CN113521046A - Application of sodium butyrate in preventing or preparing medicine for treating migraine - Google Patents

Abstract

The invention relates to the technical field of headache diseases, in particular to application of sodium butyrate in preventing or preparing a medicine for treating migraine. According to the invention, the sodium butyrate is adopted to exogenously prevent and intervene the rat model with the meningococcal stimulation, so that the sodium butyrate is observed and verified to have a treatment effect on pain caused by inflammatory stimulation, an action mechanism of the sodium butyrate is discussed, a preliminary definite result is obtained, and a certain guiding significance is provided for developing a new treatment means for clinical migraine.

Description

Application of sodium butyrate in preventing or preparing medicine for treating migraine

Technical Field

The invention relates to the technical field of headache diseases, in particular to application of sodium butyrate in preventing or preparing a medicine for treating migraine.

Background

Migraine (Migraine) is a common nervous system disease, is the second leading cause of human body disability, mostly occurs in children and adolescence, reaches the peak of morbidity in middle and young age, is common in women, has a ratio of 1: 2-3 for male and female patients, has a prevalence rate of 5% -10% in crowds, and often has a genetic background.

Frequent migraine attacks will affect the life and work of the patient, most directly sleep, because of insufficient sleep, no spirit in the daytime and work is greatly affected. And some patients often have the attack as soon as work, which is very delayed. Meanwhile, people suffer from headache diseases for a long time, and the temperament changes, so that the temperament becomes violent. Moreover, the patient can not be cured for a long time, the life is seriously affected, the mind is fragile, the confidence is lost, the heart and cerebral vessels of the human are adversely affected after the patient takes a long time, and cerebral thrombosis, hypertension and cerebral hemorrhage after the headache attack are common in clinic.

The treatment of migraine is aimed at alleviating or stopping the headache attack, alleviating the accompanying symptoms and preventing the headache recurrence. Treatment includes both drug treatment and non-drug treatment. The non-drug therapy mainly adopts physiotherapy such as magnetotherapy, oxygen therapy, psychological mediation, pressure relief, healthy living style maintenance and prevention of various migraine causes. Drug-based therapies are divided into episodic and prophylactic treatments. Treatment during the onset phase in order to achieve optimal efficacy, the drug should generally be taken immediately at the onset of symptoms. Therapeutic agents include non-specific analgesics such as non-steroidal anti-inflammatory drugs (NSAIDs) and opioids, and specific drugs such as ergot preparations and triptans.

At present, no specific treatment method is available, migraine can be eradicated, and the most effective treatment mode is to prevent induction factors in the interval period of migraine.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the application of sodium butyrate in preventing or preparing medicines for treating migraine.

With the progress of the brain-gut axis research in recent years, it has been found that there is a strong association between migraine and intestinal diseases, part of the mechanism of which is related to the enhancement of pro-inflammatory immunity. And butyric acid is used as a metabolite of intestinal flora and has the effects of resisting inflammation, regulating immunity and the like. It was found that oral butyrate treatment in chronic crush injury models as well as acute visceral pain and other pain animal models could improve pain. Under the research background, the invention adopts sodium butyrate exogenous preventive intervention in a rat model with meningococcal stimulation, observes and verifies that the sodium butyrate has a treatment effect on pain caused by inflammatory stimulation, discusses an action mechanism of the sodium butyrate exogenous preventive intervention, obtains a preliminary exact result, and has a certain guiding significance for developing a new treatment means for clinical migraine.

The migraine in the present invention is not limited to humans, but may be other mammals.

The invention adopts a single-time scleromeningitis stimulation rat model, and discusses the influence of sodium butyrate intervention on pain-related behaviors of migraine model rats through grouping and setting different time-point behavioral observations. The sodium butyrate intervention improves pain-related behaviors of rats after a single scleromeningitis stimulation, including baseline pain domains, wet dog shaking times, unilateral hair management, head scratching time and the like.

Further, the migraine is a headache caused by a scleromeningitis.

The invention researches the influence of sodium butyrate on cell inflammatory reaction of a migraine model rat TNC region, observes the expression of rat brain PAG region and TCC region Fos protein induced by inflammation soup stimulation, TCC region NF-kB pathway protein activation and inflammatory factor IL-1 beta expression change, and verifies that the intervention of the sodium butyrate can improve pain of the inflammation soup dura mater stimulated rat and an action mechanism thereof. The sodium butyrate can play a role in improving pain by inhibiting NF-kB channel activation of cells in the TNC region and inhibiting inflammatory reaction.

Further, the sodium butyrate has the effect of reducing the amount of Fos-positive expression of PAG region, TNC region, and UCSC region.

Further, the sodium butyrate inhibits inflammatory response by inhibiting NF-kB channel activation of cells in the TNC region.

Specifically, the sodium butyrate has the effects of reducing the expression of NF-kappa B p65 active fragments in the brain, increasing the expression of I kappa B in the brain and reducing the expression of p-I kappa B in the brain.

Further, the sodium butyrate has the function of reducing the high expression of IL-1 beta in the brain so as to achieve the effect of inhibiting inflammatory response.

A method of treating or preventing a migraine headache disorder, comprising administering to a subject an effective amount of sodium butyrate.

Among them, migraine is headache caused by the scleromeningitis.

Sodium butyrate is useful for treating or preventing migraine headaches.

Among them, migraine is headache caused by the scleromeningitis.

An effective amount of a therapeutic agent for treatment or prevention refers to an amount of the therapeutic agent that treats, ameliorates, or prevents the target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the symptoms, and the therapeutic agent and/or combination of therapeutic agents selected for administration. The administration can be oral, intravenous, in vivo or transdermal, and the drug is administered to the patient in need of treatment, and the specific dosage and administration can be determined by the physician according to the patient's condition.

Further, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the auxiliary materials comprise any one or more of fillers, disintegrants, diluents, excipients, binders, wetting agents, emulsifiers, lubricants, surface active agents, flavoring agents, stabilizers and the like.

Further, the dosage forms of the medicine comprise oral preparations and injections.

Further, the oral preparation comprises tablets, pills, capsules, paste, granules and syrup.

Further, the injection includes a solution type and a powder type.

The pharmaceutical dosage form provided by the invention is prepared according to the conventional method in the field. For example, in order to prepare granules, tablets, pills, etc. from sodium butyrate, various pharmaceutically acceptable excipients known in the art, such as diluents, wetting agents, disintegrants, binders, lubricants, glidants, etc. may be used; the diluent can be sucrose, starch, dextrin, microcrystalline cellulose, calcium hydrogen phosphate, calcium carbonate lactose, mannitol, xylitol, etc.; the humectant can be ethanol, water, isopropanol, etc.; the binder can be syrup, starch slurry, acacia slurry, dextrin, sodium carboxymethylcellulose, microcrystalline cellulose, gelatin slurry, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, acrylic resin, carbomer, etc.; the disintegrant may be crosslinked polyvinylpyrrolidone, sodium carboxymethyl starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be stearate, tartaric acid, liquid paraffin, talc, silicon dioxide, polyethylene glycol, and the like.

In addition, the tablets may be further formulated into coated tablets, such as enteric coated tablets, sugar coated tablets, film coated tablets, or double-layer tablets and multi-layer tablets.

Such as: in order to prepare the medicament into capsules, the effective component of the invention can be mixed with a diluent and a glidant, and the mixture is directly placed into hard capsules or soft capsules. Or mixing the effective components with binder, diluent, and disintegrating agent, making into granule or pellet, and placing into hard capsule or soft capsule. The diluents, disintegrants, glidants, binders and wetting agents used to prepare the compound tablets of the present invention may also be used to prepare the compound capsules of the present invention.

For another example: in order to prepare the effective components of the invention into injection, ethanol, water, propylene glycol or the mixture of the ethanol, the water and the propylene glycol can be used as a solvent, and proper amounts of cosolvent, solubilizer, osmotic pressure regulator and pH regulator which are commonly used in the field can be added. The solubilizer or cosolvent can be lecithin, hydroxypropyl-beta-cyclodextrin and the like; the pH regulator can be sodium hydroxide, acetate, etc.; the osmotic pressure regulator can be sodium chloride, glucose, phosphate, mannitol, acetate, etc. For example, glucose and mannitol can be added as proppant for preparing lyophilized powder for injection.

In addition, preservatives, flavors, colors, flavors, or other additives may also be added to the pharmaceutical preparation, if desired.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) the invention discovers that the sodium butyrate has good treatment or prevention effect on the migraine disease for the first time.

(2) According to research, the sodium butyrate can play a role in improving pain by inhibiting NF-kB pathway activation of cells in a TNC region and inhibiting inflammatory reaction.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a graph showing the change in mechanical pain domain values of various groups of rats at different time points in example 1 of the present invention;

FIG. 2 is a graph showing the comparison of the wet dog shaking frequency of rats in each group according to example 1 of the present invention;

FIG. 3 is a graph showing comparison between unilateral hair management and tip scratching time of rats in each group according to example 1 of the present invention;

FIG. 4 is a graph showing comparison of the expression profiles of Fos in rat PAG regions of respective groups by the immunofluorescence staining method in example 1 of the present invention;

FIG. 5 is a graph showing comparison of Fos expression levels of TNC regions in rats of various groups by immunofluorescence staining in example 1 of the present invention;

FIG. 6 is a graph showing comparison of Fos expression in the high cervical medullary region of rats in each group by immunofluorescence staining in example 1 of the present invention;

FIG. 7 is a graph showing comparison of the expression of Fos proteins in the brain of mice in each group by the WB method in example 2 of the present invention;

FIG. 8 is a graph showing comparison of the expression levels of I κ B proteins in the brains of various groups of rats by the WB method in example 2 of the present invention;

FIG. 9 is a graph showing comparison of the expression of p-I κ B proteins in the brains of various groups of rats by the WB method in example 2 of the present invention;

FIG. 10 is a graph showing the comparison of the expression of NF- κ B proteins in the brains of various groups of rats by the WB method in example 2 of the present invention;

FIG. 11 is a graph showing comparison of the levels of IL-1. beta. inflammatory factor in the brain of various groups of rats by ELISA method in example 2 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

1. Experimental Material

1.1 Experimental animals

The study was enrolled in SPF male (SD) strain rats weighing 180 + -20 grams. Purchased from the animal center of the general hospital of the people's liberation force in China. All the operation processes of animal experiments are carried out in the clean animal laboratory of the Chinese people liberation army. In the experimental process, rats freely eat drinking water, and the illumination controls the day and night alternation of the laboratory, the room temperature is 18-26 ℃, and the humidity is (55 +/-2)%.

1.2 Experimental drugs and reagents

1.2.1 sodium butyrate 303410/Sigma-Aldrich, 98% pure.

1.2.2 dural administration

Inflammation soup (inflammation soup, IS): the following components were dissolved in physiological saline at the following mass concentrations.

1) 5-hydroxytryptamine H9523/sigma, 452.36 mg/L;

2) histamine V900396/sigma, 222.3 mg/L;

3) bradykinin B3259/sigma, 2120.42 mg/L;

4) prostaglandin E2P 5640/sigma, 70.494 mg/L.

1.2.3 anesthetic drugs

Pentobarbital sodium (provided by animal experiment center of general hospital of people's liberation military in China) is prepared into 30mg/ml with physiological saline, and is preserved in dark.

1.2.4 reagents for cardiac perfusion, immunofluorescence staining

PBS powder (Beijing China fir bridge Biotechnology Co., Ltd.), acetone, OCT embedding medium (Sakura Finetek Co., Ltd.), TritonX-100 (Shanghai Solebao Biotechnology Co., Ltd.), goat serum for sealing (Beijing Bilian Yuntai Biotechnology Co., Ltd.), Fos primary antibody (ab222699/Abcam Co., Ltd.), Alexa Flour488(ab150077/Abcam Co., Ltd.).

1.3 laboratory instruments and apparatus

1.3.1 surgical articles

Disposable syringes, brain stereotaxic apparatus (shenzhen ruivor science and technology life limited), scalpels, forceps, ophthalmic scissors, dental drills and drills (korea SAESHIN PRECISION), suture needles, needle holders, tissue scissors, hemostatic forceps, gavage needles, infusion sets, and the like.

1.3.2 related devices for micro-administration

Micro-administration cannula system (Shenzhen Riwode Life technologies, Ltd.), micro-injector (10 μ l)

1.3.3 mechanical pain area determination apparatus

Von-frey fiber yarn, North Coast Medical, USA.

1.3.4 Immunofluorescent (IF) related devices

Cleaning slides and coverslips, slide racks, water baths, high temperature ovens, microtomes (Leica CM 1950/germany), immunohistochemical oil pens, micropipettes and tips, shakers, fluorescence microscopes and imaging systems (Olympus/japan), etc.

2. Experimental methods

2.1 Experimental grouping and intervention methods

Rats with consistent behavioral baselines were assigned to the Sham group (Sham group), the inflammation soup model group (IS group), the sodium butyrate high dose intervention group (SB400 group) (400mg/kg), the sodium butyrate medium dose intervention group (SB200 group) (200mg/kg), the sodium butyrate low dose intervention group (SB100 group) (100mg/kg), and the sodium butyrate intervention Sham group (Sham + SB400 group) (400mg/kg) using a random number table (random numbers generated by SPSS22.0 statistical software), with 8 per group, for a total of 48 rats.

2.2 model preparation

Placing a tube on the surgical skull: weighing and anaesthetizing a rat, and fixing the rat on a rat brain stereotaxic apparatus; after the head is preserved, the skin is disinfected for 3 times by iodophor, an incision with the length of 1.5cm is made at the position about 2cm before the connecting line of the two ears in the middle of the vertex of the head, and the skull beside the superior sagittal sinus is exposed after the subcutaneous tissue, the muscle and the periosteum are separated layer by layer. A small hole with the diameter of about 1mm is drilled at the position which is 1.5mm on the left side of the median line of the skull and is 1.5mm away from the intersection point of the median line of the skull and a coronal suture by a dental drill to serve as a cannula implantation hole, and the hole just can penetrate the skull but needs to ensure the integrity of dura mater. Inserting the cannula into the cannula implantation hole, simultaneously screwing two small screws into the screw fixing holes carefully, placing a proper amount of dental powder carefully in the triangular area, dripping a proper amount of dental water by using an injector to promote the solidification, and then suturing and disinfecting the skin. During and after operation, warm keeping is paid attention to, and the animal is prevented from dying due to low body temperature. The rats were raised in a single cage after surgery for a recovery period of 6 days.

The drug administration process through the cannula system: placing a rat cage on an operation table, opening a cage cover, after the rat adapts for a moment, fixing the head of the rat gently, unscrewing a screw cap and pulling out a catheter core, inserting an injection inner tube of a microinjector which is connected with inflammation soup/normal saline sucked in advance through a PE (polyethylene) hose into a head single tube, and fixing the injection inner tube by using a locking screw cap; under the condition that the rat can freely move, the injection is slowly injected (about 5 minutes), the reaction of the rat is noticed in the process, and the liquid extravasation caused by too fast injection is avoided; slightly waiting for a moment after injection, slightly screwing off the lock nut and inserting the catheter cap after the liquid completely enters the epidural, closing the cage cover and returning to the original position.

2.4 methods of administering sodium butyrate

The main intervention drug "sodium butyrate" in this study was a purification chemical, supplied by Sigma-Aldrich. The early research proves that the sodium butyrate has a remarkable anti-inflammatory effect and shows a good analgesic effect in certain animal models. In this section, sodium butyrate was used to prophylactically intervene in the migraine arterial model and observe the ameliorating effect of sodium butyrate on headache.

The administration method comprises the following steps: sodium butyrate is prepared into suspension by using physiological saline, and each group of rats is quantitatively perfused with the suspension every day according to the experimental design, 1 time a day and 5 days.

2.5 Experimental procedure

Firstly, the animal is adaptively trained for 3 days, the periorbital baseline pain area of the face and the ethological video are measured for 30min, the operation is performed for catheterization on the 4 th day, and the postoperative recovery period is 6 days. Rats were given 1 intragastric intervention (sodium butyrate suspension for the sodium butyrate intervention group and the same dose of saline for the other groups) periodically daily for 5 days starting on day 10; on 5 days of drug administration intervention, namely 14 days of the whole experimental process, the drug IS infused into the stomach after 1 hour of the measurement of a baseline pain area, epidural drug administration modeling IS carried out after 1 hour (the inflammation soup model group and the sodium butyrate intervention group give IS, and the sham operation group give physiological saline with the same dose), the change trend of the pain threshold value (1 hour, 2 hours, 3 hours and 4 hours) and the behavior video (30min-1 hour, 1.5-2 hours, 2.5-3 hours and 3.5-4 hours) are measured after the modeling, and at 5 hours after the modeling, animals are killed to take materials for subsequent research.

2.6 behavioral observations

2.6.1 mechanical stimulation pain Domain determination

Testing periorbital mechanical pain domain threshold values by using Von Frey, wherein the pressure values of the Von Frey fiber filaments comprise: 2g, 4g, 6g, 8g, 10g, 15g and 26 g. The pressure values of the fiber filaments were measured from low to high, and the fiber filaments were pressed vertically against the periorbital skin of the rat (5 points periorbital were selected, including two each periorbital and one in the center of the forehead) and pressure was applied until the fiber filaments were bent, and the bent state of the fiber filaments was maintained for at least 5 seconds or the rat showed a positive reaction. The positive reaction comprises the following steps: the head is retracted and avoided, the front paw repeatedly combs the head and the face, and the fiber is stimulated to make a sound. When the rat shows a positive reaction to the stimulation of the cellosilk with a certain pressure value, the cellosilk with the first grade is changed down for measuring again; if the rat is positive in at least 3 of 5 positions under the stimulation of the fiber yarn with a certain pressure value, the pressure value is the mechanical stimulation pain sense threshold value of the rat; if the weight of the fiber increases to 26g, the rats are not yet positive, and the pain threshold is recorded as 26g (peak-off value).

As a result: mechanical stimulus pain threshold comparison

The influence of different doses of sodium butyrate on the pain response of rats is monitored through the change of the pain domain of rats at different time points after the stimulation of the dura mater drug. As can be seen from FIG. 1 (comparison of IS group with sham group, # indicates P < 0.01; comparison of sodium butyrate intervention group with inflammation soup model group, # indicates P <0.01), the baseline level of rat pain domains was consistent in each group and the mean difference was not statistically significant in each group before the experiment (0 h). Pain domain values and change trends of rats in different time points (1h, 2h, 3h and 4h) after model building have difference due to difference of different intervention factors, and have statistical significance. Compared with a sham operation control group, after the rats in the inflammation soup model group receive IS dura stimulation, the pain threshold value IS obviously lower than that of a normal group (P IS less than 0.01) within 1 hour, and the threshold value of the sham operation and high-dose sodium butyrate intervention group IS not different from that of the control group; compared with the inflammation soup model group, the pain threshold values of each dose of sodium butyrate intervention group after the model is made are higher than those of the inflammation soup model group in different degrees, wherein the threshold values of the medium dose and high dose groups at each time point are higher than those of the inflammation soup model group and have statistical difference (P is less than 0.01), and the change of the threshold values and the intervention effect of the sodium butyrate show a dose-related trend.

2.6.2 behavioral observations

The behavior of the rats was recorded by a video camera and the final video was viewed and recorded by an observer blinded to the experimental group. The observation indexes comprise wet dog type shaking times and one-side hair combing and head scratching time within 4 hours after rat model making.

(1) Comparison of Wet dog shaking frequency within 4 hours after dura stimulation in rats of each group

The number of wet dog shakes was used as the pain-related behavior of the model rats and the differences between groups of rats were monitored over 4 hours after molding. As can be seen from FIG. 2(IS group, # means P <0.01 compared to sham group; sodium butyrate intervention group, # means P <0.01 compared to inflammation soup model group), the number of wet dog shakes 4 hours after molding was statistically different for each group of rats under different intervention conditions. Compared with a control group, the wet dog shaking times of rats in the inflammation soup model group are obviously higher than those in a normal group (P is less than 0.01), and the wet dog shaking times in 4h in the sham operation group and the high-dose sodium butyrate intervention group are not statistically different from those in the control group; compared with the inflammation soup model group, the wet dog shaking times of the sodium butyrate intervention group with different doses are obviously lower than that of the inflammation soup model group (P is less than 0.01), and the action effect is dose-related.

(2) Comparison of unilateral hair management and head scratching behavior time within 4 hours after dura mater stimulation of rats in each group

The unilateral hair-conditioning behavior time within 4 hours after rat model building is adopted to observe the influence of sodium butyrate on pain-related behaviors of rats after model building. As can be seen from FIG. 3(IS vs. sham, # means P < 0.05; Na butyrate intervention vs. inflammation soup model, # means P < 0.05; and # means P <0.01), the individual groups of rats had statistically significant differences in the single period of hair management behavior at 4 hours post-molding under different intervention factors. Compared with a sham operation group, the single hair management times of the rats in the inflammation soup model group are obviously higher than those in a normal group (P is less than 0.05), and the single hair management times of the sham operation group and the high-dose sodium butyrate intervention group are not statistically different from those of the sham operation group; compared with the inflammation soup model group, the wet dog shaking times of the sodium butyrate intervention group with different dosages are obviously lower than those of the inflammation soup model group (P is less than 0.05 or P is less than 0.01), and a certain dosage correlation trend is presented.

2.7 murine intracerebral Fos protein expression assay

2.7.1 method for obtaining materials

4 rats in each group are randomly extracted by a drawing method, 3% sodium pentobarbital (60mg/kg, namely 0.2ml/100g) is injected into the abdominal cavity to be anesthetized, the chest is opened, after the heart and ascending aorta are fully exposed, Phosphate Buffer Solution (PBS) precooled in advance at 4 ℃ is adopted for heart perfusion for 2 times, the whole brain tissue of the rat is quickly stripped on ice after blood is flushed, and the rat is subjected to OCT embedding and immediately placed into liquid nitrogen to be quickly frozen for about 10 s. According to a rat brain atlas, coronal sections of three parts, namely the periaqueduct gray (PAG), the caudal side of trigeminal spinal nucleus (TNC) and the superior cervical spinal cord (UCSC), are respectively carried out by using a freezing microtome, the thickness of each section is 10 mu m, 3 sections are taken out every 10 sections, the sections are attached to a prepared anti-falling glass sheet, the sections are placed into acetone precooled at the temperature of minus 20 ℃ for fixing for 10min, and the acetone is placed into a refrigerator at the temperature of minus 70 ℃ for storage and standby.

2.7.2 Observation of Fos protein expression in rat brain by immunofluorescence staining method

Fos protein expression conditions of three parts of PAG, TNC and UCSC are observed by adopting an immunofluorescence technique, and the brief steps are as follows:

firstly, taking out the slide, rewarming for 15min, soaking and washing for 5min by PBS (phosphate buffer solution) for 2 times, sealing by 10% goat serum sealing liquid for 1 hour at room temperature, adding a primary antibody (1:2000) into the sealing liquid by spin-drying, and incubating overnight at 4 ℃; taking out from a refrigerator at 4 ℃, rewarming for 30min at normal temperature, soaking for 10min in PBS (3 times), preparing a secondary antibody (1:2000) under dark room conditions, incubating for 2 hours in dark environment after dropwise adding the secondary antibody, throwing off the secondary antibody, dropwise adding a DAPI coloring agent, dropwise adding DAPI30s-1min, placing in PBS solution, soaking and washing for 10min in dark for 2 times, dropwise adding a proper amount of anti-fluorescence quenching sealing tablet, sealing, and placing the section under an OLYMPUS fluorescence microscope for observation.

The statistical method of the number of immunofluorescence Fos positive cells comprises the following steps: under a fluorescence microscope, for a sample, randomly selecting three areas under a 20-fold microscope, counting the number of positive cells in the areas, and finally taking the average value as the number of Fos positive cells in the area.

As a result: immunofluorescence staining method for determining Fos expression in each brain region

(1) Expression of Fos in PAG region

Compared with a sham operation group, the inflammation soup model group has obviously increased positive cell number of Fos in a rat PAG zone and has statistical significance (P < 0.05); the positive expression quantity of Fos in PAG region of rat in each dose intervention group of sodium butyrate is reduced in a dose-dependent trend compared with the inflammation soup model group, wherein the reduction of the medium and high dose groups is more obvious, and the positive expression quantity is statistically significant (P <0.05) compared with the inflammation soup model group (figure 4). In fig. 4, IS group, # indicates P <0.05 compared to sham group; sodium butyrate intervention group compared to inflammation soup model group indicates P < 0.05.

(2) Fos expression of TNC region

Compared with a sham operation group, the number of positive cells of Fos in the TNC region of rats in the inflammation soup model group is obviously increased and has statistical significance (P is less than 0.01); compared with the inflammation soup model group, the positive expression quantity of Fos in the TNC region of rats in each dose intervention group of the sodium butyrate is reduced, has statistical significance (P <0.01) and shows a dose-dependent reduction trend (figure 5). In fig. 5, # indicates that P <0.05 in the IS group compared with the sham group; sodium butyrate intervention group showed P <0.01 compared to inflammation soup model group.

(3) UCSC region Fos expression

Compared with a sham operation group, the inflammation soup model group has obviously increased number of positive cells of the UCSC region Fos of the rats and has statistical significance (P < 0.01); compared with the inflammation soup model group, the positive expression quantity of Fos in UCSC areas of rats in each dose intervention group of the sodium butyrate is reduced, has statistical significance (P <0.01) and shows a dose-dependent reduction trend (figure 6). In fig. 6, IS group, # # indicates P <0.01 compared to sham group; sodium butyrate intervention group showed P <0.01 compared to inflammation soup model group.

Wherein the statistical analysis described above was performed using IBM SPSS Statistics19.0 software. All data were tested for normality using the Kolmogorov-Smirnov test. For samples satisfying a normal distribution, one-way analysis of variance (ANOVA) was used for the comparisons among groups, the homologies were confirmed by the test of variance, the least difference method (LSD method) was used for the comparisons among post-test groups, and otherwise, the Dunnett' T3 method was used for post-pairwise comparisons. If the data distribution does not match the positive-too distribution, then a nonparametric test is used for the overall comparison and the interclass post comparison. Data used were recorded as Mean ± standard deviation (Mean ± SD) with P <0.05 as the standard for statistical differences.

Example 2

Inhibition of NF-kB inflammatory pathway in rat trigeminal nerve complex (TCC) cells by unilateral inflammatory dura mater

In recent years, there is increasing evidence that stress-induced immune/inflammatory responses play a potential role in migraine pathophysiology. The process and specific mechanism of the stress-induced activation of the central and peripheral immune systems and the enhancement of inflammatory responses of the body have gradually attracted attention. Studies have shown that activation of NF-. kappa.B pathways in the brain is involved in the pathogenesis of migraine. Sodium butyrate has shown strong anti-inflammatory and immunomodulatory effects in existing studies. The results of example 1 suggest that the "sodium butyrate" intervention has a significant improvement in pain-related behavior in inflammatory dural-stimulated rats, the specific mechanism of which remains to be explored. Therefore, based on example 1, the present study observed the expression of Fos protein in rat brain, NF- κ B inflammatory pathway protein in trigeminal complex (TCC) cells, and the expression of inflammatory factor IL-1 β after inflammatory dural stimulation, and explored the mechanism of pain improvement in rats with single inflammatory dural stimulation by the prophylactic treatment of "sodium butyrate".

1. Experimental Material

1.1 Experimental animals

The study was enrolled in SPF male (SD) strain rats weighing 180 + -20 grams. Purchased from the animal center of the general hospital of the people's liberation force in China. All the operation processes of animal experiments are carried out in the clean animal laboratory of the Chinese people liberation army. In the experimental process, rats freely eat drinking water, and the illumination controls the day and night alternation of the laboratory, the room temperature is 18-26 ℃, and the humidity is (55 +/-2)%.

1.2 Experimental drugs and reagents

1.2.1 surgical and perfusion sampling drugs and reagents are the same as in example 1 and will not be described again.

1.2.2 protein extraction reagent

Extraction kit of nuclear protein and cytoplasmic protein (P0028/Biyuntian biology, Inc.), PMSF (ST 506/Biyuntian biology, Inc.), BCA protein concentration determination kit (enhanced) (P0010/Biyuntian biology, Inc.), SDS-PAGE protein loading buffer 5X (P0015/Biyuntian biology, Inc)

1.2.3 Western Blot (WB) related reagents

Tris, glycine, SDS, methanol, NaCl, HCl, 30% acrylamide, ammonium persulfate, TEMED, western primary anti-diluent (P0256/Biyuntan Bio Inc.) pre-staining protein color Marker (Thermo Fisher Scientific Inc/USA), WB primary antibody (anti-Fos ab222699// Abcam, anti-I κ B sc-1643/Santa Cruz, anti-P-I κ B sc-8404/Santa Cruz, anti-P65sc-8008/Santa Cruz, anti-Actin AA128/Beyotime, anti-Histone H3 AF0009/Beyotime), skim milk, horseradish enzyme-labeled goat anti-mouse IgG (ZB-2305/Beijing Bio Inc.), horseradish-labeled goat anti-rabbit IgG (enhanced chemiluminescence assay kit in ZB-4/Beijing Biotechnology, Inc.) (P2302/Beijing Biochemical technologies, Biond 230719/Beijing Biotechnology, Biond Biotech, Inc./Biotech, detection kit (P2309/Biotech, Inc.), And western blot membrane regeneration solution (P1650/Beijing prilley Gene technology Co., Ltd.).

1.2.4 ELISA-related reagents

RatIL-1 beta ELISA kit (96T) RLB00/R & D Systems

1.3 laboratory instruments and apparatus

A cell mechanical crushing instrument, a vortex oscillator, a low-temperature high-speed centrifuge, a micro-pipetting gun and a gun head and a 2.0ml/1.5ml centrifuge tube; 96-well plate, 37 deg.C incubator, enzyme-labeling instrument, water bath, ultralow temperature refrigerator, pH value tester, micro-pipetting gun and gun head, electrophoresis apparatus, electrophoresis tank, electric transfer tank, PVDF membrane, Tanon-5200 chemiluminescence imager, etc.

2. Experimental methods

2.1 Experimental grouping and intervention methods

Rats with consistent behavioral baselines were assigned to the Sham group (Sham group), the inflammation soup model group (IS group), the sodium butyrate high dose intervention group (SB400 group) (400mg/kg), the sodium butyrate medium dose intervention group (SB200 group) (200mg/kg), and the sodium butyrate low dose intervention group (SB100 group) (100mg/kg) using a random number table (random numbers generated by SPSS22.0 statistical software), with 4 per group, for a total of 20 rats.

2.2 model preparation, administration methods and experimental procedures are the same as example 1 and will not be described herein.

2.3 method of obtaining materials

Materials obtained by ELISA method and Western Blot method

After deep anesthesia, 3% sodium pentobarbital (60mg/kg, namely 0.2ml/100g) is injected into the abdominal cavity of each group of rats, the chest is opened, after the heart and ascending aorta are fully exposed, Phosphate Buffer Solution (PBS) precooled in advance at 4 ℃ is adopted for cardiac perfusion for 2 times, the whole brain tissue of the rats is quickly stripped on ice after blood is flushed, the protein of the brain stem (containing high cervical medulla) tissue is extracted according to the instruction of a nuclear protein and cytoplasmic protein extraction kit, the whole operation process is carried out on ice, and the extracted protein is stored at-80 ℃ for later use.

2.4 index detection

2.4.1 WB assay for Fos protein expression in brain

Protein expression of Fos of brainstem (including high cervical pulp section) is detected by WB staining technology, and histone HistoneH3 is used as an internal reference.

2.4.2 WB assay of inflammatory pathway protein expression in brain

Detecting the expression of I kappa B, p-I kappa B (phosphorylated I kappa B) and NF-kappa B p65 proteins of a brain stem (including a high-position cervical marrow section) by adopting a WB staining technology, wherein in cytoplasm, the expression of I kappa B, p-I kappa B (phosphorylated I kappa B) and NF-kappa B p65 are respectively detected by taking beta-actin as an internal reference; in the nucleus, expression of NF- κ B p65 was measured using Histone Histone H3 as an internal control.

The WB brief procedure is as follows:

adding 5% of concentrated gel and 10% of separation gel, loading the gel, performing electrophoresis at constant pressure of 90V on the concentrated gel and 120V on the separation gel, performing membrane transfer (170mA constant current transfer for 70min) in a low-temperature environment, taking out a PVDF membrane, cutting, sealing with 5% of skimmed milk powder at the constant temperature of 37 ℃ for 1H, and then performing primary antibody (anti-actin 1: 4000; anti-p-I kappa B1: 200; anti-I kappa B1: 100; anti-Histone H31: 2000; anti-p 651: 100; anti-Fos1:2000) incubation at 4 ℃ overnight; the next day the membranes were removed from the primary antibody solution, washed 4 times with TBST, and incubated for a secondary antibody (1:4000) in the dark at room temperature for 1 h. Washing the membrane for 4 times by TBST, then taking out the PVDF membrane in a dark environment, immediately putting the PVDF membrane into an exposure box for exposure after dripping luminous liquid, and then operating a photographing record on a computer.

2.4.3 detection of the level of the inflammatory factor IL-1 beta in the brain by ELISA

And (3) detecting the content of the IL-1 beta in different groups of brain stem cell plasma proteins by using an enzyme-linked immunosorbent assay (ELISA) method. Immediately after the reaction was terminated, the optical density value (optical density, o.d.) was read at 450nm using a microplate reader, and the IL-1 β content in the brains of rats of different groups was compared.

3. Results

3.1 WB method for determining Fos protein expression in brain of rats in each group

As shown in fig. 7 (in the IS group, # means P <0.01 compared to the sham operation group, # means P <0.05 compared to the inflammation soup model group in the sodium butyrate intervention group (in the SB200 group and SB400 group)), the amount of Fos protein expression in the brain of rats in the inflammation soup model group was significantly increased and statistically significant (P <0.01) compared to the sham operation group; compared with the inflammation soup model group, the expression quantity of Fos in the brain of rats of each dose intervention group of sodium butyrate is reduced to different degrees, wherein the reduction of the medium and high dose groups is more obvious and has statistical significance (P is less than 0.05).

3.2 WB method for determining NF-kB pathway protein expression condition in brains of various groups of rats

As shown in FIGS. 8-10, the expression of NF-kappa B P65 active fragments in nuclei in brain of rats in the inflammation soup model group was significantly increased (P <0.01) compared with the sham operation group, the expression of NF-kappa B P65 active fragments was decreased after the sodium butyrate intervention group, and the decrease was statistically significant compared with the inflammation soup model group; compared with the control group, the expression of I kappa B (NF-kappa B inhibitor) of the rats in the inflammation soup model group is obviously reduced, the expression of p-I kappa B (phosphorylated NF-kappa B inhibitor) is obviously increased, and compared with the inflammation soup model group, the expression of I kappa B (NF-kappa B inhibitor) and the expression of p-I kappa B (phosphorylated NF-kappa B inhibitor) of each dosage group intervened by butyric acid are increased, wherein the difference of medium and high dosage groups has statistical significance.

Wherein, # in fig. 8 means that P <0.05 in the IS group compared with the sham group; sodium butyrate intervention groups (SB200 and SB400 groups) indicated P <0.05 compared to the inflammation soup model group.

In fig. 9, # indicates that P <0.05 in the IS group compared with the sham group; sodium butyrate intervention groups (SB200 and SB400 groups) indicated P <0.05 compared to the inflammation soup model group.

In fig. 10, IS group, # indicates P <0.05 compared to sham group; the sodium butyrate intervention groups (SB200 and SB400 groups) indicated P <0.05 and P <0.01, compared to the inflammation soup model group.

3.3 changes in the expression of the inflammatory factor IL-1 beta in the brains of various groups of rats

As shown in the results of fig. 11, the proinflammatory cytokine IL-1 β in the brain of rats in the inflammation soup model group is significantly increased (P <0.01) compared with the sham operation group; compared with the inflammation soup model group, the sodium butyrate intervention dose-dependently reduces the high expression of IL-1 beta in the brain of the rat, and has statistical significance. In fig. 11, # # indicates P <0.01 in the IS group compared to the sham group; the sodium butyrate intervention group showed P <0.05, and P <0.01, compared to the inflammation soup model group.

Statistical analysis was performed using IBM SPSS statisticss 19.0 software. All data were tested for normality using the Kolmogorov-Smirnov test. For samples satisfying a normal distribution, one-way analysis of variance (ANOVA) was used for the comparisons among groups, the homologies were confirmed by the test of variance, the least difference method (LSD method) was used for the comparisons among post-test groups, and otherwise, the Dunnett' T3 method was used for post-pairwise comparisons. If the data distribution does not conform to the normal distribution, then a nonparametric test is used for overall comparison and interclass post-event comparison. Data used were recorded as Mean ± standard deviation (Mean ± SD) with P <0.05 as the standard for statistical differences.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. The application of sodium butyrate in preventing or preparing medicines for treating migraine.

2. The use of claim 1, wherein the migraine is headache caused by a scleromeningitis.

3. The use according to claim 1, wherein the sodium butyrate has the effect of reducing the amount of Fos-positive expression of PAG, TNC and UCSC regions.

4. The use according to claim 1, characterized in that said sodium butyrate suppresses the inflammatory response by inhibiting the activation of the NF-. kappa.B pathway of cells in the TNC domain.

5. The use according to claim 4, wherein the sodium butyrate has the effects of reducing the expression of a fragment of NF- κ B p65 activity in the brain, increasing the expression of I κ B in the brain, and reducing the expression of p-I κ B in the brain.

6. The use according to claim 1, wherein the sodium butyrate has the effect of reducing the high expression of IL-1 β in the brain in order to inhibit the inflammatory response.

7. The use according to any one of claims 1 to 6, wherein the medicament further comprises a pharmaceutically acceptable excipient.

8. The use according to claim 7, wherein the adjuvants comprise any one or more of fillers, disintegrants, diluents, excipients, binders, wetting agents, emulsifiers, lubricants, surfactants, flavoring agents, stabilizers, and the like.

9. The use according to any one of claims 1 to 6, wherein the pharmaceutical dosage form comprises oral and injectable formulations.

10. The use according to claim 9, wherein the oral formulation comprises tablets, pills, capsules, pastes, granules, syrups;

the injection includes a solution type and a powder type.

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