Fig. 1
Chronic morphine treatments induce morphine analgesic tolerance and MIH, as well as an inflammatory response, m6A methylation and YTHDF1 in the vlPAG. a, b Time course of the daily hot-plate test and tail-immersion test before (baseline, BL) and after morphine treatment (+ 30 min) throughout a 7-d chronic morphine or saline exposure (10 mg/kg twice daily) in WT mice. Nociceptive behavior (pre-morphine BL time points only): hot plate, F1,18 = 18.094, P < 0.001; tail immersion, F1,18 = 7.472, P = 0.014. Antinociception (post-morphine + 30 min time points only): hot plate, F1,18 = 700.978, P = 0.001; tail immersion, F1,18 = 465.072, P < 0.001. c, d Antinociception tolerance. Percentage of the maximal possible effect (%MPE) for morphine antinociception from the first administration (Day 1: + 30 min) compared with the last administration (Day 7: + 30 min) (hot plate, F1,18 = 809.972, P < 0.001. ***P < 0.001 vs. Day 1 + 30 min; tail immersion, F1,18 = 446.943, P < 0.001. ***P < 0.001 vs. Day 1 + 30 min). e, f MIH. Withdrawal latency change before and after morphine administration (hot plate: F1,18 = 14.677, P = 0.001. ***P < 0.001 vs. Day 1 BL of the MT group, ### vs. Day 7 BL of the control groups; tail immersion: F1,18 = 63.115, P < 0.001. ***P < 0.001 vs. Day 1 BL of the MT group, ### vs. Day 7 BL of the control groups) (repeated measures two-way ANOVA, n = 10). g–k Immunofluorescence images and western blotting analysis of IL-1β (g, P = 0.00504), IL-6 (h, P = 0.00117), TNF-α (i, P = 0.00132), NF-κB (j, P = 0.0076), and YTHDF1 (k, P = 0.00073) in the vlPAG of the mice treated with repetitive saline or morphine administration (n = 6), Student’s t test, two-tailed. l The m6A of poly (A) + isolated from total RNA of the vlPAG after a 7-d morphine or saline treatments was indicated by m6A dot blot. Corresponding RNAs were loaded equally by twofold serial dilution with 200 ng, 100 ng, and 50 ng. Methylene blue staining served as a loading control (P = 0.0065, n = 4, Student’s t test, two-tailed). Tissues were collected on Day 7 after administration. Scale bars, 50 μm. Data are shown as the mean ± SEM