Adult male ICR mice weighing 18-22 g (Laboratory Animal Center, Nantong University, Nantong, China) were housed in groups (4-5 per cage) for at least 3 days prior to starting the experiments and acclimatized to a 12-h light/dark cycle environment with a controlled temperature of 22±1℃, and relative humidity of 50-70%. Food and water were available ad libitum except during experimental sessions. All experimental protocols were approved by the Institutional Animal Care and Use Committee, Nantong University. All procedures and the handling of animals were performed in accordance with guidelines of the International Association for the Study of Pain27 and the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington, DC). Animal studies are reported in compliance with the ARRIVE guidelines28.
Sinomenine was purchased from Aladdin Reagents (Shanghai, China). N-demethylsinomenine was chemically synthesized in our laboratory (purity > 98% as determined by HPLC) and its chemical structure was identified to be consistent with previous reports by NMR spectroscopy11,14. Naltrexone hydrochloride, WAY-100635 maleate, bicuculline were obtained from Selleck Chemicals (Houston, TX, USA). WAY-100635 maleate and bicuculline were dissolved in 10% DMSO. The other drugs were dissolved in 0.9% saline. All drugs were administered intraperitoneally (i.p.) in a volume of 10 ml/kg of body weight. Complete Freund’s adjuvant (CFA) was purchased from Sigma (St. Louis, MO, USA).
4.3 Chronic neuropathic pain model
A model of chronic neuropathic pain was induced by a chronic constriction injury (CCI) procedure, which was performed as described previously19. Briefly, mice were anesthetized with 2% isoflurane in oxygen at a flow rate of 3L/min delivered via a nose cone throughout the period of surgery. After sterile preparation, an incision was made below the right hipbone, parallel to the sciatic nerve. The right common sciatic nerve was exposed at the mid-thigh level and three ligations (4.0 chromic gut, with 1 mm spacing) were loosely tied around the nerve proximal to the sciatic trifurcation until a brief twitch in the respective hind limb was observed. After nerve ligation, the incision was sutured with silk suture and a topical antibiotic was applied. Then the mice were allowed to recover in their home cages. Mice received sham procedures that the sciatic nerve was exposed without ligation served as controls.
4.4 Chronic inflammatory pain model
Mice were temporarily anesthetized by inhalation of 3% isoflurane and then received an intraplantar injection of freshly prepared CFA (20 μL, 50% in saline) in the plantar surface of the right hind paw to induce a model of persistent inflammatory pain29. Mice that received an injection of the same volume of saline served as controls.
4.5 von Frey filament test
As described in our previous study14, mechanical allodynia in the mice was evaluated using the von Frey filament test as previously described30,31. In brief, mice were placed in a clear Plexiglas chamber with an elevated wire mesh floor and allowed to acclimatize for 20 min before testing. The mechanical paw withdrawal threshold (PWT) was measured by a series of calibrated von Frey filaments (Stoelting, Kiel, WI, USA) with bending forces ranging from 0.07 to 2 g. The filaments were applied in ascending order to the mid plantar surface of each hind paw through the mesh floor. Each filament was presented vertically against the paw until it bowed slightly, and tested three times per paw with an interval of 5 sec. The PWT value was defined as the minimal force to elicit paw withdrawal responses appearing at least twice in three consecutive trials. The cut-off value for this test was 2.0 g. Experimenters were well-trained to execute consistent behavioral tests and were blinded to the vehicle and treatment groups.
4.6 Locomotor activity
The locomotor activity of mice was measured by a commercially available apparatus (YLS-1A, Shandong Academy of Medical Sciences, China), which consists of a controller unit and five separate black acrylic locomotion chambers14. Each chamber (12×15×15 cm) was surrounded with an array of photocell beams which link to the controller unit. Mice were individually put into these chambers in a dark environment and the spontaneous locomotor activity was measured during a 60-min test period with each count indicating one beam break by the animal.
4.7 Experimental design
For the measurement of mechanical allodynia, the PWT was measured 24 h after CCI surgery or CFA injection and daily thereafter for 7 days. Prior to CCI surgery or CFA injection, all mice also received daily measures for 3 days to obtain the baseline value (BL) and to allow mice to habituate to the operators and procedures. For acute treatment studies, the effects of N-demethylsinomenine and sinomenine at three doses (10, 20, 40 mg/kg) were studied 1 day after CCI surgery or CFA injection. The PWT in all mice was measured before (0 h) and every 30 min thereafter for 4 hours after the administration (i.p.) of drugs or saline (vehicle group). For repeated dosing studies, 1 day after CCI surgery or CFA injection, mice were treated with N-demethylsinomenine (20 and 40 mg/kg) once daily for 2 weeks. The PWT was measured daily before (0 h) and 1.5 h after N-demethylsinomenine administration. In an effort to examine the pharmacological mechanisms mediating N-demethylsinomenine induced anti-allodynia against CCI-induced neuropathic pain and CFA-induced inflammatory pain, we investigated whether pretreatment with different antagonists including bicuculline (a selective GABAA receptor antagonist), WAY100635 (serotonin 5-HT1A receptor antagonist) or naltrexone (opioid receptor antagonist) affected the antinociception of 40 mg/kg N-demethylsinomenine. For antagonist studies, 1 day after CCI surgery or CFA injection, different antagonists were administered 10 min prior to 40 mg/kg N-demethylsinomenine administration respectively and the PWT was measured thereafter every 30 min for 4 hours. For locomotor activity test, the doses of 10-40 mg/kg sinomenine and N-demethylsinomenine were studied to observe the potential sedative effect in healthy mice. The drug was administered 60 min before the test sessions because this pretreatment time was adequate for the drug to produce significant anti-allodynic effects. For all the studies, a blind design was strictly followed so that different experimenters performed drug treatments and behavioral tests.
4.8 Data analyses
All data were expressed as mean ± standard error of the mean (S.E.M.) and were analyzed by the GraphPad Prism 5.01 software (San Diego, CA, USA). For mechanical allodynia test, the PWT (g) was plotted as a function of time (h or days). The statistical differences between groups were analyzed by two-way analysis of variance (ANOVA) with repeated measures (Time×Treatment) followed by Bonferroni post hoc analysis. The antinociceptive effect of each drug dose was quantified for each animal as the percentage of the maximum possible effect (% MPE) according to the following formula: % MPE = [(post-drug PWT—pre-drug PWT)/(normal baseline PWT—pre-drug PWT)]×100. The dose-effect curves for N-demethylsinomenine and sinomenine were constructed by plotting the effect (% MPE) of each dose and their individual ED50 [95% confidence limits (CL)] values were calculated respectively using nonlinear regression method by the GraphPad Prism 5.01 software (San Diego, CA, USA). A value of P<0.05 was considered statistically significant for all analyses.