Molecular mechanism research method based on chemotactic factor CCL2 regulation

Abstract

The invention discloses a molecular mechanism research method based on chemotactic factor CCL2 regulation, and relates to the field of CCL2 regulation mechanism research. Comprising the following steps: preparing experimental animals and experimental reagent equipment; animal model establishment: establishing a chronic migraine model of an experimental animal; medicine lateral ventricle injection: performing injection administration on the lateral ventricle of the experimental animal; mechanical pain threshold detection: carrying out mechanical pain detection on the animal injected with the medicine; and heat pain threshold detection: carrying out heat pain detection on the animal injected with the medicine. Through a molecular mechanism research method based on chemotactic factor CCL2 regulation, animal model establishment, RNA extraction, RT-qPCR detection, Western blot experiment, ELISA experiment and other modes are utilized, so that a mechanism for regulating chronic migraine by CCL2 through NLRP3 is established, and a foundation is laid for medication guidance and later treatment effect of migraine patients.

Description

Technical Field

[0001] The present invention relates to the research field of CCL2 regulatory mechanism, and in particular to a molecular mechanism research method based on chemokine CCL2 regulation. Background Art

[0002] Migraine is a chronic neurological disease characterized by unilateral or bilateral, throbbing pain, accompanied by autonomic symptoms such as nausea, photophobia, and phonophobia. The incidence of migraine is high, with a global incidence of 15% and an annual incidence of approximately 9.3% in my country. There are approximately more than 1 billion patients worldwide. Migraine has a high disability rate, greatly affecting the quality of life of patients and placing great economic pressure on both individuals and society. The World Health Organization ranks migraine as the third most common disease and the second most disabling neurological disorder in the world. Because the pathogenesis of migraine is currently unclear, although the diagnosis rate of migraine has been increasing in recent years, more than half of migraine patients still do not receive a corresponding diagnosis. In addition, due to insufficient preventive treatment and the abuse of acute analgesics, most migraine patients are dissatisfied with the treatment effect.

[0003] To this end, the present invention proposes a molecular mechanism research method based on the regulation of chemokine CCL2. Summary of the Invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a molecular mechanism research method based on the regulation of chemokine CCL2.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for studying the molecular mechanism of chemokine CCL2 regulation, comprising the following steps:

[0007] S1: Preparation of experimental animals, reagents and equipment;

[0008] S2: Animal model establishment: establishing a chronic migraine model in experimental animals;

[0009] S3: drug injection into the lateral ventricle of the experimental animals;

[0010] S4: Mechanical pain threshold test, mechanical pain test is performed on animals after drug injection;

[0011] S5: Thermal pain threshold test, thermal pain test is performed on animals after drug injection;

[0012] S6: RNV extraction and RT-qPCR detection;

[0013] S7: Western blot experiment;

[0014] S8: ELISA test;

[0015] S9: Immunofluorescence experiments: Rats were deeply anesthetized with pentobarbital and perfused transcardially with PBS followed by 4% paraformaldehyde. Tissue from the spinal trigeminal nucleus caudalis was obtained and fixed in 4% paraformaldehyde for 12 hours at 4°C. The tissue was cryoprotected with sucrose and sectioned coronally using a cryostat. Sections were collected and boiled in sodium citrate buffer for antigen retrieval. The sections were permeabilized with 0.3% Triton X-100 and incubated at 37°C for 10 minutes. The sections were then blocked with goat serum for 30 minutes at 37°C. Sections were incubated with primary antibodies diluted in 1% PBS overnight at 4°C. The next day, sections were incubated with fluorescent secondary antibodies and counterstained with 4',6-diamidino-2-phenylindole for nuclei. Images were acquired using a confocal laser scanning fluorescence microscope.

[0016] S10: Repeat steps S3 and S9, and perform statistical analysis on the experimental results using GraphPad Prism software. t-test was used for pairwise comparison of data within each group, and one-way analysis of variance was used to compare data between two or more groups.

[0017] Preferably: in the S1 step, the experimental animals are Sprague-Dawley rats, and the experimental reagents include: behavioral test-related equipment, RNA extraction-related reagents, qRT-PCR-related reagents, protein blotting-related reagents, ELISA detection kits, CCL2 neutralizing antibodies, rabbit anti-mouse CCL2 antibodies, NLRP3 antibodies, Tubulin antibodies, p-ERK antibodies, GFAP antibodies, Iba1 antibodies, NeuN antibodies, CD68 antibodies, and CD163 antibodies.

[0018] Preferably: in the step S1, the behavioral testing equipment includes Von-Frey fibers and a plantar heat pain detector; the protein blotting-related reagents include an SDS-PAGE gel preparation kit, electrophoresis fluid, electrotransfer fluid, TBST buffer, protein marker, transfer membrane, blocking fluid and antibodies.

[0019] Preferably, the step S2 comprises the following steps:

[0020] S21: After anesthesia, the rat was placed in a stereotaxic apparatus and the head was fixed. The hair in the surgical area on the top of the rat's head was removed and disinfected with iodine tincture.

[0021] S22: Use a scalpel to make a 1.5 cm incision along the midline of the rat's head to fully expose the skull;

[0022] S23: Without damaging the dura mater, a 1 mm diameter hole was drilled in the skull at 1.0 mm posterior to the bregma and 1.5 mm lateral to the midline using a stereotaxic instrument. A sterile cannula was placed and fixed with dental powder and water. The incision was then sutured.

[0023] S24: 5 μL of inflammatory decoction was instilled into the rat's epidural membrane via a sterile cannula once daily for seven consecutive days.

[0024] Preferably, the step S3 is to administer CCL2 mAb to the rats intracerebroventricularly 24 hours after the seventh instillation of inflammatory decoction or PBS, with the dosage of CCL2 mAb set to 100 μg, and comprises the following steps:

[0025] S31: Insert the needle tip for intraventricular drug delivery into the surgically placed sterile cannula, allowing the needle tip to penetrate approximately 4.0-4.5 mm below the hard skull.

[0026] S32: The drug was dissolved in PBS solution and injected slowly and evenly into the lateral ventricle of the rat within 10 minutes.

[0027] Preferably, the detailed operation of step S4 is: vertically placing the VonFrey monofilament on the plantar surface of the rat's hind paw until it bends for 2-5 seconds; if the rat quickly retracts the paw, licks the paw, or shakes the paw during the stimulation or after the monofilament is removed, it is considered a positive reaction.

[0028] Preferably, the detailed operation of step S5 is as follows: the rats are placed in a smooth and transparent glass cage, and the center of the hind paw of each rat is stimulated with infrared radiation emitted by a plantar tester. After the hind paw moves, the stimulation stops, and the instrument records the withdrawal latency. To prevent tissue damage, the automatic cut-off time is set to 25 seconds. Each rat is tested three times, and the average latency is calculated.

[0029] Preferably, the step S6 comprises the following steps:

[0030] S61: tissues were collected and immediately frozen in liquid nitrogen;

[0031] S62: Total RNA was isolated using Trizol reagent, and the RNA concentration was measured spectrophotometrically using a spectrophotometer, while reverse transcription was performed using AB Script III RT Master Mix and gDNA Remover Kit;

[0032] S63: RNA concentration was then quantified using 2X Universal SYBR Green Fast qPCR Mix, and mRNA expression analysis was performed on a CFX96 Touch thermal cycler;

[0033] S64: mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2^{ΔΔCT} method.

[0034] Preferably, the step S7 includes the following steps:

[0035] S71: Collect rat tissue and lyse fresh tissue using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to obtain total protein;

[0036] S72: Determine the protein concentration using a bicinchoninic acid protein assay kit, and prepare SDS-PAGE to separate equal amounts of protein samples;

[0037] S73: The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk for 2 h, and incubated with specific primary antibodies at 4°C.

[0038] S74: The membrane is then treated with a secondary antibody, and the resulting signal is detected using an imaging system and enhanced chemiluminescence (ECL) reagents.

[0039] Preferably, the step S8 comprises the following steps:

[0040] S81: Tissue samples were weighed and suspended in PBS (pH 7.2-7.4), and then subjected to multiple freeze-thaw cycles at -20°C to ensure complete tissue lysis.

[0041] S82: The mixture was centrifuged at 5000 rpm for 15 minutes to separate the supernatant;

[0042] S83: Add the sample to a microplate pre-coated with specific antibodies and incubate at 37°C for 2 hours;

[0043] S84: Biotinylated antibody working solution and HRP coupling reagent were then added, and the optical density of the sample was measured at a wavelength of 450 nm. The result was expressed in pg / mL, and the intra-assay and inter-assay coefficients of variation were less than 10%.

[0044] The beneficial effects of the present invention are:

[0045] 1. This study, based on the molecular mechanism of CCL2 regulation, uses animal model establishment, RNA extraction and RT-qPCR detection, Western blot experiments, ELISA experiments, and other methods to establish the mechanism by which CCL2 regulates chronic migraine through NLRP3, laying the foundation for medication guidance and subsequent treatment effects for migraine patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a molecular mechanism study method based on chemokine CCL2 regulation proposed by the present invention, showing that CCL2 is highly expressed in chronic migraine, in which the mechanical pain threshold and thermal pain threshold of chronic migraine rats are decreased ( Figure 1 A, B); p-ERK was used to represent central sensitization. The protein expression of p-ERK was detected by Western blot. It was found that the expression of p-ERK was significantly increased in CM rats ( Figure 1 C); In addition, Western blot and qRT-PCR were used to detect the expression changes of CCL2 and its receptor CCR2, and it was found that compared with the Sham group, the CCL2 and CCR2 in the CM group were increased at both mRNA and protein levels ( Figure 1 D, E);

[0047] Figure 2 The present invention proposes a molecular mechanism research method based on the regulation of chemokine CCL2. Inhibiting the expression of CCL2 can alleviate the hyperalgesia and central sensitization of chronic migraine. CCL2 neutralizing antibody is used to specifically inhibit the highly expressed CCL2 in vivo. Through behavioral testing, it is found that after the CCL2 neutralizing antibody is injected into the lateral ventricle, the mechanical pain threshold and thermal pain threshold of rats in the CM group + CCL2mAb group are increased compared with those in the CM group ( Figure 2 A, B). At the same time, Western blot analysis of p-ERK expression revealed that the expression of p-ERK in the CM+CCL2mAb group was lower than that in the CM group, indicating that central sensitization in CM rats was alleviated after injection of CCL2 neutralizing antibodies ( Figure 2 C). Secondly, ELISA was used to detect the levels of inflammatory factors in the caudate nucleus of the spinal trigeminal nerve in CM rats. After the application of CCL2 neutralizing antibody, the expression level of the pro-inflammatory cytokine IL-1β decreased compared with the CM group ( Figure 2 D), while the level of anti-inflammatory cytokine IL-10 increased ( Figure 2 E);

[0048] Figure 3 This is a molecular mechanism research method based on the regulation of chemokine CCL2 proposed by the present invention. NLRP3 is involved in the physiological and pathological mechanism of chronic migraine. The String database and Genecard database were used to search for CCL2-regulated signaling molecules related to chronic migraine, and it was found that NLRP3 was involved in it ( Figure 3 A, B), the expression level of NLRP3 was detected by Western blot, and it was found that the expression level of NLRP3 in the CM group was significantly increased ( Figure 3C). NLRP3 was inhibited using the NLRP3-specific inhibitor MCC950. Behavioral testing revealed that the mechanical pain threshold and thermal pain threshold of rats in the CM+MCC950 group were higher than those in the CM group ( Figure 3 D, E);

[0049] Figure 4 This is a molecular mechanism study method based on chemokine CCL2 regulation proposed in the present invention. CCL2 can participate in the physiological and pathological mechanism of chronic migraine through NLRP3. First, to detect whether CCL2 regulates NLRP3, the expression of NLRP3 was detected after specific inhibition of CCL2. It was found that after intervention with CCL2, the expression level of NLRP3 decreased compared with the CM+IgG group ( Figure 4 A), indicating that CCL2 may regulate the expression of NLRP3 as an upstream regulator of NLRP3. Secondly, behavioral analysis showed that the mechanical pain threshold and thermal pain threshold of rats increased after the application of CCL2 agonists and the use of NLRP3 specific inhibitors ( Figure 4 B, C). Western blot analysis revealed that the use of a specific NLRP3 inhibitor after application of a CCL2 agonist could inhibit the increase in p-ERK expression ( Figure 4 D). Finally, ELISA detection of inflammatory cytokines revealed that MCC950 could alleviate the increase in IL-1β expression caused by CCL2 agonist, while IL-10 had the opposite effect ( Figure 4 E, F).

[0050] Figure 5This figure shows a molecular mechanism of CCL2 regulation, based on a proposed method for studying the effects of chemokine CCL2 on microglial phenotypes. This process involves the NLRP3 inflammasome. To determine whether CCL2 affects microglia, we used CD68 to indicate the expression of pro-inflammatory microglia and CD163 to indicate the expression of anti-inflammatory microglia. First, after administration of a CCL2 neutralizing antibody, we observed changes in microglial phenotypic markers. Compared to the CM + IgG group, the CM + CCL2 mAb group showed a significant decrease in the number of CD68-positive microglia, while the number of CD163-positive microglia increased significantly. This suggests that microglia in CM rats express a pro-inflammatory phenotype, and that CCL2 inhibition can promote the transition of microglia to an anti-inflammatory phenotype. Next, we used MCC950 to inhibit the NLRP3 inflammasome. The results showed that inhibition of the NLRP3 inflammasome increased the number of CD163-positive microglia, while decreasing the number of CD68-positive microglia. This indicates that the NLRP3 inflammasome is also involved in regulating the phenotypic transition of microglia. Finally, to verify whether CCL2 regulates microglial phenotypic changes through the NLRP3 inflammasome, the NLRP3 inflammasome was blocked after the injection of CCL2 recombinant protein. The results showed that after stimulating CCL2, the number of CD68-positive microglia increased further, while after blocking the NLRP3 inflammasome, this increase was blocked, and the number of CD163-positive microglia increased. The above results are consistent with the results of the previous section that CCL2 regulates chronic migraine hyperalgesia and central sensitization. It is said that CCL2 secreted by astrocytes may participate in chronic migraine hyperalgesia and central sensitization by promoting the NLRP3 inflammasome to regulate microglial phenotypic changes. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0052] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," and "disposed" should be understood in a broad sense. For example, they may refer to fixed connection or disposition, detachable connection or disposition, or integral connection or disposition. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0053] Example 1:

[0054] A method for studying the molecular mechanism of chemokine CCL2 regulation, comprising the following steps:

[0055] S1: Preparation of experimental animals, reagents and equipment;

[0056] S2: Animal model establishment: establishing a chronic migraine model in experimental animals;

[0057] S3: drug injection into the lateral ventricle of the experimental animals;

[0058] S4: Mechanical pain threshold test, mechanical pain test is performed on animals after drug injection;

[0059] S5: Thermal pain threshold test, thermal pain test is performed on animals after drug injection;

[0060] S6: RNV extraction and RT-qPCR detection;

[0061] S7: Western blot experiment;

[0062] S8: ELISA test;

[0063] S9: Immunofluorescence experiments: Rats were deeply anesthetized with pentobarbital and perfused transcardially with PBS followed by 4% paraformaldehyde. Tissue from the spinal trigeminal nucleus caudalis was obtained and fixed in 4% paraformaldehyde for 12 hours at 4°C. The tissue was cryoprotected with sucrose and sectioned coronally using a cryostat. Sections were collected and boiled in sodium citrate buffer for antigen retrieval. The sections were permeabilized with 0.3% Triton X-100 and incubated at 37°C for 10 minutes. The sections were then blocked with goat serum for 30 minutes at 37°C. Sections were incubated with primary antibodies diluted in 1% PBS overnight at 4°C. The next day, sections were incubated with fluorescent secondary antibodies and counterstained with 4',6-diamidino-2-phenylindole for nuclei. Images were acquired using a confocal laser scanning fluorescence microscope.

[0064] S10: Repeat steps S3 and S9, and perform statistical analysis on the experimental results using GraphPad Prism software. t-test was used for pairwise comparison of data within each group, and one-way analysis of variance was used to compare data between two or more groups.

[0065] In step S1, the experimental animals are Sprague-Dawley rats, and the experimental reagents include: behavioral test-related equipment, RNA extraction-related reagents, qRT-PCR-related reagents, protein blotting-related reagents, ELISA detection kits, CCL2 neutralizing antibodies, rabbit anti-mouse CCL2 antibodies, NLRP3 antibodies, Tubulin antibodies, p-ERK antibodies, GFAP antibodies, Iba1 antibodies, NeuN antibodies, CD68 antibodies, and CD163 antibodies.

[0066] In the S1 step, the behavioral testing equipment includes Von-Frey fibers and a plantar heat pain detector; the protein blotting-related reagents include an SDS-PAGE gel preparation kit, electrophoresis fluid, electrotransfer fluid, TBST buffer, protein marker, transfer membrane, blocking fluid, and antibodies.

[0067] The S2 step comprises the following steps:

[0068] S21: After anesthesia, the rat was placed in a stereotaxic apparatus and the head was fixed. The hair in the surgical area on the top of the rat's head was removed and disinfected with iodine tincture.

[0069] S22: Use a scalpel to make a 1.5 cm incision along the midline of the rat's head to fully expose the skull;

[0070] S23: Without damaging the dura mater, a 1 mm diameter hole was drilled in the skull at 1.0 mm posterior to the bregma and 1.5 mm lateral to the midline using a stereotaxic instrument. A sterile cannula was placed and fixed with dental powder and water. The incision was then sutured.

[0071] S24: 5 μL of inflammatory decoction was instilled into the rat's epidural membrane via a sterile cannula once daily for seven consecutive days.

[0072] The step S3, 24 hours after the seventh instillation of inflammatory decoction or PBS, administers CCL2 mAb to the rats via the lateral ventricle, with the dosage of CCL2 mAb set to 100 μg, comprising the following steps:

[0073] S31: Insert the needle tip for intraventricular drug delivery into the surgically placed sterile cannula, allowing the needle tip to penetrate approximately 4.0-4.5 mm below the hard skull.

[0074] S32: The drug was dissolved in PBS solution and injected slowly and evenly into the lateral ventricle of the rat within 10 minutes.

[0075] The detailed operation of the S4 step is as follows: vertically place the VonFrey monofilament on the plantar surface of the rat's hind paw until it bends for 2-5 seconds; if the rat quickly retracts the paw, licks the paw, or shakes the paw during the stimulation or after the monofilament is removed, it is considered a positive reaction.

[0076] The detailed operation of step S5 is as follows: the rats are placed in a smooth and transparent glass cage, and the center of the hind paw of each rat is stimulated with infrared radiation emitted by the plantar tester. After the hind paw moves, the stimulation stops, and the instrument records the withdrawal latency. To prevent tissue damage, the automatic cut-off time is set to 25 seconds. Each rat is tested three times, and the average latency is calculated.

[0077] The step S6 comprises the following steps:

[0078] S61: tissues were collected and immediately frozen in liquid nitrogen;

[0079] S62: Total RNA was isolated using Trizol reagent, and the RNA concentration was measured spectrophotometrically using a spectrophotometer, while reverse transcription was performed using AB Script III RT Master Mix and gDNA Remover Kit;

[0080] S63: RNA concentration was then quantified using 2X Universal SYBR Green Fast qPCR Mix, and mRNA expression analysis was performed on a CFX96 Touch thermal cycler;

[0081] S64: mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2^{ΔΔCT} method.

[0082] The step S7 comprises the following steps:

[0083] S71: Collect rat tissue and lyse fresh tissue using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to obtain total protein;

[0084] S72: Determine the protein concentration using a bicinchoninic acid protein assay kit, and prepare SDS-PAGE to separate equal amounts of protein samples;

[0085] S73: The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk for 2 h, and incubated with specific primary antibodies at 4°C.

[0086] S74: The membrane is then treated with a secondary antibody, and the resulting signal is detected using an imaging system and enhanced chemiluminescence (ECL) reagents.

[0087] The step S8 comprises the following steps:

[0088] S81: Tissue samples were weighed and suspended in PBS (pH 7.2-7.4), and then subjected to multiple freeze-thaw cycles at -20°C to ensure complete tissue lysis.

[0089] S82: The mixture was centrifuged at 5000 rpm for 15 minutes to separate the supernatant;

[0090] S83: Add the sample to a microplate pre-coated with specific antibodies and incubate at 37°C for 2 hours;

[0091] S84: Biotinylated antibody working solution and HRP coupling reagent were then added, and the optical density of the sample was measured at a wavelength of 450 nm. The result was expressed in pg / mL, and the intra-assay and inter-assay coefficients of variation were less than 10%.

[0092] Furthermore, the results showed that CCL2 is primarily expressed in astrocytes, while its receptor is primarily expressed in microglia. Furthermore, inhibition of CCL2 can affect microglial phenotypic changes, which are also associated with the NLRP3 inflammasome. These results suggest that CCL2 may contribute to the pathophysiology of chronic migraine by promoting inflammation through NLRP3-mediated crosstalk between astrocytes and microglia.

[0093] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for studying the molecular mechanism of chemokine CCL2 regulation, characterized in that: The following steps are involved: S1: Preparation of experimental animals, reagents and equipment; S2: Animal model establishment: establishing a chronic migraine model in experimental animals; S3: drug injection into the lateral ventricle of the experimental animals; S4: Mechanical pain threshold test, mechanical pain test is performed on animals after drug injection; S5: Thermal pain threshold test, thermal pain test is performed on animals after drug injection; S6: RNV extraction and RT-qPCR detection; S7: Western blot experiment; S8: ELISA test; S9: Immunofluorescence experiment: After deep anesthesia with pentobarbital, rats were transcardially perfused with PBS and then 4% paraformaldehyde to obtain tissue of the caudate nucleus of the spinal trigeminal tract. The tissue was fixed in 4% paraformaldehyde at 4°C for 12 hours and cryoprotected with sucrose. The tissue was coronally sectioned using a cryostat and the sections were collected. The sections were boiled in sodium citrate buffer for antigen retrieval and permeabilized with 0.3% Triton X-100 and incubated at 37°C for 10 minutes. The sections were then blocked with goat serum at 37°C for 30 minutes and incubated with primary antibodies diluted in 1% PBS at 4°C overnight. The next day, the sections were incubated with fluorescent secondary antibodies and the nuclei were counterstained with 4',6-diamidino-2-phenylindole. Images were acquired using a confocal laser scanning fluorescence microscope. S10: Repeat steps S3 and S9, and perform statistical analysis on the experimental results using GraphPad Prism software. t-test was used for pairwise comparison of data within each group, and one-way analysis of variance was used to compare data between two or more groups.

2. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: In step S1, the experimental animals are Sprague-Dawley rats, and the experimental reagents include: behavioral test-related equipment, RNA extraction-related reagents, qRT-PCR-related reagents, protein blotting-related reagents, ELISA detection kits, CCL2 neutralizing antibodies, rabbit anti-mouse CCL2 antibodies, NLRP3 antibodies, Tubulin antibodies, p-ERK antibodies, GFAP antibodies, Iba1 antibodies, NeuN antibodies, CD68 antibodies, and CD163 antibodies.

3. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 2, characterized in that: In the S1 step, the behavioral testing equipment includes Von-Frey fibers and a plantar heat pain detector; the protein blotting-related reagents include an SDS-PAGE gel preparation kit, electrophoresis fluid, electrotransfer fluid, TBST buffer, protein marker, transfer membrane, blocking fluid, and antibodies.

4. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The S2 step comprises the following steps: S21: After anesthesia, the rat was placed in a stereotaxic apparatus and the head was fixed. The hair in the surgical area on the top of the rat's head was removed and disinfected with iodine tincture. S22: Use a scalpel to make a 1.5 cm incision along the midline of the rat's head to fully expose the skull; S23: Without damaging the dura mater, a 1 mm diameter hole was drilled in the skull at 1.0 mm posterior to the bregma and 1.5 mm lateral to the midline using a stereotaxic instrument. A sterile cannula was placed and fixed with dental powder and water. The incision was then sutured. S24: 5 μL of inflammatory decoction was instilled into the rat's epidural mater through a sterile cannula once a day for seven consecutive days.

5. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The step S3, 24 hours after the seventh instillation of inflammatory decoction or PBS, administers CCL2 mAb to the rats via the lateral ventricle, with the dosage of CCL2 mAb set to 100 μg, comprising the following steps: S31: Insert the needle tip for intraventricular drug delivery into the surgically placed sterile cannula, allowing the needle tip to penetrate approximately 4.0-4.5 mm below the hard skull. S32: The drug was dissolved in PBS solution and injected slowly and evenly into the lateral ventricle of the rat within 10 minutes.

6. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The detailed operation of the S4 step is as follows: vertically place the VonFrey monofilament on the plantar surface of the rat's hind paw until it bends for 2-5 seconds; if the rat quickly retracts the paw, licks the paw, or shakes the paw during the stimulation or after the monofilament is removed, it is considered a positive reaction.

7. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The detailed operation of step S5 is as follows: the rats are placed in a smooth and transparent glass cage, and the center of the hind paw of each rat is stimulated with infrared radiation emitted by the plantar tester. After the hind paw moves, the stimulation stops, and the instrument records the withdrawal latency. To prevent tissue damage, the automatic cut-off time is set to 25 seconds. Each rat is tested three times, and the average latency is calculated.

8. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The step S6 comprises the following steps: S61: tissues were collected and immediately frozen in liquid nitrogen; S62: Total RNA was isolated using Trizol reagent, and the RNA concentration was measured spectrophotometrically using a spectrophotometer, while reverse transcription was performed using AB Script III RT Master Mix and gDNA Remover Kit; S63: RNA concentration was then quantified using 2X Universal SYBR Green Fast qPCR Mix, and mRNA expression analysis was performed on a CFX96 Touch thermal cycler; S64: mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2^{ΔΔCT} method.

9. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The step S7 comprises the following steps: S71: Collect rat tissue and lyse fresh tissue using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to obtain total protein; S72: Determine the protein concentration using a bicinchoninic acid protein assay kit, and prepare SDS-PAGE to separate equal amounts of protein samples; S73: The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk for 2 h, and incubated with specific primary antibodies at 4°C. S74: The membrane is then treated with a secondary antibody, and the resulting signal is detected using an imaging system and enhanced chemiluminescence (ECL) reagents.

10. The molecular mechanism research method based on chemokine CCL2 regulation according to claim 1, characterized in that: The step S8 comprises the following steps: S81: Tissue samples were weighed and suspended in PBS (pH 7.2-7.4), and then subjected to multiple freeze-thaw cycles at -20°C to ensure complete tissue lysis. S82: The mixture was centrifuged at 5000 rpm for 15 minutes to separate the supernatant; S83: Add the sample to a microplate pre-coated with specific antibodies and incubate at 37°C for 2 hours; S84: Biotinylated antibody working solution and HRP coupling reagent were then added, and the optical density of the sample was measured at a wavelength of 450 nm. The result was expressed in pg / mL, and the intra-assay and inter-assay coefficients of variation were less than 10%.

Images