PLA extract and reagents
Piper longum was purchased from Anguo, Hebei province, China, in 2014 and identified by Rong Luo, associate professor, School of Traditional Chinese Medicine, Capital Medical University. The voucher specimens of this material have been deposited in School of Traditional Chinese Medicine, Capital Medical University.
The PLA extract was made as our previous work described [15]. The content of total alkaloids was 74.6 % determined by UV, meanwhile the contents of piperine and piperlonguminine were 53.08 and 1.73 % respectively determined by HPLC. PLA was analyzed in a previous study carried out by our laboratory. Refer to this work for information about detailed compositions and chromatogram of PLA [15].
Lipopolysaccharide (LPS, from Escherichia coli. serotype O26:B6), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), mouse anti-tyrosine hydroxylase antibody, TritonX-100 and apomorphine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-Iba-1 antibody was purchased from BOSTER (Wuhan, China). Interleukin-1β (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and reactive oxygen species (ROS) enzyme-linked immunosorbent assay (ELISA) kits were purchased from MultiScience Biotech Co., Ltd (Hangzhou, China). Nitric oxide (NO) kit was purchased from NanjingJiancheng Bioengineering Institute (Jiangsu, China).
Animals and surgery
Eighty adult male Sprague-Dawley rats (weight 260–300 g) were purchased from the Beijing Vital River Lab Animal Technology Co. Ltd. (Beijing, China) and maintained under standard conditions with a standard 12-h on/off light cycle, with food and water supplied ad libitum. After allowed to acclimate to their new surroundings for 1 week before experimental surgery, the rats were injected 2.0 μL LPS dissolved (5 mg/mL) in phosphate-buffed saline (PBS) into the right SNpc following a previous described protocol [18]. The injection position was anteroposterior −5.3 mm, lateral 2.0 mm and dorsoventral 7.8 mm from bregma. Sham-operated animals were injected 2 μL PBS into the right SNpc. Our reasearch had acquired the ethics approval by Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University and all experimental procedures were approved by the Committee. The ethics approval number is AEEI-2014-081.
Experiment design
The rats were randomly divided into five groups: the sham-operated group (n = 16), the LPS-injected group followed by vehicle treatment (model group, n = 16), the LPS-injected group followed by treatment with 25, 50 and 75 mg/kg PLA, respectively (n = 16 each group). Rats in three PLA treatment groups were intragastrically administered with PLA (dissolved in 0.5 % sodium carboxymethylcellulose) once a day after the surgery for 6 weeks. The sham-operated and model groups received 0.5 % sodium carboxymethylcellulose.
Rotational behavior assay
On the second day after treatment with PLA for 3 and 6 weeks, the rats were injected hypodermically with 0.5 mg/kg apomorphine dissolved in physiological saline to examine the rotational behavior. The number of turns performed over 30-min testing period was counted.
Rotarod test
All rats underwent a 3-day training program on a rotarod before the LPS injection, by which time a steady baseline level of rotarod performance was attained. Briefly, the rats were placed on the rod and sequentially tested at the speed accelerated from 0 to 40 rpm within 2 min. The time latency to fall from the rotarod at each speed level was recorded. At 3th and 6th week after treatment with PLA the rats were tested respectively.
Open-field test
Rats in each group were tested by the Tru Scan activity monitoring system (Coulbourn Instruments), which contains acoustic insulation and lucifugal field (60 cm × 60 cm × 65 cm). Infrared device was installed at the top of the box, which was used to accurately track the movement and the behavior. After the end of each test, 75 % ethanol was used to thoroughly clean the open-field apparatus. The rat movement was recorded for the following parameters: total movement distance (cm), total movement time (s), total rest time (s) and horizontal velocity (cm/s). Each test time was 30 min.
Preparation of tissue samples
Four rats from each group were randomly selected for morphological studies on the second day after the final behavioral tests. Decapitating all other rats, then the bilateral substantia nigra (SN) and striatum were rapidly dissected and stored at −80 °C. The SN was used for the quantification of proinflammatory cytokines, and the striatum for determination of the content of DA. For the morphological studies, rats were deeply anesthetized with chloral hydrate, then transcardially perfused with 200 mL saline followed by 200 mL of 4 % paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and post-fixed in the same fixative and then immersed in a 20 % sucrose solution and a 30 % sucrose solution. Coronal section were cut on a freezing microtome (Leica, Germany) at a thickness of 40 μm and used for immunohistochemistry as described below.
High performance liquid chromatography (HPLC)
The determination of DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) was carried out using HPLC with a Coul Array electrochemical detector (Model 5600A, ESA, USA) equipped with Waters symmetry shield RP 18 column (150 × 3.9 mm, 5 μm). The mobile phase consisted of 50 mM sodium citrate, 8 % methanol, 0.1 mM EDTA · 2Na, 0.2 mM 1-octanesulfonic acid sodium salt and was finally adjusted to pH 4.1. The flow rate was 0.8 mL/min. Striatum tissues from 6 animals of each group were used and performed as described in previous work [19].
Immunohistochemical staining of TH and Iba-1
Eight sections were selected for immunohistochemical staining of the tyrosine hydroxylase (TH). The mouse anti-TH antibody was diluted at 1:2000. Adjacent sections were used for detection of microglial marker Iba-1. The rabbit anti-Iba-1 antibody was diluted at 1:200. Sections were perforated with 0.3 % Triton-X 100 and blocked with normal horse serum (1:100 dilution), then were incubated with primary antibodies for 24 h at 4 °C. After that, sections were incubated with biotinylated anti-mouse antibody and biotinylated anti-rabbit antibody (Vector laboratories, Burlingame, CA, USA) respectively for 30 min at 37 °C, followed by avidin-biotin-peroxidase (Vector laboratories, Burlingame, CA, USA) incubation for 30 min at 37 °C. Finally, the immune complex was detected by 3, 3’ - diaminobenzidine (DAB).
To measure the numbers of TH-ir cells in the SN, stereological cell counting was performed. The optical fractionator method on a Stereo Investigator system (Micro Bright Field, USA) was used to count the total numbers of TH-ir neurons in the SN and a Leica microscope was used. The survival rate of TH-ir neurons in the SN was determined by counting the number of TH-ir neurons on LPS-injected side relative to the number of TH-ir neurons on the non-injected side. Quantitative analysis of Iba-1-stained immunohistochemical images were carried out with an Image-Pro Plus 6.0 system and positive results were expressed as average optical density value. All sections were coded and examined blindly.
IL-6, IL-1β and TNF-α immunoassay
The right SN was used to detect the proinflammatory cytokines. Tissues were made into 10 % homogenate and then the homogenate was centrifuged at 3000 g for 15 min at 4 °C. The supernatant was collected at 4 °C. IL-6, IL-1β and TNF-α were detected using the commercial enzyme-linked immunosorbent assay (ELISA) kits (MultiScience Biotech, Hangzhou, China). All experimental procedures were performed according to the manufacturer’s instructions. Besides, we used the BCA protein kit to measure protein contents in samples according to the manual. The SN tissues from 6 animals of each group were used.
Measurement of ROS and NO
The SN was also used to measure ROS and NO and tissues were made into 10 % homogenate as above. ROS was measured using enzyme-linked immunosorbent assay (ELISA) to measure absorbance value at 450 nm with ELISA kit. The content of NO was measured using NO assay kit according to the manufacturer’s guidelines by measuring the absorbance value at wavelength of 550 nm. BCA protein kit was used to measure the protein contents in samples according to the manual. The SN tissues from 6 animals of each group were used.
Statistical analysis
Data were processed by commercially available software GraphPad Prism 5.0. Results are typically presented as means ± S.E.M. Statistical significance was assessed using a one-way analysis of variance (ANOVA), followed by Dunnett post hoc test (compares all the other columns with the designated control column). Significance was set at P < 0.05.
