Dimethyl fumarate alleviates the nitroglycerin (NTG)-induced migraine in mice

Abstract

Background: Oxidative stress and inflammatory pathways are involved in migraine and endogenous antioxidant defense system has a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of the most pharmacologically effective molecules among the fumaric acid esters (FAEs), dimethyl fumarate, nuclear factor E2-related factor 2/antioxidant response element (Nrf-2/ARE) pathway-mediated, in regulating the hypersensitivity in a mouse model of nitroglycerine (NTG)-induced migraine.

Methods: Mice were orally administered with DMF at the doses of 10, 30, and 100 mg/kg, 5 min after NTG intraperitoneal injections. We performed histological and molecular analysis on the whole brain and behavioral tests after 4 h by NTG-migraine induction. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) subunit p65, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), inducible nitrite oxide synthase (iNOS), cyclooxygenase 2 (COX-2), Nrf-2, manganese superoxide dismutase (Mn-SOD), and heme-oxygenase-1 (HO-1) were detected by Western blot. Tail flick, hot plate, orofacial formalin, and photophobia tests were used to evaluate migraine-like pain and migraine-related light sensitivity. Moreover, we evaluate Nrf-2-dependent mechanism by the in vitro stimulation of cells extracted by trigeminal ganglia with diethylenetriamine/nitric oxide (DETA/NO), a nitric oxide (NO) donor. The cells were pre-treated with DMF and an antagonist of Nrf-2, trigonelline (TR) 2 h before DETA/NO stimulation.

Results: DMF treatment notably reduced histological damage as showed by cresyl violet staining; also, regulating both NF-κB and Nrf-2 pathway, DMF treatment decreased the severity of inflammation and increased the protective antioxidant action. Moreover, the headache was significantly reduced. The protective effect of DMF treatment, via Nrf-2, was confirmed in in vitro studies, through inhibition of Nrf-2 by trigonelline. Cytotoxicity, iNOS, and MnSOD expression were evaluated.

Conclusion: These results provided the evidence that DMF, by Nrf-2 modulation, has a protective effect on central sensitization induced by NTG, suggesting a new insight into the potential application of DMF as novel candidates in drug development for migraine.

Keywords: Dimethylfumarate; Migraine; NF-кb; Nrf-2.

Fig. 1

Effects of DMF treatment on NTG-induced damage. The neuronal damage in Sp5C neurons was observed in NTG-injured mice (B, B1 and F, F1) respect to control and control + sumatriptan group (respectively A, A1; C, C1; and F, F1). The treatment with DMF, mainly at the dose of 100 mg/kg, significantly restored a large number of trigeminal neurons in Sp5C area (E, E1). Data are means ± SEM of ten mice for each group. One-way ANOVA followed by Bonferroni post-test. ND not detectable. ***p < 0.001 vs. Sham; #p < 0.05 and ###p < 0.001vs. NTG, °°°p < 0.001 vs Sham. F value = 60.88

Fig. 2

Effects of DMF treatment on NTG-induced hyperalgesia. In tail flick test, DMF treatment at 30 and 100 mg/kg administered after NTG vehicle, caused a significant increase in tail curling latency (a) respect to NTG group (a); F values for treatment = 77.24. F value for time = 6.749. In Hot plate test, i.p. injection of NTG increased thermal hypersensitivity in a time-dependent manner (b), while DMF treatment, at both doses of 30 and 100 mg/kg, significantly increased the latency time to pain reaction related to the increase in time up to 240 min after NTG injection (b); F value = 8.468. In formalin test, NTG administration significantly increased the total number of flinches/shakes in phase I and II respect to control groups (c, d), while DMF treatment at 30 and 100 mg/kg significantly alleviated the nociceptive behavior induced by NTG administration during phase I and II of the test (c, d); F value (c) = 241.4; F value (d) = 148.3. Data are means ± SEM of 20 mice for each group. One-way ANOVA and two-way ANOVA followed by Bonferroni post-test. ***p < 0.001 and **p < 0.01 vs. Sham; ##p < 0.01 and ###p < 0.001vs. NTG. °°°p < 0.001, °°p < 0.01, and °p < 0.001 vs. Sham

Fig. 3

Effects of DMF treatment on NTG-induced anxiety and photophobia. EPM test was used to evaluate anxiety. NTG-injected mice showed anxiety stopping in closed arm (a), while DMF-treated mice spent more time in open arm (a); F values for treatment F (5, 798) = 4322; for time, F (6, 798) = 1360. In the same way, in NTG-injected mice, the number of entries in closed arm increased (b), while DMF treatment significantly augmented the residence time in open arm (b). In photophobia test, displaying migraine-like behaviors showed a strong tendency to stay longer in the dark zone respect to control mice (c), while NTG-injected mice, treated with DMF stayed more in the light zone (c). Data are means ± SEM of 20 mice for each group. One-way ANOVA and two-way ANOVA followed by Bonferroni post-test. ND not detectable. ***p < 0.001 vs. Sham; ##p < 0.01 and ###p < 0.001 vs. NTG. °°°p < 0.001 vs. Sham

Fig. 4

Effects of DMF on antioxidant system in NTG-induced migraine. The expression of Nrf-2, Mn-SOD, and HO-1was observed by Western blot analysis in whole brain with the rostral cervical spinal cord samples of mice, 4 h after NTG-injections. Respectively, nuclear Nrf-2 expression increased following NTG-injection as compared to sham and sumatriptan groups (A), while DMF administration, at 30 and 100 mg/kg, upregulated Nrf-2 activity (A); F value = 438.9. The same result was obtained for Mn-SOD expression (B); F value = 11.69. The levels of HO-1 were notably increased following NTG-damage compared to control groups (C), while DMF treatment, at 30 and 100 mg/kg, significantly up-regulated Mn-SOD and HO-1 expression (B and C); F value = 7.79. Data are means ± SEM of ten mice for each group. A representative blot of lysates obtained from each group is shown and densitometry analysis of all animals is reported (n = 10 mice from each group). One-way ANOVA followed by Bonferroni post-test. ##p < 0.01 and ###p < 0.01 vs. NTG; °p < 0.05, °°p < 0.01 and °°°p < 0.001 vs. Sham

Fig. 5

Effects of DMF on NF-κB inflammatory pathway in NTG-induced migraine. The expression of NF-κB and IκB-α was observed by Western blot analysis, in whole brain with the rostral cervical spinal cord samples of mice, 4 h after NTG-injections. We observed that NTG-injections caused a significant NF-κB nuclear translocation, almost completely inhibited by DMF 30 and 100 mg/kg treatment (A); F value = 165.6. Also, DMF treatment, only at the dose of 100 mg/kg, significantly reduced IκB-α cytosolic phosphorylation (B), while in NTG-injected mice the levels of IκB-α cytosolic phosphorylated were significantly increased respect to control groups (B); F value = 166.2. Data are means ± SEM of ten mice for each group. A representative blot of lysates obtained from each group is shown and densitometry analysis of all animals is reported (n = 10 mice from each group). One-way ANOVA followed by Bonferroni post-test. ***p < 0.001 vs. Sham; ##p < 0.01 and ###p < 0.001 vs. NTG. One-way ANOVA followed by Bonferroni post-test. ##p < 0.01 and ###p < 0.01 vs. NTG; °p < 0.05, °°p < 0.01, and °°°p < 0.001 vs. Sham

Fig. 6

Effects of DMF on iNOS and COX-2 expression in NTG-induced migraine. The expression of iNOS and COX-2 was observed by Western blot analysis, in whole brain with the rostral cervical spinal cord samples of mice, 4 h after NTG-injections. A significant increase in iNOS expression was observed in NTG group (A), while DMF treatment significantly reduced iNOS expression at 30 and 100 mg/kg (A); F value = 18.92. Alike, COX-2 expression was significantly elevated in NTG-injected mice in comparison to controls (B). The rise in COX-2 expression induced by NTG was considerably blocked by treatment with DMF 30 and 100 mg/kg (B); F value = 37.47. Data are means ± SEM of ten mice for each group. A representative blot of lysates obtained from each group is shown and densitometry analysis of all animals is reported (n = 10 mice from each group). One-way ANOVA followed by Bonferroni post-test. **p < 0.01 and ***p < 0.001 vs. Sham; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. NTG; °p < 0.05 and °°p < 0.01 vs. Sham

Fig. 7

Nrf-2 dependent mechanism of DMF. Cell death was assessed 24 h after treatment with increased concentrations of DMF (1–10–30–50–100 μM); the concentration of DMF at 1–10–30 μM resulted not cytotoxic (A). DETA/NO 100 μM significantly reduced cell viability compared with the control (Ctr); pre-treatment with DMF at 30 μM significantly prevented cell death (B). Also, co-incubation of DMF 30 μM with TR (1 μM), antagonized DMF cytoprotective effect (C). Western blot analysis demonstrated a significantly increasing of iNOS expression in DETA/NO stimulated cells. However, the pre-treatment with 30 μM DMF decreased iNOS expression (D), that was abolished in the presence of 1 μM TR (D). In the same way, the decreased Mn-SOD expression seen in DETA/NO-treated cells was recovered following pre-treatment with 30 μM DMF (E). TR inhibited DMF protective action (E). Data and figures are representative of at least three independent experiments. (B) ***p < 0.001 versus Ctr; ###p < 0.001 versus DETA/NO (C) ***p < 0.001 versus Ctr; ###p < 0.001 versus DETA/NO; °°°p < 0.001 versus Ctr; (D1) ***p < 0.001 versus Ctr; ###p < 0.001 versus DETA/NO; °°°p < 0.001 versus Ctr; (E1) ##p < 0.01 and ###p < 0.001 versus DETA/NO. F value (A) = 111.8; F value (B) = 101.2; F value (C) = 94.53; F value (D) = 166; F value (E) = 185.3

Fig. 8

Effects of DMF pre-treatment on NTG-induced damage. The neuronal damage in Sp5C neurons was observed in NTG-injured mice (B, B1 and F, F1) respect to control (A, A1 and F, F1). The pre-treatment with DMF did not restored damage in trigeminal Sp5C area (D, D1; E, E1 and F, F1). Data are means ± SEM of ten mice for each group. One-way ANOVA followed by Bonferroni post-test. ND not detectable. ***p < 0.001 vs. Sham; ###p < 0.001vs. NTG, °°°p < 0.001 vs. Sham. F value = 104.7