Animals

Male mice were used throughout the study to eliminate possible hormonal cycle effects. SPF-grade wild-type (C57BL/6 J) mice (weighing 18–20 g, aged 6–8 weeks) were purchased from SiPeiFu Biotechnology Co, Ltd (Beijing, China). We maintained all the animals under standard conditions of 22 ± 2°C, 50 ± 10% relative humidity, alternating light, and dark cycles for 12 h, and provided them with adequate food and water. Every effort was made to minimize the number of animals used in the experiment and to minimize animal suffering. Prior to the experiment, all animals were allowed to acclimatize for one week and were then randomly assigned to different experimental groups according to a sequence of random numbers generated by Excel software. During the treatment period, the animals were weighed daily and no adverse effects were observed (Fig. S1).

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Chinese People's Liberation Army (PLA) General Hospital, and animal experiments were conducted according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Details of the procedures (drug administration, behavioural testing, sample collection) are show in Fig. 1.

Establishment of the chronic migraine model

Nitroglycerine (NTG) is the most common and recognized experimental migraine trigger in humans and rodents. NTG induces migraine-like attacks with nociceptive hypersensitivity and striatal site aversion [35]. We used NTG to investigate potential mechanisms associated with migraine. NTG (5 mg/ml in ethanol, Beijing Yimin Pharmaceutical Co., Ltd., Beijing, China) was freshly diluted in 0.9% (wt /vol) saline to a final concentration of 0.5 mg/ml for a dose of 10 mg/kg. In brief, the chronic migraine model was simulated by an intraperitoneal injection every other day (five injections in total), each at a dose of 10 mg/kg.ce.

Drug administration

According to the experimental requirements, mice were randomly divided into the following groups: (1) Control group, (2) NTG group, (3) Control + Oil group, (4) NTG + Oil group, (5) NTG + NBP (30mg/kg) group, (6) NTG + NBP (60mg/kg) group, (7) NTG + NBP (120mg/kg) group, (8) Control + Oil + ML385 group, (9) NTG + Oil + ML385 group, (11) NTG + NBP + Vehicle group, (12) NTG + NBP + ML385 group, NBP (dis-solved in soybean oil, provided by Shijiazhuang PharmaGroup NBP Pharmaceutical Co. Ltd., Shijiazhuang, Hebei Province, China). For simulated preventive treatment, NBP or an equivalent amount of solvent was administered by gavage 5 min before each NTG injection. The dose of NBP was derived from previous literature, these studies show that the drug is neuroprotective [28, 56, 57].

ML385 (Medchem Express, Monmouth Junction, New Jersey, USA) is a specific inhibitor of Nrf2, often used to reduce Nrf2 expression in mice. Mice were injected intraperitoneally with 30 mg/kg ML385 (dis-solved in saline containing 50% PEG300) 30 min before each NTG injection or equivalent amount of solvent. The dose of ML385 was determined from the literature [31].

Sensory sensitivity testing

Central sensitization stands as a pivotal mechanism contributing to the chronic nature of migraine. This phenomenon is believed to transpire sequentially within second and third-level neurons, culminating in the development of cutaneous abnormal pain with lateral and extracranial symptoms, which exhibit a similar spatial distribution. In light of this, we conducted an assessment of periorbital and hindpaw nociceptive hypersensitivity within the NTG mouse model. All behavioral evaluations were conducted within the hours of 09:00 to 15:00, within a Controlled environment characterized by low illumination and minimal auditory disturbance. Mice were subjected to a period of environmental acclimatization spanning three days before the initiation of experiments. It's important to note that all behavioral testing and subsequent data analyses were carried out by experimenters who were blinded to the specific treatments administered to the mice. In order to replicate prophylactic treatment, assessments were conducted prior to each NTG injection.

Hindpaw mechanical threshold

The quantification of hindpaw mechanical thresholds in mice was performed as previously described [45]. Mice were individually placed on a wire rack for 30 min to allow acclimatization. Eight von Frey (Aesthesio®, Danmic Global, San Jose, CA, USA) filaments (0.04–2 g) were applied to the center of the plantar surface of the hind paw at an initial stimulus intensity of 0.16 g. We considered a positive response if we identified shaking, twitching, or paw licking after the procedure. Each filament was applied to the skin for 3 s at least 1 min intervals. Mechanical thresholds were tested and calculated using an upward and downward approach; the right hind paw was stimulated five times, and three or more positive responses out of five stimuli were considered positive responses to that filament. If there was no response to that stimulus, the filament strength increased; otherwise, the filament strength decreased.

Periorbital mechanical threshold

To quantify head allodynia, the foreheads of the mice were shaved and the mice were acclimatized for at least 3 days. We performed the test by gently placing the mouse in the palm of the hand and applying von Frey filaments vertically to the shaved skin. All the other tests were performed in the same way as for plantar thresholds. Scratching the face with the forepaw and rapid withdrawal were considered positive responses.

Light-aversive test

Photophobia is a typical accompanying symptom of migraine, so we used a light–dark box (Shanghai Xinruan Information Technology Co., Ltd., XR-XB120, Shanghai, China) to test the photophobic performance of NTG model mice.

The box (30 cm wide × 30 cm deep × 30 cm high) consisted of two compartments of equal size: the light box was painted white and illuminated with LEDs, etc. (≥ 635 lx), while the dark box was painted black and unlit (≤ 5 lx). The channel (7 cm × 7 cm) connected the two compartments and allowed the mice to move freely. Each mouse was gently placed in the middle channel on the day of the test and the time spent in the light box was recorded over a 10-min period using the manufacturer's software. Light intensity settings are from previous studies [39]. To simulate prophylactic treatment, we tested each mice 24 h after the last dose.

Elevated plus maze (EPM) test

We used an elevated plus maze (EPM; Shanghai Xinruan Information Technology Co., Ltd., XR-XG201, Shanghai, China) to assess the anxiety-like behaviour associated with migraine. The apparatus was placed 60 cm above the floor and consisted of four arms (35 cm wide × 5 cm deep), two of which have 15 cm high walls forming a 'closed' arm, while the 'open' arm acts as an open runway. Mice were placed in the centre of the maze facing one of the open arms and allowed to freely explore the EPM for 5 min. Anxious rodents were more inclined to show greater avoidance of the open arms. Record how long the mice stayed in the open arm and how many times they entered the open arm using the manufacturer's software. To simulate prophylactic treatment, we tested each mice 24 h after the last dose.

Immunofluorescence staining

The mice were anaesthetized with 1.25% avertin and a transcranial perfusion with 40 ml PBS (pH 7.2–7.4) and 20 ml 4% paraformaldehyde (PFA). Hearts were fixed in PFA (4%) for 24 h at 4 °C and then dehydrated in 15% and 30% sucrose solution. Coronal sections of 30 μm were cut using a freezing microtome (Leica Biosystems, CM1950, Heidelberger, Germany). Sections were permeabilized with 0.25% Triton X-100 (Beyotime, Shanghai, China) for 10 min at room temperature, then blocked with 10% donkey serum (Boster, Wuhan, China) for 2 h at room temperature and incubated overnight at 4 °C with primary antibody diluted in blocking solution. The next day, the secondary antibody was incubated for 2 h, and 4′,6-diamidino-2-phenylindole (DAPI) was incubated for 10 min at 37 °C. The primary antibody was incubated with 50% glycerol. After sealing with 50% glycerol, we observed the sections using a fluorescence microscope (Olympus, Japan). Negative Control sections were stained with PBS instead of primary antibody and no positive signals were identified. The antibodies used are listed in Table 1.

Table 1 Antibodies used in Western blotting and immunofluorescence analysis

For CGRP, the fluorescence signal area ratio of the SP5C was analyzed using ImageJ software (Fiji, NIH, USA). For c-Fos, the number of c-Fos positive cells was quantified by taking the same field of view in the superficial layer of the SP5C at 20 × magnification.

Western blot analysis

Fresh SP5C tissues were subjected to homogenization using a cryomill (Servicebio, Wuhan, China) and radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (PMSF, Shanghai, China). The homogenates were pre-cooled to -30°C, with homogenization cycles of 30 s each (two cycles in total, with a 15-s interval). The protein concentration was quantified utilizing a bicinchoninic acid (BCA) protein analysis kit (YangGuangBio, Beijing, China). Equal amounts of proteins (30 μg per lane) were separated on 4%-20% gradient SDS-PAGE gels (ACE Biotechnology, Shanghai, China) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were initially blocked for 1 min at room temperature using 1-min Fast Blocking Buffer for Western (YangGuangBio, Beijing, China). Following this, an overnight incubation at 4°C was performed with the primary antibodies listed in Table 1. The subsequent day, the membranes underwent a series of three 10-min washes with TBST (YangGuangBio, Beijing, China) and were then subjected to incubation with the corresponding peroxidase-linked primary antibodies for 1 h at room temperature. Immunoblotted proteins were visualized utilizing an imaging system (Tanon, Shanghai, China) coupled with a SuperFemto ECL chemiluminescence kit (Vazyme, Nanjing, China). A comprehensive list of the antibodies employed can be found in Table 1.

ROS detection of SP5C

We took 20 mg of fresh SP5C tissue and added 400 μL of buffer to make a homogenate, which was homogenized using a cryo-mill (servicebio, Wuhan, China) and centrifuged at 4°C for 10 min. 200 μL of supernatant was collected and incubated with 20 μL of BBcellProbeTM O11 ROS probe (BestBio, China) in a 96-well plate at 37°C for 30 min. A fluorescent enzyme marker (Thermo Fisher Scientific, USA) was used to detect ROS levels with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. All samples were repeated 3 times.

Enzyme‑linked immunosorbent assay (ELISA)

We used specific ELISA kits to detect CGRP in mouse plasma (Sunshine Bio, Beijing, China). Orbital blood is closer to the central nervous system, so we used orbital blood to detect CGRP. Briefly, after the mice were anaesthetized with 1.25% Avertin, the eyeballs were immediately removed and the orbital blood was collected in an anticoagulation tube, immediately centrifuged and processed (4°C, 3000r, 10 min) and then quickly frozen in a -80°C refrigerator as a backup. For the assay, samples were added to the appropriate microenzymatic plate wells and conjugated with specific antibodies according to the instructions. Enzyme reagent was added, the plate was sealed with parafilm and incubated at 37°C for 60 min. After washing the wells with Wash Buffer, we added Colour Developer A and B to each well and incubated the samples for 15 min at 37°C. Finally, we added the termination solution to each well and read the optical density (OD) at 450 nm.

A specific ELISA kit was also used to detect TNF-α, IL-6, IL-1β, SOD and MDA and levels in SP5C (Meike, Shanghai, China). Tissues were weighed and added to PBS (pH 7.2–7.4, 0.01 mol/L) at a ratio of 1:9 and homogenized at -30°C. Samples were centrifuged at 5000 rpm for 15 min and the supernatant was collected. After that, we added the samples to the appropriate microplate wells and conjugated them with specific antibodies according to the instructions. After adding the enzyme reagent, the plate was sealed with a membrane and incubated at 37°C for 60 min. Then, we washed the wells with Wash Buffer, added Chromogen A and B to each well, and incubated the samples for 15 min at 37 °C. Finally, we add Stop Solution to each well and read the optical density (OD) at 450 nm.

Statistical analysis

All experiments and data analyses were performed by experimenters’ blind to the subgroups. Sample sizes were determined on the basis of previous experience and were all biological replicates. Data are expressed as mean ± standard error of the mean (S.E.M.). GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) was used for graph generation and statistical analysis. We also relied on an independent samples t-test to assess differences between the two groups. One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test were used for multiple group comparisons. Two-way repeated-measures analysis of variance (ANOVA) was used to analyze behavioural data. Two-tailed tests were used for all statistical analyses, and p < 0.05 was considered statistically significant.