Molecular mechanism research method regulated by metabotropic glutamate receptor 5

Abstract

The invention discloses a molecular mechanism research method regulated by a metabotropic glutamate receptor 5, and relates to the field of medicine. Comprising the following steps: early-stage preparation: preparing experimental animals and experimental reagents; animal model establishment: constructing a chronic migraine animal model; animal administration: performing injection administration on the lateral ventricle of the animal; the method comprises the following steps: carrying out a behavioral experiment, carrying out mechanical pain threshold value and heat pain threshold value test, RNA extraction and RT-qPCR detection on an animal, extracting an RNA sample by using an RNAiso Plus reagent to carry out TNC part total RNA extraction, and carrying out reverse transcription on RNA into cDNA by using reverse transcription reagents ABScript III RT mix and gDNARevermix to be used for RT-qPCR detection, Westernblot experiment, immunofluorescence experiment and electron microscope experiment. The invention lays a solid foundation for diagnosis and treatment of chronic migraine.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a research method for the molecular mechanism regulated by metabotropic glutamate receptor 5. Background Art

[0002] Migraine, recognized by the World Health Organization (WHO) as a highly disabling neurovascular disease, has a global incidence rate as high as 14%, and among all patients, approximately 5% may progress to chronic migraine (CM). This disease not only seriously affects the daily life of patients but also may lead to long-term other related complications and economic burdens. Research indicates that in the pathogenesis of chronic migraine, central sensitization in the trigeminal nucleus caudalis (TNC) plays a crucial role. The emergence of this central sensitization makes the processing of pain signals more sensitive and persistent, further exacerbating the pain perception of patients.

[0003] Multiple studies have confirmed that by regulating the level of oxidative stress, the central sensitization state of chronic migraine can be effectively improved. In recent years, oxidative stress has gradually been considered an important inducer of chronic migraine, and it is closely related to the generation of reactive oxygen species (ROS) and the imbalance of antioxidant capacity. Mitochondrial hyperfission mediated by Drp1 is considered the core mechanism leading to the increase in ROS levels, thereby causing damage and dysfunction of the nervous system. Metabotropic glutamate receptor 5 (mGluR5), as a G-protein coupled receptor, has been found in recent studies that it not only plays a key role in the process of pain regulation but also can mediate the generation of ROS.

[0004] Although the diagnosis rate of migraine has been increasing in recent years, more than half of migraine patients still have not received corresponding diagnoses. Coupled with insufficient preventive treatment and abuse of acute analgesic drugs, most migraine patients are dissatisfied with the treatment effect.

[0005] Therefore, the present invention proposes a research method for the molecular mechanism regulated by metabotropic glutamate receptor 5. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose a research method for the molecular mechanism regulated by metabotropic glutamate receptor 5.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] A research method for the molecular mechanism regulated by metabotropic glutamate receptor 5, comprising the following steps:

[0009] S1: Preliminary preparation, preparing experimental animals and experimental reagents;

[0010] S2: Establishment of an animal model, constructing an animal model of chronic migraine;

[0011] S3: Administer the drug to the animal by injecting it into the lateral ventricle of the animal.

[0012] S4: Conduct behavioral experiments to test the mechanical pain threshold and thermal pain threshold of the animal.

[0013] S5: Extract total RNA from the sample and detect the expression of mGluR5 by RT-qPCR.

[0014] S6: Detect the protein expression of CGRP, mGluR5, ERK, p-ERK, p-Drp1(s616) in the TNC region by Western blot experiment.

[0015] S7: Immunofluorescence experiment. After deeply anesthetizing the animal with pentobarbital sodium by intraperitoneal injection, isolate the TNC tissue for fluorescence experiment to detect the protein expression of CGRP, c-Fos, p-Drp1(s616), and the cellular localization of mGluR5.

[0016] S8: Electron microscopy experiment. Take out the tissue in the TNC region for electron microscopy experiment to observe the morphological changes of mitochondria.

[0017] S9: Detect the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the tissue of the TNC region, and calculate the relative levels of the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) to evaluate the level of oxidative stress.

[0018] S10: After repeating the above steps at least three times, conduct data analysis.

[0019] Preferably: In the S1 step, the experimental animal is an adult male rat.

[0020] Preferably: In the S1 step, the experimental reagents include prostaglandin E2, bradykinin BK, histamine HA, 5-hydroxytryptamine, 6-methyl-2-(phenylethynyl)pyridine hydrochloride (MPEP), (RS)-2-chloro-5-hydroxyglycine (CHPG), RNA rapid extraction kit, reverse transcription reagent, RT-qPCR, reagents related to Western blot, rabbit anti-rat CGRP, c-Fos, mGluR5, p-ERK, ERK, p-Drp1(s616) antibodies, and mouse anti-rat GAPDH, β-Tubulin, NeuN antibodies, malondialdehyde MDA content detection kit, superoxide dismutase SOD activity detection kit.

[0021] Preferably: The reagents related to Western blot include SDS-PAGE gel preparation kit, electrophoresis buffer, electrotransfer buffer, TBST buffer, 5X sample buffer, protein marker, transfer membrane, blocking solution, and antibodies.

[0022] Preferably, the step S2 includes the following steps:

[0023] S21: First, achieve deep anesthesia by intraperitoneal injection of 50 mg / kg sodium pentobarbital;

[0024] S22: Subsequently, fix it on the stereotaxic apparatus; precisely incise along the midline of the scalp to fully expose the skull;

[0025] S23: After positioning, drill a hole in the skull, and the positioning coordinates are 1.0 mm behind the midline and 1.5 mm lateral;

[0026] S24: Subsequently, insert a sterile cannula with a sealing cap into the drilled hole and firmly fix it above the dura mater;

[0027] S25: After the operation, recover for 7 days, and inject 5 μL of inflammatory soup IS into the dura mater for 7 consecutive days. At the same time, inject an equal amount of sterile PBS into the rats in the sham operation group. The inflammatory soup is composed of 0.1 mM prostaglandin E2, 1 mM serotonin, 1 mM bradykinin, and 1 mM histamine.

[0028] Preferably, in the step S3, the operation steps are as follows: On the 7th day after IS injection, inject 250 nM of the drug CHPG and 2 μg of MPEP with a 5 μL injection volume through intracerebroventricular injection. The injection coordinates are 1.0 mm behind the cerebrospinal fluid, 1.5 mm lateral to the midline, and 4.5 mm subdural. The injection time is 10 minutes. The control group is injected with 5% DMSO.

[0029] Preferably, in the step S4, use von Frey filaments to test the plantar surface of the hind paw three times, with a five-minute interval each time. During the test, press the filament on the hind paw for up to five seconds or until the hind paw retracts. If the hind paw retracts, use a thinner filament in the next test; if the hind paw does not retract, use a thicker filament for the next test. According to the positive reaction of its retraction, record the corresponding value. Finally, take the average of the three measurement results to determine the mechanical pain threshold of the hind paw. The determination of the thermal pain threshold is through the following method. During the operation, let the hind paw of the rat receive radiant heat stimulation until the rat lifts its paw. If the paw does not lift within 25 seconds, stop the stimulation and record the lifting duration. This test is repeated three times, with an interval of at least five minutes each time. Finally, take the average of the three measurement results to determine the thermal pain threshold of the hind paw.

[0030] Preferably, the step S6 includes the following steps:

[0031] S61: Take out the trigeminal caudal nucleus TNC tissue and quickly place it in liquid nitrogen for cryopreservation;

[0032] S62: Subsequently, the tissue was homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors;

[0033] S63: After incubating the homogenate on ice for 1 hour, it was centrifuged at 4°C for 15 minutes; the protein concentration was measured using a BCA protein quantification kit;

[0034] S64: Equal amounts of protein samples were loaded onto a 10% SDS-PAGE gel for electrophoresis separation, and then transferred to a PVDF membrane;

[0035] S65: The transferred membrane was blocked with 5% non-fat milk at room temperature for 1 hour, and then incubated with the primary antibody overnight at 4°C;

[0036] S66: Incubated with the secondary antibody at room temperature for 1 hour. Finally, an enhanced chemiluminescence (ECL) kit and a Fusion system were used for image acquisition, and the immunoreactive bands were developed and analyzed.

[0037] Preferably, the step S7 includes the following steps:

[0038] S71: After deeply anesthetizing the rats with pentobarbital sodium by intraperitoneal injection, the rat hearts were rinsed successively with an equal volume of ice-cold phosphate-buffered saline (PBS) and 4% paraformaldehyde solution;

[0039] S72: The trigeminal caudal nucleus (TNC) tissue was isolated and fixed in 4% formaldehyde solution at 4°C for 12 hours, and then dehydrated with 20% and 30% sucrose solutions respectively;

[0040] S73: Then the TNC tissue was sectioned to obtain coronal sections with a thickness of 15 μm;

[0041] S74: Antigen retrieval was performed on the sections using sodium citrate buffer, and permeabilization was carried out with 0.3% Triton X-100 at 37°C for 10 minutes;

[0042] S75: After blocking with a solution containing 10% goat serum at 37°C for 30 minutes, the sections were incubated with the primary antibody overnight at 4°C, then incubated with the fluorescent secondary antibody at 37°C for 1 hour, and finally stained with DAPI for 10 minutes;

[0043] S76: After mounting with 50% glycerol, the sections were observed under a confocal laser scanning fluorescence microscope. For quantitative analysis, ImageJ software was used to measure the immunofluorescence intensity and count the number of c-fos immunoreactive cells.

[0044] Preferably, the step S8 includes the following steps:

[0045] S81: After the experimental animals were deeply anesthetized, they were perfused with pre-cooled PBS and then treated with 2.5% glutaraldehyde solution;

[0046] S82: After the brain tissue was quickly removed, it was cut into tissue blocks of 1 mm 3 and immersed in 4% pre-cooled glutaraldehyde for fixation;

[0047] S83: Use a JEM-1400PLUS transmission electron microscope for observation, manually depict the outline of mitochondria in the image, calculate its area and perimeter, and evaluate the degree of mitochondrial fragmentation using the ratio of area to perimeter (connectivity);

[0048] S84: Finally, perform statistical analysis on the obtained area, perimeter, and connectivity data.

[0049] The beneficial effects of the present invention are as follows:

[0050] 1. Through the research method of the molecular mechanism regulated by metabotropic glutamate receptor 5, by establishing an animal experimental model, drug administration, behavioral experiments, RNA extraction and RT-qPCR detection, Western blot experiments, immunofluorescence experiments, electron microscopy experiments, malondialdehyde content, superoxide dismutase activity detection, etc., the present invention studies the molecular mechanism by which mGluR5 promotes oxidative stress and central sensitization in chronic migraine rats through the activation of Drp1 phosphorylated by ERK. It can be known that the down-regulation of mGluR5 alleviates oxidative stress and central sensitization by inhibiting Drp1-mediated mitochondrial fission phosphorylated, and the regulatory effect of mGluR5 on mitochondrial fission may involve ERK, thus laying a solid foundation for the diagnosis and treatment of migraine. Description of the Drawings

[0051] Figure 1 This is the statistical control chart group showing enhanced hyperalgesia and up-regulated CGRP and c-Fos expression in CM rats of the present invention;

[0052] Figure 2 This is the statistical control chart group showing increased expression of mGluR5, phosphorylated ERK, and Drp1 in the TNC region of CM rats of the present invention;

[0053] Figure 3 This is the statistical control chart group showing the effects of mGluR5 on oxidative stress, hyperalgesia, and the expression levels of c-Fos and CGRP in the chronic migraine (CM) rat model of the present invention;

[0054] Figure 4 This is the statistical control chart group showing the effects of mGluR5 on the phosphorylated expression levels of ERK and Drp1 and the morphology of mitochondria of the present invention. Detailed Embodiments

[0055] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments.

[0056] In the description of the present invention, it should be noted that, unless otherwise clearly defined and limited, the terms "installation", "connection", "connection", and "setting" should be understood in a broad sense. For example, it can be fixedly connected and set, or detachably connected and set, or integrally connected and set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0057] Example 1:

[0058] A research method for the molecular mechanism regulated by metabotropic glutamate receptor 5, which includes the following steps:

[0059] S1: Preliminary preparation, preparing experimental animals and experimental reagents;

[0060] S2: Establishment of an animal model, constructing a chronic migraine animal model;

[0061] S3: Administration to animals, injecting drugs into the lateral ventricles of animals;

[0062] S4: Behavioral experiments, testing the mechanical pain threshold and thermal pain threshold of animals;

[0063] S5: Extraction of total RNA from samples and detection of the expression of mGluR5 by RT-qPCR;

[0064] S6: Western blot experiment to detect the protein expression of CGRP, mGluR5, ERK, p-ERK, p-Drp1(s616) in the TNC region;

[0065] S7: Immunofluorescence experiment, after deeply anesthetizing the animals with pentobarbital sodium by intraperitoneal injection, separating the TNC tissue for fluorescence experiment to detect the protein expression of CGRP, c-Fos, p-Drp1(s616), and the cellular localization of mGluR5;

[0066] S8: Electron microscopy experiment, taking out the tissue in the TNC region for electron microscopy experiment to observe the morphological changes of mitochondria;

[0067] S9: Detecting the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the TNC region tissue, calculating the relative levels of the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) to evaluate the oxidative stress level;

[0068] S10: After repeating the above steps at least three times, perform data analysis.

[0069] In the step S1, the experimental animals are adult male rats.

[0070] The experimental reagents include prostaglandin E2, bradykinin BK, histamine HA, 5-hydroxytryptamine, 6-methyl-2-(phenylethynyl)pyridine hydrochloride (MPEP), (RS)-2-chloro-5-hydroxyglycine (CHPG), RNA rapid extraction kit, reverse transcription reagent, RT-qPCR, reagents for Western blotting, rabbit anti-rat CGRP, c-Fos, mGluR5, p-ERK, ERK, p-Drp1(s616) antibodies, and mouse anti-rat GAPDH, β-Tubulin, NeuN antibodies, malondialdehyde MDA content detection kit, and superoxide dismutase SOD activity detection kit.

[0071] The reagents for Western blotting include SDS-PAGE gel preparation kit, electrophoresis buffer, electrotransfer buffer, TBST buffer, 5X sample buffer, protein marker, transfer membrane, blocking solution, and antibodies.

[0072] The step S2 includes the following steps:

[0073] S21: First, deep anesthesia is achieved by intraperitoneal injection of 50 mg / kg pentobarbital sodium.

[0074] S22: Subsequently, it is fixed on a stereotaxic apparatus (ST-51,603; Stoelting Company, USA); an incision is precisely made along the midline of the scalp to fully expose the skull.

[0075] S23: After positioning, a hole is drilled in the skull, and the positioning coordinates are 1.0 mm posterior to the midline and 1.5 mm lateral.

[0076] S24: Subsequently, a sterile cannula with a sealing cap is inserted into the drilled hole and firmly fixed above the dura mater.

[0077] S25: After the operation, recovery is for 7 days. Inflammatory soup IS is injected into the dura mater at 5 μL for 7 consecutive days. At the same time, an equal amount of sterile PBS is injected into the rats in the sham operation group. The inflammatory soup consists of 0.1 mM prostaglandin E2, 1 mM serotonin, 1 mM bradykinin, and 1 mM histamine.

[0078] In the step S3, the operation steps are as follows: On the 7th day after IS injection, 250 nM of the drug CHPG and 2 μg of MPEP are injected by intracerebroventricular injection at a volume of 5 μL. The injection coordinates are 1.0 mm posterior to the cerebrospinal fluid, 1.5 mm lateral to the midline, and 4.5 mm subdural. The injection time is 10 minutes. The control group is injected with 5% DMSO.

[0079] In the step S4, the plantar surface of the hind paw is tested three times using von Frey filaments, with a five-minute interval between each test. During the test, the filament is pressed against the hind paw for up to five seconds or until the hind paw retracts. If the hind paw retracts, a filament with a smaller force is used in the next test; if the hind paw does not retract, a filament with a greater force is used for the next test. According to the positive response of its retraction, the corresponding value is recorded, and finally the average value of the three measurement results is taken to determine the mechanical pain threshold of the hind paw. The determination of the thermal pain threshold is carried out in the following way. During the operation, the hind paw of the rat is subjected to radiant heat stimulation until the rat lifts its paw. If the paw is not lifted within 25 seconds, the stimulation is stopped, and the duration of paw lifting is recorded. This test is repeated three times, with an interval of at least five minutes between each test. Finally, the average value of the three measurement results is taken to determine the thermal pain threshold of the hind paw.

[0080] In the step S6, it includes the following steps:

[0081] S61: Take out the trigeminal caudal nucleus TNC tissue and quickly freeze it in liquid nitrogen for preservation;

[0082] S62: Subsequently, place the tissue in RIPA lysis buffer (Beyotime, China) containing protease (Beyotime, China) and phosphatase inhibitor (MedChemExpress, USA) for homogenization;

[0083] S63: After incubating the homogenate on ice for 1 hour, centrifuge it at 4 °C for 15 minutes; use a BCA protein quantification kit (Beyotime, China) to measure the protein concentration;

[0084] S64: Load an equal amount of protein sample onto a 10% SDS-PAGE gel for electrophoresis separation, and then transfer it to a PVDF membrane;

[0085] S65: Block the transferred membrane with 5% skim milk at room temperature for 1 hour, and then incubate it with the primary antibody overnight at 4 °C;

[0086] S66: Incubate it with the secondary antibody at room temperature for 1 hour. Finally, use a hypersensitive ECL kit (MedChemExpress, USA) and a Fusion system (Fusion, Germany) for image acquisition, and develop and analyze the immunoreactive bands.

[0087] The step S7 includes the following steps:

[0088] S71: After deeply anesthetizing the rat with intraperitoneal injection of pentobarbital sodium, rinse the rat's heart successively with an equal volume of ice-cold phosphate buffered saline PBS and 4% paraformaldehyde solution;

[0089] S72: The trigeminal caudal nucleus (TNC) tissue was isolated and fixed in 4% formaldehyde solution at 4 °C for 12 hours, and then dehydrated with 20% and 30% sucrose solutions respectively;

[0090] S73: Then the TNC tissue was sectioned to obtain coronal sections with a thickness of 15 μm;

[0091] S74: Antigen retrieval was performed on the sections using sodium citrate buffer, and permeabilization was carried out with 0.3% Triton X-100 at 37 °C for 10 minutes;

[0092] S75: After blocking with a solution containing 10% goat serum at 37 °C for 30 minutes, the sections were incubated with the primary antibody overnight at 4 °C, then incubated with the fluorescent secondary antibody at 37 °C for 1 hour, and finally stained with DAPI for 10 minutes;

[0093] S76: After mounting with 50% glycerol, the sections were observed under a confocal laser scanning fluorescence microscope. For quantitative analysis, ImageJ software was used to measure the immunofluorescence intensity and count the number of c-fos immunoreactive cells.

[0094] The step S8 includes the following steps:

[0095] S81: The experimental animals were deeply anesthetized and perfused with pre-cooled PBS, and then treated with 2.5% glutaraldehyde solution;

[0096] S82: After the brain tissue was quickly removed, it was cut into tissue blocks of 1 mm 3 and immersed in 4% pre-cooled glutaraldehyde for fixation;

[0097] S83: Observation was performed using a JEM-1400PLUS transmission electron microscope. The outlines of mitochondria in the images were manually traced, their areas and perimeters were calculated, and the degree of mitochondrial fragmentation was evaluated using the ratio of area to perimeter (connectivity);

[0098] S84: Finally, statistical analysis was performed on the obtained area, perimeter, and connectivity data.

[0099] As Figure 1 shown, CM rats showed enhanced hyperalgesia and upregulation of CGRP and c-Fos expression. A chronic migraine rat model was successfully established by continuous intradural injection of IS for seven days. During the eight-day observation period, we systematically evaluated the changes in mechanical pain and thermal pain thresholds of the rats' hind paws ( Figure 1(A-B). The research found that starting from the fourth day, both the mechanical pain threshold and the thermal pain threshold in the chronic migraine group were lower than those in the sham operation group. The above experiments confirmed the successful establishment of the chronic migraine model. The expression level of CGRP at the TNC site was detected by Western blot and immunofluorescence methods, and it was found that its expression increased in the model group ( Figure 1 (C-F), and immunofluorescence was used to detect c-fos, and it was also found that the number of its positive cells increased (Figure G-H). It indicates that central sensitization exists in CM rats.

[0100] As Figure 2 shown, the expressions of mGluR5, phosphorylated ERK, and Drp1 at the TNC site of CM rats increased. By qRT-PCR and Western blot analysis methods, we measured the mRNA and protein expressions of mGluR5 in the TNC of rats. The results of both were consistent, indicating ( Figure 2 (A-C) that the expression of mGluR5 increased in the chronic migraine group. Then we found by double immunofluorescence staining that mGluR5 was present on neurons ( Figure 2 (D). The Western blot method was used to detect the changes in the protein expressions of p-ERK and p-Drp1 (s616). We found that compared with the sham operation group, p-ERK and p-Drp1 (s616) increased in the CM group, while ERK had no obvious change ( Figure 2 (E-G). Our results indicate that phosphorylated ERK and Drp1 may be involved in CM.

[0101] As Figure 3 shown, mGluR5 has an impact on oxidative stress, hyperalgesia, and the expression levels of c-Fos and CGRP in the chronic migraine (CM) rat model. To understand the specific role of mGluR5 in chronic migraine. The method of injecting CHPG (mGluR5 agonist) and MPEP (mGluR5 antagonist) into the lateral ventricle was used in the experiment. The results showed that the mechanical pain threshold of the hind paws of rats in the CM group decreased, and CHPG further decreased the mechanical pain threshold of the CM + DMSO group, while MPEP significantly increased the mechanical pain threshold of CM. The thermal pain threshold experiment also showed a similar trend ( Figure 3 (A-B). Immunofluorescence staining showed that the expression of CGRP and the number of c-Fos positive cells in the TNC region of the CM group increased. CHPG exacerbated this phenomenon, while MPEP had an inhibitory effect ( Figure 3 (E-H). Detection of oxidative stress indicators found that the MDA level increased and the SOD activity decreased in the CM group, indicating a higher level of oxidative stress. CHPG exacerbated this change, while MPEP showed the opposite effect ( Figure 3 (I-J). The experiment confirmed that MPEP and CHPG had no effect on the pain threshold of rats in the sham operation group ( Figure 3(C-D). The research results show that mGluR5 is involved in the development of CM hyperalgesia by regulating central sensitization and oxidative stress.

[0102] As Figure 4 shown, mGluR5 has an impact on the phosphorylation expression levels of ERK and Drp1 and the morphology of mitochondria. This study confirmed through Western blot analysis that mGluR5 is involved in the regulation of the ERK signaling pathway. The expression of pERK increased in the chronic migraine model group, and CHPG enhanced while MPEP inhibited this expression change ( Figure 4 A-B). The results of immunofluorescence staining and Western blot showed that mGluR5 has a regulatory effect on the expression of p-Drp1(s616). The level of p-Drp1(s616) increased in the chronic migraine model group, and CHPG treatment further increased it while MPEP inhibited its expression ( Figure 4 A, C-E). Electron microscopy analysis found that mitochondrial fragmentation occurred under the state of chronic migraine, and CHPG exacerbated it while MPEP improved this morphological change ( Figure 4 F-I), indicating that mGluR5 plays an important regulatory role in the process of mitochondrial fission. The above results show that mGluR5 mediates the phosphorylation of dynamin-related protein 1 (Drp1) through ERK, activating mitochondrial fission

[0103] In this invention, through the research method of the molecular mechanism regulated by metabotropic glutamate receptor 5, by establishing an animal experiment model, drug administration, behavioral experiments, RNA extraction and RT-qPCR detection, Western blot experiments, immunofluorescence experiments, electron microscopy experiments, malondialdehyde content, superoxide dismutase activity detection, etc., the molecular mechanism by which mGluR5 promotes oxidative stress and central sensitization in chronic migraine rats through the activation of mitochondrial fission mediated by phosphorylated Drp1 by ERK is studied. It can be known that the down-regulation of mGluR5 alleviates oxidative stress by inhibiting mitochondrial fission mediated by phosphorylated Drp1, and the regulatory effect of mGluR5 on mitochondrial fission may involve ERK, thus laying a solid foundation for the diagnosis and treatment of migraine.

[0104] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A research method for the molecular mechanism regulated by metabotropic glutamate receptor 5, characterized in that, It includes the following steps: S1: Preliminary preparation, preparing experimental animals and experimental reagents; S2: Establishment of animal model, constructing a chronic migraine animal model; S3: Animal administration, injecting drugs into the lateral ventricle of the animals; S4: Behavioral experiments, testing the mechanical pain threshold and thermal pain threshold of the animals; S5: Total RNA extraction of samples and detecting the expression of mGluR5 by RT-qPCR; S6: Western blot experiment to detect the protein expression of CGRP, mGluR5, ERK, p-ERK, p-Drp1(s616) in the TNC region; S7: Immunofluorescence experiment, after deeply anesthetizing the animals by intraperitoneal injection of pentobarbital sodium, separating the TNC tissue for fluorescence experiment to detect the protein expression of CGRP, c-Fos, p-Drp1(s616), and the cellular localization of mGluR5; S8: Electron microscopy experiment, taking out the tissue in the TNC region for electron microscopy experiment to observe the morphological changes of mitochondria; S9: Detecting the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the TNC tissue, calculating the relative levels of MDA content and SOD activity to evaluate the oxidative stress level; S10: After repeating the above steps at least three times, perform data analysis.

2. A research method for the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that In the S1 step, the experimental animals are adult male rats.

3. A method for studying the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 2, characterized in that In the S1 step, the experimental reagents include prostaglandin E2, bradykinin BK, histamine HA, 5-hydroxytryptamine, 6-methyl-2-(phenylethynyl)pyridine hydrochloride (MPEP), (RS)-2-chloro-5-hydroxyglycine (CHPG), RNA rapid extraction kit, reverse transcription reagent, RT-qPCR, reagents related to Western blotting, rabbit anti-rat CGRP, c-Fos, mGluR5, p-ERK, ERK, p-Drp1(s616) antibodies, and mouse anti-rat GAPDH, β-Tubulin, NeuN antibodies, malondialdehyde MDA content detection kit, superoxide dismutase SOD activity detection kit.

4. A method for studying the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 3, characterized in that, The reagents related to Western blotting include SDS-PAGE gel preparation kit, electrophoresis buffer, electrotransfer buffer, TBST buffer, 5X sample buffer, protein marker, transfer membrane, blocking solution, and antibodies.

5. A research method for the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that, The S2 step includes the following steps: S21: First, achieve deep anesthesia by intraperitoneal injection of 50 mg / kg pentobarbital sodium; S22: Subsequently, fix it on the stereotaxic apparatus; precisely incise along the midline of the scalp to fully expose the skull; S23: After positioning, drill a hole in the skull, and the positioning coordinates are 1.0 mm posterior to the midline and 1.5 mm lateral; S24: Subsequently, insert a sterile cannula with a sealing cap into the drilled hole and firmly fix it above the dura mater; S25: After the operation, recover for 7 days, continuously inject 5 μL of inflammatory soup IS into the dura mater for 7 days. At the same time, inject an equal amount of sterile PBS into the rats in the sham operation group. The inflammatory soup consists of 0.1 mM prostaglandin E2, 1 mM serotonin, 1 mM bradykinin, and 1 mM histamine.

6. A research method for molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that In the step S3, the operation steps are as follows: On the 7th day after IS injection, 250 nm of the drug CHPG and 2 μg of MPEP are injected through the lateral ventricle with an injection volume of 5 μl. The injection coordinates are: 1.0 mm posterior to the cerebrospinal fluid, 1.5 mm lateral to the midline, and 4.5 mm subdural. The injection time is 10 minutes. The control group is injected with 5% DMSO.

7. A research method for the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that, In the step S4, the hind paw plantar surface is tested three times using von Frey filaments, with a 5-minute interval between each test. During the test, the filament is pressed on the hind paw for up to 5 seconds or until the hind paw retracts. If the hind paw retracts, a filament with a smaller force is used in the next test; if the hind paw does not retract, a filament with a greater force is used for the next test. According to the positive response of its retraction, the corresponding value is recorded. Finally, the average value of the three measurement results is taken to determine the mechanical pain threshold of the hind paw. The determination of the thermal pain threshold is carried out in the following way. Specifically, during the operation, the hind paw of the rat is subjected to radiant heat stimulation until the rat lifts its paw. If the paw is not lifted within 25 seconds, the stimulation is stopped, and the lifting duration is recorded. This test is repeated three times, with an interval of at least 5 minutes between each time. Finally, the average value of the three measurement results is taken to determine the thermal pain threshold of the hind paw.

8. A method for studying the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that, In the step S6, it includes the following steps: S61: Take out the trigeminal caudal nucleus TNC tissue and quickly place it in liquid nitrogen for cryopreservation; S62: Subsequently, place the tissue in RIPA lysis buffer containing protease and phosphatase inhibitors for homogenization; S63: After incubating the homogenate on ice for 1 hour, centrifuge it at 4 °C for 15 minutes; use a BCA protein quantification kit to measure the protein concentration; S64: Load an equal amount of protein sample onto a 10% SDS-PAGE gel for electrophoresis separation, and then transfer it to a PVDF membrane; S65: Block the transferred membrane with 5% skim milk at room temperature for 1 hour, and then incubate it with the primary antibody overnight at 4 °C; S66: Incubate it with the secondary antibody at room temperature for 1 hour. Finally, use a hypersensitive ECL kit and a Fusion system for image acquisition, and develop and analyze the immunoreactive bands.

9. A method for studying the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that, The step S7 includes the following steps: S71: After deeply anesthetizing the rat with pentobarbital sodium by intraperitoneal injection, rinse the rat's heart successively with an equal volume of ice-cold phosphate buffered saline PBS and 4% paraformaldehyde solution; S72: Isolate the trigeminal caudal nucleus TNC tissue and place it in a 4% formaldehyde solution at 4 °C for fixation for 12 hours, and then dehydrate it with 20% and 30% sucrose solutions respectively; S73: Then section the TNC tissue to obtain coronal sections with a thickness of 15 μm; S74: Perform antigen repair on the sections using sodium citrate buffer and permeabilize them with 0.3% Triton X-100 at 37 °C for 10 minutes; S75: After blocking the sections with a solution containing 10% goat serum at 37 °C for 30 minutes, incubate the sections with the primary antibody overnight at 4 °C, then incubate them with the fluorescent secondary antibody at 37 °C for 1 hour, and finally stain them with DAPI for 10 minutes; S76: After mounting the sections with 50% glycerol, observe the sections through a confocal laser scanning fluorescence microscope. For quantitative analysis, use ImageJ software to measure the immunofluorescence intensity and count the number of c-fos immunopositive cells.

10. A method for studying the molecular mechanism regulated by metabotropic glutamate receptor 5 according to claim 1, characterized in that, The step S8 includes the following steps: S81: After deeply anesthetizing the experimental animals, perfuse them with pre-cooled PBS, and then treat them with 2.5% glutaraldehyde solution; S82: After the brain tissue was quickly removed, it was cut into tissue blocks of 1 mm 3 and immersed in 4% pre-cooled glutaraldehyde for fixation; S83: Use a JEM-1400PLUS transmission electron microscope for observation. Manually delineate the contours of the mitochondria in the images, calculate their area and perimeter, and evaluate the degree of mitochondrial fragmentation using the ratio of area to perimeter (connectivity); S84: Finally, perform statistical analysis on the obtained area, perimeter, and connectivity data.

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