Fig 2: Schematic diagram of the experiment protocol. a. Experiment 1: To confirm the changes of pain behavior and protein expression after remifentanil exposure in RIH rats. b. Experiment 2: To explore the role of TLR4 signaling and TRPA1 after remifentanil exposure in RIH rats.

Fig 3: Effects of inhibiting TLR4 or TRPA1 on mechanical and thermal sensitivity. PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency. a-b. For rats in group TAK, TLR4 was inhibited by TAK-242. For rats in group HC, TRPA1 was inhibited by HC-030031. Compared with group RI, mechanical allodynia (a) was alleviated at 2 h, 6 h, 12 h, 24 h, and 48 h time points in group TAK and HC. However, thermal hyperalgesia (b) in group TAK and group HC showed no significant differences at most time points compared to that in group RI. All the data are means ± SD (n = 6) and analyzed by two-way ANOVA with Tukey’s post hoc comparisons. *p < .05 versus group C at the same time point; #p < .05 versus group RI at the same time point.

Fig 4: Immunofluorescence of TLR4 and TRPA1 expression after inhibiting TLR4 or TRPA1 in DRG neurons. a. Representative immunofluorescence micrographs, with the scale bar equaling to 50 µm. b. The statistical results of TLR4 expression in DRG neurons. The upregulation of TLR4 in rats with RIH was inhibited after blocking TLR4. c. The statistical results of TRPA1 expression in DRG neurons. The expression of TRPA1 in group TAK and group HC were downregulated after blocking TLR4 or TRPA1 when compared with group RI. All the data are expressed as means ± SD (n = 4) and analyzed by one-way ANOVA with Tukey’s post hoc comparisons. *p < .05, **p < .01 versus group C; #p < .05, ##p < .01 versus group RI; ^p < 0.05 versus group TAK.

Fig 5: Expression of TLR4 signaling and TRPA1 in DRG neurons after inhibiting TLR4 or TRPA1 in RIH model. a. Representative bands of Western blot for the expression of TLR4, p-NF-?B, and TPRA1 in DRG at 48 h after remifentanil exposure. b-d. When compared with group RI, inhibition of TLR4 decreased the expression of TLR4 (b), p-NF-?B (c), and TRPA1 (d). However, inhibition of TRPA1 did not affect the expression of TLR4 (b) or p-NF-?B (c). e. Consistent with the protein expression trends of TLR4 and TPRA1 after inhibiting TLR4, the mRNA expression levels of Tlr4 and Trpa1 were significantly downregulated in group TAK compared with group RI. All the data are expressed as means ± SD (n = 4) and analyzed by one-way ANOVA with Tukey’s post hoc comparisons. *p < .05, **p < .01 versus group C; #p < .05, ##p < 0.01 versus group RI; ^p < .05, ^^p < .01 versus group TAK.