Sinomenine in preparation of medicine for treating chronic pain of rheumatoid arthritis and application of sinomenine

Abstract

The invention provides sinomenine in preparation of a medicine for treating chronic pain of rheumatoid arthritis and application of the sinomenine in preparation of the medicine for treating the chronic pain of the rheumatoid arthritis, and aims at solving the problems that an existing medicine for resisting the rheumatoid arthritis is limited in action target spot and poor in treatment effect. The sinomenine takes a histamine receptor H1R on a nervous system dorsal root ganglion M1 type macrophage as the action target spot; the target spot is intervened to regulate and control polarization of macrophages, reduce expression of histamine H1R in M1 type macrophages and inhibit infiltration of the macrophages, so that chronic pain, including crymodynia, mechanical pain, heat pain and the like, of rheumatoid arthritis is effectively relieved; the sinomenine combined action target can improve pain and arthritis symptoms of a model mouse, reduce the level of inflammatory factors and inhibit LPS-induced macrophage migration and H1R expression, and the sinomenine pharmaceutical composition has various dosage forms, has unique action target and mechanism, has a remarkable treatment effect, and can be used for preparing medicines for treating chronic infectious diseases. The compound is expected to become a novel non-opioid anti-rheumatoid arthritis chronic pain medicine.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicines, in particular to sinomenine in preparation of a medicine for treating chronic pain of rheumatoid arthritis and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic inflammatory disease, and bone joint damage, deformation, redness, pain and the like are main symptoms in clinical application; although anti-rheumatic drugs (DMARDs) or surgical treatment is currently commonly used to relieve the disease, the final treatment effect on chronic pain symptoms is still unsatisfactory, and many patients still suffer from the distress caused by chronic pain, and in-depth understanding of the pathogenesis of chronic pain of rheumatoid arthritis in order to find a new target for treating chronic pain of RA is the focus of current research.

[0003] On the one hand, in the whole generation and conduction process of peripheral sensitization of chronic pain of rheumatoid arthritis (RA), the existing research finds that macrophages as key effector cells play an indispensable role; regulating the polarization, migration and function of macrophages may be a new strategy for intervening in the progression of RA disease and relieving chronic pain; sinomenine is a kind of alkaloid extracted from the dried stems of sinomenium acutum and sinomenium arboreum; sinomenine is the main component of sinomenium acutum, and has the pharmacological effects of anti-inflammatory analgesia, immunosuppression, anti-tumor and the like; the existing literature reports that sinomenine can reduce the level of pro-inflammatory factors of AA model rats, so as to change joint damage; in addition, sinomenine can also inhibit TLR4 / MyD88 and JAK2 / STATS3 and the like signal pathways, so as to inhibit inflammatory reaction; at present, the commonly used positive Qingfengtongning sustained-release tablets with sinomenine as the main component are combined with methotrexate, celecoxib and the like to treat RA, and the acute joint swelling and pain has obvious improvement, but the specific mechanism is still unclear, and it may be related to reducing local inflammatory reaction; at present, there is no report about sinomenine taking the macrophages infiltrated in the dorsal root ganglion (DRG) of the peripheral nervous system as the action target; therefore, it has high value to research and develop a new active ingredient of non-opioid drug to treat chronic pain of rheumatoid arthritis.

[0004] The application with the application publication number CN109364077A discloses the application of sinomenine in the preparation of a drug for treating rheumatoid arthritis. The dosage form of sinomenine is an injection solution, which comprises sinomenine monomer, DMSO solution and PBS buffer. The purity of sinomenine monomer is higher than 99.78%, and the mass concentration of sinomenine monomer is 30 mg / kg-300 mg / kg. In the technical solution of the application, sinomenine down-regulates the concentrations of hypoxia-inducible factor HIF-1 alpha, vascular endothelial growth factor VEGF and angiopoietin 1 ANG1 of rheumatoid arthritis through HIF VEGF-ANG axis. The sinomenine monomer with a mass concentration of 300 mg / kg can inhibit the expression of HIF-1 alpha, VEGF and ANG1. However, the application only intervenes in inflammatory factors, and it is difficult to fundamentally solve the chronic pain problem caused by rheumatoid arthritis, and it cannot explain the potential mechanism of the nervous system function.

[0005] In summary, the existing drugs usually have the following shortcomings: 1) Limited target: most of the existing drugs focus on traditional anti-inflammatory targets such as cytokines IL-1 beta, IL-6, TNF-alpha and their signaling pathways, and pay less attention to the complex neuro-immune interaction mechanism behind rheumatoid arthritis chronic pain, and lack of attention to targets such as histamine H1R on dorsal root ganglion M1 macrophages, and the treatment is not accurate and comprehensive. 2) Poor treatment effect: only intervening in inflammatory factors, it is difficult to fundamentally solve the chronic pain problem caused by rheumatoid arthritis, and the effect of relieving nerve inflammation and pain caused by macrophage polarization is limited, and it cannot effectively improve the chronic pain symptoms of patients. SUMMARY

[0006] The purpose of the present application is to solve the problems in the prior art, and to provide a sinomenine for preparing a drug for treating rheumatoid arthritis chronic pain and its application, which can solve the problems of limited target and poor treatment effect.

[0007] To achieve the above-mentioned purpose, the present application provides a sinomenine for preparing a drug for treating rheumatoid arthritis chronic pain. The active ingredient in the drug for treating rheumatoid arthritis chronic pain is sinomenine, and the sinomenine takes histamine receptor H1R on dorsal root ganglion M1 macrophages as the target.

[0008] As a preferred embodiment, the sinomenine intervenes in the polarization of rheumatoid arthritis macrophages through the mechanism mediated by histamine receptor H1R. The sinomenine reduces the expression of histamine receptor H1R in M1 macrophages, and the sinomenine inhibits the infiltration of M1 macrophages. M1 macrophages are pro-inflammatory macrophages.

[0009] As preferred, the structural formula of the sinomenine is as follows: .

[0010] As preferred, the rheumatoid arthritis is caused by abnormal macrophage function.

[0011] As preferred, the dosage form of the medicine is a pharmaceutically acceptable dosage form preparation, and the dosage form is selected from tablets, capsules, emulsions, patches or injections.

[0012] To achieve the above-mentioned purpose, the application further provides a use of sinomenine in the preparation of a medicine for treating chronic pain of rheumatoid arthritis, and the use of sinomenine in the preparation of an anti-rheumatoid arthritis chronic pain medicine by inhibiting the release of inflammatory factors of rheumatoid arthritis.

[0013] As preferred, the use of sinomenine in the preparation of a medicine for inhibiting chronic pain caused by rheumatoid arthritis, including cold pain, mechanical pain and thermal pain.

[0014] As preferred, the use of sinomenine in the preparation of an anti-rheumatoid arthritis chronic pain medicine by inhibiting LPS-induced macrophage polarization.

[0015] As preferred, the use of sinomenine in the preparation of an anti-rheumatoid arthritis chronic pain medicine by regulating the expression change of histamine receptor H1R using siRNA and overexpression plasmid.

[0016] Advantages of the application: 1) Unique action target and mechanism: the patent focuses on histamine receptor H1R on dorsal root ganglion (DRG) M1 type macrophages as an action target, and effectively regulates the macrophage polarization process related to rheumatoid arthritis by precisely intervening the target, thereby effectively solving the persistent chronic pain caused by rheumatoid arthritis, and having the advantage of good curative effect. 2) Significant therapeutic effect: by acting on rheumatoid arthritis DRG M1 type macrophages as a target, the abnormal invasion, migration and other biological behaviors of rheumatoid arthritis DRG M1 type macrophages are intervened to achieve the therapeutic effect, the expression of histamine H1R in DRG M1 type macrophages is significantly reduced, the M1 type macrophage infiltration is effectively inhibited, and the inflammatory symptoms of rheumatoid arthritis are significantly improved, including various chronic pain types such as cold pain, mechanical pain and thermal pain, and the patient's pain is effectively relieved, and the treatment effect is better than that of some existing treatment methods.

[0017] The features and advantages of the application will be described in detail in conjunction with the embodiments and the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1Figure 6 is the improvement of SIN on the pain and arthritis symptoms of CIA model mice, wherein A and B detect the effects of different amounts of SIN on the mechanical pain and cold pain threshold of CIA model mice, respectively; C shows the changes in the thickness of the hind paws of mice in each treatment group; D presents the effects of different treatments on the arthritis score of CIA model mice; E is the effect of different treatments on the mechanical withdrawal threshold of the hind paws of mice in the CFA-induced pain model; F is the effect of different treatments on the acetone stimulation and arthritis score of mice in the CFA-induced pain model, “***” represents the comparison between the Naive group and the CIA group or the CFA group, p < 0.001; “##” represents the comparison between the CIA group or the CFA group and the different dose SIN groups, p < 0.01; “###” represents the comparison between the CIA group or the CFA group and the different dose SIN groups, p < 0.001; Figure 2 Figure 7 is the effect of SIN on arthritis and immune indicators of CIA model mice, wherein A is the improvement of different amounts of SIN on the joint appearance of CIA model mice; B is the Anti-CCP antibody level of mice in each treatment group; C and D detect the levels of CD86 and CD206, respectively; E, F and G detect the levels of IL-1β, IL-6 and IL-10, respectively, “**” represents the comparison between the Naive group and the CIA group, p < 0.01, “***” represents the comparison between the Naive group and the CIA group, p < 0.001; “#” represents the comparison between the CIA group and the different dose SIN groups, p < 0.05; “##” represents the comparison between the CIA group and the different dose SIN groups, p < 0.05; “###” represents the comparison between the CIA group and the different dose SIN groups, p < 0.001; Figure 3 Figure 8 is the inhibitory effect of SIN on LPS-induced cell migration, wherein A shows the microscopic observation results of cell migration under LPS stimulation and SIN intervention; B quantifies the number of cells in each group, “**” represents the comparison between the Naive group and the LPS group, p < 0.01; “##” represents the comparison between the LPS group and the SIN group, p < 0.01; Figure 4 Figure 9 is the role of H1R in LPS-induced cell migration and the intervention mechanism of SIN, wherein A and C show the microscopic observation results of LPS-induced cell migration under H1R silencing or overexpression; B and D quantify the effects of H1R silencing and overexpression on the number of cells migrating, “*” represents the comparison between the Naive group and the H1R-OE group, p < 0.05; “**” represents the comparison between the Naive group and the LPS group, p < 0.01; “#” represents the comparison between the LPS group and the siRNA(H1R) group, p < 0.05; Figure 5Effect of SIN on LPS-induced H1R and CD86 expression, wherein A shows the expression of H1R and CD86 under different treatment conditions by fluorescence staining; B shows the relative fluorescence intensity of H1R and CD86 in each treatment group in the form of a column chart, “***” represents the comparison between the Naive group and the LPS group, p < 0.001; “##” represents the comparison between the LPS group and the medium-dose SIN group, p < 0.01; “###” represents the comparison between the LPS group and the high-dose SIN group, p < 0.001. DETAILED DESCRIPTION

[0019] The sinomenine is used for treating chronic pain of rheumatoid arthritis, and the application of the sinomenine in the preparation of the chronic pain drug for treating rheumatoid arthritis. The active ingredient in the chronic pain drug for treating rheumatoid arthritis is sinomenine, and the sinomenine takes histamine receptor H1R on the dorsal root ganglion M1 type macrophage as an action target. The sinomenine interferes with the macrophage polarization of rheumatoid arthritis through a mechanism mediated by histamine receptor H1R. The sinomenine reduces the expression of histamine receptor H1R in the M1 type macrophage, and the sinomenine inhibits the infiltration of the M1 type macrophage. The structural formula of the sinomenine is as follows: .

[0020] The rheumatoid arthritis is caused by abnormal macrophage function. The dosage form of the drug is a pharmaceutically acceptable dosage form preparation, and the dosage form is selected from tablets, capsules, emulsions, or injections.

[0021] The application of the sinomenine in the preparation of the chronic pain drug for treating rheumatoid arthritis is also provided. The sinomenine is used for preparing an anti-rheumatoid arthritis chronic pain drug for inhibiting inflammatory factors released by rheumatoid arthritis. The sinomenine is used for preparing a chronic pain drug for inhibiting chronic pain caused by rheumatoid arthritis, including cold pain, mechanical pain, and thermal pain. The sinomenine is used for preparing an anti-rheumatoid arthritis chronic pain drug for inhibiting macrophage polarization induced by LPS. The sinomenine is used for preparing an anti-rheumatoid arthritis chronic pain drug for regulating the expression change of histamine receptor H1R by using siRNA and overexpression plasmid.

[0022] Example 1: Preparation of sinomenine injection 100 ng / mL LPS solution: 1 mg of LPS powder was dissolved in 10 mL of DMEM, and stored at -20°C for standby.

[0023] DMEM containing 10% FBS: 5 mL of FBS, 100 U / mL of penicillin, and 100 U / mL of streptomycin double antibody 500 μL were added to 44.5 mL of DMEM medium.

[0024] 10 μg / ml, 20 μg / ml, 50 μg / ml of sinomenine injection: 10 μg, 20 μg, 50 μg of sinomenine powder was dissolved in 1 mL of normal saline to obtain the corresponding 10 μg / ml, 20 μg / ml, 50 μg / ml of sinomenine injection, stored at 4°C for standby.

[0025] Example 2: CFA modeling experiment in mice Purpose of the experiment: to verify the therapeutic effect of sinomenine monomer on rheumatoid arthritis (RA), especially its effect on dorsal root ganglion (DRG) M1 type macrophages.

[0026] Experimental grouping: Naive group: injection of normal saline; CFA Model group: injection of complete Freund's adjuvant (CFA) to induce arthritis; Sinomenine low, medium and high dose groups: after injection of complete Freund's adjuvant (CFA) to induce arthritis, 20 mg / kg, 50 mg / kg, and 100 mg / kg doses of sinomenine were given by gavage, respectively.

[0027] Experimental procedure: C57 mice were placed in an isoflurane gas anesthetizing machine, and the mice were induced to anesthesia at a concentration of 3% to 4%, and then maintained at a dose of 1.5% to 2.5%; during the anesthesia of the mice, the Naive group of mice was injected with normal saline into the knee joint, and the other groups of mice were injected with 15 μl of complete Freund's adjuvant (CFA) into the knee joint, and the immunization was repeated every seven days until the mice were sacrificed on the 28th day.

[0028] Example 3: Analgesic experiment in mice Purpose of the experiment: to observe the effect of different concentrations of sinomenine on the analgesic effect of CFA mice.

[0029] Experimental grouping: Naive group: daily intraperitoneal injection of normal saline (0.2 ml, qd) for 28 consecutive days; CFA Model group: daily intraperitoneal injection of normal saline (0.2 ml, qd) for 28 consecutive days; Sinomenine low dose group: daily intraperitoneal injection of 20 mg / kg sinomenine solution (0.2 ml, qd) for 28 consecutive days; Sinomenine medium dose group: daily intraperitoneal injection of 50 mg / kg sinomenine solution (0.2 ml, qd) for 28 consecutive days; Sinomenine high dose group: daily intraperitoneal injection of 100 mg / kg sinomenine solution (0.2 ml, qd) for 28 consecutive days.

[0030] Experimental result analysis: Example 4: Von-Frey test Objective: To observe the changes in mice's withdrawal of the paw using Von-Frey test, in order to determine the effect of the drug on the mechanical pain threshold of mice.

[0031] Experimental procedure: a) Place the animals in the compartment on the metal mesh for 20 minutes of adaptation; b) Start with Von-Frey fiber (0.6g) to vertically touch the left foot of the mouse, until the fiber is slightly bent, and record the response; when the mouse shows positive response such as withdrawal of the paw, use Von-Frey fiber with smaller stimulating force; if the mouse does not show response, use Von-Frey fiber with larger stimulating force, the Von-Frey fiber used is 0.04g to 4g, a total of 9; c) Repeat the above steps 5 times; d) Enter the recorded data into the up and down system for calculation.

[0032] Experimental results analysis: Example 5: Acetone cold pain test Objective: To observe the changes in mice using acetone cold pain test, in order to determine the changes in the pain threshold of mice.

[0033] Experimental procedure: a) Place the animals in the compartment on the metal mesh for 15 minutes of adaptation; b) Drop acetone on the left foot of the mouse, and observe its behavioral response within 30 seconds, record the response in the form of score, the scoring criteria are as follows: c) Repeat step twice, with an interval of two minutes each time; d) Add the scores of three times to analyze the data.

[0034] Table 1: Standard score of the reaction of acetone to the left foot of the mouse

[0035]

[0036] Experiment 6: LPS-induced macrophage polarization and migration experiment Objective: To observe the effect of different concentrations of sinomenine pretreatment on LPS-induced M1 polarization and migration of RAW264.7 macrophages.

[0037] Experimental groups: Naive group: no treatment; Model control group: cells exposed to the corresponding complete medium; Low-dose sinomenine group: cells exposed to 10 µg / ml dose of sinomenine solution; Sinomenine low-dose group: cells were exposed to a 20 µg / ml dose of sinomenine solution; Sinomenine low-dose group: cells were exposed to a 50 µg / ml dose of sinomenine solution.

[0038] Experimental procedure: Except for the Naive group, which was not intervened, the rest of the cells were exposed to the corresponding complete medium, the corresponding dose of sinomenine solution for 24 h, and then induced by 100 ng / ml of LPS solution for 24 h.

[0039] Experiment 7: Fluorescence in situ hybridization experiment Purpose of the experiment: To observe the expression of H1R / H2R on LPS-induced macrophages by fluorescence in situ hybridization technology.

[0040] Experimental procedure: According to the RNAscope kit: a) Thawing of the section: Take the section frozen at -40℃ and recover it at room temperature for 30 minutes; b) Washing: Use a 200 µl pipette to take 1×PBS solution prepared, drop it on the slide, shake gently to make the PBS fully contact the sample, and then stand for 5 minutes, repeat the above operation 3 times; c) Fixing: immerse the slide in a mixture of anhydrous methanol and 30% hydrogen peroxide prepared at a ratio of 50:1, incubate at room temperature for 10 minutes; d) Washing: Repeat step b; e) Antigen repair: immerse the slide in 1×Garget Retrieval antigen repair solution, and boil for 5 minutes; f) Washing: Repeat step b; g) Dehydration: immerse the slide in anhydrous ethanol, stand at room temperature for 3 minutes; h) Washing: Repeat step b; i) Add probe: take 1 µl of H1R probe, drop it on the sample to completely cover the sample, put the slide holder into the wet box, cover the cover, and put it into the hybridization oven, incubate at 40℃ for 2 hours; j) Washing: take 200 µl of 1×wash buffer and drop it on the slide, shake gently to make the wash solution evenly contact the sample, and then stand for 5 minutes; use absorbent paper to dry the wash solution, repeat the above operation 3 times; k) Hybridization AMP1: take 1 µl of multi-channel second-generation fluorescence AMP1, drop it on the sample to completely cover the sample; put the slide holder into the wet box, cover the cover, and put it into the hybridization oven; incubate at 40℃ for 30 minutes; l) Washing: Repeat step j; m) Hybridization AMP2: 1 μΐ of multi-channel second-generation fluorescent AMP2 was taken up and dropped on the sample to completely cover the sample. The slide holder was placed in a wet box, covered with a cover, and placed in a hybridization oven. Incubation was performed at 40°C for 30 minutes. n) Washing: Step j was repeated. o) Hybridization AMP3: 1 μΐ of multi-channel second-generation fluorescent AMP3 was taken up.

[0041] Experiment 8: Cell Transwell Experiment Purpose of the experiment: To evaluate the effect of sinomenine on the migration ability of macrophages.

[0042] Experimental groups: Naive group: No special treatment; LPS group: 100 ng / ml LPS was added; SIN+LPS group: 50 μg / ml sinomenine and 100 ng / ml LPS were added.

[0043] Experimental steps: a) Plating: The upper chamber of the Transwell was removed first, and an appropriate amount of complete medium was added to the lower chamber. The cells were digested with trypsin and centrifuged, mixed with DMEM basic medium, and transferred to the upper chamber. b) Drug administration: Sinomenine (50 μg / ml) was added to the chamber of the drug administration group, and 100 ng / mL of LPS was added to the LPS group and the drug administration group. The culture was carried out in a 37°C, 5% CO2 incubator. The blank group was not treated specially. The chamber was carefully placed back into the lower chamber containing the culture medium, and the contact surface between the lower chamber of the chamber and the culture medium in the lower chamber was free of air bubbles. c) Fixation: The upper chamber was removed and washed gently with PBS, repeated 3 times, then treated with 4% paraformaldehyde solution for 15 minutes. The PBS washing process was repeated. Then, the chamber was soaked in crystal violet dye for 30 minutes, and the PBS washing process was repeated again. d) Wiping: The FLS that did not pass through the upper chamber were gently wiped off with a cotton swab. e) Observation: Finally, the number of FLS on the membrane of the chamber was observed under a microscope.

[0044] Through the above experiments, the following data analysis results were obtained: 1. Effect of SIN on foot swelling, pain threshold and arthritis score in CIA model Combined with A-D in Figure 1 , we have: From the figure, we can see that Figure 1Figures A and B: Hindpaw Withdrawal Threshold of mice in different treatment groups (Naive, CIA Model, CIA Model + SIN different doses) respectively, detecting mechanical pain and thermal pain threshold, wherein: Hindpaw Withdrawal Threshold is the hindpaw withdrawal threshold of mice, Days after first-immunization is the number of days after the first immunization, Naive group is the normal control group, CIA Model is the collagen-induced arthritis mouse model group, CIA Model + SIN different doses is the collagen-induced arthritis mouse model group treated with different doses of sinomenine, Hindpaw thickness is the hindpaw thickness, and Arthritis scores is the arthritis score.

[0045] Figure A is the hindpaw withdrawal threshold within several days after the first immunization, detecting mechanical pain threshold: the mechanical pain threshold of the CIA Model group decreases, and the threshold increases after SIN treatment, and the higher the dose, the more obvious the effect, indicating that SIN can relieve the mechanical pain of CIA model mice.

[0046] Figure B is the hindpaw withdrawal threshold within several days after the first immunization, detecting cold pain threshold: the cold pain threshold of the CIA Model group decreases, and the threshold increases after SIN treatment, and there is a significant difference compared with the model group, indicating that SIN also has a relieving effect on cold pain.

[0047] Figure C is the hindpaw thickness within several days after the first immunization: the hindpaw thickness of the CIA Model group increases, and SIN treatment reduces the hindpaw thickness, especially in the high-dose group, indicating that SIN can reduce joint swelling.

[0048] Figure D is the arthritis score within several days after the first immunization: the arthritis score of the CIA Model group increases, and the score decreases after SIN treatment, and it is dose-dependent, further proving the improvement of SIN on the symptoms of arthritis.

[0049] Analysis of experimental results: the data shows that sinomenine relieves the cold pain and mechanical pain caused by CFA in mice in a dose-dependent manner; compared with the Naive group, the foot swelling degree of the CIA Model group gradually increases, the paw withdrawal threshold decreases, and the arthritis score increases; after treatment of the CIA Model + SIN different doses group, the foot swelling degree is relieved to some extent, the paw withdrawal threshold is improved, and the arthritis score is reduced, and the effect of the high-dose group is more obvious, further confirming the therapeutic effect of SIN in rheumatoid arthritis model.

[0050] 2. Evaluation of analgesic effect of SIN in CFA-induced inflammatory pain model Combination Figure 1 of E and F in the present application: Wherein, the ordinate Hindpaw Withdrawal Threshold is the hindpaw withdrawal threshold of mice, the ordinate Acetone Scores is the acetone score, the abscissa Time after CFA injection is the time after CFA injection, the Naive group is the normal control group, the CIA Model is the collagen-induced arthritis mouse model group, the CIA Model+SIN different amounts is the collagen-induced arthritis after adding different amounts of 20, 50, 100 mg / kg sinomenine, and the CFA Model+Meloxicam group is the collagen-induced arthritis after adding 2 mg meloxicam.

[0051] E figure shows the changes of hindpaw withdrawal threshold of mice in different treatment groups: Naive, CFA Model, CFA Model+SIN different amounts, CFA Model+Meloxicam at different time points after CFA injection; wherein the threshold of CFA Model group decreased significantly, indicating that CFA induced pain; after giving SIN or Meloxicam, the threshold increased, especially in SIN (20 mg / kg and 50 mg / kg) group and Meloxicam group, which had significant difference compared with CFA Model group, and the threshold of CFA Model+SIN (100 mg / kg) increased and approached to Naive, indicating that SIN and Meloxicam could relieve CFA-induced pain.

[0052] F figure presents the changes of acetone score of each group at different time points after CFA injection, the score of CFA Model group increased, indicating that the pain intensified; the scores of SIN treatment groups (especially 20 and 50 mg / kg groups) and Meloxicam group were lower than that of CFA Model group, indicating that SIN and Meloxicam could reduce pain-related behaviors, and SIN had analgesic effect on CFA-induced inflammatory pain.

[0053] 3. Evaluation of the effect of SIN on rheumatoid arthritis in CIA model Combination Figure 2 : Lateral is the front of the mouse, Frontal is the side of the mouse, Naive group is the normal control group, CIA Model is the collagen-induced arthritis mouse model group, CIA Model + SIN different amounts are different amounts of sinomenine added after collagen-induced arthritis 10, 20, 50 mg / kg; The ordinate Anti-CCP Antiboay is anti-cyclic citrullinated peptide antibody, which is a highly specific autoantibody index for the diagnosis of rheumatoid arthritis; CD86 is an important immune cell surface molecule and one of the markers of M1 macrophages; CD206 is a molecule mainly present on the surface of M2 macrophages, immature dendritic cells and liver sinus endothelial cells; By detecting the expression levels of CD206 and CD86, the polarization state and function of macrophages can be understood; IL-1β is interleukin-1β, a pleiotropic pro-inflammatory cytokine, and an increase in IL-1β level usually indicates that the body is inflamed or infected, such as rheumatoid arthritis, hepatitis, respiratory tract infection, etc.; IL-6 is interleukin-6, a multifunctional cytokine produced by mononuclear cells, fibroblasts, endothelial cells and other cells, and is a commonly used inflammatory marker. Its level increase can reflect the body's inflammatory and infectious conditions; IL-10 is interleukin-10, which is produced by Th2 cells and is an anti-inflammatory cytokine with immune regulation function, which can inhibit the activity of macrophages and reduce the release of inflammatory mediators. Changes in IL-10 levels can reflect the body's immune regulation state and help assess the condition of inflammatory and immune-related diseases.

[0054] A figure: CFA modeling and drug administration diagram of C57 mice, showing the appearance of the front and side joints of mice in different treatment groups (Naive, CIA Model, SIN different amounts); The joints of CIA Model group mice appear obvious redness and swelling, while after giving SIN, the degree of joint redness and swelling is reduced, and the higher the dose, the more obvious the improvement.

[0055] B-D figures: The levels of Anti-CCP antibody, CD86 and CD206 were detected respectively; B figure: The level of Anti-CCP antibody in CIA Model group is increased, and the level of antibody is decreased after SIN treatment, especially in high-dose SIN group, indicating that SIN can inhibit the production of autoantibody.

[0056] C and D figures: The level of CD86 in CIA Model group is increased, and the level of CD206 is not obviously changed, SIN treatment makes the level of CD86 decrease, and the level of CD206 in SIN (50 mg / kg) group is increased, indicating that SIN can regulate the expression of immune cell surface molecules and affect immune response.

[0057] E-G figure: The levels of cytokines IL-1β, IL-6 and IL-10 were detected.

[0058] E and F figures: The levels of IL-1β and IL-6 in the CIA Model group were significantly increased, and after SIN treatment, the levels of both were decreased in a dose-dependent manner, indicating that SIN has the effect of inhibiting the secretion of inflammatory factors.

[0059] G figure: The level of IL-10 in the Naive group is low, and in the CIA Model group, it is increased, and in the SIN treatment group, the level of IL-10 is further increased, indicating that SIN may promote the production of anti-inflammatory cytokine IL-10.

[0060] Result analysis: Compared with the normal control group (Naive), the CIA model group of mice has increased hind foot thickness, obvious joint swelling, abnormal levels of anti-CCP antibody, CD86, CD206 and other indicators in serum; E-G is to detect the expression of IL-1β, IL-6 and IL-10 inflammatory factors in mouse serum by ELISA experiment, from the data results, the sinomenine treatment dose-dependent reduces the secretion of IL-1β, IL-6, and increases the expression of IL-10, among them, the levels of pro-inflammatory factors such as IL-1β, IL-6 are increased, and the levels of anti-inflammatory factors such as IL-10 are decreased, indicating that the CIA model is successfully constructed, and after different doses of SIN administration, it can reduce the degree of joint swelling, reduce the levels of anti-CCP antibody, CD86, CD206, regulate the balance of pro-inflammatory and anti-inflammatory factors, and the effect is enhanced with the increase of dose, indicating that SIN has an improvement effect on rheumatoid arthritis.

[0061] 4. Effect of SIN on macrophage migration under LPS stimulation Combined Figure 3 : Wherein, Bright field is bright field, Naive group is normal control group, LPS is lipopolysaccharide, and it is the main component of gram-negative bacterial cell wall and has strong immune stimulating capacity;LPS as endotoxin can combine with specific receptors in the immune system, trigger a series of inflammatory cascade reactions, when LPS enters the body, first combine with LPS binding protein (LBP), then be transported to CD14 and Toll-like receptor 4 (TLR4) on the surface of immune cells, finally activate signal pathways such as nuclear factor kappa B (NF-κB), leading to the release of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6);LPS induction is an experimental method commonly used in scientific research, which uses lipopolysaccharide (Lipopolysaccharide, LPS) to induce the inflammatory response of organisms;LPS+SIN is 50ug / ml sinomenine after LPS induction;The ordinate normalized number of cells migrated is the normalized number of migrated cells.

[0062] Figure A shows the migration of cells after different treatments (Naive, LPS, LPS+SIN) under LPS stimulation, the migration of cells in the LPS group increases, while the migration of cells in the LPS+SIN group decreases, indicating that SIN may inhibit LPS-induced cell migration.

[0063] Figure B shows the quantitative results of cell migration in each group in the form of a column chart, the number of cell migration in the LPS group is significantly higher than that in the Naive group, and the number of cell migration in the LPS+SIN group is lower than that in the LPS group, further confirming the inhibitory effect of SIN on LPS-induced cell migration.

[0064] From the data in the figure, Figure 3 It can be known that the sinomenine monomer of the application can effectively inhibit LPS-induced macrophage migration, and further, Figure 3 A in the figure is the data result of using the Transwell experiment to evaluate the effect of sinomenine on macrophage migration; Figure 3 B in the figure is the statistics thereof, and compared with the blank group, it can be known from the figure that the cell migration rate and wound healing rate of the LPS group increase, and SIN treatment leads to wound healing rate and migration.

[0065] 5.Effect of different treatments on macrophage migration and H1R expression under LPS stimulation Combined with Figure 4 : Naive group is the normal control group, LPS group is the induced group using lipopolysaccharide to induce the inflammatory response of the organism, siRNA (H1R) is used to specifically silence the expression of histamine H1 receptor (H1R) gene, and LPS is used to induce inflammation. siRNA (H1R) is a small interfering RNA specifically designed to recognize and silence the mRNA of histamine H1 receptor (H1R), thereby inhibiting the expression of H1R protein. Histamine H1 receptor is a G protein-coupled receptor widely present in various tissues and cells, involved in allergic reactions, inflammatory responses and other physiological and pathological processes. The use of siRNA (H1R) combined with LPS can explore the specific role of H1R in LPS-induced inflammatory response; the ordinate Normalized cells migrated is the normalized cell migration amount; H1R-OE refers to the overexpression (Overexpression) of histamine H1 receptor (H1R), which is commonly used to study the function and mechanism of H1R in specific physiological or pathological processes. H1R-OE model is commonly used to study the role of H1R in allergic reactions, inflammatory responses, nervous system diseases and other physiological and pathological processes. By increasing the expression of H1R, researchers can observe its influence on cell signaling, physiological function and disease development.

[0066] Figure A shows the migration of cells in different treatment groups: Naive, LPS, siRNA (H1R) + LPS, where LPS (H1R) group has increased cell migration, while siRNA (H1R) + LPS group has reduced cell migration, indicating that H1R may be involved in LPS-induced cell migration.

[0067] Figure B quantifies the number of cells migrated in each group. The number of cells migrated in LPS (H1R) group is significantly higher than that in Naive group, and the number of cells migrated in siRNA (H1R) + LPS group is lower than that in LPS (H1R) group, supporting the above conclusion.

[0068] Figure C shows the migration of cells in Naive and H1R-OE groups under LPS treatment. The H1R-OE group has increased cell migration, indicating that H1R overexpression may promote cell migration.

[0069] Figure D presents the number of cells migrated in each group in the form of a column chart. The number of cells migrated in H1R-OE group is higher than that in Naive group, further confirming the role of H1R in cell migration.

[0070] After LPS stimulation, the number of cell migration increased, and the expression of H1R also increased. After using siRNA of H1R, the cell migration was inhibited, and the expression of H1R decreased; on the contrary, in the cells of H1R overexpression (H1R-OE), LPS stimulation further promoted cell migration, and the expression of H1R also increased accordingly, indicating that H1R played a key role in LPS-induced macrophage migration.

[0071] 6. Effect of SIN on the expression of H1R and CD86 under LPS stimulation Combined Figure 5 : Among them, the Naive group is the normal control group, the LPS group is the group induced by lipopolysaccharide to induce the inflammatory response of the organism, and the LPS+Sinomenine different amount group is the group induced by lipopolysaccharide to induce the inflammatory response of the organism after adding different amounts of 10, 20, 50 ug / ml sinomenine; DAPI is a commonly used fluorescent dye, which can strongly bind to DNA, and is especially suitable for cell nucleus staining. After binding to DNA, it can be excited by ultraviolet light under a fluorescence microscope, emitting blue fluorescence; Merge is a group of mixed staining agents; the ordinate Relative fluorescence intensity of H1R is the relative fluorescence intensity of H1R, and Relative fluorescence intensity of H1R relative to CD86 is the relative fluorescence intensity of H1R relative to CD86.

[0072] A figure: the expression of H1R and CD86 under different treatments (Naive, LPS, SIN different amount+LPS) is shown by fluorescence staining, wherein the expression of H1R (red) and CD86 (green), and the cell nucleus stained by DAPI is blue; Naive group: the expression level of H1R (red fluorescence) and CD86 (green fluorescence) in the cell is low, and the background signal is weak, indicating that the expression amount of these two proteins is small under normal circumstances; LPS (100 ng / ml) group: after LPS stimulation, the expression of H1R and CD86 in the cell significantly increased, and the red and green fluorescence signals significantly enhanced, and there were more co-localization (yellow signal), indicating that LPS activated the inflammatory response, and prompted the cell to express more H1R and CD86; SIN (10 μg / ml) + LPS (100 ng / ml) group: compared with the simple LPS group, the fluorescence intensity of H1R and CD86 was weakened, and the yellow signal of co-localization also decreased, indicating that low-dose SIN had a certain inhibitory effect on the expression of H1R and CD86 induced by LPS; SIN (20 μg / ml) + LPS (100 ng / ml) group: after the dose of SIN was increased, the fluorescence signals of H1R and CD86 were further weakened, and the co-localization signals were also less, indicating that the medium dose of SIN could more effectively inhibit the expression of H1R and CD86 induced by LPS; SIN (50 μg / ml) + LPS (100 ng / ml) group: after the treatment of high-dose SIN, the fluorescence intensity of H1R and CD86 was the lowest, and the yellow signal of co-localization was the least, indicating that high-dose SIN had the strongest inhibitory effect on the expression of H1R and CD86 induced by LPS.

[0073] B figure: the relative fluorescence intensity of H1R and CD86 was respectively shown in the form of column chart, the fluorescence intensity of H1R and CD86 in LPS group was higher than that in Naive group, and the fluorescence intensity in SIN treatment group was lower than that in LPS group, which was consistent with the results of A figure, further quantifying confirmed the inhibitory effect of SIN on the expression of H1R and CD86.

[0074] LPS stimulation can increase the expression level of H1R and CD86, and after the treatment of different concentrations of SIN, the expression of H1R and CD86 induced by LPS can be inhibited, and the inhibitory effect is enhanced with the increase of the concentration of SIN, indicating that SIN may play its anti-inflammatory effect by regulating the expression of H1R and CD86.

[0075] Overall analysis These experimental results as a whole show that SIN has significant relieving effect in various inflammation and pain models, it can reduce the pain behavior, joint swelling and inflammatory reaction of mice induced by CFA and CIA, inhibit the cell migration and the expression of related molecules (such as H1R and CD86) induced by LPS, and regulate the level of cytokines (reduce pro-inflammatory factors IL-1β and IL-6, and increase anti-inflammatory factor IL-10); in addition, H1R may play an important role in the cell migration and related inflammatory reaction induced by LPS, and SIN may play its anti-inflammatory effect by affecting the expression of H1R, which provides experimental basis for the application of SIN as anti-inflammatory analgesic drug.

[0076] The above examples are illustrative of the present application and are not limiting of the present application, any simple transformation of the present application also belongs to the protection scope of the present application.

Claims

1. A sinomenine used in the preparation of a drug for treating chronic pain in rheumatoid arthritis, characterized in that: The active ingredient in the drug for treating chronic pain in rheumatoid arthritis is sinomenine, which targets histamine receptor H1R on M1 macrophages of the dorsal root ganglion.

2. The preparation of a drug for treating chronic pain in rheumatoid arthritis using sinomenine as described in claim 1, characterized in that: The sinomenine intervenes in the polarization of rheumatoid arthritis macrophages through a mechanism mediated by histamine receptor H1R. The sinomenine reduces the expression of histamine receptor H1R in M1 macrophages and inhibits the migration and infiltration of M1 macrophages into the nervous system.

3. The preparation of a drug for treating chronic pain in rheumatoid arthritis using sinomenine as described in claim 1, characterized in that: The structural formula of the sinomenine is shown below: 。 4. The preparation of a drug for treating chronic pain in rheumatoid arthritis using sinomenine as described in claim 1, characterized in that: The rheumatoid arthritis mentioned above is caused by abnormal macrophage function.

5. The preparation of a drug for treating chronic pain in rheumatoid arthritis using sinomenine as described in claim 1, characterized in that: The rheumatoid arthritis mentioned above is caused by abnormal nervous system function.

6. The preparation of a drug for treating chronic pain in rheumatoid arthritis using sinomenine as described in claim 1, characterized in that: The dosage form of the drug is a pharmaceutically acceptable dosage form, selected from tablets, capsules, emulsions, patches, or injections.

7. The application of sinomenine in the preparation of a drug for treating chronic pain in rheumatoid arthritis, characterized in that: The application of the sinomenine in the preparation of an anti-rheumatoid arthritis chronic pain drug that inhibits the release of inflammatory factors by rheumatoid arthritis.

8. The application of sinomenine as described in claim 6 in the preparation of a drug for treating chronic pain in rheumatoid arthritis, characterized in that: The application of sinomenine in the preparation of drugs that inhibit chronic pain caused by rheumatoid arthritis, including cold pain, mechanical pain and heat pain.

9. The application of sinomenine as described in claim 6 in the preparation of a drug for treating chronic pain in rheumatoid arthritis, characterized in that: The application of the sinomenine in the preparation of an anti-rheumatoid arthritis chronic pain drug by inhibiting LPS-induced macrophage polarization.

10. The use of sinomenine as described in any one of claims 6 to 8 in the preparation of a drug for treating chronic pain in rheumatoid arthritis, characterized in that: The application of the sinomenine in the preparation of an anti-rheumatoid arthritis chronic pain drug by using siRNA and overexpression plasmids to regulate the expression changes of histamine receptor H1R.

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