PL was isolated from Piper longum. Preparation was performed as described in previous studies (17-19). Dried fruits (500 g) of Piper longum were extracted with ethyl acetate (EtOH; 1 liter x 3 times) at room temperature for 1 week. The combined EtOH extracts were concentrated to yield a dry residue (32.5 g), which was subsequently suspended in water (H₂O; 500 ml) and partitioned with EtOAc (3×500 ml). The partial EtOAc extract (6.0 g), which was subjected to a silica gel column chromatography (CC; 5×40 cm), was eluted with a gradient n-hexane/acetone system (20:1 to 1:1) to yield 5 fractions (F1-F5). Fractions F3 and F4 were combined and further applied to a reversed phase-C₁₈ CC (3×30 cm) with methanol (MeOH)/H₂O (1:1 to 9:1). Subfraction F34.3 (60.8 mg) was purified by high-performance liquid chromatography [mobile phase: MeOH in H₂O containing (0-40 min: 65% MeOH); flow rate: 2 ml/min; UV detection at 205 and 254 nm] to yield a compound (t_R=17.2 min, 14 mg). The chemical structure of the isolated compound was confirmed by comparison with the reported chemical structure of PL using 1D and 2D nuclear magnetic resonance spectroscopy.

Female C57BL/6J mice, at 3 months (n=7, weighing 19-22 g) and 23 months of age (n=28, 28-34 g), were obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Korea) and housed in regular polycarbonate plastic cages in an environment with a controlled temperature (21-22°C) and humidity (50-60%) and a 12-h light/dark cycle (lights on at 7 a.m.). The mice were maintained on an ad libitum diet of lab chow (Teklad 2018S, Harlan, WI, USA) with 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 in specific-pathogen-free conditions. The C57BL/6J mice at 23 months of age were randomized into the vehicle [0.5% carboxymethyl cellulose (CMC), Aged vehicle, n=14)] and PL (Aged PL, n=14) groups. The PL extract was suspended in 0.5% CMC at a concentration of 5 mg/ml as a stock solution. The 23-month-old female mice were orally administrated 10 μl/g/day of PL stock solution or 0.5% CMC for 8 weeks. The 3-month-old female mice were used as young controls (n=7). Multiple behaviour tests were performed on a single cohort of mice and the following order was obeyed: Open field test → novel object recognition test → nest-building behaviour test (17,20). All the animal experiments were approved by the Institutional Animal Use and Care Committee of the KRIBB (KRIBB-AEC-14074).

The mice were individually placed in an open field box (45×45×45 cm³) for 30 min. The horizontal locomotion of the mouse was measured using a computerized video tracking system, SMART (Panlab, Barcelona, Spain).

The novel object recognition test was performed as described in previous studies (21,22). The mice were individually habituated to a testing chamber (40×20×20 cm³) with no objects for 5 min and then placed in a testing chamber for 10 min with two identical objects (familiar, acquisition session). The mice were then returned to the home cages. One day later, the mice were placed back into 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 x 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 behaviour. A discrimination index was calculated for each animal and expressed using the following formula: [time (number) of contacts with the novel object-time (number) of contacts with the familiar object]/[time (number) of contacts with the novel object + time (number) of contacts with the familiar object] on day 2.

The nest building behaviour test was performed as described in a previous study (23). The mice were housed in single cages containing chopped wood particles for 5 days. On the first day of testing, one piece of cotton (5×5 cm; Nestlets, Ancare, Bellmore, NY, USA) was introduced into the home cage to permit nesting. The presence and quality of nesting was rated 1 day later on a 5-point scale ranging from 1 to 5 as follows: 1, nestlet not noticeably touched (>90% intact); 2, nestlet partially torn up (50-90% remaining intact); 3, mostly shredded, but often no identifiable nest site; 4, an identifiable but flat nest; and 5, a (near) perfect nest. Immediately afterward, the mice were group-housed as before.

Western blot analysis was performed as described in a previous study (21). Following 8 weeks of PL treatment, the mice were sacrificed and the hippocampal tissues were rapidly removed and homogenized in a 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 Diagnostics GmbH, Mannheim, Germany). Protein samples were resolved by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The samples 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 Chemi Luminescence kit (Intron Biotechnology, Gyeonggi-do, Korea). Western blot images were quantified using Quantity One 1-D analysis software version 4.6.1 (Bio-Rad Laboratories, Inc.). The primary antibodies used were vesicular glutamate transporter 1 (VGLUT1; 1:1,000, #135 302, SYSY, Göttingen, Germany), vesicular glutamate transporter 2 (VGLUT2; 1:1,000, #75-067 UC Davis/NIH NeuroMab Facility, Davis, CA, USA), glutamate receptor 1 (GluR1; a gift from Dr J.R. Lee, KRIBB, Daejeon, Korea, 1:1,000), N-methyl-D-aspartate receptor subtype 2B [(NR2B, 1:1,000, #4212, Cell Signaling Technology (CST), Danvers, MA, USA)], phosphorylated (p-)NR2B (p-Tyr-1472-NR2B, 1:1,000, #4208, CST), synaptophysin (1:1,000, #S5768, Sigma-Aldrich Co. LLC; Merck KGaA, Darmstadt, Germany), post-synaptic density protein 95 (PSD-95, 1:1,000, #124 014, SYSY), glutamate decarboxylase 65/67 (GAD65/67, 1:1,000, #AB1511, Merck KGaA), gephyrin (1:1,000, #147 011, SYSY), vesicular GABA transporter (VGAT, 1:1,000, #131 002, SYSY), cAMP response element binding protein (CREB, 1:1,000, #06-863, Merck KGaA), p-CREB (p-Ser133-CREB, 1:1,000, #06-519, Merck KGaA), calcium/calmodulin-dependent protein kinase type II α (CaMKIIα, 1:1,000, #sc-13141, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p-CaMKIIα (p-Thr-286-CaMKIIα, 1:1,000, #sc-12886, Santa Cruz Biotechnology, Inc.), extra cellular signal-regulated kinases 1/2 (ERK1/2, 1:1,000, #9102, CST), p-ERK1/2 (p-Thr202/Tyr204-ERK1/2, 1:1,000, #9101, CST) and β-actin (1:1,000, #MAB1501, Merck KGaA). The secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,000, #NCI1460KR, Thermo Fisher Scientific, Inc., Waltham, MA, USA) or goat anti-mouse (1:2,000, #sc-2005, Santa Cruz Biotechnology).

Immunohistochemistry and immunofluorescence staining were performed as previously described (21,24-26). Following 8 weeks of PL treatment, the mice were deeply anesthetized (250 mg/kg Avertin, intraperitoneally) and transcardially perfused with saline followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed, post-fixed overnight, and then cut into 40-μm-thick coronal sections using a vibratome (Vibratome VT1000A, Leica Microsystems GmbH, Wetzlar, Germany). The free-floating sections were then 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 then incubated with the phospho-CREB (Ser133, 1:1,000, #06-519, Merck KGaA), doublecortin (DCX, 1:1,000, #sc-8666, Santa Cruz Biotechnology), 4-hydroxy-2-nonenal (4-HNE, 1:1,000, #HNE11-S, Alpha Diagnostic, San Antonio, TX, USA), ionized calcium-binding adapter molecule 1 (Iba1, 1:1,000, #019-19741, Wako Chemicals USA, Inc., Richmond, VA, USA) and glial fibrillary acidic protein (GFAP, 1:1,000, #Z-0334, Dako, Glostrup, Denmark) primary antibodies overnight at 4°C. The sections were then washed and incubated with biotinylated secondary anti-rabbit IgG (1:200, #BA-1000, Vector Laboratories, Inc., Burlingame, CA, USA), followed by the avidin-biotinylated peroxidase complex (Vector Laboratories, Inc.) and 3,3′-diaminobenzidine (Sigma-Aldrich Co. LLC; Merck KGaA). Immunofluorescence staining was then performed with an Alexa Fluor 594 goat anti-rabbit IgG antibody (secondary antibody, 1:200, #A11012, Thermo Fisher Scientific, Inc.). Sections containing the hippocampus were selected and the number of doublecortin-positive cells in the dentate gyrus (DG) were counted under a microscope (Olympus Corp., Tokyo, Japan). The intensity of 4-HNE- and p-CREB-stained cells and the percentage area occupied by GFAP- and Iba-1-positive cells in hippocampal CA1, CA3 and DG were assessed using the MetaMorph image analyser (Molecular Devices, LLC, Sunnyvale, CA, USA).

GraphPad PRISM (GraphPad Software, Inc., La Jolla, CA, USA) software was used to perform the statistical analyses. Two-sample comparisons were performed using a Student's t-test, while multiple comparisons were made using a one-way ANOVA followed by the Tukey-Kramer's post hoc test. Associations between distance and discrimination index were examined by Pearson's correlation coefficient. All data are presented as the means ± SEM and statistical differences are accepted at the 5% level (P<0.05), unless otherwise indicated.

The aged female C57BL/6J mice (23 months old) were randomly separated into the vehicle- and PL-treated groups. PL was administered at a dose of 50 mg/kg/day for 8 weeks, from the ages of 23 to 25 months. The experimental design is presented in Fig. 1A. The aged mice (24 months of age) exhibited a significantly lower locomotor activity in the open field test than the young control mice (Fig. 1B, P<0.05). PL treatment did not markedly affect the exploratory behaviour of the aged mice compared to the aged vehicle group (Fig. 1B, P>0.05). To determine whether PL can improve cognitive function in aged mice, we performed the novel object recognition test. In the recognition session, with two different objects (one novel and the other familiar), the young control mice explored the novel object for a relatively long time period and a made contact with it a relatively high number of times, yielding a discrimination index (DI) of approximately 0.24±0.08 and 0.15±0.07, indicating that they had a memory of the familiar object (Fig. 1D and E). By contrast, the aged mice treated with the vehicle exhibited a DI that was significantly lower than that of the younger controls (−0.05±0.05 and −0.06±0.04, Fig. 1D and E), which is consistent with impaired cognition. PL treatment markedly increased the DI in aged mice to approximately 0.24±0.06 and 0.17±0.05 (Fig. 1D and E), reflecting a therapeutic effect of PL on age-related cognitive impairment. PL treatment did not alter the total exploration time (aged vehicle, 10.14±1.11 sec; aged PL, 8.97±0.48 sec, P=0.466) and total number of contacts (aged vehicle, 16.92±2.22; aged PL, 19.71±1.25, P=0.680) to both objects (familiar + novel) on day 2, indicating no influence on the total exploration activity of PL in the novel object recognition test. Additionally, we could not find any association between the distance in the open field test and the DI in the novel object recognition test in the aged mice (Fig. 1F and G, P>0.05).

Previous studies have reported that nest building, which is an indicator of well-being and social context in mice, is decreased in aging in rodent models of AD (27,28). Reduced nesting has also been observed in mice with hippocampal lesions (29). In this study, the nesting score in the nest building test was significantly lower in the aged mice than in the young control mice (Fig. 1C, P<0.05). PL significantly increased the nesting score in the aged mice (Fig. 1C, P<0.05). These results indicate that treatment with PL may improve cognitive and social decline without affecting locomotion in aged mice.

An upregulation of inflammatory responses and oxidative stress have been observed in the hippocampus in aging (30-33). An increase in inflammation in aging implicates the activation of microglia and astrocytes in the brain over this period (34). In aged brains, there is an increase in the number, size and activation of microglia (34). In this study, to investigate the effects of PL on microglia and astrocytes in aging, we measured the percentage area occupied by astrocytes (Fig. 2A and D) and microglia (Fig. 2B and E) in hippocampus through immunohistochemical assay. Additionally, immunofluorescence analysis for oxidative stress (4-HNE, an indicator of lipid peroxidation) in the hippocampus was performed (Fig. 2C and F). PL administration at a dose of 50 mg/kg/day for 8 weeks had no significant effect on glial activation and oxidative stress in the hippocampus at this point in aging.

As the results from the behavioural tests pointed to a reduction in age-related cognitive impairment with PL treatment, we examined the level of synaptic markers in the hippocampus of the aged mice treated with the vehicle or PL. As indicated by the results of western blot analysis, the expression levels of gephyrin, VGAT, GAD65/67, PSD95, VGLUT1, VGLUT2 and synaptophysin were similar between the aged vehicle and aged PL groups (Fig. 3A and B). Additionally, PL had no effect on the protein expression of the AMPA (GluR1) or NMDA (NR2B) receptors (Fig. 3C and D). Of note, the levels of phosphorylation of NR2B (Tyr1472), ERK1/2 (Thr202/Tyr204) and (Thr286) were significantly higher in the aged mice treated with PL than in the aged mice treated with the vehicle (Fig. 3C and D). There was a tendency for the phosphorylation of CREB (Ser133) to be slightly higher in the aged PL group than the aged vehicle group, although this difference was not significant. To further investigate the level of p-CREB in the areas of the hippocampus, we measured the integrated optical density (IOD) of p-CREB by immunohistochemical assay in the CA1, CA3, and DG of the aged vehicle- and aged PL-treated mice (Fig. 4). The IOD in the CA3 was markedly higher in the aged mice treated with PL than in the aged mice treated with the vehicle (Fig. 4A and C, P<0.01); however, the level of p-CREB in the CA1 and DG did not differ significantly between the groups (Fig. 4A, B and D). Taken together, these results suggest that the molecular signalling pathways involving NR2B, CaMKIIα, ERK1/2 and CREB are regulated by PL treatment in the hippocampus of the aged mice.

Neurogenesis markedly declines with aging and, thus, the maintenance of an adequate level of hippocampal neurogenesis is another important factor to consider in maintaining cognitive function (35). In this study, to investigate whether PL treatment affects hippocampal neurogenesis, we examined neuronal proliferation by immunohistochemistry using the neuroblast marker, DCX, in the DG of aged mice treated with the vehicle or PL (Fig. 5). The number of DCX-positive cells was markedly lower in the aged mice treated with the vehicle than in the young control group (Fig. 5). However, the number of DCX-positive cells was significantly higher in the PL treated aged mice than in the vehicle treated aged mice (Fig. 5, P<0.05). These results suggest that PL increases adult neurogenesis in the DG of aged mice.