Bromodomain-containing protein 4 contributes to chronic postsurgical pain via activating TLR4/NF-kappaB-dependent neuroinflammation

Abstract

Background: Chronic pain affects 1.5 billion individuals worldwide but remains woefully undertreated, particularly occurring in the populations undergoing surgical procedures. Increasing evidence indicates that epigenetic modifications are involved in the pathogenesis of chronic pain. This study aimed to investigate the role of bromodomain-containing protein 4 (Brd4) in chronification of pain after surgery.

Methods: Male Sprague-Dawley rats were subjected to skin/muscle incision and retraction surgery to induce chronic postsurgical pain. Nociceptive thresholds and locomotor activity were assessed. The spinal cord was collected to detect the expressions of Brd4, toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB) pathway, and proinflammatory cytokines and chemokines release. The BET inhibitor JQ1 was used to verify the contribution of Brd4 to chronic postsurgical pain.

Results: Significant upregulation of spinal Brd4 expression, accompanied by activation of the TLR4/NF-κB signaling cascade and increased release of proinflammatory mediators were presented in the rat model of chronic postsurgical pain. The BET inhibitor JQ1 prevented the development of chronic postsurgical pain in a dose-dependent manner. Moreover, JQ1 obviously suppressed the activation of TLR4 and the phosphorylation and translocation of NF-κB as well as subsequently cytokines and chemokines release, including IL-1β, IL-6, TNF-α, CXCL1, CXCL2 and CCL2.

Conclusions: The epigenetic regulator Brd4 initiates neuroinflammation in spinal cord through activating TLR4/NF-κB signaling pathway and enhancing cytokine and chemokines release, thereby contributing to the transition to chronic postsurgical pain. JQ1 halts chronic pain development, providing a novel therapy for patients at high risk of persistent pain.

Keywords: Brd4; Chronic postsurgical pain; Epigenetics; NF-κB; Neuroinflammation.

Fig. 1

Skin/muscle incision and retraction surgery induced chronic postsurgical pain in rats. A Illustration of the experimental procedure for surgery and behavior tests. B The L4-6 spinal cords tissues were collected for biological detection after the last behavioral test. C, D, E Time course of changes in the paw withdrawal threshold C, paw withdrawal latency D and time of paw lift E in response to mechanical and thermal stimulus. Data are presented as mean ± SEM for n = 8 rats and analyzed by the two-way repeated measures ANOVA with Bonferroni post hoc comparisons in C, D and E. ^***P < 0.001; ^**P < 0.01. SMIR, skin/muscle incision and retraction surgery

Fig. 2

Spinal Brd4 was activated in chronic postsurgical pain. A Brd4 mRNA significantly increased in SMIR rats compared with sham on postsurgical 7 d and 21 d respectively. B Brd4 protein expression in spinal cord obviously increased in SMIR rat compared with sham. C Immunostaining of spinal cord sections showed Brd4 was overexpressed in SMIR rat. Data are presented as mean ± SEM for n = 6 rats and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. ^****P < 0.0001; ^***P < 0.001; ^**P < 0.01. Brd4, bromodomain-containing protein 4; SMIR, skin/muscle incision and retraction surgery

Fig. 3

The BET inhibitor JQ1 prevented the development of chronic postsurgical pain in a dose-dependent manner. A, B, C Time course of changes in the paw withdrawal threshold A, paw withdrawal latency B and time of paw lift C in response to mechanical and thermal stimulus. D Representative tracks of rats treated with SMIR or JQ1 were recorded in open field test. F, G Summary of data from open field tests to assess spontaneous exploration and locomotor function. Data are presented as mean ± SEM for n = 8 rats and analyzed by two-way repeated measures ANOVA with Bonferroni post hoc comparisons in A, B and C, and one-way ANOVA with Bonferroni post hoc comparisons in E and F. ^###P < 0.001 vs. Sham, ^**P < 0.01 vs. SMIR, ^*P < 0.05 vs. SMIR in A-C, ^****P < 0.0001 vs. Sham, ^**P < 0.01 vs. SMIR in D-F. SMIR, skin/muscle incision and retraction surgery

Fig. 4

Brd4 regulated TLR4 and NF-κB pathway activation in chronic postsurgical pain. Western blotting and analysis data of TLR4 A, IκBα B, cytosolic p-p65 C and nuclear p65 D in spinal cord of rats receiving SMIR or JQ1. Data are presented as mean ± SEM for n = 6 rats and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. ^****P < 0.0001. TLR4, toll-like receptor 4; NF-κB, nuclear factor kappa-B; IκBα, recombinant inhibitory subunit of nuclear factor kappa-B alpha; SMIR, skin/muscle incision and retraction surgery

Fig. 5

Brd4 promoted proinflammatory cytokines and chemokines release in chronic pain after surgery. Levels of proinflammatory cytokines IL-1β A, IL-6 B, TNF-α C, and chemokines CXCL1 D, CXCL2 E, CCL2 F in spinal cord of rats receiving SMIR or JQ1 were detected. Data are presented as mean ± SEM for n = 6 rats and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. ^****P < 0.0001; ^***P < 0.001; ^**P < 0.01. IL-1β, interleukin-1 beta; IL-6, interleukin-6, TNF-α, tumor necrosis factor-alpha; CXCL1, chemokine C-X-C motif ligand 1; CXCL2, chemokine C-X-C Motif ligand 2; CCL2, chemokine C-C motif ligand 2; SMIR, skin/muscle incision and retraction surgery