C57BL/6 mice weighing 25–30 g were kept in an air-conditioned room set at 23 ± 1◦C with 50% humidity and maintained on a standard 12-12 light/dark cycle with food available ad libitum. This study design and all animal procedure were approved by the Animal Experiment Committee of the Henan University of Science and Technology, China.
Group and Treatment
Forty-eight male mice were randomly divided into the following groups (n =12, per group): control group, LPS group, LPS+Arctigenin group, Arctigenin group. Mice were orally administered Arc (50 mg·kg-1·d-1; i.g.) or its solvent for consecutive 21 days. On the 3rd week, 2 h after administration of Arc, LPS (250 µg/kg) was injected intraperitoneally for 7 consecutivedays. Arctigenin (Bellancom Chemistry, USA) was of high purity (> 99 %) as determined by HPLC analysis, and was dissolved in 0.5% carboxymethylcellulose sodium (CMC) in PBS followed by daily intragastric (i.g.) administration. LPS (Escherichia coli serotype 0111:B4, Sigma-Aldrich, St. Louis, MO, USA) was diluted in saline and intraperitoneally administered 2h after drug administration.To determin the optimum dosage, a preliminary study was performed with three different dosages of arctigenin (25, 50, 100 mg·kg-1·d-1; i.g.). Our results showed that both high dose (50 mg·kg-1·d-1; i.g.) and higher dose (100 mg·kg-1·d-1; i.g.) arctigenin administration markedly attenuated LPS-induced learning and memory, as assessed by the Morris water maze (MWM) in mice. However, no significant difference was observed between the two doses of arctigenin administration (data not shown). Therefore, the dose of arctigenin (50 mg·kg-1·d-1; i.g.) was selected as the optimum dosage for further studies.
Morris Water Maze
The Morris water maze was employed to assess spatial learning and memory ability with minor modifications (Laczó et al. 2009; Wang et al. 2017a). Briefly, the circular apparatus (100 cm diameter) was filled with white nontoxic paint and water (22 ± 1 °C), and an invisible platform (10 cm diameter) was submerged 1 cm beneath the surface of the water. During the place navigation test, each mouse was trained to escape and climb onto the hidden platform (4 trials per day × 5 days). In each trial, each mouse was given a maximum time of 60 s to find and reach the platform. If the mouse failed to find the platform within 60 s, it was guided toward it and kept there for 15 s. The probe trial was carried out on the 6th day. The mice were allowed to swim freely for 60 s (the platform was withdrawn), and the velocity and the path length and the time spent in the target quadrant were recorded. All the behavioural parameters were tracked, recorded, and analyzed by SMART systerm (Panlab, Barcelona, Spain).
Following behavioral tests, mice received transcardial perfusion with 0.9% saline and 4% paraformaldehyde. Mouse brains were collected and postfixed with 4% paraformaldehyde followed by 30% w/v sucrose in 0.1 M phosphate buffer (pH 7.4). Brain tissues were cut into serial 20 µm-thick coronal sections in coronal planes. Sections were stored at -20°C until immunostaining was performed.
Following staining in 0.1% cresyl violet solution for 5 min, brain slices were rinsed with deionized water twice and dehydrated with an ascending series of ethanol. After clearing in xylene, slides were mounted on glass coverslips. Images were obtained using a microscope, and healthy neurons containing Nissl substance were quantified from 4 randomly selected visual fields of each section (4-5 representative sections per animal) using Image-Pro Plus 6.0 software.
Following blocking in 10% goat serum for 1 h, brain slides were incubated with anti-Iba1 and anti-GFAP at 4 °C overnight. After washing three times in PBS, sections were incubated with biotinylated secondary antibodies for 1 h at room temperature. Then, they were colored and visualized by a 3, 3'-diaminobenzidine kit (DAB). Images were captured using a Nikon Eclipse Ti Microscope. The integral optical density ofimmunopositive cells was assessed from 4 randomly selected visual fields of each section (4-5 representative sections per animal) and quantified using Image-Pro Plus 6.0 software(Wang et al. 2018).
Immunofluorescence staining was carried out on brain and cell slices as previously described (Wang et al. 2017b, 2019). Following blocking in 10% goat serum at room temperature for 1 h,the slideswere incubated with the primary antibodies: Synapsin-1 (1:200; Cell Signaling Technology), PSD95 (1:300; Millipore), Aβ (1:200; Cell Signaling Technology), and NF-κB p65 (1:200; Cell Signaling Technology) at 4°C overnight. The slides were subsequently incubated with Alexa Fluor 488-or 594-conjugated secondary antibodies at room temperature for 1 h. Immunofluorescence images were obtained using a ZEISS LSM 800 confocal microscope (Carl Zeiss, Germany) and processed using ZEN software.
Cell culture and drug treatment
Immortalized murine microglial (BV-2) cells were maintained in DMEM supplemented with 10% FBS and antibiotics. BV-2 microglial cells were grown to 70–80% confluence and pretreated for 1 h in the absence or presence of arctigenin (12.5, 25, 50, or 100 μM) followed by stimulation with LPS (1µg/ml) for 24 h. After the indicated drug treatment, CCK-8 (Beyotime Institute of Biotechnology, Jiangsu, China) was employed to examine cell viability by changes in the absorbance at 450 nm. Following treatment, the cellar protein was obtained for further analyses.
AdipoR1 siRNA transfection
siRNA targeting AdipoR1 and a nontargeting control siRNA were designed and synthesized (Genepharma, Shanghai, China). Transfection was carried out as described in the manufacturer’s instructions using Lipofectamine2000 (Invitrogen, Carlsbad, CA). Following transfectionwith AdipoR1 siRNA for 48 h, BV-2 cells were pretreated with arctigenin (50 μM) before additional LPS incubation for 24 h. Finally, the cellular protein was acquired for further analyses.
Assay of proinflammatory cytokines levels
Protein from cellular lysate or brain homogenate was centrifuged at 14,000 x g for 30 min at 4 °C, and the obtained supernatants were sampled in triplicate to examine proinflammatory cytokine levels including TNF-α, IL-1β, and IL-6 using commercial ELISA kits (R&D Systems and Invitrogen).
Western blot analysis
Equal amounts of soluble protein from cellular lysate or hippocampal homogenate were electrophoretically separated using 10%-15% SDS–PAGE gel and transferred onto a nitrocellulose membrane. After blocking, membranes were incubated with primary antibodies specific for TNF-α, cleaved caspase-3, BACE1, p-IκB-α, IκB-α, p-NF-κB p65, and NF-κB p65(Cell Signaling Technology); cleaved PARP-1, APP, Iba-1, GFAP,AdipoR1, TLR4, and CD14 (Abcam) at 4°C overnight. The membranes were probed with HRP-conjugated secondary antibodies, and immunoreactive images were exhibited using a chemiluminescent detection system and collected on Bio-Rad Chemidoc Imaging System, and the intensities of the protein bands were determined using Image-Pro Plus 6.0 software.
Aliquots of the cellular lysates were incubated with anti-TLR4 antibody (Santa Cruz Biotechnology) at 4 °C overnight on a rotating device. Protein A/G Plus-Agarose beads (Santa Cruz Biotechnology) were added to the cell lysate for 4 h at 4 °C on rotation, and the beads were washed three times with iced ice-cold buffer. The washed beads were resuspended in SDS sample buffer and denatured for 5 min at 100 °C. Following centrifugation, the supernatants were subjected to the western blot analysis.
Values were presented as the mean ± SEM. All statistical procedures were performed using SPSS version 23.0. For the Morris water maze task, escape latency during the place navigation test was analyzed using two-way repeated measures ANOVA. The data that were not included in repeated measures were analyzed with one-way ANOVA followed by the Bonferroni post-hoc test for multiple comparisons. Differences were considered significant at a p value < 0.05.