Fig. 3

Glibenclamide attenuates morphine tolerance and suppresses morphine-induced microglia activation. Tail-flick method was performed to evaluate the effect of glibenclamide on the morphine tolerance. Data were shown as percentage of maximal possible effect (MPE). a Glibenclamide co-administration with morphine improved chronic morphine tolerance in mice (n = 8). Morphine (10 μg/10 μL) was intrathecally injected with different doses of glibenclamide (0.08, 0.4, and 2 μg/10 μL) once daily, and the MPE was measured 1 h after the first injection of each day. b Consecutive administration of anti-HSP70 neutralizing antibody (200 μg/kg, i.t.) once daily, partially attenuating morphine tolerance in mice (n = 6). c Immunofluorescence result showed that glibenclamide (2 μg/10 μL) significantly inhibited the activation of microglia evoked by morphine in the spinal cord (n = 4). d, e Immunoblot results demonstrated that glibenclamide (0.08, 0.4, and 2 μg/10 μL) suppressed morphine-induced upregulation of phosphorylation of p38 MAPK and NF-κB p65, but not the p38 total protein in the spinal cord. (n = 4). f, g Immunofluorescence analysis showed that glibenclamide (2 μg/10 μL) markedly inhibited the activation of neuronal c-fos and CGRP after morphine treatment in the spinal cord. The quantification of c-fos and CGRP immunofluorescence was respectively represented as number of c-fos-positive cells and mean fluorescence intensity of CGRP in dorsal horn (n = 4). Glibenclamide (0.08, 0.4, and 2 μg/10 μL) was administered once daily for 7 days. One hour after the final administration, spinal samples were collected. a, b data were analyzed by two-way ANOVA. c-g data were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. morphine-treated group. Scale bar: 75 μm