Application of limonin in preparation of medicine for resisting chronic pain of rheumatoid arthritis

Abstract

The invention relates to application of limonin in preparation of a medicine for resisting rheumatoid arthritis chronic pain, and belongs to the technical field of medicine application. In order to solve the problem that the existing medicine for treating the chronic pain of rheumatoid arthritis is difficult to control, the invention provides application of limonin in preparation of the medicine for treating the chronic pain of rheumatoid arthritis, and the limonin serves as an active ingredient in the medicine for treating the chronic pain of rheumatoid arthritis. The limonin is used for preparing the medicine for resisting the rheumatoid arthritis chronic pain, wherein the medicine intervenes the functions of rheumatoid arthritis synovial fibroblasts through a histamine H4R mediated mechanism. The traditional Chinese medicine can effectively solve the persistent chronic pain caused by rheumatoid arthritis, and has the advantage of good curative effect.

Description

Technical Field

[0001] The invention relates to application of limonin in preparing a drug for treating chronic pain of rheumatoid arthritis, belonging to the technical field of drug application. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease with erosive arthritis as the main clinical manifestation, which can occur at any age. The pathogenesis of RA is still unclear. As the main clinical manifestation of RA, persistent chronic pain has become a major cause of distress for patients and clinical visits. At the same time, with the increase and persistence of joint pain, the joint activity of patients gradually decreases, and even eventually loses function, which seriously affects the life and work of patients. Although the pain of RA is generally believed to be caused by local stimulation of joint inflammation, in clinical practice, even after the condition is improved and multiple cycles of treatment with first- and second-line drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and anti-rheumatic drugs (DMARDs), some patients still cannot effectively relieve the persistent chronic pain in multiple joints. In addition, the use of opioids alone for analgesia has significant side effects, and may even cause hyperalgesia and addiction caused by opioids, which requires strict monitoring and reduction of long-term use. Therefore, in-depth research on the intrinsic pathogenesis of RA chronic pain, discovery of new intervention targets, and search for related non-opioid analgesics are the focus and difficulty of current research.

[0003] On the one hand, existing studies have found that synovial fibroblasts (FLS) play an indispensable role as key effector cells in the entire generation and conduction process of peripheral sensitization of chronic pain in rheumatoid arthritis (RA). FLS supports and maintains local inflammation of joints by secreting proinflammatory mediators such as IL-1β and TNF-α, and further induces sensitization of pain receptors in the peripheral nervous system. On the other hand, FLS function is abnormal. Under the stimulation of inflammation, FLS is activated, showing aggressive "tumor-like" characteristics and invasive phenotypes. Excessive proliferation can lead to abnormal proliferation of synovium, local swelling and deformation of joints, invasion and damage of local joint tissues, cartilage, etc., causing local pain in joints. In addition, the migration and invasiveness of FLS are greatly increased, and they can migrate from one joint to another, forming a symmetrical rheumatoid arthritis pattern, which may also be another very important reason for the difficulty in controlling RA pain, but there is currently no targeted drug for target action treatment.

[0004] Limonin is a type of triterpenoid compound found in Rutaceae and Meliaceae plants. Its structural formula is shown below:

[0005]

[0006] It is the most abundant in citrus and is an important secondary metabolite with high biological activity in plants. As early as the late 1990s, existing literature reported that limonin has anti-inflammatory and analgesic effects, and its mechanism may be related to the inhibition of inflammatory response. Some have also proposed that limonin may be a nuclear transcription factor-κB (NF-κB) inhibitor, which can regulate the function of CD4+T cells, have a certain effect on the proliferation of NF-κB-dependent CD4+T cells, and participate in the formation of chronic pain, but its specific mechanism of action is still unclear. There is currently no report on the therapeutic effect of limonin on rheumatoid arthritis, especially on peripheral joint synovial fibroblasts as the target. Therefore, it is of great value to study and develop a new non-opioid active ingredient to treat chronic pain in rheumatoid arthritis. Summary of the invention

[0007] In view of the problems existing in the above prior art, the present invention provides an application of limonin for preparing a drug for treating chronic pain of rheumatoid arthritis, and solves the problem of how to provide a new use of limonin and treat the chronic pain of rheumatoid arthritis through a new target.

[0008] The objective of the present invention is achieved through the following technical scheme: an application of limonin for preparing a drug for treating chronic pain of rheumatoid arthritis, wherein the active ingredient in the drug for treating chronic pain of rheumatoid arthritis is the limonin, and the structural formula of the limonin is as follows:

[0009]

[0010] The limonin is used for preparing the anti-rheumatoid arthritis chronic pain drug which intervenes in the function of rheumatoid arthritis synovial fibroblasts through the histamine H4R-mediated mechanism.

[0011] In the process of studying the above-mentioned specific limonin monomer, the present invention found that the limonin of the above-mentioned structure has a good therapeutic effect on the chronic pain of rheumatoid arthritis (also known as the treatment of rheumatoid arthritis). It is also found that it has a therapeutic effect on rheumatoid arthritis through a new target (H4R), that is, by taking H4R on synovial fibroblasts (FLS) as the target, it can effectively solve the persistent chronic pain caused by rheumatoid arthritis, and has the advantage of good efficacy. More specifically, limonin can effectively reduce the expression of histamine H4R in FLS, and inhibit the migration or invasion of FLS. The present invention uses limonin to act on histamine H4R of rheumatoid arthritis synovial fibroblasts as a target, intervenes in the abnormal invasion, migration and other biological behaviors of rheumatoid arthritis synovial fibroblasts to achieve the therapeutic effect, solves the cause of chronic pain caused by rheumatoid arthritis, and has a good therapeutic effect.

[0012] In the application of the above-mentioned limonin for preparing a drug for treating chronic pain in rheumatoid arthritis, preferably, the limonin inhibits synovial hyperplasia in rheumatoid arthritis, and the rheumatoid arthritis is caused by abnormal function of synovial fibroblasts in peripheral joints. Limonin can target the synovial fibroblasts of rheumatoid arthritis as a pharmacological action target, and can significantly improve the inflammatory symptoms of rheumatoid arthritis. The administration of limonin can effectively reduce the degree of synovial hyperplasia, inflammatory cell infiltration and cartilage damage; at the same time, it can effectively inhibit the migration and invasion of synovial fibroblasts in the joint cavity, thereby inhibiting its synovial hyperplasia, and realizing that the limonin monomer can effectively relieve chronic pain caused by rheumatoid arthritis, including cold pain, mechanical pain and heat pain, etc., and has the advantage of good effect in inhibiting chronic pain.

[0013] In the application of the above-mentioned limonin for preparing an anti-rheumatoid arthritis chronic pain drug, preferably, the limonin is used to prepare an anti-rheumatoid arthritis chronic pain drug by inhibiting the inflammatory factors released by rheumatoid arthritis. Limonin can effectively improve the degree of knee joint swelling and arthritis score of mice, reduce the secretion of IL-1β, IL-6 and TNF-α in the serum of CFA model mice, and the secretion of IL-1β and IL-6 induced by TNF-α in FLS, thereby effectively reducing the inflammatory factors released by rheumatoid arthritis, and realizing that limonin monomer can effectively alleviate the inflammation of rheumatoid arthritis, thereby inhibiting the chronic pain caused by rheumatoid arthritis.

[0014] In the application of the above-mentioned limonin for preparing a drug for treating chronic pain of rheumatoid arthritis, preferably, the limonin is used to prepare a drug for treating chronic pain of rheumatoid arthritis by inhibiting the abnormal function of FLS induced by TNF-α. Limonin can inhibit the abnormal migration behavior of FLS induced by TNF-α, thereby effectively improving the synovial hyperplasia caused by abnormal function of FLS.

[0015] In the application of limonin for preparing a drug for treating chronic pain of rheumatoid arthritis, preferably, the limonin is used to prepare a drug for treating chronic pain of rheumatoid arthritis by reducing the expression of H4R induced by TNF-α. Limonin can reduce the increase of H4R in FLS induced by TNF-α in a dose-dependent manner, which also indicates that the target of limonin in regulating FLS dysfunction may be H4R.

[0016] In the application of the above-mentioned limonin for preparing an anti-rheumatoid arthritis chronic pain drug, preferably, the limonin is used to prepare an anti-rheumatoid arthritis chronic pain drug by regulating the expression changes of H4R using siRNA and overexpression plasmid. H4R-OE can antagonize the inhibitory effect of limonin on abnormal migration and function of FLS, while the use of siRNA of H4R alone can inhibit the abnormal migration of synovial fibroblasts induced by TNF-α. In addition, when siRNA (H4R) and limonin are used simultaneously, no synergistic effect occurs. It shows that limonin regulates the abnormal function of FLS by intervening H4R.

[0017] In the application of the above-mentioned limonin for preparing a drug for treating chronic pain of rheumatoid arthritis, the dosage form of the above-mentioned drug can be any pharmaceutically acceptable medicament, and preferably, the dosage form of the drug is selected from tablets, capsules, emulsions or injections. When the active ingredient in the dosage form of the above-mentioned drug is limonin, pharmaceutically acceptable adjuvants can also be selected as needed.

[0018] In summary, compared with the prior art, the present invention has the following advantages:

[0019] 1. The present invention finds that limonin plays a therapeutic role on rheumatoid arthritis by intervening in synovial fibroblasts (FLS) as the target through a new mechanism mediated by histamine H4R, thereby effectively solving the persistent chronic pain caused by rheumatoid arthritis and having the advantage of good efficacy.

[0020] 2. Limonin targets rheumatoid arthritis synovial fibroblasts and intervenes in the abnormal invasion, migration and other biological behaviors of rheumatoid arthritis synovial fibroblasts to achieve the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the CFA modeling and drug administration of mice of the present invention and a data analysis diagram of the results of limonin alleviating arthritis chronic pain in CFA mice.

[0022] Figure 2 This is an analysis chart of the effects of limonin on rheumatoid arthritis symptoms in mice.

[0023] Figure 3 This is an analysis diagram of the scratch test and Transwell test results of mouse synovial fibroblasts.

[0024] Figure 4 It is an analysis diagram of the in situ hybridization experiment for detecting H4R expression and the PCR amplification experiment of the present invention.

[0025] Figure 5 This is a result analysis diagram of the effect of limonin of the present invention on cell migration or invasion. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further specifically described below through specific embodiments and drawings, but the present invention is not limited to these embodiments.

[0027] Example 1

[0028] The structural formula of limonin is as follows:

[0029]

[0030] Preparation of 1 mg / mL limonin solution: Dissolve 1 mg of limonin powder in 1 mL of DMEM and store at -20°C for later use.

[0031] 1 mg / mL LPS solution: Dissolve 10 mg of LPS powder in 10 mL of DMEM and store at -20°C for later use.

[0032] DMEM containing 10% FBS: Add 5 mL of FBS, 500 μL of 100 U / mL penicillin and 100 U / mL streptomycin to 44.5 mL of DMEM medium.

[0033] 25 mg / mL, 50 mg / mL, 125 mg / mL limonin injection: dissolve 1.25 g, 2.5 g, 5 g limonin powder in 50 mL of normal saline, respectively, to obtain the corresponding 25 mg / mL, 50 mg / mL, 125 mg / mL limonin injection, store at 4°C for later use.

[0034] Example 2

[0035] To illustrate that limonin monomers can have therapeutic effects on rheumatoid arthritis (RA), especially the performance of peripheral synovial fibroblasts as pharmacological targets, by affecting RA behavior, pathology, and abnormal migration and invasion of RA-FLS.

[0036] An incomplete Freund's adjuvant (CFA)-induced arthritis mouse model was established, and different concentrations of limonin (10 mg / kg, 30 mg / kg, 100 mg / kg) were administered by oral gavage. Figure 1 , from the figure we can see that Figure 1 The data in A are schematic diagrams of CFA modeling and drug administration in C57 mice; Figure 1 The data in B show that limonin relieved the cold pain caused by CFA in mice in a dose-dependent manner; the data in C show that limonin relieved the mechanical pain caused by CFA in mice in a dose-dependent manner, and Figure 1 The data in D show that limonin alleviated the thermal pain induced by CFA in mice in a dose-dependent manner.

[0037] Schematic diagram of CFA modeling and drug administration in mice and experimental procedures of limonin to relieve arthritis chronic pain in CFA mice ( Figure 1 The implementation of the analysis is as follows:

[0038] 1. Construction of CFA Mouse Model

[0039] (1) C57 mice were placed in a gas anesthesia machine containing isoflurane at a concentration of 3%-4% to induce anesthesia in the mice.

[0040] (2) The mice were then maintained in an anesthetized state using a dose of 1.5%-2.5%.

[0041] (3) During the anesthesia period, the knee joints of the mice were first exposed and fully disinfected with alcohol. The needle was held vertically for injection. When a slight puncture sensation was felt, the injection was started slowly. Normal saline was injected into the knee joints of the mice in the normal group, and 20 μL of complete Freund's adjuvant (CFA) was slowly injected into the knee joints of the mice in the other groups through a microsyringe. The mice were re-immunized every seven days until the mice were killed on the 28th day.

[0042] (4) Four weeks after the injection, the knee joint was sampled, fixed, decalcified, and sectioned.

[0043] 2. Animal Experiment Grouping and Drug Administration

[0044] This study used 30 C57 mice and randomly divided them into five groups: Naive group (6 mice), CFA group (6 mice), low-dose limonin group (6 mice), medium-dose limonin group (6 mice) and high-dose limonin group (6 mice). Among them, the mice in the Naive group only received saline injection into the knee joint, while the other four groups of mice received complete Freund's adjuvant (CFA) for model construction. During the administration process, the low, medium and high-dose limonin groups were gavaged once a day at doses of 10 mg / kg, 30 mg / kg and 100 mg / kg, respectively, for a total of 28 days. The CFA group was given saline daily as a control.

[0045] 3. Von-Frey test

[0046] The Von-Frey test was used to observe the changes in the paw withdrawal of mice, thereby detecting the effects of drugs on the mechanical pain threshold of mice.

[0047] (1) Preparation: Each group of mice was placed on a suspended metal net with a transparent acrylic cover on top. They were allowed to adapt to each compartment for 20 minutes in a quiet environment until the mice stopped exploratory behavior for subsequent experiments.

[0048] (2) Use Von Frey fibers of different diameters and hardness to vertically contact the sole of the right foot of the mouse until the fiber is bent 90°, and observe the experimental animals' responses to different stimuli to determine their pain sensitivity. During the experiment, Von Frey fibers (weight 0.6 grams) were used as the initial stimulus. Once the mouse was observed to show reactions such as lifting its paw, retracting its foot, making sounds, or jumping, the experiment was terminated and recorded as "X"; if there was no reaction, it was marked as "O" and the weight of the fiber was gradually increased to a higher level, but it should be noted that the upper limit was 4 grams to prevent damage to the mouse's soft tissue. Repeat the test several times and record the results of each experiment. Leave enough time intervals between the stimulations of different fiber fibers to allow the experimental animals to return to the basic state. In this process, the grams of Von Frey fibers used ranged from 0.04g to 4g, with a total of 9 fibers.

[0049] (3) Enter the above recorded data into the up and down system for calculation.

[0050] 4. Acetone cold pain test

[0051] In this part of the experiment, the acetone cold pain test was used to observe the changes in mice in order to determine the changes in the pain threshold of mice.

[0052] (1) Place the mouse in a transparent acrylic cover on a metal mesh and allow it to acclimate for 30 minutes. Maintain a quiet environment and wait until the mouse stops exploratory behavior before conducting subsequent experiments.

[0053] (2) Acetone was dropped onto the sole of the right foot of the mouse, and its behavioral response was observed within 30 seconds and the score was recorded in a table: 0 point - no response; 1 point - rapid paw retraction, flicking or stepping on the paw; 2 points - prolonged paw retraction or repeated paw flicking; 3 points - repeated paw flicking or paw licking.

[0054] (3) Repeat the above steps twice, with an interval of two minutes between each time.

[0055] (4) Add up the three scores and perform data analysis and statistics.

[0056] 5. Hargreaves test

[0057] (1) Preparation: First, transfer the animals from the housing room to the behavior room. Gently place each animal in its own enclosure and allow the animals to acclimate for 30-60 minutes.

[0058] (2) The animals were returned to the housing room and the habituation procedure was repeated for at least 2 days before the actual Hargreaves test.

[0059] (3) Set the desired infrared intensity and place the infrared transmitter on the container just below the center of the mouse's right paw.

[0060] (4) Observe the animal's paw until the animal reacts to the heat and then withdraws.

[0061] (5) Record the reaction time. If the animal fails to respond within 20 seconds, it is recommended to terminate the test to avoid potential burns.

[0062] (6) Repeat the test at least 3 times to obtain the average reaction time.

[0063] (7) Carefully return the animals to the housing and thoroughly clean the housing and framed glass panels after each use.

[0064] The following experimental procedures correspond to the effects of limonin on rheumatoid arthritis symptoms in mice ( Figure 2 Implementation of analysis)

[0065] 6. Joint inflammation score

[0066] The severity of arthritis was determined by observing the swelling of the mouse joints and scoring them. Under the condition of unknown grouping, the joints were visually evaluated and scored according to the following criteria: 0-no swelling; 1-mild swelling of toe joints; 2-swelling of toe joints and soles; 3-swelling below the ankle; 4-swelling of the entire foot. The sum of the scores of the four limbs was the arthritis score, and ≥6 points indicated a successful modeling.

[0067] 7. Knee width detection

[0068] The transverse diameter of the mouse knee joint was measured at 0 days, 3 days, 10 days, 17 days, 24 days and 28 days. The specific operation is: first, the mouse knee joint is in a straight state, and then the widest cross-section of the knee joint is determined as the standard measurement point. Use a vernier caliper to accurately measure the measuring point to obtain the width of the knee joint. Subsequently, the difference between the width of the right (affected side) knee joint and the width of the left knee joint is calculated to evaluate the swelling of the joint.

[0069] 8. Experimental Animal Handling

[0070] The mice were anesthetized and killed, and the synovial tissue of the knee joint was removed. Fat, cartilage and other tissues were removed as much as possible. After removing the blood, the tubes were placed in 1.5EP tubes, immediately frozen in liquid nitrogen, and then moved to a -80°C refrigerator for later use.

[0071] 9. Detection of the expression of inflammatory factors in mouse blood supernatant by enzyme-linked immunosorbent assay (ELISA)

[0072] Blood was collected from the mouse's orbital cavity. When collecting blood, try to keep the blood drop vertically into the 1.5EP tube. Do not rub the tube mouth to prevent hemolysis. The blood was placed in a 1.5EP tube and allowed to stand for 2 hours at room temperature. After the serum was stratified, it was centrifuged at 4°C and 3000rpm for 15 minutes. The upper serum was aspirated into a new 1.5EP tube and stored at -80°C. Subsequent experiments were performed according to the instructions of the kit.

[0073] Scratch test and Transwell assay of mouse synovial fibroblasts ( Figure 3 Implementation of analysis)

[0074] 10. Isolation and Culture of Primary Cells

[0075] The mouse synovial tissue removed by surgery was rinsed with PBS 2-3 times, and after removing the mixed tissue, the tissue was cut into 1 mm pieces with ophthalmic scissors. 3 Size, add appropriate amount of collagenase type II (4mg / mL) and mix well, then digest for 3-4 hours. Filter through a nylon mesh with a pore size of 70μm and centrifuge, add medium containing 10% FBS and resuspend, then spread in a culture bottle, and change the medium every other day. When the cells cover 80%-90% of the bottom of the bottle, use 0.25% EDTA-trypsin to digest the cells, pass them at a ratio of 1:3, select the 3rd to 6th generation cells for experiments, and identify the cell surface markers by flow cytometry.

[0076] 11. Cell Grouping and Drug Administration

[0077] Cells in the logarithmic growth phase were taken, digested with 0.25% EDTA-trypsin, and counted after centrifugation. The cells were plated according to the cell number requirements of the specific experiment, and the following groups were established and given different drug stimulations: ① blank group; ② TNF-α group (10 ng / mL TNF-α); ③ low-dose limonin group (10 ng / mL TNF-α + 1 μM Limonin); ④ medium-dose limonin group (10 ng / mL TNF-α + 3 μM Limonin); ⑤ high-dose limonin group (10 ng / mL TNF-α + 10 μM Limonin). The cells were stimulated and cultured for 24 hours.

[0078] 12. Polymerase Chain Reaction

[0079] (1) RNA extraction: The culture medium in the 24-well plate was aspirated and discarded. The plate was gently washed three times with 1×PBS to remove the residual culture medium in the wells. 500 μL-1 mL Trizol was added to each well and allowed to stand at room temperature for 10 minutes. The cells were lysed by repeated blowing and then the Trizol and the lysed cells were aspirated into a 1.5 EP tube. Chloroform was added (Trizol: chloroform = 5:1) and the mixture was fully shaken and mixed until turbidity appeared. The plate was then allowed to stand at room temperature for 5 to 10 minutes. The plate was centrifuged at 4°C and 12,000 rpm for 15 minutes. The upper aqueous phase was aspirated into a new 1.5 mL EP tube. Next, isopropanol was added in an equal volume to the upper aqueous phase. The plate was repeatedly inverted and mixed to mix well. The plate was placed in a -20°C refrigerator for 30 minutes. The plate was centrifuged at 4°C and 12,000 rpm for 15 minutes. The supernatant was discarded and then 1 mL of pre-cooled anhydrous ethanol was added and blown evenly. Centrifuge at 4°C, 7500 rpm for 5 minutes, wash the supernatant, and place the EP tube upside down on a tissue for 15 minutes. Depending on the amount of RNA precipitate, add 20 to 50 μL of DEPC water and dissolve it completely by pipetting, and measure the RNA concentration.

[0080] (2) RNA reverse transcription: Based on the obtained concentration, reverse transcription was performed according to the instructions of HiFiScript cDNA Synthesis Kit; the obtained samples were stored in a -20°C refrigerator for subsequent use.

[0081] (3) After reverse transcription, perform q-PCR according to the instructions of the AG kit.

[0082] Primer sequences:

[0083]

[0084]

[0085] 13. Cell scratch assay

[0086] (1) Experimental groups: ① blank group; ② TNF-α group (10 ng / mL TNF-α); ③ low-dose limonin group (10 ng / mL TNF-α + 1 μM Limonin); ④ medium-dose limonin group (10 ng / mL TNF-α + 3 μM Limonin); ⑤ high-dose limonin group (10 ng / mL TNF-α + 10 μM Limonin).

[0087] (2) First, draw a straight line on the back of the 6-well plate with a marker to facilitate subsequent photography and marking.

[0088] (3) The cells were digested with trypsin and counted, with approximately 1-5×10 cells per well. 6 The cells were evenly plated in a 6-well plate by blowing the culture medium, and then shaken by the cross method. The plates were placed at 37°C and 5% CO 2 Incubate the cells in the incubator until they are fully grown (approximately 24-48 hours).

[0089] (4) Use a 1 mL pipette tip to scratch the hole perpendicular to the line drawn in (2).

[0090] (5) Add PBS along the well wall and gently wash three times to remove floating cells in the well.

[0091] (6) Add DMEM basal culture medium to the wells and administer drugs according to experimental needs.

[0092] (7) 37°C, 5% CO 2 The cells were cultured in an incubator and photographed and counted under a microscope at 0 and 24 hours.

[0093] 14. Cell Transwell Assay

[0094] (1) Experimental groups: ① blank group; ② TNF-α group (10 ng / mL TNF-α); ③ low-dose limonin group (10 ng / mL TNF-α + 1 μM Limonin); ④ medium-dose limonin group (10 ng / mL TNF-α + 3 μM Limonin); ⑤ high-dose limonin group (10 ng / mL TNF-α + 10 μM Limonin).

[0095] (2) Plating: First, remove the upper chamber of the Transwell, add an appropriate amount of complete culture medium to the lower chamber, digest the cells with trypsin, centrifuge, mix with DMEM basal culture medium, and transfer them to the upper chamber.

[0096] (3) Administration: Different doses of limonin (1 μM, 3 μM, and 10 μM) were added into the chamber of the drug administration group, and 10 ng / mL of TNF-α was added into the TNF-α group and the drug administration group. The cells were incubated at 37°C and 5% CO2 Culture in an incubator, with no special treatment for the blank group. Carefully put the chamber back into the lower chamber containing the culture medium, making sure there are no bubbles on the contact surface between the lower layer of the chamber and the culture medium in the lower chamber.

[0097] (4) Fixation: Remove the upper chamber and wash gently with PBS, repeating the operation three times. Then place it in 4% paraformaldehyde solution for 15 minutes. Repeat the PBS washing process. Next, immerse the chamber in crystal violet stain for 30 minutes and repeat the PBS washing process again.

[0098] (5) Use a cotton swab to gently wipe away the FLS that have not passed through the upper chamber. Finally, use a microscope to observe the number of FLS on the chamber membrane.

[0099] In situ hybridization assay to detect H4R expression Figure 4 Implementation of analysis)

[0100] 15. In situ hybridization assay to detect H4R expression

[0101] (1) First, sterilize the instruments required for the experiment in a clean bench under ultraviolet for 30 minutes. Carefully place the slide into a 24-well plate, add 1 mL of complete culture medium to each well, digest the cells in the culture flask with trypsin for 3 minutes, centrifuge at 800 rpm for 5 minutes, then inoculate the cell suspension into the wells and culture them in a 37°C incubator for subsequent experiments.

[0102] (2) Experimental groups: ① blank group; ② TNF-α group (10 ng / mL TNF-α); ③ low-dose limonin group (10 ng / mL TNF-α + 1 μM Limonin); ④ medium-dose limonin group (10 ng / mL TNF-α + 3 μM Limonin); ⑤ high-dose limonin group (10 ng / mL TNF-α + 10 μM Limonin). Cells were stimulated and cultured for 24 hours.

[0103] (3) Aspirate the culture medium in the 24-well plate and gently wash twice with 1× PBS.

[0104] (4) Fixation: Add 1 mL of 4% paraformaldehyde to each well and fix for 30 minutes. Discard the solution and gently wash twice with 1× PBS.

[0105] (5) Blocking: Add 1 mL of blocking solution to each well, let stand at room temperature for 1 hour, discard the solution, and wash twice with PBS. Subsequent experiments can be performed the next day.

[0106] (6) Preheating: Turn on the hybridization oven and preheat for 30 minutes. Place the H4R probe in the hybridization oven and preheat for 15 minutes. Remove the slide from the 24-well plate and spread it on a glass slide.

[0107] (7) Probe: Add 5 μL of H4R probe to each slide and spread it evenly to completely cover the sample. Place it in a wet box, cover it, and incubate it in a hybridization oven at 40°C for 2 hours.

[0108] (8) Washing: Take out an appropriate amount of wash buffer and preheat it in the hybridization oven for 15 minutes. Dilute it with double distilled water. Pipette an appropriate amount of diluted wash buffer onto each slide and shake gently to wash. Repeat 3 times and absorb the wash solution with absorbent paper.

[0109] (9) AMP1: Add 5 μL of AMP1 to each slide, spread it evenly to completely cover the sample, and incubate it in a hybridization oven at 40°C for 30 min.

[0110] (10) Repeat step (8).

[0111] (11) AMP2: Add 5 μL of AMP2 to each slide and spread it evenly to completely cover the sample. Incubate in a hybridization oven at 40°C for 30 min.

[0112] (12) Repeat step (8).

[0113] (13) AMP3: Add 5 μL of AMP3 to each slide and spread it evenly to completely cover the sample. Incubate in a hybridization oven at 40°C for 15 min.

[0114] (14) Repeat step (8).

[0115] (15) C1 channel probe labeling: add 5 μL of multi-channel second-generation fluorescent HRP-C1 to each slide, spread it evenly to completely cover the sample, put it into the hybridization oven, and incubate it at 40°C for 15 min.

[0116] (16) Repeat step (8).

[0117] (17) Staining: Add 5 μL of diluted FITC to each slide, spread evenly to completely cover the sample, and incubate in a hybridization oven at 40°C for 30 min.

[0118] (18) Repeat step (8).

[0119] (19) Blocking: Add 5 μL of HRP blocking agent to each slide, spread evenly to completely cover the sample, and incubate in a hybridization oven at 40°C for 15 min.

[0120] (20) Repeat step (8).

[0121] (21) Sealing: Add 5 μL of anti-fluorescence quenching sealing medium (containing DAPI) to each slide and cover the cell slide on the sealing medium.

[0122] (22) Storage: Keep the slides away from light at 4°C and photograph and analyze them as soon as possible using a digital pathology slide fluorescence scanner.

[0123] 16. qPCR experiment to detect the mRNA expression of H4R in cells

[0124] The specific steps are the same as 12.

[0125] Primer sequences:

[0126]

[0127] To further explore whether limonin affects FLS migration through H4R, siRNA and overexpression plasmid of H4R were used for analysis. Figure 5 Implementation of analysis)

[0128] 17. Plasmid Transfection

[0129] (1) Add 4 μg of plasmid and Opti-MEM medium to each sample, mix well, control the total volume to 250 μL, and let it stand at room temperature for 5 minutes; add Opti-MEM medium and 12 μL LipoFiter transfection reagent, mix well and let it stand for 5 minutes, the total volume is also 250 μL, then combine the two, mix well, and let it stand for 20 minutes.

[0130] (2) Mix the above two solutions in equal volumes and inject them into the cell culture wells. Place them at 37°C and 5% CO 2 The transfection was carried out in a constant temperature incubator and cultured for 24 hours for subsequent experiments.

[0131] Through the above experimental verification, the following corresponding data analysis results are obtained. Figure 1-Figure 5 ,from Figure 2 It can be seen that the limonin of the present invention significantly improves the symptoms of rheumatoid arthritis in CFA mice and can also reduce the release of inflammatory factors in serum; Figure 2 Figure A shows that limonin can reduce the knee joint swelling caused by CFA in mice in a dose-dependent manner; Figure B shows that Limonin reduces the arthritis score of mice caused by CFA in a dose-dependent manner; Figure C shows that Limonin reduces the knee joint swelling of mice caused by CFA in a dose-dependent manner; Figure DF is an ELISA experiment to detect the expression of IL-1β, IL-6 and TNF-α inflammatory factors in mouse serum; From the data results, it can be seen that the Limonin treatment of the present invention dose-dependently reduces the secretion of IL-1β, IL-6 and TNF-α.

[0132] from Figure 3The data show that the limonin monomer of the present invention can effectively inhibit the migration of FLS induced by TNF-α and reduce the release of inflammatory factors in FLS. Figure 2 A and B in the figure are the data results of evaluating the effect of limonin on FLS migration using scratch test and Transwell test; Figure 3 C and D are statistics of A and B, and compared with the blank group, it can be seen from the figure that the cell migration rate and wound healing rate of the TNF-α group increased, and Limonin treatment led to a decrease in the wound healing rate and migration efficiency in a concentration-dependent manner. Figure 3 The EF graph in the figure is the data analysis result of detecting inflammatory factors (IL-1β, IL-6) in the cell supernatant, indicating that Limonin treatment dose-dependently reduced the secretion of IL-1β and IL-6 in the cells.

[0133] from Figure 4 As can be seen from the data results, the in situ hybridization experiments in Figures AB and the q-PCR experiment in Figure C indicate that the limonin of the present invention can effectively inhibit the increase of histamine H4 receptor (H4R) in FLS induced by TNF-α.

[0134] To further explore whether limonin affects FLS migration through H4R, siRNA and overexpression plasmid of H4R were used for analysis. Figure 5 The data results show that Figure 5 The results of analysis in A and D showed that siRNA could reduce the cell migration rate induced by TNF-α through Transwell experiment, and H4R-OE antagonized the inhibitory effect of Limonin on the cell migration rate induced by TNF-α.

[0135] Figure 5 The analysis results in B and C showed that siRNA could reduce the wound healing rate induced by TNF-α through the scratch experiment, and H4R-OE antagonized the inhibitory effect of Limonin on the wound healing rate induced by TNF-α.

[0136] In general, the above data analysis results show that the limonin of the present invention can effectively target H4R on synovial fibroblasts (FLS), thereby achieving a therapeutic effect on chronic pain of rheumatoid arthritis.

[0137] The specific embodiments described in the present invention are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the attached claims.

[0138] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. A use of limonin for preparing a drug for treating chronic pain of rheumatoid arthritis, wherein the active ingredient in the drug for treating chronic pain of rheumatoid arthritis is the limonin, and the structural formula of the limonin is as follows: It is characterized in that The limonin is used for preparing the anti-rheumatoid arthritis chronic pain drug which intervenes in the function of rheumatoid arthritis synovial fibroblasts through the histamine H4R-mediated mechanism.

2. The use of limonin according to claim 1 for preparing a drug for treating chronic pain of rheumatoid arthritis, characterized in that: The limonin inhibits synovial hyperplasia in rheumatoid arthritis, which is caused by abnormal function of synovial fibroblasts in peripheral joints.

3. The use of limonin according to claim 1 for preparing a drug for treating chronic pain of rheumatoid arthritis, characterized in that: The limonin is used for preparing an anti-rheumatoid arthritis chronic pain drug by inhibiting inflammatory factors released by rheumatoid arthritis.

4. The use of limonin according to claim 1 for preparing a drug for treating chronic pain of rheumatoid arthritis, characterized in that: The limonin is used for preparing an anti-rheumatoid arthritis chronic pain drug which can inhibit the FLS dysfunction caused by TNF-α induction.

5. The use of limonin according to claim 4 for preparing a drug for treating chronic pain in rheumatoid arthritis, characterized in that: The limonin is used for preparing an anti-rheumatoid arthritis chronic pain drug which can reduce the H4R expression induced by TNF-α.

6. The use of limonin according to any one of claims 1 to 5 for preparing a drug for treating chronic pain of rheumatoid arthritis, characterized in that: The limonin is used for preparing an anti-rheumatoid arthritis chronic pain drug by regulating the expression change of H4R by using siRNA and an overexpression plasmid.

7. Use of limonin according to any one of claims 1 to 5 for preparing a drug for treating chronic pain of rheumatoid arthritis, characterized in that: The dosage form of the drug is selected from tablets, capsules, emulsions or injections.

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