Obovatol attenuates LPS-induced memory impairments in mice via inhibition of NF-κB signaling pathway

Neurochemistry International 60 (2012) 68–77

Contents lists available at SciVerse ScienceDirect

Neurochemistry International journal homepage: www.elsevier.com/locate/nci

Obovatol attenuates LPS-induced memory impairments in mice via inhibition of NF-jB signaling pathway Dong-Young Choi a, Jae Woong Lee a, Guihua Lin a, Yong Kyung Lee a, Yeon Hee Lee a, Im Seop Choi a, Sang Bae Han a, Jae Kyung Jung a, Young Hee Kim c, Ki Ho Kim c, Ki-Wan Oh a, Jin Tae Hong a,⇑, Moon Soon Lee b,⇑ a b c

College of Pharmacy and MRC, Chungbuk National University, 12 Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Republic of Korea College of Agriculture, Life and Environments, Chungbuk National University, 12 Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Republic of Korea R&D Center, Bioland Ltd., Songjeong, Byongchon, Cheonan, Chungnam 330-863, Republic of Korea

a r t i c l e

i n f o

Article history: Received 17 August 2011 Received in revised form 18 October 2011 Accepted 8 November 2011 Available online 17 November 2011 Keywords: LPS Neuroinflammation Memory impairment Obovatol Alzheimer’s disease

a b s t r a c t Neuroinflammation and accumulation of b-amyloid are critical pathogenic mechanisms of Alzheimer’s disease (AD). In the previous study, we have shown that systemic lipopolysaccharide (LPS) caused neuroinflammation with concomitant increase in b-amyloid and memory impairments in mice. In an attempt to investigate anti-neuroinflammatory properties of obovatol isolated from Magnolia obovata, we administered obovatol (0.2, 0.5 and 1.0 mg/kg/day, p.o.) to animals for 21 days before injection of LPS (0.25 mg/ kg, i.p.). We found that obovatol dose-dependently attenuates LPS-induced memory deficit in the Morris water maze and passive avoidance tasks. Consistent with the results of memory tasks, the compound prevented LPS-induced increases in Ab1–42 formation, b- and c-secretases activities and levels of amyloid precursor protein, neuronal b-secretase 1 (BACE1), and C99 (a product of BACE1) in the cortex and hippocampus. The LPS-mediated neuroinflammation as determined by Western blots and immunostainings was significantly ameliorated by the compound. Furthermore, LPS-induced nuclear factor (NF)-jB DNA binding activity was drastically abolished by obovatol as shown by the electrophoretic mobility shift assay. The anti-neuroinflammation and anti-amyloidogenesis by obovatol were replicated in in vitro studies. These results show that obovatol mitigates LPS-induced amyloidogenesis and memory impairment via inhibiting NF-jB signal pathway, suggesting that the compound might be plausible therapeutic intervention for neuroinflammation-related diseases such as AD. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease and pathologically characterized by intraneuronal neurofibrillary tangles and extraneuronal senile plaques, which are primarily composed of aggregates of the microtubule-associated protein tau, and the amyloid-beta (Ab) peptide, respectively (Castellani et al., 2007). The neurodegenerative disease affects the Abbreviations: Ab, b-amyloid; AD, Alzheimer’s disease; LPS, lipopolysaccharide; APP, amyloid precursor protein; BACE, beta-site APP-cleaving enzyme; COX, cyclooxygenase; NSAIDs, non-steroidal anti-inflammatory drugs; NOS, nitric oxide synthase; NF-jB, nuclear factor-jB; GFAP, glial fibrillary acidic protein; EMSA, elecrophoretic mobility shift assay. ⇑ Corresponding authors. Tel.: +82 43 261 2813; fax: +82 43 268 2732 (J.T. Hong), Tel.: +82 43 261 2522; fax: +82 43 271 0413 (M.S. Lee). E-mail addresses: [email protected] (D.-Y. Choi), [email protected] (J.W. Lee), [email protected] (G. Lin), [email protected] (Y.K. Lee), [email protected] (Y.H. Lee), [email protected] (I.S. Choi), [email protected] (S.B. Han), [email protected] (J.K. Jung), [email protected] (Y.H. Kim), [email protected] (K.H. Kim), [email protected] (K.-W. Oh), [email protected] (J.T. Hong), [email protected] (M.S. Lee). 0197-0186/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.11.005

entorhinal cortex, basal forebrain, hippocampus and amygdala, impairing learning and memory functions. Mechanisms underlying the clinical and pathological manifestations are poorly understood, but neuroinflammation and deposition of Ab are thought to play a critical role in AD pathogenesis (Wyss-Coray, 2006). Ab is produced from amyloid precursor protein (APP) by proteolytic actions of b- and c-secretase. Ab generation is initiated by b-site APP-cleaving enzyme (BACE) 1 which cleaves APP to form Ab N terminus, APPb and a C-terminal fragment, C99. Then, c-secretase generates Abs with two variants, Ab1–40 and Ab1–42. a-Secretase cleaves APP within the Ab domain to produce APPa and C83, precluding formation of Ab by competing with BACE1 (Cole and Vassar, 2008). Importantly, levels of BACE1 and its product (C-terminal fragment of APP) are increased in the sporadic AD brains (Holsinger et al., 2002). Thus, BACE1 has been considered as a prime therapeutic target for intervention of AD pathogenesis (Fukumoto et al., 2010). Inflammation has been described as the culprit of disease or an attempt by the immune system to contain accumulation of Ab plaques in the brain (Wyss-Coray, 2006). Although the role of inflammation in AD is still on debate, accumulating evidence

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

indicates that neuroinflammatory process significantly contributes to pathogenesis of AD (Hwang et al., 2002). Both concentrations of proinflammatory cytokines and percentage of immune cells expressing activation markers are increased in AD brains (Lombardi et al., 1999). The significance of the inflammatory process in AD pathogenesis has been highlighted by epidemiological and retrospective studies reporting a lower incidence of AD in populations who received long term treatment with non-steroidal anti-inflammatory drugs (NSAIDs) (Stewart et al., 1997). In line with the data, we have shown that systemic injection of LPS causes neuroinflammatory responses in the cortex and hippocampus, which was followed by accumulation of Ab (Lee et al., 2008a). These data suggest that inflammation might have a key, negative role in the preclinical phases of the pathology, and that its inhibition can slow or halt the cascade of neurodegeneration in AD. Expression of inflammatory proteins such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and proinflammatory cytokines is primarily regulated by nuclear factor (NF)-jB (Yamamoto and Gaynor, 2004). NF-jB is normally sequestered in the cytoplasm by a family of inhibitory proteins, IjBs, which bind NF-jB and prevent its nuclear localization when cells are not stimulated. Exposure of cells to a variety of extracellular stimuli results in the rapid phosphorylation, ubiquitination, and ultimate proteolytic degradation of IjB, which releases NF-jB to translocate to the nucleus where it regulates gene transcription (Karin and BenNeriah, 2000). Promoter region of the COX-2 (Yamamoto et al., 1995) and iNOS (Lowenstein et al., 1993) genes contains a binding site for the p65 subunit of NF-jB, and therefore signals that activate NF-jB can induce the inflammatory protein synthesis. Significantly, promoter region of BACE has NF-jB binding site, so that NF-jB may directly regulate Ab formation (Sambamurti et al., 2004). Thus, appropriate regulation of NF-jB activity would provide a putative approach to retard or diminish the neurodegenerative process in AD. It has been reported Magnolia extract contains at least 255 different ingredients, such as alkaloids, coumarins, flavonoids, lignans, neolignans, phenylpropanoids and terpenoids (Lee et al., 2011a). Among these, biphenol-structured neolignans including magnolol, honokiol, 4-O-methylhonokiol and obovatol have been the focus of studies regarding various pharmacological effects of Magnolia. A potent anxiolytic property of magnolol and honokiol was demonstrated in several studies (Kuribara et al., 1998; Maruyama et al., 1998). In addition, several compounds isolated from Magnolia family have been shown to own anti-inflammatory (Munroe et al., 2007), neuroprotective (Lin et al., 2006), and antioxidant properties (Fujita and Taira, 1994). In previous studies, we have shown antiinflammatory properties of obovatol, isolated from Magnolia obovata (Kim et al., 2008; Lee et al., 2009b, 2011a). The anti-inflammatory effects of the compound might be associated with its ability to inhibit NF-jB pathway. Thus, we speculated that obovatol may show beneficial effects when it is applied to mice with inflammation-mediated memory deficit. In this study, we examined the effects of obovatol on systemic LPS-induced neuroinflammation and deficits in memory function. 2. Material and methods 2.1. Isolation and preparation of obovatol Pure obovatol (purity P 95.0) was isolated from the leaves of M. obovata as described previously (Kwon et al., 1997). Briefly, crude extract was obtained from leaves M. obovata by extracting with MeOH for 48 h at room temperature. After the extract was filtered and concentrated, the residue was partitioned between H2O and ethyl acetate (EtOAc; 1:1, v/v) to give an EtOAc-soluble fraction. Crude obovatol was isolated from the EtOAc-soluble

69

fraction by using silica gel column. Pure obovatol was separated by being subject to a C18 column. Its chemical structure was confirmed by 1H NMR (300 MHz, CDCl3) analysis. 2.2. Animals and treatments Two months-old male ICR mice (Damool Science, Korea, Seoul, Korea) were maintained and handled in accordance with the humane animal care and use guidelines of Korean FDA. All experimental procedures were conducted according to protocol approved by IACUC of Chungbuk National University. All efforts were made to minimize animal suffering, to reduce the number of animals used. All mice were housed in a room with automatic control of temperature (21 °C), humidity (45–65%), and light:dark (12 h:12 h) cycles. Obovatol was dissolved in 100% ethanol and diluted with 20 time volume of water (final concentrations: 40, 100 and 200 lg/ml). The mice were randomly divided into three treatment groups and they received three different doses of obovatol (0.2, 0.5, 1.0 mg/kg/ day) for 21 days. Obovatol was treated until they were sacrificed. Vehicle (5% ethanol)-treated mice served as control group. Lipopolysaccharide (LPS, Escherichia coli, serotype 055:B5, Sigma, St. Louis, MO, USA) was dissolved in sterile saline (0.9% NaCl) and injected intraperitoneally (0.25 mg/kg). A timeline in Fig. 1 describes obovatol administration and behavioral tests. 2.3. Behavioral tests 2.3.1. Morris water maze test A spatial memory test was performed as previously described with minor modifications (Lee et al., 2009a). The Morris water maze is a white circular pool (diameter: 100 cm and height: 35 cm) with a featureless inner surface. The circular pool was filled with nontoxic black-dyed water and kept at 22–25 °C. The pool was divided into four quadrants of equal area. A black platform (4.5 cm in diameter and 14.5 cm in height) was centered in one of the four quadrants of the pool and submerged 0.5–1.0 cm below the water surface so that it was invisible at water level. The pool was located in a test room, which contained various prominent visual cues. The swimming route of mouse, from the start position to the platform, was monitored and analyzed by a video tracking system (SMART-LD Program, Panlab, Barcelona, Spain). Six habituation trials (3 times/ day) were performed 1 and 2 day(s) before LPS challenge, and test trials were conducted 1, 3 and 7 day(s) posterior to LPS injection. For each daily trial, the mouse was placed into the water maze at one of three randomly determined locations and released allowing the animal to find the hidden platform. After the mouse found and climbed onto the platform, the trial was stopped and the escape latency was recorded. The maximum trial length was 150 s. If animals did not locate the platform within 150 s, the experimenter guided the mouse by hand to the platform and an escape latency of 150 s was recorded. The inter-trial time was 20 s. During the interval, the mouse was kept on the escape platform before starting the next trial. The animal was then placed in the pool again, but at a different location. After the third trial, the mouse was returned to its cage. In order to assess the spatial retention of the location of the hidden platform, a probe trial was conducted 24 h after the last acquisition session. During this trial, the platform was removed from the maze, and each mouse was allowed to search the pool for 60 s before being removed. The time spent in the target quadrant was used as a measure of consolidated spatial memory. 2.3.2. Passive avoidance performance test The passive avoidance test is used for the evaluation of emotional memory based on contextual fear conditioning. The passive avoidance response was determined using a step-through apparatus (Med Associates, Inc., Georgia, VT, USA) that is consisted of an illumi-

70

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

Fig. 1. The chemical structure of obovatol (A) and timeline depicting treatment of obovatol and assessments of cognitive functions of mice (B). Arrow heads represent days on which acquisition tests were conducted.

nated and a dark compartment (each 20.3  15.9  21.3 cm) adjoining each other through a small gate with a grid floor, 3.175 mm stainless steel rod, set 8 mm apart. The learning trials were performed 1 and 2 days before LPS challenge, and test trials were conducted 1, 3 and 7 days following LPS injection. For the learning trials, the animals were placed in the illuminated compartment facing away from the dark compartment and allowed to acclimatize for 1 min. Then, the door between the two boxes was opened, and each animal was able to move freely into the dark compartment. When the mice moved completely into the dark compartment, the door was closed and they received an electric shock (1 mA, 3 s duration). The mice were removed from the chamber and then returned to their home cages. After the learning trial, the mice were subject to the same condition without electric shock. The amount of time that each animal spent until they enter the dark compartment was recorded automatically, and described as step-through latency. 2.4. Perfusion and harvest of brain tissues After the behavioral tests, animals were perfused with 4% paraformaldehyde (in 0.1 M PBS) under anesthetization of pentobarbital (100 mg/kg). The brains were taken out from skull and postfixed in 4% paraformaldehyde for 24 h. The brains were transferred to 30% sucrose solutions in PBS. Subsequently, brains were frozen on a cold stage and sectioned in a cryostat (40 lm), followed by immunohis- tochemistry. For biochemical assays, the brains were immediately removed from skull. The cortex and hippocampus were dissected on ice, and then immediately stored at 80 °C until biochemical analysis. 2.5. Cell culture 2.5.1. Primary astrocytes The cortex dissected from neonatal rat brain (day 2) were incubated for 15 min in Ca2+- and Mg2+-free Hanks’ balanced

saline solution (Life Technologies, Inc.) containing 0.2% trypsin (Gibco BRL, Grand Island, NY, USA). The brain tissue was dissociated by trituration using the cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) in Dulbecco’s modified Eagle’s medium (DMEM) containing F12 nutrient mixture (Invitrogen, Carlsbad, CA). The resultant was centrifuged (1500 rpm, 5 min), resuspended in serum-supplemented culture media, and plated into 100 mm dishes. Serum-supplemented culture media was composed of DMEM supplemented with F12, FBS (5%), NaHCO3 (40 mM), penicillin (100 units/ml), and streptomycin (100 lg/ ml). The cells were incubated in a 5% CO2 incubator at 37 °C for 9 days. At confluence, the cells were subjected to shaking for 16–18 h at 37 °C, then treated with cytosine arabinoside for 48 h, and the medium was replaced with DMEM/F12HAM containing 10% FBS. The monolayer was treated with 1.25% trypsin–EDTA for a short duration after which the cells were dissociated and plated into uncoated glass coverslips. The astrocyte cultures formed a layer of process-bearing, glial fibrillary acidic protein (GFAP)-positive cells. The purity of astrocyte cultures was assessed by GFAP-immunostaining. Under these conditions, over 95% of the cells were astrocytes. The cultured cells were treated with obovatol and/or siRNA targeting p50 or p65 30 min before LPS (10 lg/ml) addition, and cells were harvested for the assay of Ab and Western blotting.

2.5.2. Glioma cells C6 glioma cells were cultured in modified Ham’s F12 medium containing 2 mM L-glutamine and 1.5 g/l sodium bicarbonate. The medium was then supplemented with 15% horse serum, 2.5% fetal bovine serum, 0.5 lg/ml fungizone and 0.02 mg/ml gentamicin. Cells were maintained in 100 mm culture dishes (Sarstedt, Newton, USA) at 37 °C in a humidified 5% CO2 incubator. Cells were treated with obovatol and/or siRNA for p50 or p65 30 min before LPS, and collected for biochemical assays.

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

2.6. Biochemical assays 2.6.1. Western blotting Cells or brain tissues were homogenized with lysis buffer (50 mM Tris pH 8.0, 150 mM sodium chloride (NaCl), 0.02% sodium azide, 0.2% sodium dodecyl sulfate (SDS), 1 mM phenylmethanesulphonylfluoride (PMSF), 10 ll/ml aprotinin, 1% igapel 630, 10 mM sodium fluoride (NaF), 0.5 mM ethylenediamine tetraacetic acid (EDTA), 0.1 mM ethylene glycol tetraacetic acid (EGTA) and 0.5% sodium deoxycholate), and centrifuged at 15,000g for 15 min. Equal amount of proteins (40 lg) were electrophoresed on a 10% or 15% SDS–polyacrylamide gel, and then transferred to a PVDF membrane (Hybond ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Blots were blocked for 2 h at room temperature in 5% (w/v) non-fat dried milk in Tris-buffered saline (10 mM Tris (pH 8.0) and 150 mM NaCl) containing 0.05% Tween-20. The membrane was then incubated for 3 h at room temperature with specific antibodies. Rabbit polyclonal antibodies against APP (1:500), BACE (1:500), C99 (1:500), Ab (1:1000), iNOS (1:2000), COX-2 (1:500), GFAP (1:2000) and b-actin (1:5000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used in this study. The blots were then incubated in the corresponding horseradish peroxidase–conjugated anti-rabbit, anti-mouse and anti-goat immunoglobulin G (Santa Cruz Biotechnology Inc.). Immunoreactive proteins were detected with the ECL Western blotting detection system. The relative density of the protein bands was quantified by densitometry using Electrophoresis Documentation and Analysis System 120 (Eastman Kodak Com., Rochester, NY, USA). 2.6.2. Electrophoretic mobility shift assay (EMSA) The DNA binding activity of NF-jB was determined using an EMSA. The assay was performed according to the manufacturer’s instruction (Promega, Madison, USA). Briefly, homogenates of brain tissue and cells were spun down at 13,000 rpm for 5 min, and the resulting supernatant was removed. The pellets were suspended in 400 ll of solution A containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenyl methyl sulfonyl fluoride, vigorously vortexed, allowed to incubate on ice for 10 min, and finally centrifuged at 12,000 rpm for 6 min. The pelleted nuclei were resuspended in solution C (solution A, 420 mM NaCl, and 20% glycerol) and allowed to incubate on ice for 20 min. Next, the nuclei solution were centrifuged at 15,000 rpm for 15 min, and the resulting nuclear extract supernatant was collected in a chilled micro tube. NF-jB-corresponding oligonucleotides were endlabeled using T4 polynucleotide kinase and [c-32P] ATP for 10 min at 37 °C. Two microliter of each nuclear extract was mixed with 1 ll (50,000–200,000 cpm) of 32P end-labeled NF-jB oligonucleotide and incubated at room temperature for 20 min. Subsequently, 1 ll of gel loading buffer was added, and the DNA–protein complex was separated on 6% nondenaturing acrylamide gel until the dye was four-fifths of the way down the gel. The gel was dried at 80 °C for 1 h and exposed to film overnight at 70 °C. The relative densities of the DNA–protein binding bands were scanned by MyImage (SLB) and quantified by Labworks 4.0 software (UVP, Inc.). 2.6.3. a-, b- and c-Secretases activity The total activities of a-, b- and c-secretase present in cortical and hippocampal regions were determined using a commercially available a-secretase activity kit (R&D Systems, Wiesbaden, Germany), b-secretase fluorescence resonance energy transfer (BACE 1 FRET) assay kit (PANVERA, Madison, USA) and c-secretase activity kit, (R&D systems, Wiesbaden, Germany) according to the manufacturer’s instructions, respectively. Each tissue was homogenized in cold 1 cell extraction buffer (a component of the kit) to a final protein concentration of 1 mg/ml. To determine a (or c)secretase activity, 50 ll of lysate was mixed with 50 ll of reaction

71

buffer. The mixture was incubated for 1 h in the dark at 37 °C after 5 ll of substrate was added. Substrate conjugated to the reporter molecules EDANS and DABCYL was cleaved by a (or c)-secretase and released a fluorescent signal. This fluorescence was measured using a Fluostar galaxy fluorometer (excitation at 355 nm and emission at 510 nm) equipped with Felix software (BMG Labtechnologies, Offenburg, Germany). The level of a (or c)-secretase enzymatic activity was proportional to fluorescence intensity which was expressed as fluorescence units. To determine b-secretase, 10 ll of lysate was mixed with 10 ll of BACE1 substrate (Rh-EVNLDAEFKQuencher). The reaction mixture was then incubated for 1 h at room temperature in a black 96-microwell plate. The reaction was stopped by adding 10 ll of BACE1 stop buffer (2.5 M sodium acetate). Fluorescence was determined using a Fluostar galaxy fluorometer (excitation at 545 nm and emission at 590 nm) equipped with Felix software. Enzyme activity was linearly related to fluorescence increases, and the activity was expressed as fluorescence units. All controls, blanks and samples were run in triplicate. 2.6.4. Measurement of Ab levels Ab1–42 and Ab1–40 levels in the cortex and hippocampus were determined using ELISA kits (IBL, Immuno-Biological Co., Ltd., Japan). ELISA was conducted as described in manufacturer’s manual. Briefly, 100 ll of sample was pipetted into the primary coated-plate and was incubated overnight at 4 °C. After washing each well with washing buffer, 100 ll of secondary antibody solution was added and the mixture was incubated for 1 h at 4 °C in the dark. After rinse, chromogen was added and the mixture was incubated for 30 min at room temperature in the dark. After the addition of stop solution, the resulting color was assayed at 450 nm using a microplate absorbance reader (Sunrise™, TECAN, Switzerland). 2.7. Immunohistochemistry Immunohistochemical staining was performed using the avidin– biotin peroxidase method. The sections were incubated overnight at 4 °C with anti-GFAP (1:2000, Covance, Berlely, CA, USA). After washing in PBS, the sections were incubated in biotinylated goat anti rabbit IgG (1:2000 dilution, Vector Laboratories, Burlingame, CA) for 1 h at room temperature. The sections were subsequently washed and incubated with avidin-conjugated peroxidase complex (ABC kit, 1:200 dilution, Vector Laboratories) for 30 min followed by PBS washing. The peroxidase reaction was conducted in PBS using 3,30 diaminobenzidine tetrahydrochloride (0.02%) as a chromogen. Finally, the sections were rinsed, mounted on poly-glycine-coated slides and microscopical analysis was performed. 2.8. Statistical analysis All statistical analysis was performed with GraphPad Prism 4 software (Version 4.03, GraphPad software, Inc., San Diego, CA). Group differences in the escape distance, latency, velocity in the Morris water maze task or step-through latency in passive avoidance task were analyzed using two-way ANOVA with repeated measures, the factors being treatment and testing day. Otherwise were analyzed by one-way ANOVA followed by Dunnette’s post hoc test. All values are presented as mean ± S.E.M. Significance was set at p < 0.05 for all tests. 3. Results 3.1. Effects of obovatol on memory impairments To investigate the effects of obovatol on systemic LPS-induced memory impairments, we performed the Morris water maze and

72

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

passive avoidance test (step-through test). In our previous studies, we have shown that beta-amyloid-induced cognitive dysfunction was attenuated by treatment with 4-O-methylhonokiol, a biphenolic compound isolated from Magnolia officinalis (Lee et al., 2010b, 2011b). In the studies, we treated mice with the compound (1 mg/ kg, p.o.) for 3 and 5 weeks respectively, and found long-term administration with the compound effectively stabilized cognitive function in mice without adverse effects. Although we did not have pharmacokinetic data for obovatol, we assumed that obovatol might own similar pharmacokinetic parameters to those of 4-Omethylhonokiol, since chemical structure of obovatol is close to that of 4-O-methylhonokiol. Thus, the compound (0.2, 0.5 and 1 mg/kg) was orally administered for 3 weeks prior to LPS injection. The animals were then trained for 2 days (3 times/day) to allow them to learn the location of the hidden platform. The mice were then examined 1, 3 and 7 day after LPS injection, and their profiles for spatial learning were recorded (Fig. 2). In comparison to control mice, the escape distance and latency of LPS-injected mice were significantly increased on day 1 and 3, and the impairment was recovered to the normal on day 7. The LPS-mediated deficit in memory function was prevented by obovatol treatment in a dose-dependent manner (Fig. 2A and B). However, there was no statistical significant difference in average speed among groups

(Fig. 2C). Twenty-four-hours after the last acquisition session, the probe tests were carried out to reflect memory consolidation. Before the test, the hidden platform was removed and the amount of time animals spent in target quadrant was measured. It was revealed that LPS injection significantly induced deficit in spatial memory, while treatment with obovatol significantly improved consolidated the memory in the animals (Fig. 2D). We also evaluated contextual memory of mice by the passive avoidance test. In the passive avoidance test, there was no significant difference in the learning trial. However, injection of LPS significantly decreased the step-through latency compared to the control group, which was sustained by day 7. These memory impairments were efficiently blocked by obovatol treatment (Fig. 2E). 3.2. Effects of obovatol on Ab accumulation in the cortex and hippocampus A single intraperitoneal injection of LPS (0.25 mg/kg) increased the Ab1–42 level in the cortex and hippocampus, which was suppressed by a high dose of obovatol (0.5 or 1.0 mg/kg, Fig. 3A and B). In contrast, LPS decreased the Ab1–40 level in the cortex and hippocampus, and the alteration was abolished by obovatol (Fig. 3). To

Fig. 2. Inhibitory effect of obovatol on LPS-induced memory impairments. Mice were treated with LPS (0.25 mg/kg, i.p.) after 3 weeks treatment of obovatol (0.2, 0.5 and 1 mg/kg). The Morris water maze tests and passive avoidance tests were performed as described in the method section. (A)–(C) LPS injection elongates escape distance and time without affecting average swimming speed. (D) Probe test reveals that LPS injection induces spatial memory deficit which is ameliorated by obovatol treatment. (E) LPS decreases the latency to enter the dark compartment. The memory deficit induced by LPS was attenuated by obovatol treatment. Values are presented as mean ± S.E. from 10 mice. OB = obovatol, ⁄p < 0.05 vs. control, ⁄⁄p < 0.01 vs. control, ⁄⁄⁄p < 0.001 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS, ###p < 0.001 vs. LPS.

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

clarify how Ab1–42 deposition occurred, we analyzed the concentrations of APP (a substrate of BACE1), BACE1, C99 (a product of BACE1) and Ab by Western blot analysis. LPS injection significantly elevated the levels of the proteins, and obovatol exhibited its inhibitory effects on the elevations, suggesting that Ab accumulation results from induction of APP and its cleavage enzyme, and inhibitory capacity of obovatol prevents Ab accumulation (Fig. 3C). To confirm the speculation, we next assayed a-, b-, and c-secretases activities. The activity of b-secretase was significantly increased in the hippocampus (Fig. 4B), while the activity of c-secretase was increased in both the cortex and hippocampus by LPS injection (Fig. 4C). However, the activity of a-secretase was decreased in mice brains (Fig. 4A). All of the alterations in secretase activity were prevented by obovatol. This result further explains how LPS injection induces Ab accumulation, and obovatol prevents the abnormality. 3.3. Effects of obovatol on neuroinflammation In the previous study, we found that systemic LPS induced neuroinflammatory responses, which were associated with Ab deposition and memory impairment. Therefore, we examined the inhibitory effects of obovatol on systemic LPS-mediated neuroinflammatory reactions including inductions of iNOS, COX-2 and GFAP by Western blot analysis. As shown in Fig. 5A, expressions of iNOS, COX-2 and GFAP were markedly increased in response to LPS. Treatment with obovatol ameliorated the increases in a dose-dependent manner. This result was consistent with the profile of the inhibitory effect of obovatol on Ab elevation. Immunostainings for astrocytes indicated that obovatol treatment did not affect astroglial morphology (data not shown), while LPS caused astroglial activation in the cortex and hippocampus, as determined by their thick and short processes. Obovatol markedly attenuated the astrogliosis (Fig. 5B).

73

Because NF-jB is a critical inducer of COX-2, iNOS and other proinflammatory cytokines, we determined whether obovatol might suppress NF-jB activation in vivo and in vitro. Nuclear extracts from brain tissues were prepared and EMSA was performed to assay NF-jB DNA binding activity. In the cortex and hippocampus, LPS injection resulted in a strong NF-jB activation, which was markedly relieved by obovatol treatment (Fig. 5C). 3.4. Effects of obovatol on LPS-treated astrocytes and glioma cells In order to test whether the results from in vivo studies can be replicated in vitro, we employed two different cells including rat primary astrocytes and C6 glioma cells. Cells were treated with 1, 2.5, and 5 lM of obovatol 1 h before LPS addition. Consistent to in vivo studies, the compound attenuated LPS-induced increase in BACE1 expression and its products generation (Fig. 6A). In addition, obovatol ameliorated inflammatory responses including iNOS, COX-2, and GFAP inductions (Fig. 6B). Next, we examined whether cotreatment of obovatol and siRNA targeting p50 or p65 synergically suppresses the expressions of iNOS and BACE1. Obovatol alone significantly attenuated LPS-induced rise in iNOS and BACE1 (Fig. 7A). LPS-induced DNA binding activity of NF-jB was also decreased by obovatol (Fig. 7B). However, inhibitory effect of obovatol on iNOS expression and NF-jB activation was diminished by treatment of the siRNAs (Fig. 7A and B). Cotreatment with obovatol and siRNA for p50 or p65 blocked LPS-induced increase in BACE1, but not synergically. 4. Discussion Increasing evidence suggests that neuroinflammation and Ab deposition play a key role in pathogenesis of AD (Akiyama et al., 2000; Selkoe, 1998). Our present study shows that obovatol, a

Fig. 3. Inhibitory effects of obovatol on LPS-induced increase in Ab formation. Mice were treated with LPS (0.25 mg/kg, i.p.) after 3 weeks treatment of obovatol (0.2, 0.5 and 1 mg/kg). The levels of Ab1–42 and Ab1–40 were measured by ELISA. We also employed Western blot analysis to determine expression of APP, BACE, C99 and Ab. (A) and (B) Single injection of LPS (0.25 mg/kg) elevates Ab1–42 concentration, whereas LPS decreases Ab1–40 in the cortex and hippocampus, which is attenuated by administration of obovatol. (C) LPS injection increases expressions of APP, BACE, C99 (a product of enzymatic action of BACE) and Ab in the cortex and hippocampus. Obovatol reduces the elevations in a dose-dependent manner. OB = obovatol, ⁄p < 0.05 vs. control, ⁄⁄p < 0.01 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS.

74

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

Fig. 4. Effects of obovatol on LPS-mediated alteration in secretase activity. Mice were treated with LPS (0.25 mg/kg, i.p.) after 3 weeks treatment of obovatol (0.2, 0.5 and 1 mg/kg). a-, b- and c-secretase activity was measured by using enzyme assay kit for each secretase. (A) LPS injection decreases a-secretase activity in the cortex and hippocampus, while obovatol attenuates the reduction. (B) b-secretase activity is elevated by LPS injection in the hippocampus and it is inhibited by obovatol. (C) c-secretase activity is up-regulated by LPS injection in both cortex and hippocampus, and it is ameliorated by obovatol. OB = obovatol, ⁄p < 0.05 vs. control, ⁄⁄p < 0.01 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS.

biphenolic compound isolated from M. obovata improves systemic LPS-induced memory impairment. Significantly, obovatol ameliorates LPS-mediated amyloidogenesis and neuroinflammatory reactions in the cortex and hippocampus, and such pharmacological effects might be related to the memory improvement in the animals. Our data also indicates that the anti-inflammatory and anti-amyloidogenic effects of obovatol result from blunting NFjB signaling pathway, since the transcription factor is a positive regulator for inflammation as well as b-secretase. We did not observe any adverse effects of obovatol during the present study. Consistent to our observation, Liu et al. described that administration of Magnolia bark extract (480 mg/kg/day for 21 days, or 240 mg/kg/day for 90 days) with rats did not cause any mortality or signs of toxicity (Liu et al., 2007). It is assumed that the Magnolia extract contains around 0.1–0.3% obovatol (Lee et al., 2011a). And the dose of 480 mg Magnolia extract/kg/day is approximately equivalent to 0.48–1.44 mg obovatol/kg/day, which is close to the dose we used. According to report from the National Institute of Chinese Medicine, the oral LD50 for Magnolia extract is

higher than 50 g/kg in mice. Thus, the dose we used for this study seems to be safe to use for clinical study. Chronic inflammation is associated with a broad spectrum of neurodegenerative diseases including AD (Glass et al., 2010). It has been shown that there is increase in markers of neuroinflammation in AD brains (Wyss-Coray, 2006). Elevated cytokines and chemokines as well as the accumulation of activated glial cells are found in or near the pathologic lesions of AD (Wyss-Coray, 2006). Significantly, LPS-induced systemic inflammation has been shown to exacerbate tau pathology in animal models for AD (Lee et al., 2010a). Although the exact role of inflammation in neurodegenerative processes for AD has not reached to the consensus, our data are in agreement with the argument that neuroinflammation may drive the neurodegenerative cascade in AD. Pathogenic role of inflammation was further emphasized by retrospective epidemiological studies that various NSAIDs may significantly reduce lifetime risk of developing AD (Combs et al., 2000). According to meta-analysis of epidemiological studies, NSAIDs reduce AD incidence by an average of 58% (Szekely et al., 2004). In support of the studies, NSAIDs also have been shown to reduce levels of highly amyloidogenic Ab1–42 peptide and Ab deposition in a mouse model of AD (Lim et al., 2000). Thus, anti-inflammatory compounds including obovatol might be eligible for a therapeutic intervention for AD. In this study, we observed astrocytic activation and concomitant increase in iNOS and COX-2 expression after LPS injection (Fig. 5). The activation of astrocytes might be detrimental to adjacent neurons. Activated astrocytes release neurotoxic substance including NO, cytotoxic cytokines, and superoxide radicals. The molecules can attack and modify bio-active molecules such as proteins, lipids and DNA, causing dysfunction of cellular organelles. In support of the notion, Noble et al. reported that acute systemic inflammation induces central mitochondrial damage and amnesic deficit in adult Swiss mice (Noble et al., 2007). Several groups also reported that injection of LPS causes neuroinflammation resulting in AD-like neuronal malfunction (Milatovic et al., 2003; Sparkman et al., 2005). It is also true that amelioration of glial activation by anti-inflammatory molecules protects neurons from LPS-caused neuronal abnormality (Marchalant et al., 2007). In agreement with the studies, present investigation exhibits anti-inflammation and consequent cognitive stabilization by obovatol. It is believed that the fibrillar form of Ab peptide is generated in the early stages of AD (Drouet et al., 2000), which causes neuronal dysfunction and death (Yankner, 1996). In support of the suggestion, neurotoxicity of Ab peptide has been demonstrated (Emre et al., 1992). Current study also showed that Ab1-42 is increased by systemic LPS injection (Fig. 3). This increase in neurotoxic molecule may be associated with memory impairments in mice. Indeed, previous study revealed that LPS injection caused Ab deposition and memory impairments in mice brains, and treatment of sulindac, an anti-inflammatory drug significantly attenuated the amyloidogenesis and deficits in memory function, suggesting that inflammation-induced increase in Ab might be involved in the memory impairments (Lee et al., 2008a). NF-jB is a positive regulator in the expression of a variety of rapid-response genes involved in inflammatory and immune reactions. Importantly, the transcriptional factor controls expression of BACE1, which enhances Ab formation (Sambamurti et al., 2004). Therefore, therapeutic intervention targeting NF-jB would likely be of benefit in the treatment of AD. There is abundant evidence that NF-jB activity is associated with amyloidogenesis. For instance, ()-epigallocatechin-3-gallate, a compound from green tea modifies cognitive function and secretase activity through inhibition of NF-jB pathway in preseniline 2 mutant mice (Lee et al., 2009a). Further support comes from a report that the AD brains contain increased levels of both BACE1 and NF-jB p65, and NF-jB p65 expression leads to increase of BACE1 promoter

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

75

Fig. 5. Anti-neuroinflammatory effects of obovatol in mice brains. (A) Western blot analysis shows that LPS injection causes significant increase in inflammatory proteins including iNOS, COX-2 and GFAP, which is attenuated by obovatol treatments. (B) Immunostainings for GFAP, a marker for astrocytes reveal that systemic LPS injection induces astroglial activation in the cortex and hippocampus and the gliosis is relieved by obovatol treatment. (C) DNA binding activity of NF-jB is measured by EMSA. The assay indicates that LPS injection increases NF-jB activity and obovatol attenuates the activity of the transcription factor. OB = obovatol, Scale bars: 10 lm (high magnification), 25 lm (low magnification). ⁄p < 0.05 vs. control, ⁄⁄p < 0.01 vs. control, ⁄⁄⁄p < 0.001 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS, ###p < 0.001 vs. LPS.

Fig. 6. Anti-inflammatory and anti-amyloidogenic effects of obovatol in astrocytes and glioma cells. (A) Western blot analysis exhibits that LPS treatment increases APP, BACE and C99 in both type of cells. Obovatol attenuates the alterations. (B) LPS injection elevates the expressions of inflammatory proteins such as NF-jB, GFAP, COX-2 and iNOS in both cells. All of the changes in inflammatory molecules are inhibited by obovatol treatment in a dose-dependent manner. OB = obovatol, ⁄p < 0.05 vs. control, ⁄⁄p < 0.01 vs. control, ⁄⁄⁄p < 0.001 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS.

activity and BACE1 transcription, while knockout of NF-jB p65 decreases BACE1 gene expression in cells (Chen et al., 2011). In this investigation, we observed that obovatol inhibits LPS-induced

DNA binding activities of NF-jB (Fig. 5) and Ab formation (Fig. 3), indicating that obovatol may reduce LPS-induced amyloidogenesis by way of blocking NF-jB signaling pathway.

76

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77

NF-jB signaling pathway. Therefore, this study supports the speculation that obovatol might be a putative therapeutic intervention of neuroinflammatory diseases such as AD. Acknowledgments This work was supported by the National Research Foundation (NRF) of Korea funded by the Korean Government (MRC, 2009-0091433), Priority Research Centers Program of the NRF funded by the Ministry of Education, Science and Technology (2010-0029709) and the Korea Ministry of Education, Science and Technology (The Regional Core Research Program/Chungbuk BIT Research-Oriented University Consortium). References

Fig. 7. Effects of NF-jB silencing on anti-inflammation of obovatol. (A) LPS induces expressions of iNOS and BACE in astrocytes and C6 glioma cells. The induction is blocked by obovatol. (B) DNA binding activity of NF-jB is markedly increased by LPS in both cells, and obovatol reduces the activity of the inflammatory transcriptional factor. Inhibitory effects of obovatol on NF-jB signaling pathway are diminished by siRNA targeting p50 or p65 subunit of NF-jB. OB = obovatol, ⁄ p < 0.05 vs. control, #p < 0.05 vs. LPS.

The NF-jB activation process is complex where the endogenous inhibitor of NF-jB, IjB, is phosphorylated, ubiquitinated and subsequently degraded, allowing migration of NF-jB to the nucleus (Karin and Ben-Neriah, 2000). The inhibitory effects could be exhibited via the attenuation of IKK phosphorylation and subsequent IjB degradation (Kim et al., 2008; Yang et al., 2010). Alternatively, amelioration of biosynthesis of the NF-jB subunit (p65 or p50) can suppress the activity. In this study, we have observed that obovatol attenuates LPS-induced increase in expression of p65 subunit (Fig. 6B). Other studies demonstrated the NF-jB inhibition by natural compounds including obovatol via blocking of IjB phosphorylation (Choi et al., 2007; Lee et al., 2008b). Thus, anti-inflammatory effects of obovatol might be achieved by inhibition of nuclear translocation of NF-jB and/or its expression. We expected that cotreatment with obovatol and siRNA for p50 or p65 synergically inhibits activation of NF-jB. However, inhibitory effect of obovatol on LPS-induced NF-jB activation was diminished by siRNAs. There are two distinct NF-jB pathways; classic and alternative pathways (Bonizzi and Karin, 2004). The former is mediated by nuclear translocation of p65 and p50 heterodimer. The latter is triggered by heterodimer of RelB and p52. They are usually activated concurrently, but it might be assumed that silencing of p65 and p50 synthesis may stimulate the latter pathway and inhibitory effect of obovatol might be masked. This might explain the unexpected phenomenon. However, further study is required to elucidate the results.

5. Conclusion Systemic LPS caused neuroinflammation and elevation of Ab1–42 which may result in memory deficit. Long-term treatment of obovatol prevented the detrimental effects of systemic LPS. The beneficial property of obovatol might be conferred by blocking

Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I.R., McGeer, P.L., O’Banion, M.K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-Coray, T., 2000. Inflammation and Alzheimer’s disease. Neurobiol. Aging 21, 383–421. Bonizzi, G., Karin, M., 2004. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25, 280–288. Castellani, R.J., Zhu, X., Lee, H.G., Moreira, P.I., Perry, G., Smith, M.A., 2007. Neuropathology and treatment of Alzheimer disease: did we lose the forest for the trees? Expert Rev. Neurother. 7, 473–485. Chen, C.H., Zhou, W., Liu, S., Deng, Y., Cai, F., Tone, M., Tone, Y., Tong, Y., Song, W., 2011. Increased NF-kappaB signalling up-regulates BACE1 expression and its therapeutic potential in Alzheimer’s disease. Int. J. Neuropsychopharmacol. 1, 14. Choi, M.S., Lee, S.H., Cho, H.S., Kim, Y., Yun, Y.P., Jung, H.Y., Jung, J.K., Lee, B.C., Pyo, H.B., Hong, J.T., 2007. Inhibitory effect of obovatol on nitric oxide production and activation of NF-kappaB/MAP kinases in lipopolysaccharide-treated RAW 264.7 cells. Eur. J. Pharmacol. 556, 181–189. Cole, S.L., Vassar, R., 2008. The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology. J. Biol. Chem. 283, 29621–29625. Combs, C.K., Johnson, D.E., Karlo, J.C., Cannady, S.B., Landreth, G.E., 2000. Inflammatory mechanisms in Alzheimer’s disease: inhibition of beta-amyloidstimulated proinflammatory responses and neurotoxicity by PPARgamma agonists. J. Neurosci. 20, 558–567. Drouet, B., Pincon-Raymond, M., Chambaz, J., Pillot, T., 2000. Molecular basis of Alzheimer’s disease. Cell. Mol. Life Sci. 57, 705–715. Emre, M., Geula, C., Ransil, B.J., Mesulam, M.M., 1992. The acute neurotoxicity and effects upon cholinergic axons of intracerebrally injected beta-amyloid in the rat brain. Neurobiol. Aging 13, 553–559. Fujita, S., Taira, J., 1994. Biphenyl compounds are hydroxyl radical scavengers: their effective inhibition for UV-induced mutation in Salmonella typhimurium TA102. Free Radic. Biol. Med. 17, 273–277. Fukumoto, H., Takahashi, H., Tarui, N., Matsui, J., Tomita, T., Hirode, M., Sagayama, M., Maeda, R., Kawamoto, M., Hirai, K., Terauchi, J., Sakura, Y., Kakihana, M., Kato, K., Iwatsubo, T., Miyamoto, M., 2010. A noncompetitive BACE1 inhibitor TAK-070 ameliorates Abeta pathology and behavioral deficits in a mouse model of Alzheimer’s disease. J. Neurosci. 30, 11157–11166. Glass, C.K., Saijo, K., Winner, B., Marchetto, M.C., Gage, F.H., 2010. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934. Holsinger, R.M., McLean, C.A., Beyreuther, K., Masters, C.L., Evin, G., 2002. Increased expression of the amyloid precursor beta-secretase in Alzheimer’s disease. Ann. Neurol. 51, 783–786. Hwang, D.Y., Chae, K.R., Kang, T.S., Hwang, J.H., Lim, C.H., Kang, H.K., Goo, J.S., Lee, M.R., Lim, H.J., Min, S.H., Cho, J.Y., Hong, J.T., Song, C.W., Paik, S.G., Cho, J.S., Kim, Y.K., 2002. Alterations in behavior, amyloid beta-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer’s disease. FASEB J. 16, 805– 813. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18, 621–663. Kim, J.C., Lee, S.Y., Kim, S.Y., Kim, J.K., Kim, H.J., Lee, H.M., Choi, M.S., Min, J.S., Kim, M.J., Choi, H.S., Ahn, J.K., 2008. HSV-1 ICP27 suppresses NF-kappaB activity by stabilizing IkappaBalpha. FEBS Lett. 582, 2371–2376. Kuribara, H., Stavinoha, W.B., Maruyama, Y., 1998. Behavioural pharmacological characteristics of honokiol, an anxiolytic agent present in extracts of Magnolia bark, evaluated by an elevated plus-maze test in mice. J. Pharm. Pharmacol. 50, 819–826. Kwon, B.M., Ro, S.H., Kim, M.K., Nam, J.Y., Jung, H.J., Lee, I.R., Kim, Y.K., Bok, S.H., 1997. Polyacetylene analogs, isolated from hairy roots of Panax ginseng, inhibit Acyl-CoA: cholesterol acyltransferase. Planta Med. 63, 552–553. Lee, J.W., Lee, Y.K., Yuk, D.Y., Choi, D.Y., Ban, S.B., Oh, K.W., Hong, J.T., 2008a. Neuroinflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation. J Neuroinflammation 5, 37.

D.-Y. Choi et al. / Neurochemistry International 60 (2012) 68–77 Lee, S.Y., Yuk, D.Y., Song, H.S., Yoon do, Y., Jung, J.K., Moon, D.C., Lee, B.S., Hong, J.T., 2008b. Growth inhibitory effects of obovatol through induction of apoptotic cell death in prostate and colon cancer by blocking of NF-kappaB. Eur. J. Pharmacol. 582, 17–25. Lee, J.W., Lee, Y.K., Ban, J.O., Ha, T.Y., Yun, Y.P., Han, S.B., Oh, K.W., Hong, J.T., 2009a. Green tea ()-epigallocatechin-3-gallate inhibits beta-amyloid-induced cognitive dysfunction through modification of secretase activity via inhibition of ERK and NF-kappaB pathways in mice. J. Nutr. 139, 1987–1993. Lee, S.Y., Cho, J.S., Yuk, D.Y., Moon, D.C., Jung, J.K., Yoo, H.S., Lee, Y.M., Han, S.B., Oh, K.W., Hong, J.T., 2009b. Obovatol enhances docetaxel-induced prostate and colon cancer cell death through inactivation of nuclear transcription factorkappaB. J. Pharmacol. Sci. 111, 124–136. Lee, D.C., Rizer, J., Selenica, M.L., Reid, P., Kraft, C., Johnson, A., Blair, L., Gordon, M.N., Dickey, C.A., Morgan, D., 2010a. LPS-induced inflammation exacerbates phospho-tau pathology in rTg4510 mice. J Neuroinflammation 7, 56. Lee, J.W., Lee, Y.K., Lee, B.J., Nam, S.Y., Lee, S.I., Kim, Y.H., Kim, K.H., Oh, K.W., Hong, J.T., 2010b. Inhibitory effect of ethanol extract of Magnolia officinalis and 4-Omethylhonokiol on memory impairment and neuronal toxicity induced by betaamyloid. Pharmacol. Biochem. Behav. 95, 31–40. Lee, Y.J., Lee, Y.M., Lee, C.K., Jung, J.K., Han, S.B., Hong, J.T., 2011a. Therapeutic applications of compounds in the Magnolia family. Pharmacol. Ther. 130, 157– 176. Lee, Y.K., Choi, I.S., Ban, J.O., Lee, H.J., Lee, U.S., Han, S.B., Jung, J.K., Kim, Y.H., Kim, K.H., Oh, K.W., Hong, J.T., 2011b. 4-O-Methylhonokiol attenuated beta-amyloidinduced memory impairment through reduction of oxidative damages via inactivation of p38 MAP kinase. J. Nutr. Biochem. 22, 476–486. Lim, G.P., Yang, F., Chu, T., Chen, P., Beech, W., Teter, B., Tran, T., Ubeda, O., Ashe, K.H., Frautschy, S.A., Cole, G.M., 2000. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer’s disease. J. Neurosci. 20, 5709–5714. Lin, Y.R., Chen, H.H., Ko, C.H., Chan, M.H., 2006. Neuroprotective activity of honokiol and magnolol in cerebellar granule cell damage. Eur. J. Pharmacol. 537, 64–69. Liu, Z., Zhang, X., Cui, W., Li, N., Chen, J., Wong, A.W., Roberts, A., 2007. Evaluation of short-term and subchronic toxicity of magnolia bark extract in rats. Regul. Toxicol. Pharmacol. 49, 160–171. Lombardi, V.R., Garcia, M., Rey, L., Cacabelos, R., 1999. Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer’s disease (AD) individuals. J. Neuroimmunol. 97, 163– 171. Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, A.M., Snyder, S.H., Russell, S.W., Murphy, W.J., 1993. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90, 9730–9734.

77

Marchalant, Y., Rosi, S., Wenk, G.L., 2007. Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation. Neuroscience 144, 1516–1522. Maruyama, Y., Kuribara, H., Morita, M., Yuzurihara, M., Weintraub, S.T., 1998. Identification of magnolol and honokiol as anxiolytic agents in extracts of saiboku-to, an oriental herbal medicine. J. Nat. Prod. 61, 135–138. Milatovic, D., Zaja-Milatovic, S., Montine, K.S., Horner, P.J., Montine, T.J., 2003. Pharmacologic suppression of neuronal oxidative damage and dendritic degeneration following direct activation of glial innate immunity in mouse cerebrum. J. Neurochem. 87, 1518–1526. Munroe, M.E., Arbiser, J.L., Bishop, G.A., 2007. Honokiol, a natural plant product, inhibits inflammatory signals and alleviates inflammatory arthritis. J. Immunol. 179, 753–763. Noble, F., Rubira, E., Boulanouar, M., Palmier, B., Plotkine, M., Warnet, J.M., Marchand-Leroux, C., Massicot, F., 2007. Acute systemic inflammation induces central mitochondrial damage and mnesic deficit in adult Swiss mice. Neurosci. Lett. 424, 106–110. Sambamurti, K., Kinsey, R., Maloney, B., Ge, Y.W., Lahiri, D.K., 2004. Gene structure and organization of the human beta-secretase (BACE) promoter. FASEB J. 18, 1034–1036. Selkoe, D.J., 1998. The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol. 8, 447–453. Sparkman, N.L., Kohman, R.A., Scott, V.J., Boehm, G.W., 2005. Bacterial endotoxininduced behavioral alterations in two variations of the Morris water maze. Physiol. Behav. 86, 244–251. Stewart, W.F., Kawas, C., Corrada, M., Metter, E.J., 1997. Risk of Alzheimer’s disease and duration of NSAID use. Neurology 48, 626–632. Szekely, C.A., Thorne, J.E., Zandi, P.P., Ek, M., Messias, E., Breitner, J.C., Goodman, S.N., 2004. Nonsteroidal anti-inflammatory drugs for the prevention of Alzheimer’s disease: a systematic review. Neuroepidemiology 23, 159–169. Wyss-Coray, T., 2006. Inflammation in Alzheimer disease: driving force, bystander or beneficial response? Nat. Med. 12, 1005–1015. Yamamoto, Y., Gaynor, R.B., 2004. IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem. Sci. 29, 72–79. Yamamoto, K., Arakawa, T., Ueda, N., Yamamoto, S., 1995. Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells. J. Biol. Chem. 270, 31315–31320. Yang, J., Splittgerber, R., Yull, F.E., Kantrow, S., Ayers, G.D., Karin, M., Richmond, A., 2010. Conditional ablation of Ikkb inhibits melanoma tumor development in mice. J. Clin. Invest. 120, 2563–2574. Yankner, B.A., 1996. Mechanisms of neuronal degeneration in Alzheimer’s disease. Neuron 16, 921–932.