Anti-c-Jun N-terminal kinase (JNK), MAPK 14 (p38) and extracellular signal-regulated kinase (ERK), phosphorylated (p)-p38, p-JNK, and p-ERK rabbit antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. nos. 9252, 9212, 4695, 9211, 9251 and 9101, respectively). Anti-β-actin rabbit polyclonal antibody was purchased from Beyotime Institute of Biotechnology (Haimen, China; cat. no. AA128). Protease inhibitor cocktail and extraction buffer [radioimmunoprecipitation assay (RIPA) lysis buffer; cat. no. P0013B] were purchased from the Beyotime Institute of Biotechnology. Enhanced chemiluminescence (ECL) reagents were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). LPS was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and NBP was obtained from CSPC Pharmaceutical Co., Ltd. (Shijiazhuang, China).

Adult male C57BL/6 mice (n=30) weighing 18–20 g were purchased from the Comparative Medicine Center at Yangzhou University [Yangzhou, China; cat. no. scxk(Su) 2012-004] and housed under pathogen-free conditions with a 12-h light/dark cycle, 60–70% relative humidity and temperature of 20–22°C, and received food and water ad libitum in their cages. All animals were ~10 weeks of age at the start of administration. Prior to the experiment, the animals were allowed 10 days to adapt to the new environment. All mice were randomly separated into 3 groups (n=10/group) as follows: i) The control group, which was treated with saline; ii) the LPS-treatment group, which received a single intraperitoneal injection of LPS at 5 mg/kg to generate the PD model; and iii) the NBP-treatment group, which received gastric perfusions of NBP (120 mg/kg) dissolved in soybean oil once a day for 30 days, following injection with LPS (similar to the LPS-treatment group). All experiments commenced following 7 months of treatment. The present study was approved by the ethics committee of Bengbu Medical College (Bengbu, China).

The rotarod test is widely used to assess motor and coordination abilities, particularly bradykinesia in mice (14). In the present study, a rotarod (3-cm diameter) was utilized at a fixed speed of 20 rpm. The retention time for each mouse resting on the revolving rod was recorded. The rotarod test was repeated 3 times for each mouse, with 30-min rest periods in between the tests. The retention time of each mouse was determined and used for comparison.

Following treatment, motor behavior was analyzed in an open-field test. The apparatus consisted of a square (40×40 cm) with a surrounding wall (height, 30 cm). The square floor was divided into 16 (4×4) sub-squares using transverse and longitudinal segments. Mice were placed in the center of the structure and then the behavior of the mice was recorded for 5 min. When each mouse repeated the test 3 times, their spontaneous activity was analyzed for 30 min. The line crossings, grooming and rearing of each mouse were recorded and analyzed.

Following treatment, 3 mice were randomly selected from each group for the subsequent study. Mice were deeply anesthetized with 10% chloral hydrate (3 ml/kg; Sigma-Aldrich) at room temperature and perfused through the ventriculus sinister with 100 ml saline, followed by 4% paraformaldehyde for 20 min. The midbrains were collected and fixed with 4% paraformaldehyde at 4°C for 24 h. Subsequently, the midbrains were embedded in paraffin and cut (35-µm-thick) and processed for immunohistochemistry. The sections were blocked with 5% bovine serum albumin (cat. no. P0007; Beyotime Institute of Biotechnology) dissolved in PBS at room temperature for 1 h.

The sections were incubated with primary monoclonal antibodies at 37°C overnight [rabbit anti-tyrosine hydroxylase (TH); 1:1,000; cat. no. 2791; Cell Signaling Technology, Inc.]. Then, the sections were rinsed with PBS, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 50 min (goat anti-rabbit Immunoglobulin G, 1:800 (cat. no. GB23303; Goodbio Technology Co., Ltd., Wuhan, China). Sections were subsequently incubated for 30 min in peroxidase substrate mixing liquid and then washed and detected with a DAB staining kit (cat. no. K5007; Dako; Agilent Technologies, Santa Clara, CA, USA) at room temperature for 3–10 min, terminating the coloration when brown granules (positive cells) were observed.

Finally, the sections were counterstained with hematoxylin at room temperature for 3 min, dehydrated with deionized water, dried and sealed. The immunopositive cells in the stained sections were analyzed under an optical microscope (Nikon Corporation, Tokyo, Japan). The number of TH-positive cells (magnification, ×40) in every sixth section was counted by two researchers blinded to the treatment.

RIPA lysis buffer was used to extract proteins from the ventral mesencephalon. Tissues were lyzed in RIPA buffer supplemented with protease inhibitor cocktail. Then, the lysates were centrifuged (8,000 × g for 10 min at 4°C) and the protein concentration in the extracts was determined using the Bradford assay.

Equal amounts (30 µg) of protein samples were electrophoresed on 12% denaturing polyacrylamide gels and then transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MD, USA) using a semi-dry blotting apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 2 h at 4°C. The membrane was then blocked with 5% bovine serum albumin (cat. no. P0007; Beyotime Institute of Biotechnology) dissolved in TBS containing Tween [TBST; 10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% Tween-20] at room temperature for 1 h. Following washing with TBST buffer 3 times, membranes were incubated with primary antibodies against JNK, ERK, p38, p-JNK, p-ERK and p-p38 (all 1:1,000) at 4°C overnight. Then, the membranes were washed 3 times with TBST for 10 min and incubated with the HRP-conjugated secondary antibodies (cat. no. GB23303; Goodbio Technology Co., Ltd.) at room temperature for 1 h (1:5,000). An anti-β-actin antibody (1:1,000) was used as an internal control.

Band intensities were detected with ECL reagents (cat. no. KGP1123; Nanjing KeyGen Biotech Co., Ltd.) and measured with Quantity one Software version 25.0.0 (Bio-Rad Laboratories, Inc.).

All values were expressed as the mean ± standard deviation. Results were analyzed using one-way analysis of variance followed by Tukey post-hoc tests using SPSS software version 18.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate whether NBP protects mice from LPS-induced neurotoxicity, the behavioral tests were conducted, including the rotarod and the open-field test.

Rotarod test results demonstrated that the retention time of the control group was 298.61±2.94 sec (Fig. 1A). Compared with the control group, the LPS-treatment group (103.78±46.01 sec) had a significantly reduced retention time (P<0.01). However, the NBP-treatment group, who demonstrated marked improvement in retention time (Fig. 1A), had significantly enhanced retention times (221.83±53.88 sec) compared with mice in the group treated with LPS (P<0.01).

As presented in Fig. 1B-D, the open field test, which assessed the degree of locomotory capacity demonstrated that the number of line crossing, rearing and grooming actions in the control group was 205.56±26.82, 27.06±3.59 and 5.50±0.84, respectively. In the LPS-treatment group, the line crossing (96.00±28.65), rearing (3.83±1.87) and grooming (1.44±0.27) activities were decreased significantly (all P<0.01; Fig. 1B-D). In the NBP treatment group, the line crossing (156.67±37.23), rearing (16.78±4.64) and grooming (3.94±1.22) behaviors were significantly increased compared with the LPS-treatment group (all P<0.01; Fig. 1B-D). Therefore, the mice treated with NBP demonstrated a significant improvement in behavioral performance in the open field test compared with the LPS-treatment group.

These results demonstrated that NBP could protect mice from LPS-induced behavioral impairment.

TH-positive cells are an important pathological indicator for assessing whether a drug exhibits protective effects on dopaminergic neurons (15). Immunoblotting was also used to analyze the number of TH-positive cells in the SNpc (Fig. 2). In the LPS-treatment group, the number of TH-positive cells in the SNpc (122.33±30.29) was significantly reduced compared with the control group (242.33±19.14; P<0.01). Compared with the LPS-treatment group, the number of TH-positive cells in the NBP treatment group (191.67±23.12) were significantly increased (P<0.01), indicating that NBP could inhibit the loss of TH.

A previous study demonstrated that the MAPK signaling pathway serves an important role in the pathogenesis of PD (4). To evaluate whether the MAPK pathway is involved in the protective effect exerted by NBP, the alterations in JNK, p38 and ERK in mice treated with LPS and NBP were investigated.

As demonstrated in Fig. 3, the levels of p-JNK, p-p38 and p-ERK in the control group were 0.23±0.08, 0.28±0.08 and 0.99±0.24, respectively. The levels of p-JNK, p-p38 and p-ERK in the LPS-treatment group were 0.56±0.13, 0.52±0.17, and 1.0±0.27, respectively, which indicated phosphorylation of JNK, p38, and ERK, although no significant difference was observed with ERK. The levels of p-JNK, p-p38 and p-ERK in the NBP-treatment group were 0.31±0.09, 0.34±0.11, and 0.85±0.21, respectively. Compared with the LPS-treatment group, treatment with NBP significantly inhibited phosphorylation of JNK and p38 (P<0.05), but had little effect on ERK. Compared with the control group, treatment with NBP had no significant effect on the phosphorylation of JNK and p38 (P>0.05).

Therefore, these results indicated that inhibition of the JNK and p38 pathway is involved in the protective effect of NBP against LPS-induced neurotoxicity.