear translocation. Simultaneously, schizandrin C suppressed the phosphorylation and degradation of IκB-α (Fig. 5A). The inhibitory effects of schizandrin C on the nuclear translocation of NF-κB p65 were confirmed by immunofluorescence confocal microscopy (Fig. 5B). Furthermore, we examined the effects of schizandrin C on NF-κB and AP-1 transactivity in LTA-stimulated BV 2 microglial cells. Cells were transiently transfected with NF-κB and AP-1-luc reporter plasmid, and reporter activities were regulated by schizandrin C. The LTA-induced increases in NF-κB and AP-1 reporter activities were suppressed by schizandrin C in a dose-dependent manner (Fig. 5C). We further investigated whether treatment of BV-2 microglial cells with schizandrin C affected LTA-stimulated NF-κB binding to the iNOS promoter using a ChIP-PCR assay. As shown in Fig. 5D, when cells were treated with schizandrin C at 20 μM and stimulated with LTA, the in vivo binding of NF-κB to the iNOS promoter was reduced when compared to LTA-stimulated cells. These results indicated that schizandrin C inhibited LTA-stimulated expression of neuroinflammatory molecules by blocking activation of NF-κB and AP-1. 3.5. The inhibitory effect of schizandrin C on LTA-mediated phosphorylation of STATs and MAPKs STATs and MAPKs comprise another key signaling pathway involved in cytokine-induced neuroinflammatory responses [23]. Specifically, this pathway transmits signals from cytokines to the nucleus and regulates the mechanisms of cellular responses. STAT-1 and STAT-3 are related to cellular responses to neuroinflammatory cytokines. Therefore, we clarified whether STAT-1 and STAT-3 are activated in LTA-stimulated BV2 microglial cells, and investigated whether various doses of schizandrin C influenced activated STATs. Western blot analysis revealed that schizandrin C remarkably inhibited LTA-induced phosphorylation of STAT-1 and STAT-3 (Fig. 6A). Next, the binding activity of STAT-1 to the iNOS gene promoter was investigated by a ChIP-PCR assay. A low level of STAT-1 binding activity to the iNOS gene promoter was observed in unstimulated cells, whereas a large quantity of STAT-1 binding activity was induced by LTA treatment. This increased STAT-1 binding activity was dramatically inhibited by schizandrin C (Fig. 6B). To investigate whether MAPKs are regulated by schizandrin C in LTA-stimulated

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p-ERK ERK p-JNK JNK p-p38 p38 Fig. 6. Effect of schizandrin C on LTA-induced activation of JAT-STATs and MAPK. (A) BV-2 microglial cells were treated with increasing doses of schizandrin C for 1 h followed by LTA for 2 h. Phosphorylation of STAT-1 and STAT-3 was confirmed by western blotting. Western blot detection of STAT-1 or STAT-3 was estimated by comparison to the protein-loading control for each lane. (B) Cells were incubated with schizandrin C (20 μM) for 1 h and then with LTA for 4 h. DNA was immunoprecipitated using an anti-p-STAT-1 antibody and the precipitated iNOS promoter region was amplified by PCR. The input represents PCR products from chromatic pellets prior to immunoprecipitation. (C) Cells were treated with increasing doses of schizandrin C for 1 h followed by LTA for 0.5 h. An equal amount of cell extract was analyzed by western blotting with anti-p-ERK1/2, anti-JNK, or anti-p-p38 antibodies. ERK1/2, JNK, or p38 bands indicated that the induction of total ERK1/2, JNK, or p38 protein was not changed.

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3.6. Inhibition of the cAMP/PKA/CREB and Nrf-2 pathway abolished the anti-neuroinflammatory potential of schizandrin C Experiments were carried out to investigate the role of the cAMP/PKA/ CREB and Nrf-2 pathway in the observed anti-neuroinflammatory effect of schizandrin C. Briefly, BV-2 microglia cells were transiently transfected with HO-1 and Nrf-2 siRNA, after which the effects of schizandrin C on LTA-induced ROS generation and iNOS promoter activity were investigated. Transfection with HO-1 and Nrf-2 siRNA significantly inhibited schizandrin C-mediated inhibition of iNOS promoter activity and ROS production when compared to control siRNA (Fig. 7A and B). Next, we pre-incubated cells with schizandrin C in the presence or absence of ddAdo or PKA inhibitor (H-89) and then treated cells with LTA. We found that ddAdo or PKA inhibitor (H-89) reversed the inhibitory effects of schizandrin C on iNOS promoter activity and ROS production (Fig. 7C and D). These results suggested that activation of the cAMP/PKA/CREB and Nrf-2 pathway was responsible for the antineuroinflammatory effect of schizandrin C. 4. Discussion Accumulating evidence indicates that phase II detoxifying/antioxidant enzymes may contribute to retrogressive neurodegenerative disorders in pathological processes of dementia [19]. Interestingly,

cAMP/PKA/CREB and Nrf-2 signaling activate phase II detoxifying/ antioxidant enzymes such as HO-1 and NQO-1. These enzymes play key roles in neurodegenerative disorders by enhancing the reduction of ROS and neuroinflammatory responses [24]. Natural products are particularly promising sources of induction of the Nrf-2-mediated phase II detoxifying/antioxidant enzyme pathway. Polypenols, alkaloids, flavonoids, and terpenoids have previously been reported to induce the Nrf-2-mediated phase II detoxifying/antioxidant enzyme pathway [5]. However, the effects of schizandrin C on the Nrf-2-mediated phase II detoxifying/antioxidant enzyme pathway are currently poorly understood. Here, we present the first evidence that schizandrin C isolated from S. chinensis inhibits the neuroinflammatory response in LTA-stimulated microglia through phase II detoxifying/antioxidant enzyme induction via cAMP/PKA/CREB and Nrf-2 signaling. There is sufficient evidence that over activation of microglia is associated with various neurodegenerative disorders. Microglia are activated by stress inducers, including pathogen invasion, protein aggregation, and signals from damaged cells. Upon activation, they promote the production of pro-inflammatory cytokines and mediators to remove abnormal factors and recover the injured site. However, uncontrolled activation of microglia in the nervous system results in chronic neuroinflammatory responses that lead to further neurodegeneration [25,26]. Numerous microglia cells are activated in the brain during the course of several neurodegenerative diseases, including Alzheimer's disease, resulting in the

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Fig. 7. Effect of cAMP/PKA/CREB and Nrf-2 disruption on schizandrin C-mediated anti-neuroinflammatory effects. V-2 microglial cells were transfected with si-control and si-Nrf-2 or si-HO-1. Twenty-four hours after transfection, cells were treated with schizandrin C (20 μM) for 1 h and were then stimulated with LTA for 24 h, after which iNOS promoter activity (A) and ROS (B) levels were determined. Cells were pre-treated with schizandrin C (20 μM) in the absence or presence of ddAdo (100 μM) or H-89 (5 μM) for 1 h and were then stimulated with LTA for 24 h. iNOS promoter activity (C) and ROS (D) levels were determined. *P b 0.05 vs the LTA-treated group; #P b 0.05 vs the schizandrin C plus LTA-treated group.

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Activation process Inhibition process

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Fig. 8. The proposed mechanisms underlying the anti-neuroinflammatory effects of schizandrin C on LTA-induced microglial activation. Schizandrin C inhibited the expression of pro-inflammatory mediators, and attenuated the activation of MAPK, NF-κB, AP-1 and STAT-1,3, which was accompanied by inducing HO-1 and NQO-1 through the cAMP–PKA–CREB pathway.

release of pro-inflammatory cytokines and retention of chronic inflammation [9]. In other words, neuroinflammation, microglia activation, and up-regulation of pro-inflammatory cytokines represent pathological features of neurodegenerative diseases. Upon activation of microglia cells, the cellular response initiates from the activation of the membrane receptor. TLRs are well-characterized pattern recognition receptors that recognize pathogen-associated molecular patterns, including LPS, LTA, or bacterial DNA, or damage associated molecular patterns, including heat shock proteins and amyloid β. TLRs are present in all of the major cells of the CNS, and their activation leads to the expression of various pro-inflammatory mediators and anti-microbial factors. TLR2 and TLR4 are believed to be involved in chronic inflammatory responses in vivo,

as well as in several autoimmune related diseases such as atherosclerosis and rheumatoid arthritis [27,28]. Therefore, to investigate whether schizandrin C might decrease neuroinflammatory molecules through down-regulation of TLR-2, its effects on the expression of TLR-2 in LTA-stimulated BV-2 microglial cells were examined. Real-time PCR showed that schizandrin C did not affect the expression of TLR-2 in LTA-stimulated BV-2 microglial cells (data not shown), suggesting that schizandrin C-mediated inhibition of neuroinflammatory molecules is not a result of inhibition of TLR-2 expression. Next, we examined the effect of schizandrin C on microglia using Gram-positive endotoxin LTA, which is known to act on TLR2. First, we demonstrated that schizandrin C has an anti-neuroinflammatory

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effect by inhibiting inflammation inducers, including ROS, NO, and PGE2, pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1β, IL-6, and MCP-1, and pro-inflammatory mediators, iNOS, COX-2, and MMP-9, in LTA-stimulated microglia. Schizandrin C also showed a strong ability to suppress the upstream cellular pathways involved in the progression of TLR-triggered inflammatory responses in microglia. The primary kinase-mediated signal pathways, MAPK pathway, and transcription factor NF-κB and AP-1 pathway, which has its binding site in the promoter region of pro-inflammatory cytokines, are engaged upon TLR activation, which induces the transduction of extracellular signals for cellular reactions [22]. In the present study, we found that schizandrin C diminished the phosphorylation of MAPKs, including ERK, JNK, and P38, in response to LTA. In addition, schizandrin C decreased the nuclear translocation and transactivation of NF-κB and AP-1 caused by LTA stimulation. Another group of key transcription factors, STATs, are believed to strengthen the pro-inflammatory environment. STAT-1 and STAT-3 have been shown to play important roles in cytokine responses, inflammatory gene expression, and apoptotic cell death. Indeed, a study using LTA-stimulated macrophages showed that STAT-3 phosphorylation was crucial for the production of the pro-inflammatory cytokines IL-1β and IL-6, and an in vivo study revealed that STAT-1 knock-out mice did not have IL-6 [12]. Results of the present study showed that LTA-induced phosphorylation of STAT-1 and STAT-3 significantly increased after 4 h, which was later than the occurrence of MAPK activation. However, the phosphorylation levels of STAT-1 and STAT-3 decreased with schizandrin C treatment in dose-dependent manners. The later induction time of STAT-1 and STAT-3 relative to that of MAPKs also suggests that induction of STAT-1 and STAT-3 in microglia was downstream of MAPK, and that schizandrin C inhibited MAPK activation and subsequent STAT-1 and STAT-3 signal factors. The suppressed production of these neuroinflammatory molecules was reversed in cells treated with Nrf-2 and HO-1 siRNAs and with ddAdo and PKA inhibitor (H-89) [29]. Although many studies have focused on the protective effects of phase II detoxifying/ antioxidant enzymes against oxidative stress, these enzymes have recently received attention due to their potent immune-modulators and anti-inflammatory effects. Phase II detoxifying/antioxidant enzymes are primarily regulated by the cAMP/PKA/CREB and Nrf-2 signal pathway, which promotes transcription of anti-oxidant genes, including phase II detoxifying/antioxidant enzymes, and exhibits cytoprotective effects. In the progression of Parkinson's disease, Nrf-2-knockout mice showed significant activation of microglia when compared to wild type cells, underlining the importance of the cAMP/PKA/CREB and Nrf-2 signal pathway in the modulation of microglia function [2]. Moreover, over-expression of phase II detoxifying/antioxidant enzyme genes has been shown to have beneficial effects in several experimental animal models of inflammation, as well as in microglia [2,30]. In accordance with results of these previous studies, we here demonstrated that schizandrin C exhibits anti-neuroinflammatory effects in microglia through up-regulation of cAMP/PKA/CREB and Nrf-2-mediated HO-1 and NQO-1, and down-regulation of various transcription factors involved in pro-inflammatory responses.

5. Conclusion Schizandrin C isolated from S. chinensis activated the cAMP/PKA/CREB and Nrf-2 mediated phase II detoxifying/antioxidant enzyme signaling pathway in microglia. The cAMP/PKA/CREB and Nrf-2 mediated phase II detoxifying/antioxidant enzyme signaling pathway of schizandrin C was mediated by disruption of the LTA-induced neuroinflammatory response via inactivation of the MAPK, NF-κB, AP-1 and STAT signaling pathways (Fig. 8). Therefore, schizandrin C isolated from S. chinensis represents a novel inducer of the cAMP/PKA/CREB and Nrf-2 mediated phase II detoxifying/antioxidant enzyme-signaling pathway, and is a potential

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nutraceutical for the prevention and treatment of neurodegenerative diseases. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A3010601). This study was supported by a grant (code #7-19-42) from the Rural Development Administration, Republic of Korea. References [1] Lai HC, Wu MJ, Chen PY, Sheu TT, Chiu SP, Lin MH, et al. Neurotrophic effect of citrus 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone: promotion of neurite outgrowth via cAMP/PKA/CREB pathway in PC12 cells. PLoS One 2011;6:e28280. [2] Lee IS, Lim J, Gal J, Kang JC, Kim HJ, Kang BY, et al. Anti-inflammatory activity of xanthohumol involves heme oxygenase-1 induction via NRF-2-ARE signaling in microglial BV2 cells. Neurochem Int 2011;58:153–60. [3] Manandhar S, You A, Lee ES, Kim JA, Kwak MK. 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