Adult CD-1 mice (18–22 g) were purchased from the Experimental Animal Center at Nanjing Medical University, Nanjing, China. Five to six mice per cage were housed under pathogen-free conditions with soft bedding under controlled temperature (22 ± 2 °C) and a 12-h light/dark cycle (lights on at 8:00 a.m.). For each group of experiments, the animals were matched by age and body weight. Behavioral testing was performed during the light cycle (between 9:00 a.m. and 5:00 p.m.). Mice were allowed to acclimate to these conditions for at least 2 days before inclusion in experiments.
All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) unless stated otherwise. IL-1β was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ionized calcium binding adapter molecule 1 (IBA-1) were from Sigma-Aldrich (St. Louis, MO, USA) and Wako Pure Chemical Industries (Osaka, Japan). The p65/RelA and immunofluorescence IBA-1 antibodies were from Abcam (Cambridge, MA, USA). Immunofluorescence c-fos and calcitonin gene-related peptide (CGRP) antibodies were from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory, Northeast Pharmaceutical Group Company (Shenyang, China). Fetal bovine serum (FBS) and other cell culture media and supplements were purchased from Hyclone (Logan, UT, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sunshine Biotechnology (Nanjing, China).
Cell preparation and stimulation
BV-2 cells mouse brain endothelial cells bEND3 were cultured in humidified 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, penicillin (100 U/ml), and streptomycin (100 U/ml) (KeyGEN). For inducing inflammasome activation, 105 cells were plated in 6-well plate overnight, and the medium were changed to serum-free medium next morning and then the cells were treated with morphine (200 μM) with or without metformin for 6 h. Metformin (4, 20, or 100 μM) was administrated 15 min before morphine treatment. Cell extracts and precipitated supernatants were analyzed by immunoblotting.
Cell viability assessment
The cell viability was evaluated by CCK-8 assay (Dojindo Molecular Technologies, Inc.). BV-2 cells were plated in the 96-well plates (2.0 × 104 cell per well) and incubated for 24 h before experiments. The cells were washed with D-Hanks buffer solution. Two hundred microliters of CCK-8 solution was added to each well and incubated for an additional 1 h at 37 °C. The optical density (OD) of each well at 450 nm was recorded on a Microplate Reader (Thermo, Varioskan Flash). The cell viability (% of control) is expressed as the percentage of (ODtest − ODblank) / (ODcontrol − ODblank), where ODcontrol is the optical density of the control sample and ODblank is the optical density of the wells without BV-2 cells.
Tolerance models and behavioral analysis
Animals was habituated in the testing environments for 2 days and carried out behavioral testing in a blinded manner. For the test of chronic tolerance, mice were injected with saline or morphine (10 mg/kg) subcutaneously every 12 h for 7 days and analgesia was assessed 30 min later by the tail-flick assay . The test was performed by gently holding the mouse in a terry cloth towel and immersing between 2 and 3 cm from the tip of the tail into warm water (52 °C). A cutoff time of 10 s was set to avoid tissue damage. Data were calculated as percentage of maximal possible effect (% MPE), which was calculated by the following formula: 100% × [(Drug response time − Basal response time) / (10 s − Basal response time)] = % MPE. The experimenters were blinded to the treatment. Metformin (50, 100, or 200 mg/kg) was dissolved in saline and administered intraperitoneally 15 min before morphine treatment twice a day from day 1 to day 7.
NF-κB activation assay
Cells (BV2) were plated in class bottom cell culture dishes and treated with morphine (200 μM) for 2 h with or without metformin (100 μM). Cells were fixed with ice-cold methanol and were permeabilized with 0.25% Triton X-100/PBST. After blocking with 1% bovine serum albumin (BSA) in PBST for 1 h, the coverslips with BV-2 cells were incubated for 2 h at room temperature with the p65/RelA antibody diluted in 1% BSA (1:50). Then, the coverslips were exposed to the fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG (1:100, at room temperature for 1 h) and then were rinsed three times with PBS. Finally, the coverslips were stained with 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole, a fluorescent DNA dye to mark nucleus) for 1 min. Confocal microscopy analyses were carried out using Olympus FV1000 confocal system.
Analysis of mRNA levels by quantitative real-time polymerase chain reaction (PCR)
Cells samples were homogenized in Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and total RNA was treated by DNaseI and subjected to quantitative PCR, which was performed with ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green I dye. The specific primer sequences for IL-1β, IL-6, TNF-α, TLR4, and GAPDH are listed as follows: IL-1β sense 5′-TCATTGTGGCTGTGGAGAAG-3′, antisense 5′-AGGCCACAGGTATTTTGTCG-3′, TNF-α sense 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, antisense 5′-TGGGAGTAGACAAGGTACAACCC-3′, IL-6 sense 5′-ATCCAGTTGCCTTCTTGGGACTGA-3′, antisense 5′-TAAGCCTCCGACTTGTGAAGTGGT-3′, IL-4 sense 5′-CGAGGTCACAGGAGAAGG-3′, antisense 5′-TGAGGACGTTTGGCACAT-3′, TGF-β sense 5′-ATGGTGGACCGCAACAAC-3′, antisense 5′-GCACTGCTTCCCGAATGTC-3′, Toll-like receptor-4 (TLR-4) sense 5′-ACTGTTCTTCTCCTGCCTGACA-3′, antisense 5′-CCTAGTCTTTGAGTCGTTTCAGG-3′, IL-10 sense 5′-AACATACTGCTAACCGACTC-3′, antisense 5′-GGATCATTTCCGATAAGG-3′, GAPDH sense 5′-CAAAAGGGTCATCTCC-3′, and antisense 5′-CCCCAGCATCAAAGGTG-3′ GAPDH. Gene was used as an endogenous control to normalize for differences in the amount of total RNA in each sample.
To identify temporal expression level of IBA-1, GAPDH, IL-1β, TNF-α, and the phosphorylated protein levels of p38 MAPK, N-methyl-d-aspartic acid receptor NR1 (NMDAR-NR1), PKCγ, protein samples were analyzed as described before. In brief, samples (cells or spinal cord tissue segments at L1-L6) were collected and washed with ice-cold PBS before being lysed in radio immunoprecipitation assay (RIPA) lysis buffer [Beyotime, Shanghai, China; 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/L phenylmethylsulfonyl fluoride, 0.15 U/mL aprotinin, and 1 mg/mL pepstatin] and then sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 10% whole milk in TBST (Tris-Hcl, NaCl, Tween 20) for 2 h at room temperature, probed with primary antibodies at 4 °C overnight [GAPDH, 1:8000; IBA-1, 1:1000; IL-1β, 1:1000; TNF-α, 1:1000; p-p38 (Tyr182), 1:1000; p-NR1(Ser896), 1:1000; p-PKCγ, 1:1000] and then incubated with horseradish peroxidase-coupled secondary antibodies from Cell Signaling Technology (Beverly, MA, USA). Data were acquired with the Molecular Imager (Gel DocTM XR, 170–8170) and analyzed with Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA, USA).
After anesthesia by intraperitoneal injection of sodium pentobarbital (100 mg/kg), the animal was perfused with normal saline followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.2–7.4, for 20 min. Then, L4 and/or L5 lumbar segment were dissected out and post-fixed in the same fixative. The embedded blocks were sectioned as 30 μm thick and processed for immunofluorescence assay. Sections from each group (five mice in each group) were incubated with primary antibody (IBA-1, 1:200; c-fos, 1:200; CGRP, 1:200). Then, the free-floating sections were washed with PBS and incubated with the secondary antibody (1:300; Jackson Laboratories, USA) for 2 h at room temperature. After using PBS to wash three times, the samples were investigated with a confocal microscope (Leica TCS SPEII, Leica Biosystems, Wetzlar, Germany) for morphologic details. Images were randomly coded and transferred to a computer for analysis.
SPSS Rel 15 (SPSS Inc., Chicago, IL, USA) was used to conduct all the statistical analyses. Data were statistically evaluated by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. The mean fluorescent pixels of IBA-1 and CGRP were measured by Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). Results were represented as mean ± SEM of three independent experiments. Results described as significant were based upon a criterion of p < 0.05.