Tg-APPswe/PS1dE9 transgenic mice (Tg-APP/PS1 mice) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained in a hemizygote state by crossing transgenic C57BL6 x C3H F1 hybrid mice and non-transgenic C57BL6 x C3H F1 mice (23,24). Mice not expressing the transgene were used as wild-type controls. The mice were housed in regular polycarbonate plastic cages in an environment with controlled temperature (21–22°C) and humidity (50–60%) with a 12-h light/dark cycle (lights on at 7 a.m.). Furthermore, the animals were maintained on an ad libitum diet of laboratory chow (2018S; Harlan Teklad, Madison, WI, USA) and had free access to water. The cages were filled to an approximate depth of 1.5 cm with bedding made of chopped wood particles (JSBio, Daejeon, Korea). All materials used were autoclaved and gamma-irradiated. The animal room was maintained under specific-pathogen-free (SPF) conditions. The Tg-APP/PS1 mice at 5 months of age were randomized into the control (n=10) and HJ (n=10) groups. The methanolic extract of HJ at 500 mg/kg/day or the vehicle were administered to the mice by oral gavage for 2.5 months from 5 months of age. All animal experiments were approved by the Institutional Animal Use and Care Committee of the Korea Research Institute of Bioscience and Biotechnology and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, 8th edition, 2011).

HJ was purchased from Gangwon Herbs, Gangwon, Republic of Korea, on July, 2014. The voucher specimen was identified by Professor W.K. Oh, and a voucher specimen (SNU-2014-0004) was deposited at the College of Pharmacy, Seoul National University, Korea. The HJ extract was then prepared and supplied by the Korea Bioactive Natural Material Bank (Seoul, Korea). Briefly, the dried aerial parts of HJ were soaked in 100% methanol in an extraction container for 2 days at room temperature. The methanol-soluble extract was filtered through a cheesecloth, concentrated exhaustively and dried to produce a methanolic extract under reduced pressure. This HJ extract was suspended in 0.5% carboxymethylcellulose (CMC) at a concentration of 50 mg/ml as a stock solution. The 5-month-old male and female mice were administered orally 500 mg/kg/day of HJ-methanol extract or 0.5% CMC (vehicle) for 10 weeks.

BV-2 microglial cells (a gift from Dr B.S. Han, KRIBB, Daejeon, Korea) were maintained in Dulbecco's modified Eagle's medium (DMEM, Cat. no. SH30243.01; HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (both from Gibco, Grand Island, NY, USA). The cells were incubated at 37°C under 5% CO₂ and 95% relative humidity. They were seeded in 12-well plates (1×10⁵ cells/well), incubated overnight, and thereafter co-treated with differently diluted HJ extracts (100 and 500 µg/ml) for 24 h. In this preliminary experiment, HJ concentrations of 50, 100, 200 and 500 µg/ml were not shown to have cytotoxic effects on the BV-2 cells (data not shown). Subsequently, two different concentrations of HJ (100 and 500 µg/ml) were selected for use in further experiments, and the BV-2 cells were cultured under the same conditions with additional LPS (1 µg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 24 h.

Nitrite in the culture supernatants was measured by the Griess reaction as an indicator of NO production. The BV2 cells were seeded in 12-well plates (1×10⁵ cells/well), incubated overnight, and thereafter co-treated with various concentrations of HJ (100 and 500 µg/ml) and LPS (1 µg/ml) for 24 h. Following LPS stimulation, a 100 µl volume of culture supernatants from each sample were mixed with the same volume of Griess reagent (G4410; Sigma-Aldrich) and then incubated at room temperature for 15 min. The NO concentration was determined by measuring the absorbance at 540 nm on a microplate reader (Thermo Multiskan Spectrum; Thermo Fisher Scientific, Waltham, MA, USA). The nitrite concentration was calculated with reference to a sodium nitrite standard curve generated with known concentrations.

RNA preparation and qPCR were performed as previously described (25). Following 24 h of stimulation with LPS or treatment with LPS + HJ, the mRNA expression levels of TNF-α, IL-1β, IL-6, iNOS and β-actin in BV2 microglial cells were evaluated by RT-qPCR. In addition, following the administration of HJ for 2.5 months in vivo, mice were anesthetized with Avertin (0.3 g/kg, i.p.) and the mouse brain was extracted and dissected using Rodent Brain Matrix (ASI Instruments, Warren, MI, USA). The cortex was removed and the expression levels of TNF-α, IL-1β and IL-6 were evaluated by RT-qPCR. Total RNA was purified from the cells and tissues using TRI reagent (Sigma-Aldrich). Reverse transcription was carried out using a Promega RT-PCR kit (Promega, Madison, WI, USA). PCR reaction was prepared with a mix of 5 µl of 2X SYBR-Green mix and performed using the StepOne Real-Time PCR system (both from Applied Biosystems, Foster City, CA, USA). The cycle number at which the fluorescence emission exceeds the fixed threshold was defined as threshold cycle (CT). The cycling conditions were as follows: 10 min at 95°C, followed by 41 cycles of 20 sec at 95°C, 30 sec at 60°C, and 20 sec at 72°C. The difference in amplification fold was calculated based on qPCR amplification of the target gene vs. β-actin as a reference using built-in software of the Gene Expression Analysis for StepOne Software version 2.1 (Applied Biosystems). qPCR was repeated 4 times. The following primer sets were used: TNF-α forward, 5′-ATCCGCGACGTGGAACTG-3′ and reverse, 5′-ACCGCCTGGAGTTCTGGAA-3′; IL-1β forward, 5′-CTACAGGCTCCGAGATGAACAAC-3′ and reverse, 5′-TCCATTGAGGTGGAGAGCTTTC-3′; IL-6 forward, 5′-TTCCATCCAGTTGCCTTCTTG-3′ and reverse, 5′-GGGAGTGGTATCCTCTGTGAAGTC-3′; iNOS forward, 5′-GTTCTCAGCCCAACAATACAAGA-3′ and reverse, 5′-GTGGACGGGTCGATGTCAC-3′; and β-actin forward, 5′-TATTGGCAACGAGCGGTTCC-3′ and reverse, 5′-GGCATAGAGGTCTTTACGGATGT-3′.

The levels of TNF-α, IL-1β, and IL-6 produced were measured using ELISA kits (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, the BV2 microglial cells were seeded in 12-well plates (1×10⁵ cells/well), incubated overnight and thereafter co-treated with various concentrations of HJ (100 and 500 µg/ml) and LPS (1 µg/ml) for 24 h. A total of 100 µl of culture supernatants was collected to determine the TNF-α, IL-1β, and IL-6 concentrations by ELISA.

Novel object recognition is a validated and widely used test for assessing recognition memory (26,27). The mice were placed individually in a 40×20×20 cm³ testing chamber for 10 min with two identical objects (familiar, acquisition session). The mice were then returned to home cages and one day they later placed back in the testing chamber in the presence of one of the original objects and one novel object (novel, recognition session) for 10 min. The original objects were cylindrical wooden blocks 10 cm high × 2 cm in diameter. The novel object was a 10×2.5×2 cm rectangular wooden block. The acquisition and recognition sessions were video-recorded and an observer who was blinded to the drug treatment scored the time spent exploring the objects. The chambers and objects were cleaned with ethanol between trials. Exploration was defined as sniffing and touching the object with the nose and/or forepaws. Sitting on the object was not considered exploratory behavior. In preliminary experiments, wild-type mice exhibited no significant preference for the cylindrical or rectangular block. The time spent exploring both objects was calculated. A discrimination index was calculated for each animal and expressed by the ratio of time spent exploring the novel object − time spent exploring the familiar object/(time spent exploring the novel object + time spent exploring the familiar object) on day 2, as previously described (28).

The Y-maze test was conducted as previously described (29). A Y-maze with three identical arms of plexiglass (51.5×11.5×12 cm) 120° apart was placed in the center of a room. The walls of each arm had a distinct design that provided visual cues. Each mouse was placed at the end of one arm facing the center and allowed to explore the maze for a period of 10 min. The sessions were video-recorded and scored for entries into arms. The percentage of spontaneous alternation was calculated as the ratio of the actual to possible alternations (defined as the total number of arm entries minus 2) multiplied by 100: alternation (%) = [(number of alternation)/(total arm entries − 2)] ×100.

Immunohistochemistry was conducted as previously described (30). The mice were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. The perfused brains were dissected, post-fixed overnight and then cut into 40-µm-thick coronal sections on a vibratome (Vibratome VT1000A; Leica, Mannheim, Germany). Free-floating sections were incubated in PBS containing 3% H₂O₂ (v/v), rinsed 3 times in PBS, and blocked with serum for 1 h at room temperature. The sections were incubated overnight at 4°C with the primary antibodies. The primary antibodies were mouse monoclonal antibodies against Bam10 (4% BSA, Cat. no. A5213; Sigma-Aldrich), rabbit polyclonal antibodies against ionized calcium-binding adapter molecule 1 (Iba-1, 5% HS, Cat. no. 019-19741; Wako, Richmond, VA, USA), glial fibrillary acidic protein (GFAP, 5% GS, Cat. no. Z0334; Dako) and mouse polyclonal antibody against AT8 (phospho-tau, 5% GS, Cat. no. MN1020; Thermo Fisher Scientific). Immunohistochemistry was then performed using biotinylated secondary anti-rabbit IgG, and avidin-biotinylated peroxidase complex (ABC kit) (both from Vector Laboratories, Burlingame, CA, USA), and 3,3′-diaminobenzidine (Sigma-Aldrich). Sections containing the parietal cortex were selected and the number of plaques in the cortex was counted under a microscope (Olympus Corp., Tokyo, Japan). The assessments of Bam10-stained areas in the cortex were performed using the MetaMorph image analyzer (Molecular Devices Inc., Sunnyvale, CA, USA).

Western blot analysis was performed as described in a previous study (25). The mice were sacrificed and the brain tissues were rapidly removed and homogenized in homogenization buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 0.1% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). Protein samples were resolved by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were then transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were incubated with primary antibodies followed by secondary antibodies, and specific signals were visualized using an enhanced chemiluminescence kit (Intron Biotechnology, Seoul, Korea). The western blot images were quantified using Quantity One 1-D analysis software version 4.6.1 (Bio-Rad Laboratories, Inc.). The primary antibodies were C-terminal fragments (CTFs, Cat.no. A8717; Sigma-Aldrich), phospho-amyloid precursor protein (p-Thr668 APP, Cat.no. 6986; Cell Signaling Technology, Inc., Danvers, MA, USA), a disintegrin and metalloproteinase 10 (ADAM10, Cat. no. AB1997) and beta-site APP-cleaving enzyme 1 (BACE1, Cat. no. AB2077) (both from Abcam, Cambridge, UK), neprilysin (NEP, Cat. no. SC-9149; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), insulin degrading enzyme (IDE, Cat. no. 97-273; Calbiochem, Darmstadt, Germany), vesicular glutamate transporter 1 (vGlut1, Cat. no. 135 302; Synaptic Systems, Göttingen, Germany), N-methyl-D-aspartate receptor subtype 2B (NR2B, Cat. no. 06-600; Merck Millipore, Darmstadt, Germany), synaptophysin (SYP, Cat. no. S5768; Sigma-Aldrich), β-actin (Cat. no. MAB1501; Merck Millipore) and post-synaptic density protein 95 (PSD-95, a gift from Dr J.-R Lee; KRIBB). The secondary antibodies were goat anti-rabbit IgG (Cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) and goat anti-mouse IgG (Cat. no. sc-2005; Santa Cruz Biotechnology, Inc.).

Two-sample comparisons were carried out using the Student's t-test, while multiple comparisons were made using one-way ANOVA followed by Tukey-Kramer's post hoc test. All data are presented as the means ± SEM and statistical differences were accepted at the 5% level (p<0.05) unless otherwise indicated.

To examine the inhibitory effects of HJ on LPS-induced NO production by BV2 cells, the NO levels in the culture medium were measured by Griess reaction. Stimulation of the BV2 cells with LPS caused a marked induction of NO production. However, treatment with HJ resulted in the significant inhibition of LPS-induced NO production (Fig. 1A). We also performed RT-qPCR to determine whether the inhibition of NO production by HJ in the LPS-stimulated BV2 cells is associated with the decreased levels of iNOS, which produces NO as a key mediator of inflammation. Treatment with HJ resulted in a significant decrease in the mRNA expression of iNOS in the LPS-stimulated BV2 cells (Fig. 1B). These results indicated that the HJ-induced decrease in the expression of iNOS is responsible for the inhibition of NO production.

In order to investigate whether HJ inhibits the expression of LPS-induced pro-inflammatory cytokines in the murine microglial cell line, BV2, the cells were co-treated with LPS and HJ for 24 h and the mRNA levels of TNF-α, IL-1β and IL-6 were then analyzed by RT-qPCR. As shown in Fig. 2A–C, the LPS-induced increase in the expression of TNF-α, IL-1β and IL-6 was significantly decreased by treatment with HJ. The production of TNF-α, IL-1β and IL-6 induced by LPS in the culture supernatants was evaluated by ELISA. The levels of TNF-α, IL-1β and IL-6 were significantly increased in the culture medium of LPS-stimulated BV2 cells; however, co-treatment with HJ resulted in a marked decrease in the release of these pro-inflammatory cytokines (Fig. 2D–F). These results suggest that HJ is effective in the suppression of pro-inflammatory cytokine production through the alteration of the transcript levels of TNF-α, IL-1β and IL-6 in activated BV2 microglial cells.

Based on the in vitro anti-inflammatory effects of HJ, we examined whether HJ has a therapeutic effect on an animal model of AD. Tg-APP/PS1 mice were administered HJ at a dose of 500 mg/kg/day for 2 months from 5 months of age. The body weight of the HJ group during the treatment period was similar to that of the controls (data not shown). To investigate whether the treatment of Tg-APP/PS1 mice with HJ functionally attenuates AD-type cognitive deterioration, we evaluated object recognition memory (27) and spatial working learning and memory (31) using the object recognition test and spontaneous Y-maze alteration test in HJ- or vehicle-treated mice. The HJ-treated Tg-APP/PS1 mice exhibited increased exploration preference of the novel object during the recognition session of the object recognition test compared with vehicle-treated Tg-control mice (Fig. 3A). In the spontaneous Y-maze alteration test, the HJ-treated Tg-APP/PS1 mice exhibited increased alteration percentage compared with the vehicle-treated Tg-control mice (Fig. 3B). This increase in the percentage of spontaneous alterations was statistically significant (p<0.05). These results indicated that treatment with HJ for 2 months improved the cognitive decline of Tg-APP/PS1 mice.

Subsequently, to investigate whether HJ can attenuate the levels of Aβ and tau hyperphosphorylation in Tg-APP/PS1 mice, we measured the percentage area occupied by plaques and phospho-tau in the cortex by immunohistochemical assay. The Tg-APP/PS1 mice were administered HJ at a dose of 500 mg/kg/day for 2.5 months from 5 months of age. The administration of HJ effectively decreased the percentage area occupied by amyloid plaques in the cortex (Fig. 4A–C; p<0.05). Tau hyperphosphorylation was also significantly decreased in the HJ-treated Tg-APP/PS1 mice compared with the vehicle-treated Tg-control mice (Fig. 4D–F; p<0.01).

In order to further investigate which factors contributed to the reduction of Aβ in HJ-treated Tg-APP/PS1 mice, the changes in APP processing and Aβ clearance-related factors were analyzed using brain tissues from both groups of mice. As shown by the results of western blot analysis, HJ had no effect on the protein expression of total APP (Fig. 5A and B), phospho-APP (Fig. 5A and C), and the β-cleavage product (CTFβ) and α-cleavage product (CTF α) (Fig. 5A, D and E). In addition, treatment with HJ did not alter the levels of ADAM10 (α-secretase) and BACE1 (β-secretase) (Fig. 5A, F and G). Moreover, since the proteolytic degradation of Aβ is known to be a major route for the clearance of plaques and neprilysin, and insulin degrading enzyme and major Aβ-degrading metalloproteases are involved in the downregulation of the levels of Aβ secreted extracellularly (32,33), the protein levels of NEP and IDE in brain tissues were determined in both groups. Immunoreactivity assessed by western blot analysis of IDE expression was significantly increased in the HJ-treated Tg-APP/PS1 mice (Fig. 5A and I), but the level of NEP was not altered (Fig. 5A and H). These results suggest that HJ has an effect on Aβ clearance through the increased level of IDE, but not by APP processing.

Previously, it has been reported that Aβ is able to initiate inflammation, which implicates the activation of microglia and the recruitment of astrocytes, and the release of cytokines, such as TNF-α, interferon-γ, IL-1β, IL-1α and COX-2, and ROS that are involved in neuronal and synaptic damage (4,34). Thus, to investigate whether HJ can reduce the activation of microglia and astrocytes in Tg-APP/PS1 mice, we measured the number of Iba-1 immunoreactive cells and the areas of GFAP immunoreactivity in the cortex (Fig. 6). The number of Iba-1-positive cells was significantly decreased in the cortex tissues of the HJ-treated Tg-APP/PS1 mice (Fig. 6A–C; p<0.05). Moreover, the percentage area occupied by active astrocytes was also significantly decreased in the cortex tissues of the HJ-treated Tg-APP/PS1 mice (Fig. 6D–F; p<0.05). Additionally, the mRNA levels of TNF-α and IL-6 in the cortex homogenates in the HJ-treated Tg-APP/PS1 group were significantly lower than those in the Tg-APP/P1 control group (Fig. 7A and B; p<0.05 and p<0.01). However, the mRNA expression levels of IL-1β in the HJ-treated Tg-APP/PS1 group were not significantly altered (Fig. 7C). These findings indicated that the activation of microglia and astrocytes, and the expression levels of the pro-inflammatory cytokines, TNF-α and IL-6, were markedly reduced by treatment with HJ for 2.5 months in the animal model of AD. These results suggest that treatment with HJ effectively attenuates neuroinflammation in the brains of Tg-APP/PS1 mice.