Male C57BL/6 mice weighing 18–20 g were obtained from JXJ Experimental Animal Co., Ltd., Shanghai, China (2010002601739) and kept in a room maintained on a 12 h light/dark cycle and temperature of 20–22°C with food and water available ad libitum. To minimize discomfort and pain for the animals, experimental procedures were carried out in accordance with the European Community's Council Directive of 24 November 1986 (86/609/EEC). The mice were randomly divided into 3 groups (n=9): normal saline-treated controls (NS), piperine-treated 10 mg/kg body weight MPTP-induced group (P+M), and the group treated with MPTP alone (MPTP). Piperine was dissolved in 5% carboxymethylcellulose sodium solution. The mice were treated with piperine (10 mg/kg; Selleck Chemicals, Houston, TX, USA) by daily intragastric administration for 15 days. For the MPTP treatment, the mice received intraperitoneal injection of MPTP hydrochloride (30 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) dissolved in normal saline once daily for 7 successive days, starting the 8th day of piperine treatment. After the behavioral testing was conducted, the animals were sacrificed and their brains were dissected and prepared for immunohistochemical staining (4 mice from each group were sacrificed by cervical dislocation) or western blot analysis (the remaining mice were sacrificed by perfused fixation) (for experimental schedules, see Fig. 1).

To measure motor coordination, the mice were assessed on the rotarod apparatus (27). Prior to the test session, each mouse received 30 min daily training for two successive days (speed 0.17 × g). The rotational speed was increased to 0.21 × g on the third day. Each mouse was placed in a separate lane of the rotarod (3 cm in diameter) and the time they remained on the rotating bar was recorded. The maximum time was 6 min/trial. Each mouse was given three trials on the rotating bar, and the average retention time for each mouse was used for comparison. The examiner conducting the rotarod test was blind to the treatment.

For the MWM test, a stainless steel cylindrical tank (120 cm in diameter) surrounded by a wall 40 cm high and filled with homothermal water (22°C) was used. A plastic platform, 8 cm in diameter, was submerged 1.5 cm below the water surface with its base fixed to the floor of the tank. Four large unique navigation markers were placed above the edge of each quadrant of the tank as geographical cues prior to releasing the animals into the water.

In the hidden platform acquisition test, on each of four consecutive days mice underwent four swimming sessions. For each session, the mice were placed facing the wall of the pool and released from a starting point pseudo-randomly chosen from the four predetermined positions into the water. The time mice spent to reach the platform was recorded as the escape latency. If any mouse failed to reach the platform within 60 sec, they were guided and placed on the platform for 20 sec by the experimenter and the escape latency was recorded as 60 sec. The platform location remained constant throughout the test. For a particular day for an individual mouse, the average time spent over the four sessions was utilized as the latency score. The 4 day averages were then measured for each group to evaluate spatial learning ability.

On the fifth day, an additional 1 min session occurred with the platform removed (probe session). The mice were placed in the diagonal quadrant of the hidden platform originally located. Site crossings (the number of times animals crossed the original platform location) were recorded and used to indicate the degree of memory maintenance.

The results were analyzed with the analysis system of Morris water Maze (Huaibei Zhenghua Biological Equipment, Anhui, China). Two series of MWM tests were conducted by two professional technicians who were blind to the treatments.

Brain tissue was prepared for immunohistochemical staining. Briefly, the mice were perfused under chloral hydrate anesthesia through the ventriculus sinister with normal saline, followed by 4% ice-cold paraformaldehyde for ~20 min. After antigen retrieval and phosphate-buffered saline (PBS) rinse, the brain sections were incubated at 37°C overnight with rabbit monoclonal anti-tyrosine hydroxylase (TH) (1:600), rabbit anti-IL-1β (1:200) or rabbit anti-Iba-1 (1:200) (all from Abcam, Qatar, Kingdom of Saudi Arabia). The following day, the brain sections were again rinsed with PBS and incubated with the appropriate biotinylated secondary antibody, goat anti-mouse/rabbit HRP-labeled (K5007; Dako, Glostrup, Denmark) for 50 min. The sections were then washed and stained with a DAB staining kit (Dako). After Harris hematoxylin staining and dehydration, the stained sections were mounted and analyzed under an optical microscope (Nikon, Tokyo, Japan).

Activity of the lipid peroxidation product MDA was measured by using a thiobarbituric acid reactive substances assay kit (Jiancheng Bioengineering, Nanjing, China) according to manufacturer's instructions. The assay was performed using a homogenate of midbrain tissues in physiological saline according to the given protocol. The supernatant was prepared by centrifugation at 9,184 × g for 10 min at 4°C (Smart R17; Hanil Science Inc., Incheon, Korea). Absorbance at 532 nm was measured using a microplate reader (Synergy HT; BioTek, Winooski, VT, USA). The MDA content was calculated according to the equation in the protocol and expressed in nanomoles per milligram protein.

The aforementioned midbrain tissue homogenates were also used to study SOD activity. Total SOD activity was determined spectrophotometrically using a SOD assay kit (Jiancheng Bioengineering) and calculated according to the manufacturer's instructions. In this study, a SOD unit was defined as the amount that reduced the absorbance at 550 nm by 50%. The result was expressed as SOD units per milligram protein.

Brain tissues were prepared as previously described (28–30). Briefly, the midbrain tissues were homogenized overnight in an ice-cold lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). Homogenates were centrifuged for 30 min at 13,225 × g at 4°C (Smart R17; Hanil Science Inc.) and the resulting supernatant contained the cytosolic fraction. Protein concentration was measured with a BCA Protein assay kit (Biosharp, Shanghai, China). After boiling the samples at 95°C for 5 min, 35 µg of protein was loaded in each lane with a gel loading buffer containing 10% glycerol, 62.5 mM Tris-HCl, pH 6.8, 50 mM dithiothreitol, 2% sodium dodecyl sulfate (SDS) and 0.1% (w/v) bromophenol blue. Western blot analysis was conducted as described by Burnette (31), with little modification. Proteins were resolved using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% acrylamide resolving gel for IL-1β, Bcl-2 and Bax, and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in TNE containing 5% skim dry milk in Tris-buffered saline and 0.05% Tween-20 detergent for 4 h. The primary antibodies were diluted in blocking buffer. Anti-IL-1β (1:500; rabbit monoclonal), anti-Bax (1:500; mouse monoclonal), and anti-Bcl-2 (1:500; mouse polyclonal) (all from Abcam) were used. The nitrocellulose membranes were then incubated overnight with primary antibodies at 4°C. After washing three times, the membranes were incubated with secondary antibodies, goat-anti-mouse-HRP-labeled (1:1,000; Boster Biongineering, Wuhan, China) for 2 h at room temperature. The color was revealed by recovering DAB (50 mg/100 ml + 30% H₂O₂) reagent on the membranes. Conserved protein (β-actin) was measured to assay equal loading of protein. The pixels were measured using the Chemidoc XRS imaging system (Universal Hood II; Bio-Rad Laboratories, Hercules, CA, USA).

Results are presented as mean ± standard deviation. The comparisons among groups were evaluated by one-way ANOVA, followed by LSD tests using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered statistically significant.

The aim of the antioxidant therapy in PD is to decrease functional impairments (32). Thus, in the present study, the rotarod was used to directly assess motor coordination. As expected, MPTP treatment significantly decreased the latency to fall off the rotating rod relative to NS mice, an effect that was significantly prevented by pretreatment with piperine (10 mg/kg) (Fig. 2A).

Various cognitive impairments including deficits in learning and memory are common clinical symptoms of PD, so the MWM was used to assess learning and recall. The mean escape latency decreased gradually over repeated days in all the groups (Table I and Fig. 3), and the mean escape latencies of the NS and piperine groups were significantly shorter compared to the MPTP group. These results indicated that MPTP impairs learning ability in the MWM test and piperine pretreatment was able to protect against this impairment. In the probe trial, the MPTP group showed decreased site crossings (P<0.05), suggesting that piperine is capable of improving the spatial memory ability of PD mice in the MWM test (Table I).

Brain sections were immunostained for TH, a marker for DA neurons. The result was expressed as the number of TH-positive neurons in the SNpc. TH-positive cells were significantly decreased in the SNpc after MPTP administration relative to the NS group (P<0.05), demonstrating that MPTP induced dopaminergic neuronal toxicity. However, piperine treatment (10 mg/kg) clearly protected against MPTP-induced dopaminergic neuronal death in the SNpc (Fig. 4).

Previous findings have shown that MPTP treatment induces degeneration of DA neurons due to the induction of pro-inflammatory cytokine secretion by activated microglia (33,34). Therefore, we examined the expression of IL-1β, a representative pro-inflammatory cytokine, and Iba-1 as a marker of activated microglia. Immunohistochemical data (Fig. 5B) and western blot analysis (Fig. 5C and D) demonstrated that piperine significantly suppressed the expression of IL-1β in the SNpc of MPTP-treated brains. The Iba-1-positive cells showed that activated microglia were apparently present in the SNpc of the MPTP group. The number and morphological phenotype of activated microglia were obviously alleviated following piperine pretreatment (Fig. 5A).

MDA is a marker of lipid peroxidation and oxidative stress. The level of lipid peroxidation indicated by MDA was significantly increased in the midbrain of MPTP-treated mice compared to the NS group (Fig. 3). The increase was significantly prevented by piperine pretreatment (Fig. 2B). Similarly, treatment with piperine markedly increased SOD activity in the mouse midbrain samples while MPTP only induced modest increases compared to NS-treated mice (Fig. 2C). These results suggested that piperine attenuated the oxidative stress induced by MPTP.

To confirm the anti-apoptotic potential of piperine, we evaluated the expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 in the midbrain tissue. The balance of pro-apoptotic and anti-apoptotic signals from the Bcl-2 family can be influenced by members of the caspase family, which can determine whether neurons undergo apoptosis. Following MPTP treatment, Bcl-2 expression was reduced in the midbrain compared to the NS group, while piperine pretreatment prevented this decrease (Fig. 6A and B). Similarly, MPTP treatment alone increased Bax expression relative to the NS group, an effect significantly attenuated by pretreatment with piperine (Fig. 6A and C). The ratio of the anti-apoptotic/pro-apoptotic proteins demonstrated that piperine significantly attenuated MPTP-induced apoptosis by a reduced expression of pro-apoptotic proteins (Fig. 6D). These results suggested that piperine had an anti-apoptotic effect on MPTP-induced neuronal cell death.