Drugs and reagents
DS was synthesized and gastrodin was extracted and purified by the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, and the purity was validated as > 95% using high-performance liquid chromatography. Stock solution of both reagents were prepared in dimethyl sulfoxide (DMSO) and subsequently diluted in sterile 0.9% saline. The cell culture reagents were purchased from Invitrogen Corporation (Thermo Fisher Scientific, Carlsbad, CA, USA). Antibodies for Western blotting and immunofluorescence were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Alexa 546-conjugated goat anti-rabbit and Alexa 488-conjugated goat anti-mouse secondary antibodies were from Life Technologies (Thermo Fisher Scientific, Carlsbad, CA, USA). Goat serum, Mito-Tracker Green, Lyso-Tracker Red, and Rh123 were purchased from Beyotime Biotechnology (Shanghai, China). Nigericin, 3-methyladenine (3-MA), and MCC950 were purchased from MedChemExpress (Monmouth Junction, NJ); lipopolysaccharide (LPS) and tritonX-100 from Sigma-Aldrich (St. Louis, MO); and recombinant IL-1Ra from PeproTech (Rocky Hill, NJ, USA). CellROX® Deep Red Reagent was purchased from Invitrogen (Carlsbad, CA, USA), annexin-V/7aad staining kit from BD Biosciences (Franklin Lakes, NJ, USA), and FAM-FLICA® caspase assay kit from Immunochemistry Technologies (Bloomington, MN, USA).
Experimental animals
Male ICR mice and C57BL/6 mice (weighing 18–20 g) were obtained from Beijing Huafukang Experimental Animal Institute (Beijing, China). The mice were housed 5–6 per cage at room temperature (22 ± 2 °C) in specific pathogen-free conditions under a 12/12-h reversed light-dark cycle, with food and water provided ad libitum. The mice were acclimatized for 3–4 days prior to the experiments, and randomly divided into the different groups. All procedures were approved by the Experimental Animal Care and Use Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College.
Establishment of somatic pain model and behavioral assessment
Acetic acid-induced somatic pain model was established to preliminarily evaluate the analgesic effect of DS. The ICR mice were given a single intraperitoneal injection of DS (1, 3, or 10 mg/kg), normal saline (10 mL/kg), or gastrodin (positive control; 80 mg/kg), followed by 1% acetic acid (10 mL/kg, i.p.) 30 min later. The numbers of writhing and stretching in mice were counted over a period of 15 min after acetic acid injection.
Establishment of neuropathic pain model and behavioral analysis
Chronic neuropathic pain following peripheral nerve injury was simulated via chronic constrictive injury (CCI) of the unilateral sciatic nerve. Briefly, the C57BL/6 mice were anesthetized with isoflurane, and randomly divided into the sham-operated, untreated CCI model, DS-treated (1, 3, and 10 mg/kg) and gastrodin (80 mg/kg) groups (n = 8 mice per group). The left sciatic nerve trunk was exposed by blunt dissection at mid-thigh level, and 4 ligatures (4-0 chromic catgut) were tied loosely around the nerve with 1 mm spacing. In the sham-operated mice, the sciatic nerve was only exposed but not ligated. On the ninth day after surgery, the mice were given a single intrathecal injection of the suitable dose of DS or saline. Briefly, the mice were anesthetized with isoflurane (4% for induction and 1% for maintenance), and a 100 μL micro-injector was inserted from the intervertebral space between the L5 and L6 discs into the spinal subarachnoid space. After confirming proper intrathecal injection via tail flicking, 100 μL normal saline or drug was microinjected followed by a 100 μL normal saline flush.
The sensitivity of mechanical nociception was measured by the Von Frey withdrawal test (Von Frey filaments, IITC Life Science Inc, California, USA) after 30 min, 1 h, and 2 h of intrathecal injection over a period of 4 days. The animals were acclimatized in boxes set on an elevated metal mesh floor for at least 30 min. Pressure values were pressed vertically on the sole of the hind paws with increasing force till the animal withdraw the hind limb. The procedure was repeated 3 times for an average data, and the value of paw withdrawal threshold was recorded. All behavioral analyses were performed by an investigator blinded to the experimental grouping.
The half-effective dose (ED50) of DS was determined by Von Frey withdrawal test on CCI-induced neuropathic pain model mice. Model mice were treated with a serious dose of DS (1, 3, 10, 30, 100 mg/mg) to test the paw withdrawal threshold at 0.5 h, 1 h, and 2 h after administration. The values of peak time of paw withdrawal threshold were selected to calculate the ED50 of DS on CCI-induced model mice.
BV-2 cells culture and treatment
BV-2 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2 and 95% humidity. The cells were pre-stimulated with LPS (100 ng/mL) for 3.5 h, and then with 5 μM nigericin for 30 min, 6 h, 12 h, and 24 h as appropriate. In addition, the cells were treated with varying concentrations of DS (0.3, 3, and 30 μM) or 3-MA (5 mM) for 1 h and 30 min respectively prior to nigericin.
Extraction and separation of cell supernatant protein
Cell supernatant protein was extracted by the standard methanol/chloroform method. The cell supernatants were layered with 1/4 volume of chloroform, and the same volume of methanol was added. After evenly mixing the solutions on a vortex shaker, the mixture was centrifuged at 12,000 rpm for 5 min at room temperature. The upper aqueous phase (methanol) was carefully removed, and the intermediate protein layer was aspirated and extracted once with methanol as described above.
Western blotting analysis
Proteins were quantified by BCA Protein Assay Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Equal amount of proteins per sample were separated by 8% and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the bands were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp., Bedford, MA, USA). After blocking with 5% bovine serum albumin (BSA) in tris-based saline-Tween 20 (TBST) at room temperature for 1 h, the blots were incubated overnight with primary antibodies against IBA-1, NLRP3, pro-caspase-1, caspase-1, pro-IL-1β, IL-1β, LC3II/I, Beclin 1, protein 62 (p62), and β-tubulin (all diluted 1:1000) at 4 °C. Following incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies, the blots were developed using enhanced chemiluminescence reagents (Perkinelmer, USA). The positive bands were visualized with Tanon 2000 Imaging System (Beijing, China) and their intensities were quantified using ImageJ Software (NIH, USA).
Enzyme-linked immunosorbent assay
The level of IL-1β in the supernatant from cultured BV-2 cells was measured by enzyme-linked immunosorbent assay (ELISA) kit (Cat. No. 432606, BioLegend) according to the manufacturer’s instructions. The optical density (OD) at 450 nm was obtained by an ELISA plate reader (Synergy H1, BioTek, USA).
Immunofluorescence
BV-2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.2% triton X-100 in phosphate buffered saline (PBS) containing 10% goat serum for 30 min. The cells were then incubated overnight with anti-NLRP3 (1:200) and anti-ASC (1:200) primary antibodies at 4 °C, washed with PBS, and probed with donkey anti-goat Alexa Fluor 488- and goat anti-rabbit Alexa Fluor 546-conjugated secondary antibodies (1:200) for 1 h at 37 °C. The nuclei were counterstained with DAPI (1 μg/mL), washed thrice with PBS, and observed under the Leica TCS SP8 confocal microscope (Leica Microsystems GmbH, Mannheim, Germany). Spinal cord tissues from the CCI mice were also stained with anti-NLRP3 and anti-IBA-1 antibodies as described above.
Mitochondrial and lysosomal imaging
BV-2 cells were seeded in laser confocal plate (Nest, Jiangsu, China), and incubated with 75 nM Lyso-Tracker Red and 150 nM Mito-Tracker Green at 37 °C for 1 h. The nuclei were counterstained with DAPI (1 μg/mL), washed thrice with PBS, and observed under the Leica TCS SP8 confocal microscope.
Mitochondrial membrane potential and reactive oxygen species measurement
Mitochondrial membrane potential (MMP) was measured using the cationic fluorescent probe Rh123 which rapidly translocated from the mitochondrial membrane to the matrix following membrane depolarization. The reactive oxygen species (ROS) levels were detected using CellROX Deep Red, which emits a strong fluorescence signal under oxidizing conditions. The suitably treated cells were incubated with 10 μM Rh123 or 5 μM CellROX Deep Red at room temperature for 30 min, and 1 μg/mL DAPI was added 10 min before the end of staining. After washing with PBS, the cells were observed under the Leica TCS SP8 confocal microscope to determine the fluorescence intensities of the respective probes.
Flow cytometry
BV-2 cells were cultured and prepared to detect by flow cytometric staining with propidium iodide (PI)-PerCP and caspase-1-FITC (FAM-YVAD-FMK, Immunochemistry Technologies) for NLRP3 inflammasome activation; PI-PerCP and caspase-3/7-FITC (FAM-DEVD-FMK, Immunochemistry Technologies), and 7aad-PerCP and Annexin-V-APC for apoptosis (BD Biosciences). Briefly, the cultured cells at 37 °C were stimulated by LPS (100 ng/mL) for 3.5 h, incubation with or without 3-MA (5 mM) for another 30 min, and then incubation with DS (0.3, 3, and 30 μM for apoptosis measurement; 30 μM for NLRP3 inflammasome activation measurement) for 1 h. After stimulating with nigericin (5 μM), the harvested cells were washed twice with ice-cold PBS, and stained with 7aad or PI for 5 min at room temperature, Annexin-V for 15 min at room temperature, or FAM-YVAD-FMK (caspase-1) and FAM-DEVD-FMK (caspase-3/7) for 1 h at 37 °C, respectively. The samples were acquired in a FACS BD verse cytometer (BD Biosciences), and data were analyzed using FlowJo software (Version 10.0; Three Star).
Statistical analysis
Data were presented as the mean ± SEM of at least 3 independent experiments. The paw withdrawal threshold (PWT (g)) was measured with Von Frey test and compared by two-factor analysis of variance (ANOVA) followed by Tukey’s post hoc test. The percentage of analgesia was calculated using the formula: (post-DS threshold in ipsilateral paw − baseline threshold in ipsilateral paw) / (baseline threshold in contralateral paw − baseline threshold in ipsilateral paw) × 100. Analysis of ED50 was performed with GraphPad best projected by the non-linear least-squares method in which data is normalized according to the maximal possible effect. The other parameters were analyzed using one-way ANOVA followed by an appropriate post hoc test, and P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 8.01 software (GraphPad Software Inc., CA, USA).