Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat

Abstract

Background: MyD88 is the adaptor protein of MyD88-dependent signaling pathway of TLRs and IL-1 receptor and regulates innate immune response. However, it was not clear whether and how MyD88 and related signaling pathways in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) are involved in neuropathic pain.

Methods: Chronic constriction injury (CCI) was used to induce neuropathic pain in the rat. The expression of MyD88, TRIF, IBA1, and GFAP was detected with immunofluorescent staining and Western blot. The expression of interleukin-1 beta (IL-1β), high mobility group box 1 (HMGB1), NF-κB-p65, phosphorylated NF-κB-p65, ERK, phosphorylated ERK, and tumor necrosis factor-alpha (TNF-α) was detected with Western blot. Pain-related behavioral effects of MyD88 homodimerization inhibitory peptide (MIP) were accessed up to 3 weeks after intrathecal administration.

Results: Peripheral nerve injury significantly increased the protein level of MyD88 in the DRG and SDH, but had no effect on TRIF. MyD88 was found partly distributed in the nociceptive neurons in the DRGs and the astrocytes and microglia in the SDH. HMGB1 and IL-1β were also found upregulated in nociceptive pathways of CCI rats. Intrathecal application of MIP significantly alleviated mechanical and thermal hyperalgesia in the CCI rats and also reversed CCI-induced upregulation of MyD88 in both DRG and SDH. Further investigation revealed that suppression of MyD88 protein reduced the release of TNF-α and glial activation in the SDH in the CCI rats.

Conclusions: MyD88-dependent TIR pathway in the DRG and SDH may play a role in CCI-induced neuropathic pain. MyD88 might serve as a potential therapeutic target for neuropathic pain.

Keywords: CCI; Dorsal root ganglion; MyD88; Neuropathic pain; Spinal dorsal horn; TRIF.

Fig. 1

CCI increase expression MyD88 in rat DRG and SDH. a and b Western blot analyses the time course for MyD88 expression in DRG (a) and SDH (b) (n = 4 in each). Western blot analysis is shown on the top; quantification of protein levels (relative to naive group) is shown on the bottom. One-way ANOVA, *P < 0.05 versus naive and sham. c Cellular distribution of MyD88 in DRGs. MyD88 was expressed in the large-, medium-, and small-sized neurons (white arrows) sham to the CCI (top row). Double immunostaining shows coexpression of MyD88 with CGRP- and IB4-positive neurons, respectively (middle and bottom rows). d Distribution and cellular of MyD88 in the SDH. MyD88 was distributed predominantly in the superficial layers (top row). MyD88 was coexpression with astrocytes (GFAP, red) and microglial cells (IBA1, red) (middle and bottom rows). Scale bar: 20 μm (c, d)

Fig. 2

CCI increase expression IL-1β and HMGB1 in rat DRG and SDH. a and b Western blot analyses the time course for Pro-IL-1β and IL-1β expression in DRG (A) and SDH (B). c-d Western blot analyses the time course for HMGB1 expression in DRG (C) and SDH (D). Western blot analysis is shown on the top; quantification of protein levels (relative to naive group) is shown on the bottom. N = 4 in each group, One-way ANOVA, *P < 0.05 vs. naive or sham

Fig. 3

CCI induce NF-κB p65 and ERK signaling activation in rat DRG and SDH. a-b Western blot analysis shows time course for the expression of total NF-κB p65 and activated phospho-NF-κB p65 in DRG (A). Quantification of protein levels of total NF-κB p65 and activated phospho-NF-κB p65 in DRG (B). One-way ANOVA, *P < 0.05 versus naive and sham. c-d Western blot analysis shows time course for the expression of total NF-κB p65 and activated phospho-NF-κB p65 in SDH (C). Quantification of protein levels of total NF-κB p65 and activated phospho-NF-κB p65 in SDH (D). e-f Western blot analysis shows time course for the expression of total ERK and activated pERK in DRG (E). Quantification of protein levels of total ERK and activated pERK in DRG (F). g-h Western blot analysis shows time course for the expression of total ERK and activated pERK in SDH (G). Quantification of protein levels of total ERK and activated pERK in SDH (H). N = 4 in each group, One-way ANOVA, *P < 0.05 vs. naive or sham

Fig. 4

Attenuated neuropathic pain by of MIP after CCI treatment. a-b CCI-induced mechanical allodynia (a) and thermal hyperalgesia (b). Two-way ANOVA, *P < 0.05 versus sham. c-d Pre-intrathecal injection of MIP attenuated CCI-induced mechanical allodynia (c) and thermal hyperalgesia (d) (each administration is indicated by an arrow on the 3, 2, and 1 days before CCI). e-f Mechanical allodynia (e) and thermal hyperalgesia (f) is attenuated by MIP in the early phase of CCI operation (each administration is indicated by an arrow on the 3, 4, and 5 days after CCI). MIP (500 mM) was administrated i.t.in a volume of 20 μl; The control peptide was used in the control (Ctrl) group. Nine rats were included in each group. Two-way ANOVA, *P < 0.05 vs. Sham + Ctrl or Sham + MIP, #P < 0.05 vs. Sham + Ctrl, Sham + MIP or CCI + Ctrl

Fig. 5

Intrathecal administration of MIP decreases MyD88 protein expression in rat DRG and SDH after CCI. a-b Western blot showing inhibitory effects of MIP on CCI-increased protein level of MyD88 in DRG (A) and SDH (B). c Data summary of B. d-e Immunofluorescence showing inhibitory effects of MIP on expression of MyD88 in DRG (D) and SDH (E). Scale bar: 20 μm (d, e). MIP (i.t., in a volume of 20 μl, 500 mM) was given once a day on postoperative days 3, 4, and 5, respectively. The control peptide was used in the control (Ctrl) group. Tissues were collected on postoperative days 14 (n = 4 each group).One-way ANOVA, *P < 0.05 vs. Sham, #P < 0.05 vs. CCI + Ctrl

Fig. 6

Suppressed of NF-κB p65, ERK signal by inhibition of Myd88 in rat SDH after CCI. a Western blot showing inhibitory effects of MIP on CCI-induced increased protein level of NF-κB p65 and pNF-κB p65. b Data summary of A. c Western blot showing inhibitory effects of MIP on CCI-induced increased protein level of ERK and pERK. d Data summary of C. Others are the same as Fig. 5

Fig. 7

Suppressed activation of glial cells and TNF-α production by inhibition of MyD88 in rat SDH after CCI. a Immunostaining showing inhibitory effects of MIP on activation of microglial cells (IBA1), and astrocytes (GFAP). Scale bar: 20 μm. b Western blot showing inhibitory effects of MIP on CCI-induced increased protein level of IBA1, GFAP, and TNF-α. c Data summary of B. Others are the same as Fig. 5

Fig. 8

Schematic illustration demonstrates MyD88-dependent signaling pathways of neuropathic pain induced by CCI. Nerve injury produces abundant HMGB1and IL-1β in the DRG and SDH. The binding of HMGB1 and IL-1β to their receptors (TLR2/4 andIL-1R, respectively) activates MyD88 in the DRG and SDH, which phosphorylate NF-κB p65 and ERK. Phosphorylated NF-κB p65 subsequently enter the nucleus to regulate the expression of proinflammation cytokines such as TNF-α. Phosphorylated ERK enter the nucleus to induce transcription factors such as AP-1, which regulates the expression of certain cytokines and activates glial cells. All these signaling events consequently result in central and peripheral sensitizations that produce neuropathic pain