NF-κB, MAPKs and Akt signaling pathways.

Neuropharmacology 79 (2014) 642e656

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Pseudoginsenoside-F11 (PF11) exerts anti-neuroinflammatory effects on LPS-activated microglial cells by inhibiting TLR4-mediated TAK1/ IKK/NF-kB, MAPKs and Akt signaling pathways Xiaoxiao Wang a, Chunming Wang a, Jiming Wang b, Siqi Zhao a,1, Kuo Zhang a, Jingmin Wang a, Wei Zhang a, Chunfu Wu a, *, Jingyu Yang a, * a b

Department of Pharmacology, Shenyang Pharmaceutical University, Box 31, 103 Wenhua Road, 110016 Shenyang, PR China Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute at Frederick, National Institutes of Health, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2013 Received in revised form 10 January 2014 Accepted 13 January 2014

Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been shown to possess significant neuroprotective activity. Since microglia-mediated inflammation is critical for induction of neurodegeneration, this study was designed to investigate the effect of PF11 on activated microglia. PF11 significantly suppressed the release of ROS and proinflammatory mediators induced by LPS in a microglial cell line N9 including NO, PGE2, IL-1b, IL-6 and TNF-a. Moreover, PF11 inhibited interaction and expression of TLR4 and MyD88 in LPS-activated N9 cells, resulting in an inhibition of the TAK1/IKK/ NF-kB signaling pathway. PF11 also inhibited the phosphorylation of Akt and MAPKs induced by LPS in N9 cells. Importantly, PF11 significantly alleviated the death of SH-SY5Y neuroblastoma cells and primary cortical neurons induced by the conditioned-medium from activated microglia. At last, the effect of PF11 on neuroinflammation was confirmed in vivo: PF11 mitigated the microglial activation and proinflammatory factors expression obviously in both cortex and hippocampus in mice injected intrahippocampally with LPS. These findings indicate that PF11 exerts anti-neuroinflammatory effects on LPSactivated microglial cells by inhibiting TLR4-mediated TAK1/IKK/NF-kB, MAPKs and Akt signaling pathways, suggesting its therapeutic implication for neurodegenerative disease associated with neuroinflammation. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: PF11 Microglia TLR4 NF-kB MAPKs Akt

1. Introduction

Abbreviations: PF11, pseudoginsenoside-F11; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; IL, interleukin; PGE2, prostaglandin E2; LPS, lipopolysaccharide; MAPKs, mitogen-activated protein kinases; TLR4, toll-like receptor 4; MyD88, myeloid differentiation factor 88; TRAF6, tumor necrosis factor receptorassociated factor 6; IRAK1, interleukin-1 (IL-1) receptoreassociated kinase 1; TAK1, transforming growth factor b activated kinase 1; IKK, IkB kinases; NF-kB, nuclear factor kB; ERK1/2, extracellular signal regulated kinase; JNK, c-Jun N-terminal protein kinase; PI3K, phosphatidylinositol 3-kinase; NADPH, nicotinamide adenine dinucleotide phosphate; AD, Alzheimer’s disease; PD, Parkinson’s disease. * Corresponding authors. Tel./fax: þ86 24 23986339. E-mail addresses: [email protected] (X. Wang), [email protected]. cn (C. Wang), [email protected] (J. Wang), [email protected] (S. Zhao), [email protected] (K. Zhang), [email protected] (J. Wang), 798891145@ qq.com (W. Zhang), [email protected] (C. Wu), [email protected] (J. Yang). 1 Current address: State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China. 0028-3908/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2014.01.022

Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside contained in Panax quinquefolium L., was isolated from leaves of Panax pseudoginseng subsp. Himalaicus HARA (Himalayan Panax) (Wang et al., 2011). As a typical member of saponins in ginseng family, PF11 was able to reduce memory impairment in scopolamine-induced dementia (Li et al., 1999) and improved spatial and non-spatial memory in mice treated with morphine or methamphetamine (Hao et al., 2007; Li et al., 2000; Wu et al., 2003). Especially, PF11 could also improve cognitive impairment in mice treated with Ab1-42, as well as APP/PS1 mice through its inhibitory effect on amyloidogenesis, oxidative stress and beneficial effects on neuronal functions (Wang et al., 2013). Therefore, PF11 is a promising candidate for the development of neuroprotective drugs. Microglia, the resident macrophage-like cells in the brain, have been proposed to play a pivotal role in the immune surveillance of the central nervous system (CNS) (Lue et al., 2010). Furthermore, lines of evidence showed that microglia-mediated neuroinflammation

X. Wang et al. / Neuropharmacology 79 (2014) 642e656

contribute to the pathology of both acute pathologies such as stroke, traumatic brain injury and chronic neurodegenerative diseases (Gu et al., 2012; Hagberg et al., 2012; Wu et al., 2009). Although the etiology of these diseases is complicated, brain inflammation plays an important role, and it has been confirmed that blocking inflammation could either delay onset or alleviate the symptoms of these diseases (Brown and Neher, 2010). Enzymes associated with inflammation, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), have been identified postmortem in AD and PD brains (Collins et al., 2012; Zilka et al., 2012). When subjected to abnormal stimulation, microglia become activated, with the release of proinflammatory cytokines (Choi et al., 2011; Siopi et al., 2013) and reaction oxygen species (ROS) due to catalysis by NADPH oxidase, which further aggravate neuroinflammatory injury (Dohi et al., 2010). Toll like receptor 4 (TLR4), an important Pattern Recognition Receptor (PRR) is expressed by microglial cells (Okun et al., 2009) and is responsible for inflammatory cascade in microglia upon binding with lipopolysaccharide (LPS) (Verstak et al., 2009). Thus, inhibiting the activation of microglia mediated by TLR4 in inflammatory responses represents a potentially neuroprotective treatment strategy. Until now, the effect of PF11 on microglia-mediated neuroinflammation has not been investigated. This study was designed to investigate the capability of PF11 on LPS-induced microglial activation in vitro and in vivo. Here, we report that PF11 is able to inhibit the activation of microglia by inhibiting TLR4-mediated TAK1/IKK/ NF-kB, MAPKs and Akt signaling pathways which resulting in neuron protection. 2. Materials and methods 2.1. Reagents and antibodies Iscove’s Modified Dulbecco’s Medium (IMDM), Dulbecco’s Modified Eagel Medium (DMEM), Neurobasal Medium, B27 supplement, fetal bovine serum (FBS) and 0.25% trypsin were purchased from Gibco BRL (Grand Island, NY, USA). Lipopolysaccharide (LPS) from Escherichia coli 026:B6, 20 ,70 -dichlorodihydrofluororescein diacetate (DCF-DA), Nitrotetrazolium Blue chloride (NBT), 2,2-diphenyl-1picrylhydrazyl (DPPH), minocycline hydrochloride (MINO), dephenylene iodonium (DPI), N-acetyl-Lcysteine (NAc) and pyrrolidine dithiocarbamate (PDTC), poly-LLysine hydrobromide, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and normal horse serum were purchased from Sigma Chemical Co. (St. Louis, MO, USA); LPS-RS (LPS-derived from the photosynthetic bacterium Rhodobacter sphaeroides) was from InvivoGen (SanDiego, CA, USA); Trizol reagent was from Invitrogen Co. (Carlsbad, CA, USA); RevertAidÔ First Strand cDNA Synthesis Kit was from Fermentas (Burlington, Ontario, Canada); Taq polymerase was from the Takara Biotechnology (Dalian, China). TNF-a, IL-1b, IL-6 and PGE2 ELISA kits were from R&D Systems (Abingdon, UK); Antibodies see Table 1.

2.2. Microglial cell culture The murine microglia cell line N9 was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). The cells were similar to primary microglia in producing NO and various cytokines after stimulation. N9 cells were cultured in IMDM supplemented with 5% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in humidified 5% CO2 (Iribarren et al., 2005).

2.3. Animals Thirty-six male C57BL/6 mice were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). The animals were housed under conventional conditions at 22 C, 50e60% humidity and a 12 h light/ 12 h dark cycle. Mice were provided free access to food and water. All experiments were conducted to minimize the suffering of animals caused by any procedures according to the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China.

2.4. PF11 treatments PF11 (Fig. 1A) was isolated from the aerial parts of Panax quinquefolium L. by Department of Chemistry for Nature Products of Shenyang Pharmaceutical University (China). The purity of PF11 detected with HPLC was more than 98%.

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Table 1 Primary antibodies used in this study. Primary antibody

Dilution

Source

Toll-like Receptor 4 Antibody (Rodent Specific) IKKb (2C8) Rabbit Antibody IKKa Rabbit Antibody TAK1 Rabbit Antibody Phospho-IKKa/b (Ser176/180) (16A6) Rabbit Antibody Phospho-TAK1 (Thr184/187) (90C7) Rabbit Antibody iNOS Rabbit Antibody COX-2 Rabbit Antibody p38 MAPK Rabbit Antibody SAPK/JNK (56G8) Rabbit Antibody p44/42 MAPK (Erk1/2) (137F5) Rabbit Antibody Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XPÔ Rabbit Antibody Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) XPÔ Rabbit Antibody Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit Antibody p-Akt (S473) Rabbit Antibody Akt Rabbit Antibody Anti Iba1 Rabbit Antibody NF-kB p65 Rabbit Antibody MyD88 (HFL-296) Rabbit Antibody NF-kB p50(E-10) Mouse Antibody b-actin Mouse Antibody Naþ/K þ -ATPase a(H-3) Mouse Antibody HDAC1 (10E2) Mouse Antibody Phospho-IkBa (Ser32/36) (5A5) Mouse Antibody IkBa (L35A5) Mouse mAb (Amino-terminal Antigen) MAP2 Antibody

1:1000

Cell Signaling Technology (CST)

1:1000 1:1000 1:1000 1:500 1:500 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000

1:2000

1:1000 1:500 1:1000 1:1000 1:1000 1:500 1:1000 1:500 1:1000 1:1000 1:1000

Wako Santa Cruz

CST

1:1000 1:1000

Millipore

2.4.1. Treatment procedures of PF11 in vitro PF11 was initially dissolved in dimethyl sulfoxide (DMSO) and diluted with phosphate-buffered saline (PBS). The highest concentration of DMSO present in experimental conditions (0.1%) was not toxic to the cells. The endotoxin content of purified PF11 was less than 0.2 EU/mg. To determine interactions of TLR4 with MyD88 or TAK1 by immunoprecipitation, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 10 min. For determination phosphorylation of IKKa/b, TAK1 by Western blot, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 15 min. To detect phosphorylation of MAPKs, NF-kB and IkB-a by Western blot, and nuclear translocation of NFkB p65 by High Content Screening System, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 30 min. For detection of mRNA expression of proinflammatory factors by RT-PCR and ROS production, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 6 h. In NADPH oxidase activity assay, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 12 h. To investigate NO production by Griess reaction, proinflammatory factors release by ELISA, and expression of TLR4, MyD88, iNOS and COX-2 by western blot, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 24 h. Generally, the MTT reduction assay was performed after treatment with LPS for 24 h.

2.4.2. Treatment procedures of PF11 in vivo The mice were divided into three groups randomly (n ¼ 12 in each group): (1) saline group; (2) LPS group; and (3) PF11 8 mg/kg group (Hao et al., 2007; Wang et al., 2013; Wu et al., 2003). Each mouse in the PF11 8 mg/kg group was treated with PF11 at a dose of 8 mg/kg orally once daily for 21 days, while each mouse in the saline group and LPS group received an equal volume of saline. On day 8, the mice in the LPS group and PF11 8 mg/kg group were slowly injected with LPS 40 mg into hippocampus bilaterally (1 ml on each site over 5 min) using the following coordinates: AP -2.4 mm, ML 2.2 mm and DV -2.4 mm from the bregma (Paxinos and Franklin, 2001), the mice of saline group were injected with equal volume of saline. When the injection was finished, the needle was left in situ for 5 min to avoid reflux along the injection track before withdraw.

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Fig. 1. Effects of PF11 on production of NO and PGE2 and expression of iNOS and COX-2 induced by LPS in N9 microglial cells. (A) The chemical structure of PF11. (B) N9 microglial cells were pretreated with PF11 (1e100 mM) or minocycline (MINO, 20 mM) and then stimulated with LPS (1 mg/ml) for 24 h. NO production was measured by Griess reagents. (C) N9 microglial cells were pretreated with PF11 (1e100 mM) or MINO (20 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 24 h. The production of PGE2 in supernatant was analyzed. (D) N9 microglial cells were pretreated with PF11 (1e100 mM) or MINO (20 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 24 h. Cell viability was detected by MTT assay. (E, G) N9 microglial cells were pretreated with PF11 (1e100 mM) or MINO (20 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 6 h. The mRNA levels of iNOS and COX-2 were measured by RT-PCR. (F, H) N9 microglial cells were pretreated with PF11 (1e100 mM) or MINO (20 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 24 h. The protein levels of iNOS and COX-2 were analyzed by Western blot. ###p < 0.001 vs. control group (cultured in medium alone); **p < 0.01, ***p < 0.001 vs. LPS group.

2.5. Measurement of cell viability Cell viability was evaluated by the MTT reduction assay (Lee et al., 2010a). In brief, N9 cells at 1 105 cells/well were seeded into 96-well plates. After various treatments for 24 h, the medium was removed and the cells were incubated with MTT (0.25 mg/ml) for 3 h at 37 C. The formazan crystals in the cells were solubilized with DMSO. The level of MTT formazan was determined by measuring its absorbance at 490 nm using a microplate reader (Synergy HT, BioTek, USA).

2.6. Nitrite assay Accumulation of nitrite (NO 2 ), an indicator of NO synthase activity, in cell culture supernatant was measured by the Griess reaction (Chen et al., 2009). N9 cells (1 105 cells/well) were seeded into 96-well plates and treated with PF11 for 2 h then stimulated with 1 mg/ml LPS. After incubation for 24 h, fifty microliter of culture supernatant was mixed with equal volume of Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylene diamide dihydrochloride and 2% phosphoric acid) at room temperature. Fifteen minutes later, the absorbance was determined at 540 nm using a microplate reader (Synergy HT, BioTek, USA).

2.7. Enzyme-linked immunosorbent assay (ELISA) N9 microglial cells were plated into 96-well plates (1 105 cells/well). Cells were pretreated with PF11 or minocycline for 2 h and then stimulated with 1 mg/ml LPS for

24 h. The levels of PGE2, TNF-a, IL-1b and IL-6 in the culture medium were measured by ELISA kits according to the manufacturer’s instructions. 2.8. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA of N9 microglial cells, cortex and hippocampus of mice were extracted using Trizol and converted to cDNA by using a cDNA first-strand synthesis system (Fermentas, Canada). PCR amplification was carried out in 25 ml PCR reaction mixture containing 10 mM TriseHCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 20 pmol primer sets, 1 units of Taq DNA polymerase (Takara, Dalian), 0.2 mM dNTPs, and 1 mg cDNA. Nucleotide sequences of the primers and PCR conditions were described in Table 2. PCR products were separated on 1.2% agarose gels, and visualized with ethidium bromide (EB). Expression level of the GAPDH gene was used for standardization. 2.9. Immunofluorescence staining The nuclear localization of NF-kB p65 was examined by indirect immunofluorescence assay using High Content Screening System (Molecular Devices, USA) (Ock et al., 2010). N9 microglial cells were cultured in 96-well plates for 24 h. The cells were pretreated with PF11 or minocycline for 2 h. After stimulation with LPS for 30 min, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and blocked with 1% normal horse serum. The polyclonal antibody to NF-kB p65 was applied overnight, followed by 1 h of incubation with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit

Table 2 Primer sequences and experimental conditions used for RT-PCR. Gene

Forward primer

Reverse primer

PCR conditions

iNOS

50 -ACA ACA GGA ACC TAC CAG CTC A-30

50 -GAT GTT GTA GCG CTG TGT GTC A-30

COX-2

50 -CCA GAT GCT ATC TTT GGG GAG AC-30

50 -CTT GCA TTG ATG GTG GCT G-30

IL-1b

50 -ATG GCA ACT GTT CCT GAA CTC AAC T-30

50 -CAG GAC AGG TAT AGA TTC TTT CCT TT-30

IL-6

50 -GCT ATG AAG TTC CTC TCT GC-30

50 -CTA GGT TTG CCG AGT AGA TC-30

TNF-a

50 -CCA GAC CCT CAC ACT CAG AT-30

50 -AAC ACC CAT TCC CTT CAC AG- 30

GAPDH

50 -ACC ACA GTC CAT GCC ATC AC-30

50 -TCC ACC ACC CTG TTG CTG TA-30

Denaturation: 94 Extension: 72 C, Denaturation: 94 Extension: 72 C, Denaturation: 94 Extension: 72 C, Denaturation: 94 Extension: 72 C, Denaturation: 94 Extension: 72 C, Denaturation: 94 Extension: 72 C,

C, 45 s; Annealing:64 C, 45 s; 1 min; 30 cycles C, 45 s; Annealing: 56 C, 45 s; 1 min; 30 cycles C, 30 s; Annealing: 55 C, 45 s; 45 s; 28 cycles C, 45 s; Annealing: 52 C, 45 s; 1 min; 30 cycles C, 30 s; Annealing: 55 C, 45 s; 45 s; 28 cycles C, 30 s; Annealing: 55 C, 45 s; 45 s; 28 cycles

X. Wang et al. / Neuropharmacology 79 (2014) 642e656 IgG. After being washed with PBS, the cells were visualized and photographed. Briefly, using a 40 objective, images of 9 sites per well (3 3) were captured for high-content analysis. Images were acquired via two independent channels with fixed exposure times. Two wavelengths could be imaged together for the same site. For NF-kB p65, a FITC filter set was used. For corresponding fluorescent images of Hoechst-stained cell nuclei, a UV filter set was used (Liu et al., 2012; Smith et al., 2013). 2.10. Western blot analysis

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2.15. Neuronal cell culture Primary neuronal cells were from the cerebral cortex of embryonic day 16 (E16) C57BL/6 mice (Pan et al., 2008). After dissection and removal of the meninges, the cortices were minced and dissociated with 0.05% trypsin at 37 C for 15 min. The reaction was stopped by addition of 1% horse serum, and the tissue was then mechanically dissociated with a Pasteur pipette. Cells were seeded into 96-well plates (1 105 cells/well) pre-coated with 0.01% poly-L-lysine, and cultured in Neurobasal Medium supplemented with 2% B27, 100 U/ml penicillin and 100 mg/ml streptomycin. Human SH-SY5Y neuroblastoma cell line was obtained from the American Type Culture Collection (ATCC) and cultivated in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. SHSY5Y cells seeded at a density of 1 105 cells/well in 96-well plates and allowed to settle at 37 C for 24 h before replacement with conditioned medium.

Western blot analysis was performed as previously described (Lee et al., 2009) with slight modifications. For determination of IKKa/b and TAK1, N9 microglial cells were pretreated with PF11 for 2 h, and then exposed to LPS for 15 min. To determine phosphorylation of MAPKs and IkB-a, N9 microglial cells were pretreated with PF11 or minocycline for 2 h, and then exposed to LPS for 30 min. In order to detect TLR4, MyD88, iNOS, COX-2, N9 microglial cells were pretreated with PF11 for 2 h, and then exposed to LPS for 24 h. To make whole cell lysates, cells were washed with ice-cold PBS and lysed for 10 min in RIPA lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 1 mM phenylmethyl-sulfonylfluoride, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mg/ml aprotinin). Nuclear and cytoplasmic proteins were obtained with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, USA) according to manufacturer’s protocol. The protein concentration in the supernatant of the lysate was measured by BCA protein assay (Beyotime, China). Equal amounts (60 mg) of proteins were separated electrophoretically by SDS-PAGE and then transferred to 0.45 mm polyvinylidene fluoride membrane (PVDF: Millipore, Bedford, MA). The membranes were soaked in blocking buffer (5% skimmed milk or BSA) and then incubated overnight with corresponding primary antibody, followed by horseradish peroxidase conjugated secondary antibody. Following three washes in Tris-buffered saline-Tween 20 (TBST), immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) (Thermo, USA). Band pattern was analyzed with Quantity One 4.1.0 software (Bio-Rad).

N9 microglial cells were pretreated with different concentrations of PF11 (1e 100 mM) for 2 h, then stimulated with LPS (1 mg/ml) for 48 h. The culture medium was collected as conditioned medium (CM) and clarified by centrifugation at 12,000 g for 5 min to remove cellular debris. The conditioned medium were then transferred to neuronal cells, which were further incubated at 37 C for 24 h. Cell viability was measured by MTT assay. To observe morphological changes such as the dendritic processes of primary cortical neurons, cells were stimulated as described above and fixed in 4% paraformaldehyde at 37 C for 30 min. After rinsing, cells were blocked with 5% BSA in PBS for 1 h and incubated with anti-MAP2 antibody overnight at 4 C. After being washed, cells were incubated with Alexa Fluor 488-conjugated anti-mouse IgG for 1 h. Neurons were then photographed under a fluorescence microscope (Olympus IX71, Japan).

2.11. Immunoprecipitation (IP)

2.17. Brains collection and detection

To examine proteineprotein interactions, N9 microglial cells cultured on 100 mm dishes were pretreated with PF11 for 2 h and then exposed to LPS for 10 min. The cells were lysed in 1 mL buffer consisting of 50 mM TriseHCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 10 mM NaF, 1 mM Na3VO4, 10 mg/mL leupeptin, 10 mg/mL aprotinin and 20 mM PMSF after harvesting. Aliquots of the cellular lysates (containing 500 mg proteins) were incubated with proper primary anti-TLR4 and anti-MyD88 antibodies with rocking overnight at 4 C. The immune complexes were allowed to bind to 40 ml of Recombinant Protein G Agarose beads (Invitrogen, USA) at 4 C for 2 h, and the beads were washed three times with lysis buffer. The washed beads were resuspended in electrophoresis sample buffer and boiled for 10 min. After centrifugation, the supernatants were obtained as immunoprecipitates for Western blot analysis (Lin et al., 2013; Zhou et al., 2008).

After PF11 treatment, all the brains were collected. For immunofluorescence staining, six mice in each group were anesthetized and perfused transcardially with 25 ml of PBS, followed by 25 ml of PBS containing 4% paraformaldehyde, and sacrificed by decapitation. The head was cut off and grasped by one hand, the scalp and muscle was cut with a scissor. Beginning at upper edge of the foramen magnum, small bites of bone progressing were taken dorsally and laterally with rongeurs. Pressure away from the brain surface was maintained to ensure that slippage of the rongeurs would not penetrate the brain. Two channels of removed bone along both lateral surfaces at a level just above the junction of the zygomatic arch were created, then a flap of skull bone remains was carefully elevated and broken off near the junction of sutures that form the bregma point. To remove the dura mater, a slit along the midline of cortical surface was made with a scissor, and the dura mater was elevated and pushed aside. The conjunction site between brainstem and medulla was also cut off. The brainstem was elevated and the remaining attached blood vessels and optic nerve was cut, then the brain was removed from the cranium entirely and postfixed for 24 h in the same fixative at 4 C. The brains were subsequently cytoprotected overnight in 20% and 30% sucrose prepared in PBS respectively at 4 C. After sinking to bottom, all the brains were taken out from the sucrose solution. The water was absorbed with filter paper quickly. The brains were subsequently put into ziplock bag individually and transferred to 80 C until frozen section (Herber et al., 2004; Lee et al., 2010b). For RT-PCR, the rest of mice in each group were anesthetized and perfused transcardially with 50 ml of ice-cold PBS, and sacrificed by decapitation. The brain was removed from the cranium carefully as mentioned above and dissected to collect cortex and hippocampus on the ice, then stored at 80 C immediately until solubilized with Trizol (Rogers et al., 2011). To determine the effect of PF11 on microglial activation in mice treated intrahippocampally with LPS, the expression of Iba-1 in cortex and hippocampus was detected. Serial 20 mm-thick coronal sections containing the hippocampus and cortex (between 1.0 and 3.0 mm from bregma) were cut with a freezing microtome (Shandon, USA) and mounted on gelatin-coated slides. Antigen retrieval was performed by immersion of the slides into 10 mM trisodium citrate buffer pH 6.0 in a water bath at 85e95 C for 20 min, 3% H2O2 was used to quench endogenous peroxidase, nonspecific binding was blocked by incubating in PBS containing 5% goat serum for 60 min at room temperature. After overnight incubation at 4 C with the primary antibody for Iba-1 (1:1000, Serotec), the sections were incubated with biotinylated secondary antibody for 1 h at room temperature, washed with PBS, and subsequently incubated with streptavidin-conjugated peroxidase complex (Vector Laboratories, USA) for 30 min followed by washing with PBS. The peroxidase reaction was performed using 3, 30 -diaminobenzidine tetrahydrochloride as the chromogen. Finally, the sections were dehydrated, and cover-slipped for light microscopy and photography (Olympus IX71, Japan). The number of Iba-1 positive microglia of each slice section was counted by one person blinded to the

2.12. Stable free radical scavenging activity (DPPH reduction assay) The stable free radical scavenging activity was determined according to the reported method (Jeong et al., 2007). In brief, a 100 mM DPPH radical solution was dissolved in 100% ethanol. The mixture was shaken vigorously and allowed to stand for 10 min in the dark. The test materials (100 ml each) were added to 900 ml of DPPH radical solution. After incubation at room temperature for 30 min, the absorbance at 517 nm was measured, using a microplate reader (Synergy HT, BioTek, USA). 2.13. Measurement of ROS production Production of ROS was measured by DCF-DA assay (Yang et al., 2006). N9 microglial cells were pretreated with PF11 (1e100 mM), DPI (an NADPH oxidase inhibitor, 1 mM) or NAc (a potent antioxidant compound, 5 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 6 h. DCF-DA (10 mM) was then added and incubated for 1 h at 37 C, and fluorescence was measured using a microplate reader with excitation at 485 nm and emission at 528 nm. 2.14. NADPH oxidase activity assay NADPH oxidase activity was determined using the NBT assay (Park et al., 2011), based on the reduction of NBT to formazan by superoxide oxide. The cells (1 105 cells/well) were cultured in 24-well plates and then treated with 1e100 mM PF11 for 2 h followed by LPS (1 mg/ml) for 12 h. 0.1% NBT was added to the media at the end of the treatment periods. After incubation for 2 h at 37 C, the cells were washed twice with PBS fixed with methanol and air-dried. The cells were photographed under a microscope by a digital camera (Olympus IX71, Japan). The NBT deposited inside the cells was then dissolved with 240 ml of 2 M potassium hydroxide (KOH) and 280 ml of DMSO, and gently shaken for 10 min at room temperature. The dissolved NBT solution was then transferred to a 96-well plate and the absorbance was measured using a microplate reader at 630 nm.

2.16. Microglia-conditioned medium preparation, treatments and detection

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treatment group identities with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). 2.18. Data analysis Results were expressed as mean S.E.M. of three independent experiments performed in triplicates. One-way ANOVA followed by Dunnett’s t-test was used for statistical analysis (SPSS 15.0 software, SPSS, USA).

PF11 (Fig. 2A, B & C). Minocycline (20 mM) also significantly inhibited LPS-induced IL-1b, IL-6 and TNF-a released by N9 cells. Furthermore, PF11 and minocycline attenuated pro-inflammatory cytokine mRNA expression induced by LPS in N9 cells (Fig. 2D, E & F).

3.3. PF11 inhibited LPS-induced interactions of TLR4 with MyD88 or TAK1 and expressions of TLR4 and MyD88 in N9 microglia

3. Results 3.1. PF11 inhibited NO and PGE2 production in LPS-stimulated microglial cells through suppression of iNOS and COX-2 expression As shown in Fig. 1B and C, exposure of N9 microglial cells to LPS (1 mg/ml) for 24 h markedly increased nitrite (a stable oxidized product of NO) and PGE2 production by approximately 5 and 2 folds, which were inhibited by PF11 (30e100 mM) or minocycline (20 mM). PF11 alone did not induce the production of NO and PGE2 in N9 cells. N9 cells treated with PF11 (1e100 mM) for 24 h did not show reduction in viability (Fig. 1D), excluding the cytotoxicity of PF11. As shown by Western blot analysis, LPS treatment significantly increased the production of iNOS and COX-2 proteins by N9 microglial cells, which was also markedly attenuated by PF11 (30e100 mM) (Fig. 1F and H). RT-PCR analysis also showed that PF11 (3e100 mM) attenuated the increase of iNOS and COX-2 mRNA expression induced by LPS in N9 cells (Fig. 1E and G). PF11 alone had no effect. 3.2. PF11 inhibited the production of IL-1b, IL-6 and TNF-a and down-regulates the expression of cytokine mRNA in LPS-stimulated N9 microglial cells Stimulation of N9 microglial cells with LPS led to the increases in the production of IL-1b, IL-6 and TNF-a. However, the production of these proinflammatory mediators was significantly inhibited by

Since TLR4 is an important receptor of LPS and the interaction of TLR4 with adaptor molecule MyD88 is critical for TLR4 to activate downstream signaling pathways and induce inflammatory response, the effect of PF11 on interactions of TLR4 with its adaptor molecules was investigated. As shown in Fig. 3A, LPS significantly increased the formation of TLR4/MyD88 complex in the precipitates 10 min after LPS treatment. LPS stimulated N9 microglia pretreated with PF11 showed a reduction in the intensity of the MyD88 band co-immunoprecipitated using anti-TLR4 antibody compared to LPS treated group. Furthermore, the ligand association of TLR4 with TAK1 was detected after stimulation with LPS. PF11 significantly attenuated the LPS-stimulated formation of TLR4/ TAK1 complex. Likewise, the reverse co-immunoprecipitation of TLR4 using the MyD88 antibody was also diminished in N9 microglia pretreated with PF11 compared to LPS treated group. The decreases in the complexes were not caused by the reduction of these molecules as the levels of total TLR4 and MyD88 in the cell lysate were not changed in 0.5 h after LPS treatment (Fig. 3B). As shown in Fig. 3B, LPS stimulation of N9 cells for 24 h caused a relative increase in TLR4 and MyD88 expressions. So to investigate whether PF11 could modulate of TLR4 and MyD88 expressions, the effects of PF11 on LPS-induced upregulation of these proteins in N9 cells were examined. As shown in Fig. 3C, PF11 exhibited a statistically inhibitory effect on the increased expression of TLR4 and MyD88. These data revealed that pretreated with PF11 was not only

Fig. 2. Effects of PF11 on LPS-induced IL-1b, IL-6 and TNF-a production in N9 microglial cells. N9 microglial cells were incubated with PF11 (1e100 mM) or minocycline (MINO, 20 mM) for 2 h then stimulated with LPS (1 mg/ml) for 24 h, and extracellular levels of IL-1b (A), IL-6 (B) and TNF-a (C) in culture media were measured using commercial ELISA kits. Total RNA was isolated 6 h after LPS treatment, the mRNAs levels of IL-1b (D), IL-6 (E) and TNF-a (F) were determined by RT-PCR. ###p < 0.001 vs. control group (cultured in medium alone); *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group.


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Fig. 3. Effects of PF11 on LPS-induced interactions of TLR4 with MyD88 or TAK1 and expression of TLR4 and MyD88 in N9 microglial cells. (A) N9 cells were pretreated with 100 mM PF11 for 2 h and stimulated with LPS (1 mg/ml) for 10 min. The complexes of TLR4/MyD88 and TLR4/TAK1 were precipitated by antibody against TLR4 first and then analyzed by Western blot for MyD88 and TAK1. The protein levels of TLR4 and MyD88 in whole cell lysate were analyzed by Western blot. (B) N9 cells were treated with LPS (1 mg/ml) for the indicated time points, and expression of TLR4 and MyD88 was determined by Western blot. (C) N9 cells were pretreated with PF11 (3e100 mM) for 2 h and then incubated with LPS (1 mg/ml) for 24 h. TLR4 and MyD88 expression was analyzed by Western blot. ###p < 0.001 vs. control group (cultured in medium alone); *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group.

capable of blocking interactions between TLR4 and MyD88 but also decreasing expression of TLR4 and MyD88 in N9 microglia. 3.4. PF11 attenuated the TAK1-IKKa/b-IkB-NF-kB inflammatory signaling pathway in LPS-activated N9 microglia As shown in Fig. 4AeC, LPS markedly induced the phosphorylation of TAK1, IKKa/b and IkB-a, but reduced IkB-a expression in N9 cells. However, these effects were significantly reversed by PF11, indicating that PF11 was able to inhibit LPS-induced TAK1, IKKa/b activation and IkB-a degradation in N9 microglia. We further examined the effect of PF11 on NF-kB activation by LPS in microglia. Fig. 4D illustrated that the nuclear levels of NF-kB p65 and p50 subunits were significantly increased by LPS treatment of N9 cells. However, PF11 abolished LPS-induced NF-kB p65 and p50 translocation into nuclei. The findings were supported by immunofluorescence staining of the intracellular NF-kB p65 subunit (Fig. 4E). As a control, PDTC, a specific inhibitor of NF-kB, also significantly inhibited nuclear translocation of NF-kB p65 and p50 subunits in LPS-activated N9 microglia (Fig. 4D). 3.5. PF11 suppressed the phosphorylation of MAPKs and Akt in LPSstimulated N9 microglial cells Stimulation of N9 cells with LPS resulted in increasing phosphorylation of p38, JNK and ERK1/2 MAPKs (Fig. 5A). Pretreatment with PF11 for 2 h significantly attenuated the phosphorylation of ERK1/2, p38 and JNK induced by LPS. Furthermore, PF11

significantly inhibited LPS-induced phosphorylation of Akt in N9 cells (Fig. 5B).

3.6. LPS-RS inhibited TLR4 mediated TAK1-IKKa/b-NF-kB, MAPKs and Akt inflammatory signaling pathways in LPS-stimulated N9 microglia To confirm the contribution of TLR4 in LPS-activated microglia, LPS-RS, a special TLR4 inhibitor was used to demonstrate role of TLR4 in NF-kB, MAPKs and Akt pathways. It was shown that LPSinduced TAK1 and IKKa/b phosphorylation were significantly inhibited by LPS-RS (Fig. 6A and B). Moreover, the levels of NF-kB p65 and p50 protein were decreased in the nucleus but increased in the cytoplasm of LPS-stimulated N9 cells with the treatment of LPSRS, indicating that LPS-RS inhibited the translocation of NF-kB p65 and p50 subunits from cytosol to the nucleus (Fig. 6C). Additionally, LPS-RS pretreatment significantly inhibited the phosphorylation levels of ERK1/2, p38, JNK and Akt in LPS-stimulated N9 microglial cells (Fig. 6D and E). LPS-RS per se did not induce increase in ERK1/ 2, p38, JNK and Akt phosphorylation. These finding suggested that LPS-RS inhibited TLR4 mediated signaling cascades leading to NFkB, MAPK and Akt activation. PF11 exerted the comparable effects on inhibition of the TAK1IKKa/b-IkB-NF-kB, MAPKs and Akt signaling pathways activation mediated by TLR4 in LPS-activated N9 microglial cells, suggesting blocking TLR4 might be the basis for the anti-inflammatory effect of PF11 in microglia.

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Fig. 4. Effects of PF11 on the TAK1-IKKa/b-IkB-NF-kB inflammatory signaling pathway in LPS-stimulated N9 microglial cells. (A, B) N9 microglial cells were pretreated with PF11 (3e 100 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 15 min. (C) Cells were pretreated with PF11 (3e100 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 30 min. Cell lysates were analyzed by Western blot. (D) Cells were pretreated with PF11 (3e100 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 30 min. Nuclear (N) and cytosolic (C) extracts were isolated and the levels of p65 and p50 in each fraction were determined by Western blot. HDAC1 and b-actin were used as internal controls. (E) Cells were pretreated with PF11 (30 mM) for 2 h, then activated with LPS (1 mg/ml) for 30 min. The translocation of p65 subunit of NF-kB was determined by immunocytochemistry. Scale bar ¼ 50 mm ### p < 0.001 vs. control group (cultured in medium alone); *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group.

3.7. PF11 attenuated LPS-induced intracellular ROS production and activation of NADPH oxidase in N9 microglial cells As shown in Fig. 7A, PF11 had no eliminating effect on DPPH free radical at the concentrations from 1 to 100 mM, indicating that PF11 per se has no direct free radical scavenging effect. Stimulation of N9 cells with LPS resulted in increasing intracellular ROS which were significantly attenuated by PF11 (3e100 mM) in a concentration-dependent manner. The effect was similar to antioxidant compound NAc (5 mM) and NADPH oxidase inhibitor DPI (1 mM) (Fig. 7B). Furthermore, exposure of N9 cells to LPS for 12 h increased an NBT positive population, suggesting that LPS treatment significantly up-regulated NADPH oxidase activity. PF11 also inhibited the activation of NADPH oxidase induced by LPS in N9 cells (Fig. 7C and D). 3.8. PF11 suppressed microglia-mediated neurotoxicity Both SH-SY5Y neuroblastoma cells and primary neuronal cells were used to examine the effect of PF11 on neurotoxicity of LPSactivated microglia. As shown in Fig. 8B and C, after exposure to conditioned medium from LPS-stimulated microglia for 24 h, LPSstimulated microglial-conditioned medium (L-CM) resulted in approximately 40% reduction in the viability of SH-SY5Y neuroblastoma cells and approximately 50% reduction in the primary

cortical neurons. There were no significant changes in the survival ratio of SH-SY5Y cells and primary cortical neurons when treated with LPS (0.1e1 mg/ml) alone (Fig. 8A). It suggested that the toxic action of the LPS-stimulated microglial-conditioned medium was mostly dependent on microglial secreted products but not on LPS. As expected, when using conditioned medium from N9 microglial cells co-treated with LPS and PF11 (PF11-CM) or minocycline (MINO-CM), the neuronal toxicity was markedly reduced. The group of PF11 (100 mM)-CM resulted in a 30% and 38% increase in the survival of SH-SY5Y cells and primary cortical neurons, respectively, compared with the groups of C-CM. Direct incubation of neuronal cells with PF11 had no effect on cell viability (data not shown). Furthermore, as shown in Fig. 8D, primary cultured cortical neurons treated with L-CM showed a marked loss of MAP-2 positive neurons and their dendrites. However, MAP-2 positive neurons were obviously observed in the group of PF11 (30 mM)-CM, and the dendritic processes surrounding the cortical neurons were preserved. These results indicated that PF11 exerted neuroprotective effect predominantly by suppressing microglial activation by LPS. 3.9. PF11 inhibited the activation of microglia in mice induced by LPS The effect of PF11 on microglial activation in mice induced by intrahippocampal administration of LPS was performed to


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distribution of microglia were normalized significantly after PF11 treatment, and hyper-ramified microglia predominated, but ameboid microglia were rare (Fig. 9A, B & C). To further confirm the inhibitory effect of PF11 on neuroinflammation in mice, the quantitative measurement of mRNA levels of iNOS, COX-2, IL-1b, IL-6 and TNF-a was performed. RT-PCR analysis indicated that upregulated expression of the iNOS, COX-2, IL-1b, IL-6 and TNF-a was induced by LPS, which was mitigated by PF11 (Fig. 9D). Furthermore, neither LPS nor PF11 exhibited any effect on expression of NeuN in cortex and hippocampus (data not shown). 4. Discussion

Fig. 5. Effects of PF11 on LPS-induced phosphorylation of ERK-1/2, JNK, p38 MAPKs and Akt in N9 microglial cells. (A, B) N9 microglial cells were pretreated with PF11 (3e 100 mM) or MINO (20 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 30 min. Cell lysates were prepared and analyzed by Western blot. ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group.

determine the anti-neuroinflammation effect of PF11 in vivo. Iba-1 is expressed by both resting and activated microglia. Compared with saline group, the number of Iba-1 positive microglia in cortex and hippocampus was increased significantly in LPS group. PF11 8 mg/kg reduced LPS-induced microglial activation in cortex and hippocampus markedly as evidenced by the decrease in the number of Iba-1 positive cells. Furthermore, microglia were wellorganized and hyper-ramified with long branches in saline group. But microglia was amorphous or ameboid cells with thicker and shorter processes in LPS group, and bushy microglial morphology was appeared sometimes. However, the morphology and

Neuroinflammation, characterized by abnormal activation of microglia (Tweedie et al., 2009) contributes to progressive neuronal degeneration (Agostinho et al., 2010). Overactive state of microglia can lead to neuronal death due to the secretion of inflammatory and oxidative stress molecules (Dai et al., 2011; Ock et al., 2010). Therefore, inhibition of excessive microglia activation may be a potential therapeutic strategy to reduce neuronal cell death. The present study firstly demonstrated that PF11, a constituent of Panaxa quinquefolium, exhibited an inhibitory effect on LPSinduced TAK1/IKK/NF-kB, MAPK and Akt pathways in microglial cells, therefore reduced proinflammatory cytokine production and oxidative reaction. Furthermore, PF11 protected neuronal cells from the cytotoxicity of conditioned medium from LPS-stimulated N9 microglia and mitigated microglial activation in mice. NO, an important regulatory mediator involved in cell survival and death exerts a number of pro-inflammatory effects during several physiological and pathological processes (Boje, 2004). Excessive production and accumulation of NO by activated microglial cells is believed to contribute to neuronal death during ischemia, trauma and neurodegenerative diseases in vivo and in vitro (Heneka and O’Banion, 2007; Liu et al., 2002; Saha and Pahan, 2006). Studies using selective and non-selective inhibitors of NOS have afforded significant neuroprotective effect (Park et al., 2011). PGE2 in particular has potent pro-inflammatory effects and is involved in all the processes leading to the classic signs of inflammation (Fattahi and Mirshafiey, 2012). The biosynthesis of NO and PGE2 is regulated by iNOS and COX-2 signaling molecules respectively (Dewapriya et al., 2013). Upregulation of iNOS and COX-2 contributes to the development of many chronic inflammatory diseases (Kim et al., 2013). The inhibitors of iNOS and COX-2 have been shown to block microglial activation and subsequent events and exert neuroprotective effects (Li et al., 2003; Park et al., 2011; Pawlak et al., 2012). In the present study, PF11 suppressed not only NO and PGE2 production but also iNOS and COX-2 mRNA and protein expression in LPS-activated N9 microglial cells. TNF-a, IL-1b and IL-6 are three main pro-inflammatory cytokines produced by activated microglia during CNS inflammation (Dai et al., 2011). TNFa plays a central role in initiating and regulating the cytokine cascade during an inflammatory response (Rubio-Perez and Morillas-Ruiz, 2012). IL-1b promotes the cascade of glial cell reactions and contributes directly to ischemic, traumatic brain injury and neurodegenerative disease, which all lead to neuronal loss (Rothwell, 2003; Rothwell and Luheshi, 2000; Simi et al., 2007). IL6 is a multifunctional cytokine that plays an important role in host defense, with major regulatory effects upon the inflammatory response (Burton et al., 2013; Rubio-Perez and Morillas-Ruiz, 2012). The results in the present study indicated that PF11 significantly inhibited LPS-induced TNF-a, IL-1b and IL-6 secretions and mRNA expressions. Inhibition of microglial activation by PF11 was further confirmed by measurement the expression of proinflammatory genes in brain tissues, the results indicated that PF11 could also suppress those mRNA upregulation induced by LPS in mice.

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Fig. 6. LPS-RS inhibited TLR4-mediated activation of NF-kB, MAPKs and Akt signaling pathways in LPS-stimulated N9 microglial cells. (A, B) Cells were pretreated with LPS-RS (1 mg/ ml) or PF11 (100 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 15 min. (C) Cells were pretreated with LPS-RS (1 mg/ml) or PF11 (100 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 30 min. Nuclear (N) and cytosolic (C) extracts were isolated. (D, E) Cells were pretreated with LPS-RS (1 mg/ml) or PF11 (100 mM) for 2 h and then stimulated with LPS (1 mg/ml) for 30 min. Cell lysates were prepared and analyzed by Western blot. ###p < 0.001 vs. control group; ***p < 0.001 vs. LPS group.

Interestingly, the suppressive effects of PF11 on IL-1b or IL-6 production were greater than its effect on TNF-a, which supported the possibility that PF11 had a greater inhibitory effect on the production of secondary cytokines. These findings suggest that PF11 may be a promising candidate which can inhibit the primary steps in the neuro-inflammatory pathway. Minocycline, a tetracycline derived antibiotic with anti-inflammatory properties in the CNS, was shown to cross the blood brain barrier readily and attenuate inflammation associated with microglial activation, such as blocking the deleterious effects of neuroinflammation on neurogenesis, long-term potentiation, and neuronal survival (Liu et al., 2006; Tikka et al., 2001). Furthermore, minocycline attenuates neuroinflammation in a number of experimental models of brain injury, including cerebral focal and global ischemia, Amyotrophic Lateral Sclerosis (ALS), Experimental Autoimmune Encephalomyelitis (EAE) and MPTP-induced PD (Henry et al., 2008; Liu et al., 2006). Moreover, minocycline reduces LPS-induced BV2 microglial activation and cytokine production (Stolp et al., 2007). So minocycline was used as a positive control in the present study and the results suggested that minocycline inhibited the release of proinflammatory cytokines and expression of iNOS and COX-2 in LPSstimulated N9 microglial cells. Members of the TLR family play critical roles as regulators of innate and adaptive immune responses (Aravalli et al., 2007). To date, 11 human TLRs and 13 murine TLRs have been identified (Okun et al., 2009). TLR4, an important member of TLR family, is highly expressed on macrophages and microglia and is able to recognize LPS associated with gram-negative bacteria (Glass et al.,

2010). Stimulation of the TLR4 extracellular domain by LPS sequentially triggers the intracellular association of MyD88 with its cytosolic domain. Therefore, MyD88 serves as a key TLR4 adaptor protein, linking the receptors to downstream kinases, suggesting that TLR4 and MyD88 act as specific targets for inflammatory responses. In the present study, PF11 was shown to decrease the formation of the complexes of TLR4 with MyD88 or TAK1 on the membrane, which indicated that PF11 could disturb the association of TLR4 with its adaptors, leading to inactivation of TLR4. This was confirmed by the findings that pretreatment of N9 microglial cells with PF11 significantly blocked the activation of NF-kB and release of proinflammatory mediators induced by LPS, in association with decreased formation of the complex of TLR4 with MyD88, even the levels of total TLR4 and MyD88 were not changed. It had been reported that expression of TLR4 was up-regulated upon brain inflammation both in vivo and in vitro (Block et al., 2007), and increased expression of TLR4 induced by LPS was time-dependent and TLR4 expression was significantly increased after exposure of LPS for 24 h in BV2 microglial cells (Park et al., 2011; Yoon et al., 2013). Time-dependent increase of TLR4 and MyD88 expression was found in present study, and PF11 inhibited the increase markedly at 24 h after LPS treatment, which might contributed to the anti-neuroinflammatory effects of PF11, but remained to be determined in further research. Activation of TLR4 signaling at the plasma membrane by LPS stimulates NF-kB and MAPK signaling through the MyD88-IRAKTRAF6-TAK1 signaling complex. As LPS engages the TLR4 complex, the intracellular TIR domain of the receptor recruits the adaptor


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Fig. 7. Radical scavenging effect of PF11 on DPPH and effect of PF11 on LPS-induced ROS production. (A) DPPH solution (100 mM) in 100% ethanol was incubated alone or with PF11 (1e100 mM) at room temperature for 30 min. The quantification of relative band intensities was determined by measuring the absorbance at 517 nm. (B) N9 cells were treated with 1e100 mM PF11 for 2 h, followed by LPS (1 mg/ml) for 6 h. Intracellular reactive oxygen species (iROS) accumulation was measured by DCF-DA. (C) N9 cells were treated with 1e 100 mM PF11 for 2 h, followed by LPS (1 mg/ml) for 12 h. Intracellular ROS accumulation was measured by NBT. (D) The NBT positive cells were observed under microscope. Scale bar ¼ 50 mm ###p < 0.001 vs. control group; **p < 0.01, ***p < 0.001 vs. LPS group.

protein MyD88 and initiates downstream inflammatory signaling (Lin et al., 2013). At first, TAK1 was recruited to TLR4 after stimulation with LPS, after that it dissociated from the receptor presumably to bifurcate the signal into two important branches, that was, the MAPK pathway and the IKK-dependent cascade (MaderaSalcedo et al., 2013). In brief, TLR4 activation by LPS triggers the MyD88, which leads to the activation of a signalosome consisting of TRAF6, IRAK1, and TAK, and subsequently, to activation of TAK1 by autophosphorylation. Phosphorylation of TAK1 leads to activation of the IKK complex consisting of IKK-a, IKK-b and IKK-g. The IKK complex phosphorylates IkB, leading to its ubiquitylation and subsequent degradation, which allows NF-kB translocation and its target genes expression. TAK1 also phosphorylates MAPKs with subsequent AP-1 activation and gene transcription (Gatheral et al., 2012; Kim et al., 2012). The present results showed that PF11 significantly suppressed the LPS-induced phosphorylation of TAK1. The phosphorylation of IKKa/b, an important regulator of NF-kB (Perkins, 2007), was inhibited by PF11, followed by reduction in the degradation of IkB-a, thus blocked NF-kB p50 and p65 translocation into nucleus. MAPKs are a group of molecules that play key roles in inflammatory processes (Jung et al., 2010b). Consistent with previous reports (Zhang et al., 2010), it was observed that MAPKs such as ERK1/2, JNK and p38 were involved in LPS-stimulated activation of N9 microglia in the present study, and the phosphorylation of ERK1/2, JNK and p38 in response to LPS was decreased by PF11 treatment. It appears that MAPKs regulate NF-kB-dependent gene transcription. So it suggested that PF11-mediated inhibition of NF-

kB activation by LPS might be one of the possible mechanisms underlying its inhibitory actions on iNOS, COX-2 and proinflammatory cytokines production by microglial cells. TLR4mediated signaling has been found to activate various intracellular signaling molecules, such as PI3K/Akt (Kogut et al., 2012; Liu et al., 2008). PI3K/Akt signaling has been shown to participate in the regulation of gene expression of iNOS and COX-2 in microglia activated by different stimulus including LPS (Lee et al., 2012; Nam et al., 2008). PF11 inhibited the activation of PI3K/Akt signaling in LPS-stimulated N9 microglia and the production of inflammatory factors, possibly partially through the inhibition of Akt phosphorylation. PI3K has been shown to positively regulate cytokine expression through inducing formation of the p85 regulatory subunit, TLR4 and MyD88 complex, and contribute to TLR4mediated NF-kB activation and cytokine release (Endale et al., 2013). Although the co-immunoprecipitation between TLR4 and p85 subunit was not performed in the present study, LPS-induced TLR4 activation in MyD88 precipitated proteins was found to be inhibited by PF11. It has been reported that PI3K binding with the Cterminus of MyD88, and MyD88 association with the p85 subunit is increased by the LPS challenge. Thus, PF11 treatment may disrupt the association of PI3K to MyD88 and limit TLR4/MyD88/PI3K complex formation in response to LPS-stimulation. The molecules involved in TLR4 signaling through different mechanisms are required for TLR4-mediated activation of microglia. Here, molecules participated in TLR4 signaling were further clarified. Previous studies reported that lipid A chain, the

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Fig. 8. PF11 protected against microglial-mediated neurotoxicity. (A) SH-SY5Y cells and primary cortical neurons were treated with LPS (0.1, 1, 10 mg/ml) for 48 h (B) SH-SY5Y cells and (C) primary cortical neuron cells were treated with conditioned medium (CM) from N9 microglial cells exposed to LPS (1 mg/ml) and/or PF11 (1e100 mM) for 24 h, cell viability was assessed by MTT assay. (D) The morphological changes of primary cortical neurons treated with conditioned medium were detected by immunocytochemistry. Scale bar ¼ 100 mm ###p < 0.001 vs. C-CM group; ***p < 0.001 vs. L-CM group.

biologically active core of LPS, has structural similarity with LPS-RS (Kobayashi et al., 1998). Lipid A derivatives can inhibit the acute inflammatory response to LPS, such as LPS-dependent induction of p38 MAPK activation, NF-kB activation in human endothelial cells, and LPS-stimulated release of TNF-a, IL-1b, and IL-6 in human monocytes, as well as ICAM-1 expression in human gingival fibroblasts (Coats et al., 2005; Kawata et al., 1999). LPS-RS, one of lipid A derivatives, is a potent antagonist of toxic LPS in both human and murine cells and also prevents LPS-induced shock in mice (Rodgers et al., 2009). LPS-RS has been used to block LPSdependent activation of TLR4. The present study showed that LPS-RS inhibited LPS-induced phosphorylation of TAK1, IKKa/b, MAPKs and Akt, and blocked NF-kB p50 and p65 translocation, which demonstrated that the antagonistic effect of LPS-RS against TLR4 resulted in the suppression of downstream signaling pathways. Therefore, TLR4 was responsible for activation of TAK1-IKKa/ b-NF-kB, MAPK and PI3K/Akt pathways in LPS-stimulated N9 microglial cells. PF11 exhibited the similar effects on TLR4mediated signaling pathways with LPS-RS in present study, suggesting the inhibitory effect of PF11 on LPS-stimulated N9 microglial cells was predominant ascribe to antagonism to TLR4. TLR4 activated major signal transduction pathways through distinct adaptor proteins capable of contributing to cellular responses (Buchanan et al., 2010). Based on this, it was presumed that PF11 might interfere with the subsequent cell events after LPS binding to TLR4, including TLR4 interaction with MyD88 and TAK1. Since different cell events should initiated at different time, so different detection time was used in present study. Co-IP assay was performed 10 min after LPS treatment and showed that LPS significantly increased the formation of the complex of TLR4/ MyD88 and TLR4/TAK1 in the precipitates, which was inhibited by PF11, indicating that PF11 might inhibit the interaction of MyD88 with TLR4, thus resulting in a block on LPS-induced TAK1

activation. Western blot analysis was also carried out to detect phospho-forms of TAK1, IKK and the three MAPKs at different times after LPS treatment in N9 microglial cells. In preliminary tests, the phosphorylation levels of TAK1 and IKKa/b were increased most significantly at 15 min after LPS treatment in N9 microglia, and the phosphorylation of MAPKs peaked at 30 min (data not shown). Therefore, for determination of TAK1 and IKKa/b, N9 microglial cells were pretreated with PF11 for 2 h, and then were exposed to LPS for 15 min. Furthermore, maximal MAPK phosphorprotein expression levels are known to occur 30e60 min after LPS treatment in human and murine monocytes/macrophages (Kim et al., 2012; Ock et al., 2010). So N9 microglial cells were pretreated with PF11 or minocycline for 2 h, and then were exposed to LPS for 30 min in MAPKs analysis, and PF11 exerted obviously inhibitory effect on phosphorylation of TAK1, IKKa/b and MAPKs. The pathogenesis of inflammation is complex and regulated by cytokine networks, many pro-inflammatory genes, such as iNOS and COX-2 are involved. Furthermore, it has been found that iNOS and COX-2 are responsible for the high-output production of NO and PGE2 after being exposed to LPS for 24 h according to our previous studies (Zhang et al., 2010), so the expression of iNOS and COX-2 were determined 24 h after LPS treatment in N9 microglial cells in present study, and PF11 significantly inhibited the expression of iNOS and COX-2. In all, it could conclude that PF11 exerted antiinflammatory effects at least in part via interfering with the interaction of the TLR4 and TAK1, and in part by modulating TAK1-IKKNF-kB and MAPKs signaling pathways. ROS generation and oxidative stress have been implicated in neuronal cell death following neurodegeneration and TBI (Combs et al., 2012; Huo et al., 2011). First, high concentrations of ROS released extracellularly may exert direct toxicity to neurons, and second, the increase in concentrations of intracellular ROS (iROS) may modify the signaling events leading to the activation of


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Fig. 9. PF11 inhibited the neuroinflammation in cortex and hippocampus of mice after intrahippocampal administration of LPS. The representative images of Iba-1 positive microglia in cortex (A) and hippocampus (B) of mice in saline group (A1, A4, B1 and B4), LPS group (A2, A5, B2 and B5), and PF11 8 mg/kg group (A3, A6, B3 and B6) were captured at low magnification (A1, A2, A3, and B1, B2 and B3) and high magnification (A4, A5, A6, and B4, B5 and B6) respectively. The high magnification image illustrated the microglia of respective boxed area in the low magnification image. Scale bars: (A1, B1 ¼ 100 mm); (A4, B4 ¼ 50 mm). Typical Iba-1 positive microglia were marked by black arrowheads. (C) The histogram represented the number of Iba-1 positive microglia in cortex and hippocampus. (D) The mRNA expression levels of iNOS, COX-2, IL-1b, IL-6 and TNF-a in cortex and hippocampus were detected by RT-PCR, expression level of the GAPDH gene was used for standardization. ###p < 0.001 vs. saline group; **p < 0.01, ***p < 0.001 vs. LPS group.

microglia (Block et al., 2007; Roy et al., 2008). Accumulation of iROS triggers the release of inflammatory mediators in microglia through the activation of signaling molecules such as MAPKs and NF-kB (Asehnoune et al., 2004; Pan et al., 2008). In this study, the levels of iROS were significantly increased in N9 microglia after LPS treatment, which was effectively suppressed by PF11. This may be attributed to the capacity of PF11 to suppress MAPKs and NF-kB in LPS-activated microglia. The attenuation of microglial activation following NADPH oxidase inhibition could potentially reduce inflammation and b-amyloid-induced neurotoxicity, and facilitate neuronal survival following TBI (Choi et al., 2012; Combs et al., 2012). NADPH oxidase is the major enzyme for the production of ROS in immune cells and is highly expressed in microglia (Peterson and Flood, 2012). We have found that LPS markedly increased the activity of NADPH oxidase in N9 microglia, and PF11 significantly inhibited the activity of NADPH oxidase, which might contribute to the decreased release of iROS. Several studies have demonstrated that microglial activation is a key contributor to neuronal death by releasing inflammatory mediators (Pineda et al., 2012). In the present study, it was confirmed that the conditioned medium from LPS-stimulated microglia were potently toxic to SH-SY5Y cells and primary cortical neurons. PF11 exerted neuroprotective effects on neuronal cells exposed to conditioned medium of LPS-stimulated microglia. But PF11 did not induce the proliferation of neuronal cells, indicating that PF11 protected neurons from injury by inhibiting microglia originated inflammatory mediators. To overcome the limitation of the in vitro conditioned medium experiments, effect of PF11 on microglial activation was further investigated in a mouse neuroinflammation model. Under the current experimental conditions, PF11

significantly inhibited microglial activation evidenced by reduction of Iba-1 positive cells and inflammatory gene expression in cortex and hippocampus in mice after intrahippocampal administration of LPS, but the expression of NeuN, a specific marker of neuron, was not influenced by LPS, which was consistent with a previous report (Block et al., 2007; Tanaka et al., 2006). Although the toxic effect on neurons of LPS mediated by microglia in vitro is not consistent with that in vivo, the neuronal damage induced by conditioned medium of LPS-stimulated microglia is a popular model which could mimic the pathological condition in neurodegenerative disease associated with microglial activation (Pan et al., 2008; Park et al., 2011; Zhang et al., 2012). Some ginsenosides have been reported to exert antiinflammatory effects. Rh1 and Rg3 as well as Rb1 reduced the expression of COX-2, iNOS and the release of TNF-a and IL-1b through inhibition of MAPKs and NF-kB phosphorylation in LPSstimulated BV2 microglial cells (Jung et al., 2010a; Park et al., 2012; Smolinski and Pestka, 2003). In this study, PF11 also exhibited the similar anti-inflammatory activity. Since the Rh1, Rg3, Rb1 and PF11 are the dammarane type ginsenosides, it is speculated that the dammarane skeleton is important for the antiinflammatory activities of ginsenosides. However, ginsenosides exhibit weak membrane permeability in general (Tawab et al., 2003), which means oral bioavailability of ginsenosides would be influenced (Wang et al., 2011). Therefore, the plasma concentration of ginsenosides in the body may limit their pharmaceutical efficacy to treat CNS disorders. While PF11, an ocotillol-type ginsenoside, may display a higher bioavailability because of its more lipophilic property, better membrane permeability compared to Rb1 (Wang et al., 2011; Xu et al., 2003), and better Blood Brain Barrier (BBB) permeability. Bae et al. reported that ginsenoside Rg3 was

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Fig. 10. Proposed signaling mechanism for the effects of PF11 on LPS-induced neuroinflammation in N9 microglial cells. Activation of TLR4 with LPS leads to interaction of TLR4 with MyD88, and subsequently activation of TAK1-IKK complex, MAPKs and Akt pathways. The TAK-IKK complex phosphorylates IkBa, which leads to the degradation of IkB-a and the subsequent nuclear translocation of NF-kB. Similarly, ERK, p38 and JNK MAPK phosphorylate and activate AP-1. Phosphorylation of Akt also leads to activation of NF-kB. Activation of NF-kB and AP-1 results in the expression and production of pro-inflammatory mediators. PF11 disrupts the neurotoxic pathways and protects against LPS-induced toxicity through antagonism to TLR4 and inhibition of NF-kB, MAPKs and Akt signaling.

metabolized to ginsenoside Rh2 by human intestinal bacteria. The transformed ginsenoside Rh2 was shown to protect against ischemic brain injury. Moreover, the anti-inflammatory effect of ginsenoside Rg3 against LPS/IFN-g-activated BV-2 cells was less potent than that of ginsenoside Rh2, which suggested that the in vivo anti-ischemic effect of Rg3 might originate from Rh2 (Bae et al., 2002, 2006). Therefore, it is speculated that the antiinflammatory effects of PF11 might be ascribed to the prototype or its metabolite or both of them, which needed further research. In summary, the present study showed that PF11 inhibited neuroinflammation induced by LPS in N9 microglia through inhibiting the activation of TLR4 mediated TAK1-IKK-NF-kB, MAPK and PI3K/Akt pathways, attenuating the translocation and expression of NF-kB and expression of iNOS, COX-2 and NADPH oxidase, and release of NO, PGE2, IL-1b, IL-6, TNF-a and iROS (Fig. 10). Furthermore, PF11 exerted significant protective effects against microglial-mediated neuron injury and anti-neuroinflammatory effects in mice. Taken together, these results demonstrated that PF11 possessed obviously neuroprotective anti-inflammatory property, strongly suggesting its potential as a drug candidate for neurodegenerative diseases associated with neuroinflammation such as AD and PD. Conflict of interest statement No competing financial interests exist. Acknowledgments This work was supported by grants from National Science Foundation of China (30973890), and by the National Key Scientific Project for New Drug Discovery and Development, P. R. China (2010ZX09401-304).

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AKT signaling pathways.

Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK, p38 MAPKs and Akt signaling pathways.

Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells.

Recombinant Human Endostatin Suppresses Mouse Osteoclast Formation by Inhibiting the NF-κB and MAPKs Signaling Pathways.

Akt Signaling Pathways.

17β-estradiol activates mTOR in chondrocytes by AKT-dependent and AKT-independent signaling pathways.

Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1.

p38 signaling pathways.

Protocatechuic Acid Inhibits Inflammatory Responses in LPS-Stimulated BV2 Microglia via NF-κB and MAPKs Signaling Pathways.

Salusin-β induces smooth muscle cell proliferation by regulating cyclins D1 and E expression through MAPKs signaling pathways.

Schisantherin A protects lipopolysaccharide-induced acute respiratory distress syndrome in mice through inhibiting NF-κB and MAPKs signaling pathways.

mTOR signaling pathways, ovarian dysfunction, and infertility: an update.

Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival.

Akt and ERK signaling pathways.

GCN5 Potentiates Glioma Proliferation and Invasion via STAT3 and AKT Signaling Pathways.

ERK Signaling Pathways.

GSK3β and MAPK Signaling Pathways.

AKT signaling pathways.

ERK Signaling Pathways in Melanocytes.

TIPE3 protein promotes breast cancer metastasis through activating AKT and NF-κB signaling pathways.

Inhibition of the PI3K-Akt and mTORC1 signaling pathways promotes the elongation of vascular endothelial cells.

mTOR signaling pathways.

CXCR7 signaling induced epithelial-mesenchymal transition by AKT and ERK pathways in epithelial ovarian carcinomas.

Hyperthermia induced HIF-1a expression of lung cancer through AKT and ERK signaling pathways.