TLR3 mediates central sensitization in a chronic migraine model induced by repeated nitroglycerin through the ERK signaling pathway

Abstract

Background: Studies have demonstrated that Toll-like receptor 3 (TLR3) plays a crucial role in neuropathic pain. However, there have been no relevant reports regarding the role of TLR3 in migraine chronification. This study aims to investigate the molecular mechanisms of TLR3 in the central sensitization of chronic migraine (CM).

Methods: C57BL/6 male mice were used as models for chronic migraine (CM) disease, receiving an intraperitoneal injection of nitroglycerin (NTG) every other day. Calibrated von Frey filaments were employed to measure the pain threshold in the hind paw sole and periorbital region, enabling the assessment of mechanical allodynia. Western blot was employed to detect the expression changes of TLR3, TRAF6, TAK1, c-Fos, calcitonin gene-related peptide (CGRP), and the extracellular signal-regulated kinase (ERK) signaling pathway. Immunofluorescence was used to detect the cellular localization of TLR3 and the expression changes of central sensitization-related indicators, such as c-Fos and CGRP. In addition, we investigated the effects of TLR3 inhibitor (CU CPT4a), MEK inhibitor(PD98059), TRAF6 inhibitor(C25-140), and TAK1 inhibitor (Takinib) on chronic migraine-like behavior, and activation of the ERK pathway in the Trigeminal nucleus caudalis (TNC).

Results: Recurrent injections of NTG resulted in a significant increase in the expression of TLR3, TRAF6, TAK1, CGRP, and c-Fos proteins, as well as the activation of the ERK signaling pathway. Concurrent inhibition of TLR3 function, TRAF6, TAK1, and the ERK pathway counteracted these changes and alleviated hyperalgesia in CM mice.

Conclusions: Our findings suggest that TLR3 may play a role in central sensitization in CM mice by TRAF6-TAK1 axis modulating the ERK signaling pathway.

Keywords: ERK; TLR3; central sensitization; chronic migraine; neuron.

Figure 1.

Schematic diagram of mouse TNC site. TNC: Trigeminal nucleus caudalis. The black circle represents the TNC site of the mouse, and below is an enlarged cross-sectional view of TNC.

Figure 2.

After NTG administration, the expression of TLR3 in TNC neurons increased. (a) Double immunofluorescence labeling of TLR3, Iba-1, GFAP, and NeuN in the TNC after NTG administration. The first image on the far left shows a higher magnification image. Scale bar: 50 μm. The images on the right show an image with a higher magnification. Scale bar: 10 μm. (b) Quantitative analysis of the number of TLR3 with NeuN in the TNC within a 212 × 212 μm² visual field of each section per mouse after NTG injection. Data are presented as mean ± SEM; n = 4 per group, three sections from each mouse. ***p < 0.001 compared to the vehicle group.

Figure 3.

TLR3 antagonist CU CPT4a inhibited NTG-induced mechanical allodynia. Repeated treatment with CU CPT4a or vehicle (equal volumes of DMSO) every other day before NTG injections for 9 days (a). Repeated treatment with CU CPT4a inhibited basal hind paw mechanical (b) and periorbital mechanical allodynia (d). Post-treatment responses, including hind paw mechanical (e) and periorbital mechanical allodynia (c), were estimated 2 h after NTG administration. Data are presented as mean ± SEM; two-way ANOVA and Turkey post hoc analysis; n = 10 per group. **p < 0.01, ***p < 0.001 compared to the vehicle group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the NTG group.

Figure 4.

TLR3 antagonist CU CPT4a reduced the expression of TLR3 in TNC. (a, d) The first image on the far left shows the location of the TNC, marked with a white dashed box. Scale bar: 50 µm. The images on the right show images with higher magnification. Scale bar: 10 µm (a). Quantitative analysis of the number of TLR3+Neuron in the TNC in a 212 × 212 µm² visual field of each section per mouse after NTG injection (d); n = 4 per group, three sections from each mouse; (b, c) Immunoblot analysis of TLR3 (n = 5). Data are presented as the mean ± SEM; one-way ANOVA and Tukey’s post hoc tests. **p < 0.01, ***p < 0.001 compared to the VEH group; ##p < 0.01 compared to the NTG group.

Figure 5.

TLR3 blockade results in the downregulation of c-Fos and CGRP expression. (a–d) In the TNC, quantitative analysis of CGRP immunofluorescence intensity; Scale bars: 100 μm (a, b) and c-Fos immunoreactive cells; Scale bars: 10 μm (c, d). n = 4 for each group, each mouse was selected for three slices for analysis. (e–h) Immunoblot analysis of c-Fos and CGRP (n = 5). Data are presented as the mean ± SEM; one-way ANOVA and Tukey’s post hoc tests. **p < 0.01, ***p < 0.001 compared to the VEH group; ##p < 0.01 compared to the NTG group.

Figure 6.

TLR3 regulates the expression of c-Fos and CGRP by activating the ERK signaling pathway. (a–d) Immunoblot analysis was performed in the VEH, NTG, NTG+DMSO, and NTG+CU CPT4a groups for p-MEK1/2 to MEK1/2 ratio, p-ERK1/2 to ERK1/2 ratio, p-TAK1 to TAK1 ratio, and TRAF6 (n = 5). (e–f) Immunoblot analysis was performed in the VEH, NTG, NTG+DMSO, and NTG+PD98059 groups for p-ERK1/2 to ERK1/2 ratio, CGRP and c-Fos (n = 5). Data are the mean ± SEM; one-way ANOVA and Tukey’s post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001 versus the VEH group, and #p < 0.05, ##p < 0.01, ###p < 0.001 versus the NTG group.

Figure 7.

ERK inhibition attenuates NTG-induced pain hypersensitivity. PD98059 effectively prevented the decrease in both basal and acute mechanical withdrawal thresholds in the periorbital area (a, b) and the hind paw (c, d). Acute hypersensitivity reactions were detected 2 h after each NTG treatment. Data are presented as mean ± SEM; two-way ANOVA and Turkey post hoc analysis; n = 10 per group. **p < 0.01, ***p < 0.001 compared to the VEH group; #p < 0.05, ##p < 0.01 and ###p < 0.001 compared to the NTG group.

Figure 8.

TLR3 regulates the ERK signaling pathway by TRAF6/TAK1 axis. (a) Immunoblot analysis of p-TAK1 to TAK1 ratio, p-MEK to MEK ratio, p-ERK to ERK ratio, and c-Fos were conducted in the indicated groups (n = 5). (b) Immunoblot analysis of p-MEK to MEK ratio, p-ERK to ERK ratio, and c-Fos were conducted in the indicated groups (n = 5). Data are the mean ± SEM; one-way ANOVA and Tukey’s post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001 versus the VEH group, and #p < 0.05, ##p < 0.01, ###p < 0.001 versus the NTG group.

Figure 9.

Blocking TRAF6 and TAK1 alleviates CM-related pain hypersensitivity in CM mice. (a–h) C25-140 and takinib effectively prevented the decrease in both basal and acute mechanical withdrawal thresholds in the periorbital area and the hind paw. Acute hypersensitivity reactions were detected 2 h after each NTG treatment. Data are presented as mean ± SEM; two-way ANOVA and Turkey post hoc analysis; n = 10 per group. **p < 0.01, ***p < 0.001 compared to the VEH group; #p < 0.05, ##p < 0.01 and ###p < 0.001 compared to the NTG group.

Figure 10.

Schematic diagram of how TLR3 promotes central sensitization by TRAF6-TAK1 axis modulating the ERK signaling pathway.