Korean J Pain.  2021 Jan;34(1):47-57. 10.3344/kjp.2021.34.1.47.

Toll-like receptor 4/nuclear factor-kappa B pathway is involved in radicular pain by encouraging spinal microglia activation and inflammatory response in a rat model of lumbar disc herniation

• Zhu L
• Huang Y
• Hu Y
• Tang Q
• Zhong Y

• KMID: 2510311
• DOI: http://doi.org/10.3344/kjp.2021.34.1.47

Abstract

Background
Lumbar disc herniation (LDH) is a common cause of radicular pain, but the mechanism is not clear. In this study, we investigated the engagement of toll-like receptor 4 (TLR4) and the nuclear factor-kappa B (NF-κB) in radicular pain and its possible mechanisms.
Methods
An LDH model was induced by autologous nucleus pulposus (NP) implantation, which was obtained from coccygeal vertebra, then relocated in the lumbar 4/5 spinal nerve roots of rats. Mechanical and thermal pain behaviors were assessed by using von Frey filaments and hotplate test respectively. The protein level of TLR4 and phosphorylated-p65 (p-p65) was evaluated by western blotting analysis and immunofluorescence staining. Spinal microglia activation was evaluated by immunofluorescence staining of specific relevant markers. The expression of proand anti-inflammatory cytokines in the spinal dorsal horn was measured by enzyme linked immunosorbent assay.
Results
Spinal expression of TLR4 and p-NF-κB (p-p65) was significantly increased after NP implantation, lasting up to 14 days. TLR4 was mainly expressed in spinal microglia, but not astrocytes or neurons. TLR4 antagonist TAK242 decreased spinal expression of p-p65. TAK242 or NF-κB inhibitor pyrrolidinedithiocarbamic acid alleviated mechanical and thermal pain behaviors, inhibited spinal microglia activation, moderated spinal inflammatory response manifested by decreasing interleukin (IL)-1β, IL-6, tumor necrosis factor-α expression and increasing IL-10 expression in the spinal dorsal horn.
Conclusions
The study revealed that TLR4/NF-κB pathway participated in radicular pain by encouraging spinal microglia activation and inflammatory response.

Keyword

Cytokines; Hyperalgesia; Intervertebral Disc; Microglia; Neuralgia; NFkappa B; Nucleus Pulposus; Pain; Toll-Like Receptor 4

Figure

• 

  Fig. 1 The protein level of (A, B) toll-like receptor 4 (TLR4) and (C, D) phosphorylated-p65 (p-p65) in spinal dorsal horn of rats with nucleus pulposus (NP) implantation (*P = 0.002 or 0.001, **P < 0.001, n = 5/group). Error bars indicate standard error of means.

• 

  Fig. 2 (A-C) Expression of toll-like receptor 4 (TLR4) in bilateral spinal dorsal horn of rats with nucleus pulposus (NP). (D) Specificity of antibody is tested by negative control (n = 2). (E-I) Expression of TLR4 in ipsilateral spinal dorsal horn of different groups (*P = 0.001, **P < 0.001, n = 4/group). (J-R) Double immunofluorescence staining of TLR4 (red) with cellular markers (green): ionized calcium binding adaptor molecule-1 (Iba-1, microglia marker), glial fibrillary acidic protein (GFAP, astrocyte marker) and neuronal specific nuclear protein (NeuN, neuronal marker) respectively. (H) The histogram shows Iba-1 positive area of spinal dorsal horn in different groups. Scale bar (A) = 400 μm, Scale bars (B, C) = 200 μm, Scale bars (D-H, J-R) = 100 μm. Error bars indicate standard error of means.

• 

  Fig. 3 (A, B) Nucleus pulposus (NP) implantation decreased mechanical and thermal thresholds in ipsilateral but not contralateral hindpaws. Toll-like receptor 4 antagonist TAK242 (TAK) (C, D) and nuclear factor-kappa B inhibitor pyrrolidinedithiocarbamic acid (PDTC) (E, F) alleviate mechanical and thermal pain behaviors of rats with NP (vs. sham, *P = 0.001, **P < 0.001; vs. NP + vehicle [Veh], #P = 0.002; n = 10/group). Error bars indicate standard error of means. PWT: paw withdrawal threshold, PWL: paw withdrawal latency, ipsi: ipsilateral hindpaws, contra: contralateral hindpaws.

• 

  Fig. 4 TAK242 (TAK) decreases spinal phosphorylated-p65 (p-p65) expression (C, D) without changing toll-like receptor 4 (TLR4) expression (A, B). Error bars indicate standard error of means. NP: nucleus pulposus, Veh: vehicle. **P < 0.001; n = 5/group.

• 

  Fig. 5 TAK242 (TAK) or pyrrolidinedithiocarbamic acid (PDTC) reduces microglia specific marker ionized calcium binding adapter molecule-1 (Iba-1) expression in rats with nucleus pulposus (NP) (vs. sham, **P < 0.001; vs. NP + vehicle [veh], ##P < 0.001; n = 4/group). Scale bars (A-G) =100 μm. (H) The histogram shows the Iba-1 positive area of spinal dorsal horn in different groups. Error bars indicate standard error of means.

• 

  Fig. 6 Both TAK242 (TAK) and pyrrolidinedithiocarbamic acid (PDTC) reduce spinal expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), increase IL-10 expression of rats with nucleus pulposus (NP) (**P < 0.001; n = 4/group). Error bars indicate standard error of means. Veh: vehicle.

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