EA (purity>95%), LPS (Escherichia coli O111:B4) and 6-hydroxydopamine (6-OHDA) were obtained from Sigma Aldrich (St. Lewis, CA, USA). Enzyme-linked Immunosorbant assay (ELISA) kits for TNF-α, IL-1β and IL-18 were bought from Elabscience Biotechnology Co., Ltd (Wuhan, China). MTT assay kit was from Beijing Solarbio science and Technology Co., Ltd. (Beijing, China). The small interfering RNA (siRNA) against NLRP3 was purchased from GenePharma (Shanghai, China). Anti-CR3 complement receptor (OX-42 Catalog No. Ab1211) and tyrosine hydroxylase (TH, Catalog No. Ab113) antibodies were bought from Abcam (Cambridge, MA, USA). Anti-caspase-1 (Catalog No. 22915-1-AP), ionized calcium-binding adapter molecule-1 (Iba-1, Catalog No. 10904-1-AP), TNF-α (Catalog No. 17590-1-AP), IL-1β (Catalog No. 66737-1-Ig), IL-18 (Catalog No. 10663-1-AP), β-actin (Catalog. No. 20536-1-AP), rabbit IgG (Catalog No. SA00001-2) and mouse IgG (Catalog No. SA00001-1) antibodies were purchased from Proteintech Group (Chicago, IL, USA). Anti-NLRP3 (Catalog No. orb101128) antibody was purchased from Biorbyt (Cambridge, United Kingdom).
Animal and Treatment
Male Wistar rats (200–250 g, 8–10 weeks old) were bought from the Experimental Animal Center in the Third Military Medical University. All experimental procedures were carried out in accordance with Chinese Guidelines of Animal Care and Welfare and this study received an approval from the Animal Care and Use Committee of Zunyi Medical University (Zunyi, China). Rats were acclimated to their environment for 1 week before the experiments. All the animals were randomly allocated to five experimental groups with six rats in each group: control, EA alone (50 mg/kg), LPS, LPS+EA (10 mg/kg) and LPS+EA (50 mg/kg). With anesthetized by 7% chloral hydrate (0.5 ml/100 g, v/w), rats received a single LPS (10 µg in 5 µl PBS) unilateral injection into the SN pars compacts followed by the coordinates 5.2 mm posterior to bregma, 1.9 mm lateral to the midline, and 8.0 mm ventral to the surface of the skull . Daily intragastric injection of EA (10 and 50 mg/kg) was administrated for 7 consecutive days after LPS injection. Control animals were injected with a single PBS into SN pars compacts. Afterwards, animals were sacrificed and the biochemical analysis was performed.
Rotarod test was performed to study the muscular coordination. It contained cylindrical arrangement of the thin steel rods with two parts by compartmentalization to permit the detection of two rats at the same time. Prior to the start of the test, rats were allowed to remain stationary at 0 rpm. Then, the rotational speed was steadily increased to 10 rpm in 30 s interval till rats fell off the rungs. The duration time each rat stayed on the rod was recorded and calculated for analyzing the behavior changes of rats .
Immunohistochemical Analysis and Cell Counting in the SN
Rat brains were cut with a horizontal sliding microtome into 35 μm transverse free-floating sections and immunostained with the corresponding antibodies. Briefly, brain slices were incubated with 0.3% Triton X-100 and blocked with goat serum. Subsequently, brain slices were incubated with anti-TH (1:500), OX-42 (1:800), and NLRP3 (1:800) antibodies at 4 °C overnight, respectively . Digital images of TH-positive neurons, OX-42-positive microglia and NLRP3-positive inflammasome in midbrain SN were acquired by an Olympus microscope (Olympus, Tokyo, Japan). Quantification of TH-positive neurons was determined through visually counting the number of TH-positive neuronal cell bodies blindly by two investigators and results were analyzed from the average. The mean value for SN TH-positive neuronal numbers was then deduced by averaging the counts of 6 sections for each rat. Representative fluorescence images of OX-42 and NLRP3 inflammasome were obtained and the fluorescence intensity was calculated using ipwin32 software.
Cell Culture and Treatment
Mouse microglial BV-2 cell lines were obtained from China Center for Type Culture Collection (Wuhan, China). Cultures were maintained in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in the humidified atmosphere of 5% CO2 and 95% air . DA neuron MN9D cell lines were purchased from the Cell Culture Center in the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China). MN9D cells were cultured in DMEM medium with 10% FBS and 1% penicillin-streptomycin on an atmosphere with 5% CO2 at 37 °C in the humidified atmosphere of 5% CO2 and 95% air .
Cell viability was evaluated by MTT assay. BV-2 and MN9D cells were cultured in 1×105/well in 96-well plates for 24 h. Afterwards, cells were treated with different concentrations of EA for 30 min followed by LPS (100 ng/ml) or 6-OHDA (100 μM) treatment for another 24 h and then incubated with MTT solution (5 g/l) for 4 h. Formazan crystals in the cells were solubilized using 200 μl dimethyl sulfoxide (DMSO) and the absorbance was detected by an automated microplate reader within 490 nm wavelength .
Western Blot Analysis
Total protein content was extracted from rat midbrain tissue and BV-2 cells using a lysis buffer containing protease inhibitors. Protein levels were quantified by BCA assay . Protein (10 μg) from each sample was subjected to SDS-PAGE gel under the reduced conditions. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 2 h at room temperature and then incubated overnight at 4 °C with the primary antibodies: Iba-1 (1:800), NLRP3 (1:800), caspase-1 (1:800), TNF-α (1:1000)，IL-1β (1:500), IL-18 (1:800) and β-actin (1:2000). Next, the membranes were incubated for 1 h with a horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG at 1:2000 dilution. The blot films were developed with enhanced ECL Reagent.
The levels of TNF-α, IL-1β, and IL-18 were measured by ELISA according to the manufacturer’s instructions. The microplate reader was used to measure the absorbance at 450 nm .
Activated microglia were identified with an anti-OX-42 antibody. Cells were fixed with paraformaldehyde (4%) for 30 min. Later, cells were permeabilized using triton X-100 (0.3%) for 15 min. Then, cells were blocked using goat serum blocking solution for 60 min at 37 °C. Thereafter, cells were incubated with 1:800 dilution of anti-OX-42 antibody overnight at 4 °C. Following overnight incubation, cells were incubated in dark for 30 min with goat anti-rabbit secondary antibody (1:1000) or goat anti-mouse secondary antibody (1:1000). Cells were also counterstained with DAPI for 5 min . After rinsing cells with PBS, representative fluorescence images were obtained using EVOS® Floid® Cell imaging station. The fluorescence intensity was calculated by using ipwin32 software.
BV2 cells were cultured and seeded in a 6-well plate at a density of 1 × 105 cells/ml. The transfection of siRNA was performed complying with the manufacturer's protocol. Briefly, NLRP3 siRNA (2 μl) was diluted into 18 μl of transfection medium. GP-siRNA-Mate plus (180 μl) was used to transfect with siRNAs dilution (20 μl), then made up 2 ml with MEM medium. After 6 h of transfection, the transfection solution was removed. Cells were rinsed with PBS and replaced with MEM medium containing 2% FBS. Then, cells were treated with EA for 30 min followed by LPS (100 ng/ml) stimulation for another 24 h . The gene sequences were as follows: Sense 5’-UUC UCC GAA CGU GUC ACG UTT-3; antisense 5’-ACG UGA CAC GUU CGG AGA ATT-3’.
Results were indicated as mean ± standard error of the mean (SEM). Statistical significance was analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Upon ANOVA demonstrating the significant differences, pairwise comparison between means was evaluated by Bonferroni's post hoc test with correction. A value of p<0.05 was considered statistically significant.