MicroRNA-155-5p promotes neuroinflammation and central sensitization via inhibiting SIRT1 in a nitroglycerin-induced chronic migraine mouse model

Abstract

Background: Previous studies have confirmed that the microglial activation and subsequent inflammatory responses in the trigeminal nucleus caudalis (TNC) are involved in the central sensitization of chronic migraine (CM). MicroRNA-155-5p has been shown to modulate the polarization of microglia and participate in inflammatory processes in a variety of neurological diseases. However, its role in CM remains unclear. The purpose of this study was to determine the precise role of miR-155-5p in CM.

Methods: A model of CM in C57BL/6 mice was established by recurrent intraperitoneal injection of nitroglycerin (NTG). Mechanical and thermal hyperalgesia were evaluated by Von Frey filaments and radiant heat. The expression of miR-155-5p was examined by qRT-PCR, and the mRNA and protein levels of silent information regulator 1(SIRT1) were measured by qRT-PCR, Western blotting (WB) and immunofluorescence (IF) analysis. The miR-155-5p antagomir, miR-155-5p agomir, SRT1720 (a SIRT1 activator) and EX527 (a SIRT1 inhibitor) were administered to confirm the effects of miR-155-5p and SIRT1 on neuroinflammation and the central sensitization of CM. ELISA, WB and IF assays were applied to evaluate the expression of TNF-α, myeloperoxidase (MPO), IL-10, p-ERK, p-CREB, calcitonin gene-related peptide (CGRP), c-Fos and microglial activation. The cellular localization of SIRT1 was illustrated by IF.

Results: After the NTG-induced mouse model of CM was established, the expression of miR-155-5p was increased. The level of SIRT1 was decreased, and partly colocalized with Iba1 in the TNC. The miR-155-5p antagomir and SRT1720 downregulated the expression of p-ERK, p-CREB, CGRP, and c-Fos, alleviating microglial activation and decreasing inflammatory substances (TNF-α, MPO). The administration of miR-155-5p agomir or EX527 exacerbated neuroinflammation and central sensitization. Importantly, the miR-155-5p agomir elevated CGRP and c-Fos expression and microglial activation, which could subsequently be alleviated by SRT1720.

Conclusions: These data demonstrate that upregulated miR-155-5p in the TNC participates in the central sensitization of CM. Inhibiting miR-155-5p alleviates neuroinflammation by activating SIRT1 in the TNC of CM mice.

Keywords: Central sensitization; Chronic migraine; Inflammation; MiR-155-5p; Microglia; SIRT1.

Fig. 1

Time schedule of drug administration, behavioral testing, and sample collection. A The Sham and NTG group. B The NTG + miR-155-5p antagomir and NTG + agomir group. C The NTG + SRT1720 and NTG + EX527 group. D The NTG + miR-155-5p agomir + SRT1720 group. E The label of the symbols. Behavioral assessments were conducted before NTG injection or 3 days after antagomir/agomir administration. Tissues were collected within 24 h after the last NTG injection or 3 days after miR-155-5p antagomir/agomir administration

Fig. 2

Recurrent NTG injection induced hyperalgesia and upregulated the expression of CGRP, c-Fos. A The periorbital and paw mechanical threshold and thermal withdrawal latency during the injection of NTG. B, C The protein expression of CGRP and c-Fos in the TNC. D Counting of c-Fos was restricted to laminae I and II of the TNC region. E, F Immunofluorescence staining images of CGRP and c-Fos in the TNC. (n = 6–8 in each group; scale bar = 20/100 μm; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group)

Fig. 3

The administration of NTG increased miR-155-5p level and decreased the expression of SIRT1. A The expression level of miR-155-5p. B The mRNA expression of SIRT1. C The protein level of SIRT1. D The immunofluorescence intensity of SIRT1. E, F SIRT1 was partly coexpressed with Iba1, NeuN and GFAP in the TNC. After SRT1720 (a SIRT1 activator) treatment, the coexpression of SIRT1 and Iba1 increased, while the co-localization of SIRT1 and GFAP did not change. (n = 6–8 in each group; scale bar = 20 μm; **p < 0.01 and ****p < 0.0001 compared with the Sham group)

Fig. 4

The effects of miR-155-5p antagomir and agomir on hyperalgesia and the expression of CGRP, c-Fos. A The mechanical and thermal pain thresholds in different groups. Compared with the Sham group, pain thresholds significantly decreased in the NTG group. Compared with the NTG + anta NC group, the pain thresholds were notably increased in the NTG + miR-155-5p antagomir group. The pain thresholds in the NTG + miR-155-5p agomir group were reduced compared to NTG + ago NC group. B, C, D, E The protein expression of CGRP and c-Fos. Compared with the Sham group, the protein levels of CGRP and c-Fos notably increased in NTG group. Compared with the NTG + anta NC group, the expression of CGRP and c-Fos were decreased in the NTG + miR-155-5p antagomir group. The expression of CGRP and c-Fos in the NTG + miR-155-5p agomir group was significantly increased compared to NTG + ago NC group. F, G, H Immunofluorescence staining images of CGRP and c-fos. Compared with the Sham group, the immunofluorescence intensity of CGRP and number of c-Fos-positive cells evidently increased in NTG group. Compared with the NTG + anta NC group, the immunofluorescence intensity of CGRP and number of c-Fos-positive cells were markedly decreased in the NTG + miR-155-5p antagomir group. The immunofluorescence intensity of CGRP and number of c-Fos-positive cells in the NTG + miR-155-5p agomir group were increased compared to NTG + ago NC group. There were no significant differences among the NTG, NTG + anta NC and NTG + ago NC groups. (n = 6–8 in each group; scale bar = 20/100 μm; **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 5

The effects of miR-155-5p antagomir and agomir on the protein levels of p-ERK and p-CREB. A, B, C, D, E, F Compared with the Sham group, the expression of p-ERK and p-CREB-S133 increased in the NTG group. Compared with the NTG + anta NC group, the expression of p-ERK and p-CREB-S133 were notably decreased in the NTG + miR-155-5p antagomir group. The expression of p-ERK and p-CREB-S133 in the NTG + miR-155-5p agomir group were significantly increased compared to NTG + ago NC group. The total amount of ERK and CREB remained stable among the Sham, NTG, NTG + anta NC, NTG + antagomir, NTG + ago NC and NTG + agomir groups. (n = 6–8 in each group; ****p < 0.0001 compared with the Sham group; ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 6

The effects of miR-155-5p antagomir and agomir on the expression of SIRT1. A, B, C Compared with the Sham group, the protein level of SIRT1 notably decreased in NTG group. Compared with the NTG + anta NC group, the expression of SIRT1 was increased in the NTG + miR-155-5p antagomir group. The expression of SIRT1 in the NTG + miR-155-5p agomir group was significantly decreased compared to NTG + ago NC group. D, E The immunofluorescence intensity of SIRT1 in the NTG group was lower compared to the Sham group. MiR-155-5p antagomir and agomir resulted in similar effects on SIRT1 compared with the WB results. There were no significant differences among the NTG, NTG + anta NC and NTG + ago NC groups. (n = 6–8 in each group; scale bar = 20 μm; **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; #p < 0.05, ##p < 0.01 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 7

The effects of miR-155-5p antagomir and agomir on proliferation and morphological changes of microglia. A Immunofluorescence analysis of Iba1 was restricted to laminae I, II and III of the TNC region. The NTG group was selected to present the partition more clearly. B, C, D, E, F Compared with the Sham group, the number of Iba1-immunoreactive cells increased, while the total and mean length decreased in NTG group. Compared with the NTG + anta NC group, the Iba1-immunoreactive cells distinctly decreased, while the total and mean length were higher in NTG + miR-155-5p antagomir group. The total and mean length decreased in the NTG + miR-155-5p agomir group compared to NTG + ago NC group, and no significant difference was observed in Iba1-immunoreactive cells between the two groups. There were no significant differences among the NTG, NTG + anta NC and NTG + ago NC groups. (n = 6–8 in each group; *p < 0.05, **p < 0.01 and ****p < 0.0001 compared with the Sham group; #p < 0.05 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 8

The effects of miR-155-5p antagomir and agomir on microglial polarization and inflammatory substances. A, B, C, D The mRNA expression of M1-associated markers (CD86, iNOS) and M2-associated markers (CD206, Arg1). Compared with the Sham group, the mRNA levels of CD86 and iNOS notably increased, while the mRNA levels of CD206 and Arg1 decreased in NTG group. Compared with the NTG group, the mRNA expression of CD86 and iNOS distinctly decreased, while the CD206 and Arg1 levels increased in NTG + miR-155-5p antagomir group. The expression of CD86 and iNOS significantly was higher, while the CD206 and Arg1 levels were lower in the NTG + miR-155-5p agomir group than that in NTG group. E, G, F Compared with the Sham group, the expression of TNF-α,MPO increased, while the expression of IL-10 decreased in NTG group. Compared with the NTG + anta NC group, the expression of TNF-α and MPO decreased, while the IL-10 level increased in miR-155-5p antagomir group. The expression of TNF-α and MPO were higher in NTG + miR-155-5p agomir group than in the NTG + ago NC group, while the IL-10 level was lower. There were no significant differences among the NTG, NTG + anta NC and NTG + ago NC groups. (n = 5–8 in each group; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 9

The effects of SRT1720 and EX527 on pain thresholds, and the expression of CGRP. A, B The mechanical thresholds and thermal withdrawal latency in different groups. A Compared with the NTG + vehicle group, 5 mg/kg SRT1720 did not effect, while 20 mg/kg and 100 mg/kg SRT1720 both increased the pain thresholds. B Compared with the NTG + vehicle group, 5 mg/kg EX527 did not effect, while 10 mg/kg and 50 mg/kg EX527 both downregulated the pain thresholds. C, D The protein expression of CGRP in different groups. C Compared with the NTG + vehicle group, 5 mg/kg, 20 mg/kg and 100 mg/kg SRT1720 all downregulated the expression of CGRP. D Compared with the NTG + vehicle group, 5 mg/kg EX527 did not effect, while 10 mg/kg and 50 mg/kg EX527 both increasing the expression of CGRP. There were no significant differences between the NTG + SRT1720 (20 mg/kg) and NTG + SRT1720 (100 mg/kg) groups, as well as NTG + EX527 (10 mg/kg) and EX527 (50 mg/kg) groups. E Mechanical and thermal pain thresholds after miR-155-5p antagomir, agomir, SRT1720, EX527 treatment in the Sham group. No significant difference was observed in the mechanical and thermal pain thresholds among the NTG, NTG + vehicle, NTG + miR-155-5p antagomir, NTG + miR-155-5p agomir, NTG + SRT1720, NTG + EX527 groups. (n = 5–6 in each group; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 10

The effects of SRT1720 and EX527 on CGRP and c-Fos expression levels in the TNC. A, B, C Compared with the Sham group, the expression of CGRP and c-Fos increased in NTG group. The CGRP and c-Fos levels in the NTG + SRT1720 group were significantly reduced compared with that in the NTG + vehicle group. The CGRP and c-Fos expression levels were higher in the NTG + EX527 group than that in the NTG + vehicle group. D, E, F The average immunofluorescence intensity of CGRP and the relative number of c-Fos-positive cells both were both higher in the NTG group than in the Sham group. Compared with the NTG + vehicle group, the fluorescence intensity of CGRP and number of c-Fos-positive cells were markedly decreased in the NTG + SRT1720 group. The fluorescence intensity of CGRP and the number of c-Fos-positive cells in the NTG + EX527 group were significantly higher than that in the NTG + vehicle group. There was no significant difference between the NTG and NTG + vehicle groups. (n = 5–8 in each group; scale bar = 20/100 μm; **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 11

The effects of SRT1720 and EX527 on the phosphorylation of ERK and CREB. A, B, C, D The expression of p-ERK and p-CREB-S133 were both higher in the NTG group than in the Sham group. Compared with the NTG + vehicle group, the protein levels of p-ERK and p-CREB-S133 were markedly decreased in the NTG + SRT1720 group. The p-ERK and p-CREB-S133 expression levels were higher in the NTG + EX527 group than in the NTG + vehicle group. The total amount of ERK and CREB remained stable among these groups. There was no significant difference between the NTG and NTG + vehicle groups. (n = 6–8 in each group; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the Sham group; ##p < 0.01 and ###p < 0.001 compared with the NTG + NC groups)

Fig. 12

The effects of SRT1720 and EX527 on microglial activation and inflammatory responses. A, B, C, D Compared with the Sham group, the number of Iba1-immunoreactive cells increased, while the mean and total length of microglia processes decreased in NTG group. Compared with the NTG + vehicle group, the Iba1-immunoreactive cells markedly decreased, while the total and mean length were higher in NTG + SRT1720 group. There was a little difference in Iba1-immunoreactive cells, total and mean length between the NTG + vehicle and NTG + EX527 groups, without statistically significance. No significant difference was observed between the NTG and NTG + vehicle groups. E The expression of Iba1 and iNOS were both higher in the NTG group than in the Sham group. Compared with the NTG + vehicle group, the protein levels of Iba1 and iNOS were markedly decreased in the NTG + SRT1720 group. The Iba1 and iNOS expression levels were higher in the NTG + EX527 group than in the NTG + vehicle group. F, G, H Compared with the Sham group, the expression of TNF-α, MPO notably increased, and the expression of IL-10 decreased in NTG group. Compared with the NTG + vehicle group, the expression levels of TNF-α and MPO were lower, while the IL-10 level was higher in NTG + SRT1720 group. The expression of TNF-α and MPO were higher in NTG + EX527 group than that in the NTG + vehicle group, and the level of IL-10 decreased in NTG + EX527 group. There was no significant difference between the NTG and NTG + vehicle groups. (n = 6–8 in each group; *p < 0.05, **p < 0.01 and ****p < 0.0001 compared with the Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)

Fig. 13

SRT1720 attenuated the miR-155-5p agomir-evoked elevation of CGRP and c-Fos in the TNC. A, B, C The protein levels of CGRP and c-Fos were higher in the NTG + miR-155-5p agomir group than in the NTG group. Compared with the NTG + miR-155-5p agomir group, the expression of CGRP and c-Fos were significantly decreased in the NTG + miR-155-5p agomir + SRT1720 group. D, E, F The immunofluorescence analysis was consistent with WB results. The administration of SRT1720 dismissed the effort of miR-155-5p agomir on the expression of CGRP and c-Fos. (n = 6 in each group; **p < 0.01 and ****p < 0.0001 compared with the Sham group; #p < 0.05, ##p < 0.01 and ####p < 0.0001 compared with the NTG + NC groups; $$$$p < 0.0001 compared with the NTG + miR-155-5p agomir group)

Fig. 14

SRT1720 attenuated the miR-155-5p agomir-evoked upregulation of p-ERK, TNF-α, MPO and upregulation of IL-10. A, B Compared with the NTG group, the p-ERK protein levels were notably increased in the NTG + miR-155-5p agomir group. The p-ERK levels in the NTG + miR-155-5p agomir + SRT1720 group were significantly reduced compared to NTG + miR-155-5p agomir group. C, D Compared with the NTG group, the TNF-α and MPO levels were slightly increased in the NTG + miR-155-5p agomir group. The TNF-α and MPO levels in the NTG + agomir + SRT1720 group were substantially reduced compared to NTG + miR-155-5p agomir group. E The expression of IL-10 in the NTG + miR-155-5p agomir group was lower than that in the NTG group. Compared with the NTG + miR-155-5p agomir group, the IL-10 expression in the NTG + agomir + SRT1720 group was markedly increased. (n = 6 in each group; **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; #p < 0.05 and ###p < 0.001 compared with the NTG + NC groups; $$$$p < 0.0001 compared with the NTG + miR-155-5p agomir group)