The P2Y13 receptor-mediated microglial morphological transformation through the p38MAPK signaling pathway contributes to central sensitization in a murine model of chronic migraine

Abstract

Background: Central sensitization is a crucial pathophysiological mechanism of chronic migraine (CM), and neuroinflammation mediated by activated microglia contributes significantly to the development of central sensitization. The P2Y13 receptor (P2Y13R), belonging to the G protein-coupled receptor family, is expressed in microglia and actively participates in the intricate pathophysiological process underlying chronic neuropathic pain. However, the precise relationship between the P2Y13R and CM remains largely unclear.

Methods: The CM mouse model was established by repeatedly injecting nitroglycerin (NTG) intraperitoneally at intermittent intervals, and the pain threshold was assessed using von Frey fiber and hot plate tests. Specific interventions were conducted on the P2Y13R and p38 MAPK signaling pathways in the Trigeminal Nucleus Caudalis (TNC) of mice through stereotactic injection. Western blotting and immunofluorescence were employed to assess the expression and localization of P2Y13R, c-Fos, calcitonin gene-related peptide (CGRP), components of the p38 MAPK signaling pathway, and inflammatory factors.

Results: The expression of P2Y13R was significantly upregulated upon NTG administration and exhibited a predominant distribution within the microglia in the TNC in mice with CM. Pharmacological inhibition of P2Y13R effectively reduced hyperalgesia in CM mice, lowered CGRP and c-fos levels, thereby improving central sensitization. Furthermore, inhibiting P2Y13R suppressed microglial activation and pro-inflammatory cytokine production. Additionally, activation of the p38MAPK pathway was observed in the TNC of CM mice, with P2Y13R inhibition significantly reducing p38MAPK pathway activity. Pharmacological inhibition of p38MAPK significantly ameliorates central sensitization in CM mice, while suppressing microglial activation and pro-inflammatory cytokine levels.

Conclusions: This research emphasizes the significance of P2Y13R in mediating central sensitization in CM mice through the p38MAPK pathway, thereby suggesting that targeting P2Y13R holds promise as a potential therapeutic strategy for effectively managing CM.

Keywords: Central sensitization; Microglia; P2Y13R; Stereotactic injection; p38MAPK.

Fig. 1

P2Y13R is present in microglia and shows significant upregulation after repeated NTG injections. a, b Western blotting analysis of P2Y13R on different days after NTG injection (n = 5 mice / group). c The yellow dotted box delineates the area of TNC. d Dual immunofluorescence staining of P2Y13R with Iba1, GFAP, NeuN (n = 4 mice / group). Scale bar: 20 μm. The values represent the mean ± SEM; statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc tests; ***p < 0.001 vs. the sham group

Fig. 2

P2Y13R Inhibition alleviates mechanical and thermal hyperalgesia caused by NTG. Measurement of hind paw (a, b), periocular (c, d) mechanical threshold, and thermal withdrawal latency (e, f). n = 10, the values represent the mean ± SEM; statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc tests. ***p < 0.001 vs. the VEH group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the NTG group

Fig. 3

P2Y13R inhibition led to a notable reduction in the levels of c-fos and CGRP expression. a Schematic diagram of microinjection in the TNC region in mice. b, c Western blotting analysis of P2Y13R (n = 5 mice per group). d-f Western blotting analysis of c-fos and CGRP (n = 5 mice / group). g, h Immunofluorescence analysis of CGRP (n = 4 mice / group). i, j Immunofluorescence analysis of c-fos (n = 4 mice / group). The values represent the mean ± SEM; statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc tests; ***p < 0.001 vs. the VEH group; ^###p < 0.001 vs. the NTG group

Fig. 4

P2Y13R affects the morphological alterations of activated microglia and the production of inflammatory factors. a Immunofluorescence staining of Iba1 in different groups. b Enlarged image of microglia in the white dotted box in a. c-g Quantification of the number of Iba1⁺ microglia, immunoreactivity of Iba1, total length of initial processes, average length of initial processes and soma size in microglia (n = 4 mice / group). h-k. Western blotting analysis of IL-1β, IL-6, TNF-α (n = 5 mice / group). Values are presented as the mean ± SEM; statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc tests; ***p < 0.001 vs. the VEH group; ^###p < 0.001 vs. the NTG group

Fig. 5

P38MAPK inhibition significantly reduced the upregulation of c-fos, CGRP induced by NTG. a Dual immunofluorescence staining of p-p38 with Iba1 (n = 4 mice / group). Scale bar: 50 μm. b Immunofluorescence analysis of p-p38⁺ cells among Iba1⁺ cells. c-f Western blotting analysis of p-p38 to p38 ratio (n = 5 mice / group). g-i Western blotting analysis of c-fos and CGRP (n = 5 mice / group). j, k Immunofluorescence analysis of CGRP (n = 4 mice / group). l, m Immunofluorescence analysis of c-fos (n = 4 mice / group). Values are presented as the mean ± SEM; statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc tests; **p < 0.01, ***p < 0.001 vs. the VEH group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the NTG group

Fig. 6

The inhibition of p38MAPK significantly improved pain hyperalgesia. Measurement of hind paw (a, b), periocular (c, d) mechanical threshold, and thermal sensitivity (e, f). n = 10, values are presented as the mean ± SEM; statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc tests; ***p < 0.001 vs. the VEH group; ^##p < 0.01, ^###p < 0.001 vs. the NTG group

Fig. 7

The p38MAPK signaling pathway regulates microglial activation and inflammatory responses induced by NTG. a Immunofluorescence staining of Iba1 in different groups. b Enlarged image of microglia in the white dotted box in a. c-g Quantification of the number of Iba1⁺ microglia, immunoreactivity of Iba1, total length of initial processes, average length of initial processes and soma size in microglia (n = 4 mice / group). h-k Western blotting analysis of IL-1β, IL-6, TNF-α (n = 5 mice / group). Values are presented as the mean ± SEM; statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc tests; ***p < 0.001 vs. the VEH group; ^###p < 0.001 vs. the NTG group