Spinal NLRP3 inflammasome activation mediates IL-1β release and contributes to remifentanil-induced postoperative hyperalgesia by regulating NMDA receptor NR1 subunit phosphorylation and GLT-1 expression in rats

Abstract

Background: Trafficking and activation of N-methyl-D-aspartate (NMDA) receptors play an important role in initiating and maintaining postoperative remifentanil-induced hyperalgesia (RIH). Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been linked to the development of inflammatory and neuropathic pain. We hypothesized that activation of NLRP3 inflammasome mediates IL-1β release and contributes to RIH in rats by increasing NMDA receptor NR1 (NR1) subunit phosphorylation and decreasing glutamate transporter-1 (GLT-1) expression.

Methods: Acute exposure to remifentanil (1.2 μg/kg/min for 60 min) was used to establish RIH in rats. Thermal and mechanical hyperalgesia were tested at baseline (24 h before remifentanil infusion) and 2, 6, 24, and 48 h after remifentanil infusion. The levels of IL-1β, GLT-1, phosphorylated NR1 (phospho-NR1), and NLRP3 inflammasome activation indicators [NLRP3, Toll-like receptor 4 (TLR4), P2X purinoceptor 7 (P2X7R), and caspase-1] were measured after the last behavioral test. A selective IL-1β inhibitor (IL-1β inhibitor antagonist; IL-1ra) or three different selective NLRP3 inflammasome activation inhibitors [(+)-naloxone (a TLR4 inhibitor), A438079 (a P2X7R inhibitor), or ac-YVADcmk (a caspase-1 inhibitor)] were intrathecally administered immediately before remifentanil infusion into rats.

Results: Remifentanil induced significant postoperative hyperalgesia, increased IL-1β and phospho-NR1 levels and activated the NLRP3 inflammasome by increasing TLR4, P2X7R, NLRP3, and caspase-1 expression, but it decreased GLT-1 expression in the L4-L6 spinal cord segments of rats, which was markedly improved by intrathecal administration of IL-1ra, (+)-naloxone, A438079, or ac-YVADcmk.

Conclusion: NLRP3 inflammasome activation mediates IL-1β release and contributes to RIH in rats by inducing NMDA receptor NR1 subunit phosphorylation and decreasing GLT-1 expression. Inhibiting the activation of the NLRP3 inflammasome may be an effective treatment for RIH.

Keywords: N-methylessentialate receptors; NOD-like receptor protein 3 inflammasome; Remifentanil; glutamate transporter-1; postoperative hyperalgesia.

Figure 1.

Experimental design. Adult male Sprague-Dawley rats (240-260 g) were subjected to normal saline or remifentanil infusion. Animals in the Remifentanil group were infused intravenously with remifentanil hydrochloride (dissolved in normal saline) at a rate of 1.2 μg/kg/min for 60 min. Animals in the Normal saline group received the same volume of saline under identical conditions. The withdrawal threshold and latency to mechanical and thermal stimulation, respectively, were evaluated at the baseline (24 h before remifentanil infusion) and 2, 6, 24, and 48 h after remifentanil infusion. The L4-L6 segments of the spinal cord were collected after the last behavioral test. To verify the roles of IL-1β and NLRP3 inflammasome activation in the development and maintenance of RIH, a selective IL-1ra or three different selective NLRP3 inflammasome activation inhibitors [(+)-naloxone (a TLR4 inhibitor), A438079 (a P2X7R inhibitor), or ac-YVADcmk (a caspase-1 inhibitor)] were intrathecally administered immediately before remifentanil infusion in rats. RIH, remifentanil-induced hyperalgesia; IL-1ra, IL-1β inhibitor; NLRP3, NOD-like receptor protein 3; P2X7R, P2X purinoceptor 7.

Figure 2.

Behavioral changes after remifentanil infusion in rats. Under 3% sevoflurane anesthesia, remifentanil (1.2 \x{03bc}g/kg/min) or saline was intravenously infused for 60 min. The (A) PWT to von Frey filament stimulation and (B) PWL to thermal stimulation were evaluated 24 h before the incision and 2, 6, 24, and 48 h after the remifentanil infusion. The data are presented as the mean ± SD (n = 8/group). *P < 0.05 vs. Normal saline group. PWL, paw withdrawal latency; PWT, paw withdrawal threshold.

Figure 3.

IL-1β, p-NR1 and GLT-1 expression after remifentanil infusion in rats. The L4-L6 segments of the spinal cord were harvested right after the last behavioral test. (A) mRNA and (B) protein levels of IL-1β were detected by RT-qPCR and ELISA, respectively. (C) The p-NR1 and GLT-1 expression were measured by western blotting, and quantitative analysis of (D) p-NR1 and (E) GLT-1 are shown as the ratio of protein relative density to β-actin. The data are presented as the mean ± SD (n = 4/group). *P < 0.05 vs. Normal saline group. RT-qPCR, reverse quantitative transcription PCR; p-NR1, phospho-NMDA receptor NR1; GLT-1, glutamate transporter-1.

Figure 4.

IL-1ra on behavioral changes, p-NR1 and GLT-1 expression after remifentanil infusion in rats. IL-1ra (a selective IL-1β inhibitor; 100 μg dissolved in 10 μl vehicle) or vehicle (0.15 μl DMSO dissolved in 10 μl saline) was intrathecally administered into the rat before remifentanil infusion. The L4-L6 segments of the spinal cord were harvested right after the last behavioral test. The (A) PWT to von Frey filament stimulation and (B) PWL to thermal stimulation were evaluated 24 h before the incision and 2, 6, 24, and 48 h after remifentanil infusion. The data are presented as the mean ± SD (n = 8/group). (C) The p-NR1 and GLT-1 expression were measured by western blotting, and the quantitative analysis values of (D) p-NR1 and (E) GLT-1 are presented as the ratio of relative protein density to β-actin. The data are presented as the mean ± SD (n = 4/group). *P < 0.05 vs. Normal saline + Vehicle group, #P < 0.05 vs. Remifentanil + Vehicle group. IL-1ra, IL-1β inhibitor antagonist; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; p-NR1, phospho-NMDA receptor NR1; GLT-1, glutamate transporter-1.

Figure 5.

Expression of proteins associated with NLRP3 inflammasome activation after remifentanil infusion in rats. The L4-L6 segments of the spinal cord were harvested right after the last behavioral test. (A) The NLRP3, TLR4, P2X7R, and caspase-1 expression were measured by western blotting, and the quantitative analysis values of (B) NLRP3, (C) TLR4, (D) P2X7R, and (E) caspase-1 are presented as the ratio of relative protein density to β-actin. The data are presented as the mean ± SD (n = 4/group). *P < 0.05 vs. Normal saline group. NLRP3, NOD-like receptor protein 3; TLR4, Toll-like receptor 4; P2X7R, P2X purinoceptor 7.

Figure 6.

After remifentanil infusion, the effects of NLRP3 inflammasome activation inhibitors on behavioral changes, IL-1β, p-NR1, and GLT-1 expression in rats. Three different selective NLRP3 inflammasome activation inhibitors, (+)-Naloxone (a TLR4 inhibitor; 1,200 μg dissolved in 10 μl vehicle), A438079 (a P2X7R inhibitor; 600 ng dissolved in 10 μl vehicle), and ac-YVAD-cmk (a caspase-1 inhibitor; 20 μg dissolved in 10 μl vehicle) or vehicle (0.15 μl DMSO dissolved in 10 μl saline) was intrathecally administered into the rats respectively before remifentanil infusion. The L4-L6 segments of the spinal cord were harvested immediately after the last behavioral test. The (A) PWT to von Frey filament stimulation and (B) PWL to thermal stimulation were evaluated 24 h before the incision and 2, 6, 24, and 48 h after remifentanil infusion. The data are presented as the mean ± SD (n=8/group). (C) mRNA and (D) protein levels of IL-1β were detected by RT-qPCR and ELISA, respectively. (E) The p-NR1 and GLT-1 expression were measured by western blotting, and the quantitative analysis values of (F) p-NR1 and (G) GLT-1 are presented as the ratio of relative protein density to μ-actin. The data are presented as the mean ± SD (n=4/group). *P<0.05 vs. Normal saline + Vehicle group; #P<0.05 vs. Remifentanil + Vehicle group. NLRP3, NOD-like receptor protein 3; TLR4, Toll-like receptor 4; P2X7R, P2X purinoceptor 7; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; p-NR1, phospho-NMDA receptor NR1; NMDA receptor NR1; GLT-1, glutamate transporter-1; RT-qPCR, reverse quantitative transcription PCR.