Use of kynurenine acid for the manufacture of a medicament for alleviating and / or treating rheumatoid arthritis

Abstract

The present application relates to the application of kynurenic acid in the preparation of drugs for relieving and / or treating rheumatoid arthritis. The present application proves that kynurenic acid plays an important role in the immune regulation of rheumatoid arthritis, which realizes the effect of relieving inflammation by affecting the metabolic reprogramming of macrophages under inflammatory environment and repairing the function of mitochondria under inflammatory state. And kynurenic acid can relieve the arthritis induced by collagen in the animal body and reduce the production of inflammatory factors, which has important significance for the treatment, symptom relief and life quality improvement of patients with rheumatoid arthritis, and has a broad application prospect in the preparation of products for relieving and / or treating rheumatoid arthritis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of kynurenic acid in preparation of a medicine for relieving and / or treating rheumatoid arthritis. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease mainly with inflammatory synovitis. The patient has clinical manifestations of joint swelling and deformity, can be accompanied by inflammatory damage outside the joints, and even can lead to high disability in severe cases. The pathological mechanism of RA is related to the joint action of various cells such as macrophages, T cells, B cells and fibroblast-like synoviocytes, and inflammatory cytokines such as interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). The patient's body usually produces autoantibodies against self antigens, which eventually leads to synovial inflammation and cartilage destruction. The imbalance of immune homeostasis in the pathogenesis of RA has been a hot spot widely concerned by researchers, and the interaction between metabolism and immunity is complex and close. Metabolic disorders can lead to immune dysfunction. Active metabolites can act as organic ligands and substrates for coordinating cell communication and immune function, and play a key role in the immune response process. Therefore, it is important for the prevention and treatment of RA to explore the key metabolites and related metabolic pathways of the immune regulation of RA as drug targets for specific regulation of the metabolic reactions in the over-activated immune cells (macrophages, T cells, B cells) to achieve the effect of moderate regulation of the immune response of the body.

[0003] Tryptophan (TRP) is an essential amino acid required for protein biosynthesis and is also a precursor of various bioactive molecules in the body. It is basically metabolized through the kynurenine pathway (KP) to produce at least about one hundred kinds of metabolites, such as kynurenine (KYN), kynurenic acid (KynA), anthranilic acid (AA), xanthurenic acid (XanA), quinolinic acid (QUIN), picolinic acid (PICO), quinaldic acid (QL-Dic) or 3OH-kynurenine (Widner B et al., 1997), and the rest of the free TRP is mainly used for protein synthesis and the synthesis of neurotransmitters such as serotonin and neuromodulators such as tryptamine. Studies have found that TRP metabolism is an important part of the cell and body communication strategy, and TRP and its metabolites play an important role in various different physiological processes, including maintaining cell growth and proliferation as a component of proteins, or coordinating the body's response to external influences such as environment and diet as neurotransmitters and signaling molecules.

[0004] Kynurenic acid, as one of the products of tryptophan metabolism, is widely distributed in the body fluids and central nervous system of mammals, can antagonize excitatory amino acids (EAA) and nicotine receptors, and is associated with some diseases of the nervous system, ophthalmology, kidney, etc. Because of the complicated pathogenesis and treatment plan of RA, relevant researchers have not fully studied it, and there is no relevant report on relieving or treating RA by administering tryptophan metabolites such as KynA. SUMMARY

[0005] In order to overcome the above-mentioned deficiencies in the prior art, the application provides the use of kynurenic acid in the preparation of a medicine for relieving and / or treating rheumatoid arthritis.

[0006] According to the above-mentioned application provided by the application, the medicine uses kynurenic acid as the active ingredient and is prepared into a preparation with pharmaceutically acceptable excipients.

[0007] According to the above-mentioned application provided by the application, the preparation is selected from one of injection, tablet, powder, granule, capsule, oral liquid, pill, and sustained-release preparation.

[0008] The application also provides a medicine for relieving and / or treating rheumatoid arthritis, wherein the medicine contains kynurenic acid.

[0009] Further, the medicine provided by the application further contains pharmaceutically acceptable excipients.

[0010] Preferably, the excipients include anti-adhesion agents, penetration promoters, buffers, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH adjusters, adhesives, disintegrants, fillers, lubricants, wetting agents, integrating agents, osmotic pressure adjusters, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filtration aids, or release retardants.

[0011] Preferably, the application object of the medicine provided by the application includes but is not limited to human or animal.

[0012] Preferably, the medicine provided by the application has a dosage form of injection, tablet, powder, granule, capsule, oral liquid, pill, or sustained-release preparation.

[0013] Preferably, the medicine provided by the application relieves the inflammatory response of RA by participating in the metabolic reprogramming of macrophages in the inflammatory environment.

[0014] More preferably, the medicine provided by the application relieves the inflammatory response of RA by repairing the function of mitochondria in the inflammatory state.

[0015] The present application also provides use of kynurenic acid in the preparation of a food, drink, health product, enteral nutritional agent, and / or dietary supplement for improving and / or relieving symptoms of rheumatoid arthritis.

[0016] In another aspect, the present application provides a food, drink, health product, enteral nutritional agent, and / or dietary supplement for improving and / or relieving symptoms of rheumatoid arthritis, characterized in that it comprises kynurenic acid.

[0017] The present application has the beneficial effects of:

[0018] The present application first discovers and proves that kynurenic acid, a tryptophan metabolite, plays an important role in immune regulation of rheumatoid arthritis, which achieves the effect of relieving inflammation by affecting the metabolic reprogramming of macrophages in the inflammatory environment and repairing the mitochondrial function in the inflammatory state. And KynA can relieve arthritis induced by collagen in animals and reduce the production of inflammatory factors. It has important significance for the treatment, symptom relief, and improvement of life quality of patients with rheumatoid arthritis, and has a broad application prospect in the preparation of products for relieving and / or treating RA. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The effects of TRP metabolites in CIA mice are shown, wherein: A. arthritis clinical scores of mice treated with TRP (n=6), KynA (n=6), XanA (n=6) and solvent control (n=7); B. changes in body weight of mice; C. serum anti-type II collagen antibody titer levels of mice; D. serum IL-6 levels of mice; *P≤0.05, **P≤0.01.

[0020] Figure 2A The effects of KynA on macrophages are shown.

[0021] Figure 2B The effects of KynA on Th17 cells are shown.

[0022] Figure 3 IHC staining of joints of CIA mice is shown, wherein: the black square indicates the high-power magnification area, and the brown color indicates F4 / 80 positivity; **P≤0.01.

[0023] Figure 4The effects of TRP metabolites in primary bone marrow-derived macrophages BMDM are shown, wherein: A. TNF-a level in BMDM culture supernatant after TRP metabolite treatment; B. IL-6 level in BMDM culture supernatant after TRP metabolite treatment; C. IL-1b level in BMDM culture supernatant after TRP metabolite treatment; D. TNF-a level in BMDM culture supernatant after KynA treatment at different concentrations; E. IL-6 level in BMDM culture supernatant after KynA treatment at different concentrations; F. IL-1b level in BMDM culture supernatant after KynA treatment at different concentrations; *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0024] Figure 5 The results of BMDM transcriptome after KynA treatment are shown.

[0025] Figure 6 The KynA prevents the accumulation of dysfunctional mitochondria is shown. DETAILED DESCRIPTION

[0026] The inventors found that the levels of tryptophan metabolites, particularly TRP, KynA and XanA, were different between rheumatoid arthritis patients and healthy controls. Further, by early intervention of the three metabolites TRP, KynA and XanA in a mouse model of collagen-induced arthritis (CIA), the inventors found that the arthritis clinical score of the KynA group was significantly lower than that of the control group, the TRP group and the XanA group. The anti-type II collagen antibody titer of the KynA group was significantly reduced, while the antibody titers of the TRP group and the XanA group had no significant difference with the control group. The IL-6 concentration of the KynA group was significantly lower than that of the control group, indicating that KynA can alleviate CIA arthritis inflammation.

[0027] To further understand how KynA inhibits arthritis inflammation in CIA mice, the changes of several major immune cell subsets related to the pathogenesis of RA in the control group and the KynA group were analyzed by flow cytometry. It was found that the macrophages in the DLNs of the KynA group mice were reduced compared with the solvent control group, while the macrophages in the MLNs had no significant difference between the two groups. In addition, it was also found that the proportion of CD4+IL-17+ cells (Th17) in the DLNs and MLNs of the KynA group mice was reduced compared with the solvent control group, indicating that KynA alleviates arthritis inflammation by inhibiting Th17 cells.

[0028] To understand the effect of KynA on joint local macrophages, F4 / 80 immunohistochemical staining was performed on mouse joint sections. It was found that the expression of F4 / 80 in the control group was significantly higher than that in the KynA group, indicating that KynA alleviates local joint inflammation by affecting CIA synovial macrophages.

[0029] On this basis, combined with in vitro cell experiments, TRP, KynA and XanA were used to treat primary bone marrow-derived macrophages BMDM, and LPS was used to activate macrophages to simulate an inflammatory environment. The inflammatory factors TNF-α, IL-6 and IL-1β secreted by macrophages pretreated with KynA decreased significantly, and the inhibitory ability of KynA on inflammatory factors increased with the increase of concentration.

[0030] To further explore how KynA regulates macrophage-mediated inflammation, transcriptome sequencing analysis found that the differentially expressed genes in the KynA-treated macrophage group were mainly enriched in cytokine chemokine signaling pathways, G protein receptor internalization pathways and oxidoreductase regulation pathways. This indicates that KynA may mediate the reprogramming of macrophage metabolism in an inflammatory environment.

[0031] The technical solutions of the present application will be described in detail below in combination with the embodiments and the accompanying drawings. The following examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are commercially available reagents and materials unless otherwise specified.

[0032] Example 1

[0033] Experimental methods

[0034] 1. Experimental animals

[0035] A total of 25 healthy 7-week-old DBA / 1J female mice, weighing 10-15 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were raised in a specific pathogen-free (SPF) environment, with a 12 h light / 12 h dark environment, an environmental temperature controlled at (25±1 ℃), a relative humidity of 50-70%, and free access to food and water.

[0036] 2. Collagen-induced arthritis (CIA) mouse modeling and treatment

[0037] (1) Grouping: The DBA / 1J mice were numbered using ear pins, and they were randomly divided into 4 groups (TRP group 6, KynA group 6, XanA group 6, and solvent control group vehicle 7).

[0038] (2) Gavage: Gavage with TRP, KynA, XanA and solvent every other day starting 1 week before the first immunization, with a gavage amount of 0.1 mL per mouse, and compound doses of TRP 0.15 mg / mouse, KynA 0.24 mg / mouse, and XanA 0.15 mg / mouse, which continued until the end of the experiment.

[0039] (3) First immunization:

[0040] 1) Prepare 0.05 M glacial acetic acid.

[0041] 2) Weigh 6 mg of collagen type II with an electronic balance.

[0042] 3) Dissolve the weighed collagen in 3 mL of prepared 0.05 M glacial acetic acid on ice until the collagen is completely dissolved. At this time, the final concentration of collagen acetic acid solution is 2 mg / mL.

[0043] 4) On ice, mix 3 mL of complete Freund's adjuvant (CFA, 1 mg / mL) and 3 mL of prepared collagen type II acetic acid solution (2 mg / mL) in equal volumes, and repeatedly blow with a 5 mL syringe until completely emulsified.

[0044] 5) Keep the operation low temperature, transfer the emulsified liquid to a 1 mL syringe, and remove air bubbles. Fix the mouse and inject the emulsion subcutaneously as close to the tail base as possible. Each mouse is injected with 0.1 mL, i.e. 100 μg of collagen type II per mouse. Observe the state of the mice and record the body weight every week.

[0045] (4) Second boost: 21 days after the first immunization, the mice are given a second boost. Mix equal volumes of Freund's incomplete adjuvant IFA and collagen type II acetic acid solution (2 mg / mL) and emulsify by blowing, as described above. After complete emulsification, transfer the liquid to a 1 mL syringe, remove air bubbles, and inject the emulsion subcutaneously into the tail, avoiding the inflammatory tissue caused by the first immunization. Each mouse is injected with 0.1 mL, i.e. 100 μg of collagen type II per mouse. About 1 week after the second immunization, the mice begin to develop symptoms, mainly redness and swelling of the toes and soles, and in severe cases, lameness. The researcher scores and records the arthritis of each mouse paw every other day, and records the body weight every week.

[0046] 3. Experimental materials

[0047] (1) Blood collection and subsequent processing:

[0048] 1) After anesthetizing the mouse with gas, press the mouse's neck on both sides, and insert the capillary blood collection tube obliquely into the inner corner of the eye at 2-3 mm, so that the blood flows into the 5 mL EDTA anticoagulant blood collection tube.

[0049] 2) After blood collection, release the pressure on the mouse's neck, and remove the capillary blood collection tube after the eyeball is reset, and stop bleeding with a dry cotton ball.

[0050] 3) Centrifuge the collected mouse peripheral blood at 1500 rpm for 10 min, and collect the supernatant in a 1.5 mL EP tube, and store it at -80°C for testing.

[0051] (2) Mesenteric lymph node isolation and subsequent processing:

[0052] 1) Sacrifice the mouse, fix the four limbs, disinfect the skin with 75% alcohol, cut open the mouse skin along the midline of the abdomen, bluntly dissect the peritoneum to open the abdominal cavity, and take the lymph nodes extending in the mesenteric adipose tissue and transfer them into pre-cooled DPBS.

[0053] 2) Remove the fat, mesentery and other tissues around the mesenteric lymph nodes, gently grind with a glass slide, and rinse with DPBS until the grinding solution is clear.

[0054] 3) Collect the grinding solution through a 70 μm cell sieve into a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 minutes.

[0055] 4) Discard the supernatant, resuspend the cells with DPBS, and divide the single-cell suspension into 4 equal parts and store in a 4°C refrigerator for testing.

[0056] (3) Joint drainage area lymph node isolation and subsequent processing:

[0057] 1) Sacrifice the mouse, fix the four limbs, disinfect the skin with 75% alcohol, cut open the mouse skin along the midline of the abdomen, bluntly dissect the peritoneum to open the abdominal cavity, and take the lymph nodes extending in the mesenteric adipose tissue and transfer them into pre-cooled DPBS.

[0058] 2) Carefully remove the fat tissue around the DLN, gently grind with a glass slide, and rinse with DPBS until the grinding solution is clear.

[0059] 3) Collect the grinding solution through a 70 μm cell sieve into a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 minutes.

[0060] 4) Discard the supernatant, resuspend the cells with DPBS, and divide the single-cell suspension into 4 equal parts and store in a 4°C refrigerator for testing.

[0061] (4) Joint fixation and subsequent processing: Cut off the mouse hind paw from the ankle joint, peel off the skin, and then flatten each joint in the embedding box. Immerse it completely in general tissue fixative, fix it at 4°C for 48 h, decalcify it in 10% ethylenediaminetetraacetic acid, and embed it in paraffin for subsequent experiments.

[0062] 4. Flow detection

[0063] (1) Th1 / Th17 cell staining:

[0064] 1) Prepare a single-cell suspension, count the cell amount, resuspend with 1 mL complete medium (RPMI 1640 + 10% FBS + 1% two-way antibiotic), and transfer the cells into a 24-well cell culture plate.

[0065] 2) Thaw the cell stimulation mixture completely in a 37°C water bath.

[0066] 3) Add 2 μL of the cell stimulation mixture (cell concentration is about 1 x 10 6 cells / mL) per well, and incubate the cells in a 37°C CO2 incubator for 6 h.

[0067] 5) Collect the stimulated cells in a flow tube, centrifuge at 350 G for 5 min, and discard the supernatant.

[0068] 6) Dead / live cell staining: Resuspend the cells in each tube with 100 μL of DPBS, add 1 μL of Zombie NIR dye, and incubate at room temperature for 20 min in the dark.

[0069] 7) Add 1 mL of staining buffer to each tube of cells, centrifuge at 350 G for 5 min, and discard the supernatant.

[0070] 8) Cell surface marker staining: Resuspend the cells in each tube with 100 μL of staining buffer, add 1 μL of mouse anti-CD4 PerCP, and incubate at 4°C for 30 min in the dark.

[0071] 9) Repeat step 7

[0072] 10) Fix and permeabilize: Resuspend the cells in each tube with 100 μL of 1 x fix and permeabilization buffer, and fix at 4°C for 60 min in the dark.

[0073] 11) Add 1 mL of 1 x permeabilization buffer to each tube of cells, centrifuge at 500 G for 5 min, and discard the supernatant.

[0074] 12) Add 100 μL of 1 x permeabilization buffer to each tube of cells, add 1 μL of mouse anti-IFN-γ FITC and 1 μL of mouse anti-IL-17A PE, respectively, and incubate at room temperature for 60 min in the dark.

[0075] 13) Add 1 mL of staining buffer to each tube of cells, centrifuge at 500 G for 5 min, and discard the supernatant.

[0076] 14) Add 300 μL of staining buffer to each tube of cells, filter with a 70 μm cell strainer, and then perform detection on the machine.

[0077] (2) Treg cell staining:

[0078] 1) Cell surface marker staining: Take 1 part of the single-cell suspension prepared in the above “3. Experimental sampling”, resuspend the cells in each tube with 100 μL of staining buffer, add 1 μL of mouse anti-CD4 PerCP and 1 μL of mouse anti-CD25 PE, respectively, and incubate at 4°C for 30 min in the dark.

[0079] 2) Add 1 mL staining buffer to each tube of cells, centrifuge at 350 G for 5 min, discard the supernatant.

[0080] 3) Fix the membrane: add 100 μL of 1x membrane fixation solution to each tube of cells, resuspend, and fix at 4°C in the dark for 60 min.

[0081] 4) Add 1 mL of 1x permeabilization buffer to each tube of cells, centrifuge at 500 G for 5 min, discard the supernatant.

[0082] 5) Add 100 μL of 1x permeabilization buffer to each tube of cells, add 1 μL of mouse anti-FOXP3 APC, incubate at room temperature in the dark for 60 min.

[0083] 6) Add 1 mL of staining buffer to each tube of cells, centrifuge at 500 G for 5 min, discard the supernatant.

[0084] 7) Add 300 μL of staining buffer to each tube of cells, filter through a 70 μm cell strainer, and then load onto the machine for detection.

[0085] (3) Macrophage staining:

[0086] 1) Cell surface marker staining: take 1 part of the single cell suspension prepared in the above "3. Experimental sampling", resuspend each tube of cells with 100 μL of staining buffer, add 1 μL of mouse anti-MHCII FITC and 1 μL of mouse anti-CD11b PE, respectively, incubate at 4°C in the dark for 30 min.

[0087] 2) Add 1 mL of staining buffer to each tube of cells, centrifuge at 350 G for 5 min, discard the supernatant.

[0088] 3) Add 300 μL of staining buffer to each tube of cells, filter through a 70 μm cell strainer, and then load onto the machine for detection.

[0089] (4) Plasma cell staining:

[0090] 1) Cell surface marker staining: take 1 part of the single cell suspension prepared in the above "3. Experimental sampling", resuspend each tube of cells with 100 μL of staining buffer, add 1 μL of mouse anti-B220 FITC and 1 μL of mouse anti-CD138 PE, respectively, incubate at 4°C in the dark for 30 min.

[0091] 2) Add 1 mL of staining buffer to each tube of cells, centrifuge at 350 G for 5 min, discard the supernatant.

[0092] 3) Add 300 μL of staining buffer to each tube of cells, filter through a 70 μm cell strainer, and then load onto the machine for detection.

[0093] 5. Plasma anti-type II collagen antibody and plasma IL-6 inflammatory factor detection, according to the operating instructions.

[0094] 6. Joint H&E pathology

[0095] (1) Paraffin section deparaffinization: place the section in each solution in the following order: xylene 20 min-xylene 20 min-anhydrous ethanol 5 min-anhydrous ethanol 5 min-75% alcohol 5 min, and then wash with deionized water.

[0096] (2) Hematoxylin staining: stain with hematoxylin dye for 3-5 min, wash the section with deionized water, add differentiation solution, wash the section with deionized water again, add blue return solution for blue return, and then wash with deionized water.

[0097] (3) Eosin staining: place the section in 85% and 95% gradient alcohol for 5 min each, and then place the section in eosin dye for 5 min.

[0098] (4) Dehydration and mounting: place the section in each solution in the following order: anhydrous ethanol 5 min-anhydrous ethanol 5 min-anhydrous ethanol 5 min-xylene 5 min-xylene 5 min, and then mount with neutral balsam.

[0099] (5) Microscope examination, image acquisition, read the image with caseviewer software, and score the joint pathology according to the scoring standard shown in the following table.

[0100] 7. Joint immunohistochemistry

[0101] (1) Paraffin section deparaffinization: place the section in each solution in the following order: environmentally friendly deparaffinizing solution 10 min-environmentally friendly deparaffinizing solution 10 min-environmentally friendly deparaffinizing solution 10 min-anhydrous ethanol 5 min-anhydrous ethanol 5 min-anhydrous ethanol 5 min, and then wash with deionized water.

[0102] (2) Heat repair antigen, after cooling, place the slide in PBS (PH 7.4), and wash on a decoloring shaker for 3 times, 5 min each time.

[0103] (3) Block endogenous peroxidase: place the section in 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 min, place the slide in PBS (PH 7.4), and wash on a decoloring shaker for 3 times, 5 min each time.

[0104] (4) Serum blocking: add 3% BSA in the histology circle to evenly cover the tissue, and block at room temperature for 30 min.

[0105] (5) Add primary antibody: Gently shake off the blocking solution, add F4 / 80 primary antibody on the section, and place the section in a wet box for incubation at 4°C overnight.

[0106] (6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash on a shaking table for 3 times, 5 min each time. After gently shaking off the section, add HRP-labeled secondary antibody in the circle to cover the tissue, and incubate at room temperature for 50 min.

[0107] (7) DAB color development: Place the slide in PBS (pH 7.4) and wash on a shaking table for 3 times, 5 min each time. After gently shaking off the section, add DAB color developing solution in the circle. Under the microscope, the positive part is brownish yellow, and the section is washed with deionized water to stop the color development.

[0108] (8) Counterstain the nucleus: Counterstain with hematoxylin staining solution for about 3 min, wash with deionized water, differentiate with hematoxylin differentiation solution for a few seconds, wash with deionized water, and then return to blue with hematoxylin return blue solution and wash with deionized water.

[0109] (9) Dehydration and mounting: Place the section in 75% alcohol for 5 min, 85% alcohol for 5 min, absolute ethanol for 5 min, absolute ethanol for 5 min, n-butanol for 5 min, and xylene for 5 min, and then dry and mount with mounting medium.

[0110] (10) Microscope examination and image acquisition, then read the image with caseviewer software. The hematoxylin-stained nucleus is blue, and the F4 / 80-positive cells expressed by DAB are brownish yellow. ImageJ is used to calculate the average optical density (AOD) of representative images.

[0111] 8. Statistical analysis

[0112] The experimental data was statistically analyzed using GraphPad Prism 9 software. For data of measurement, if it conforms to normal distribution, it is expressed in the form of mean (standard deviation SD), and the statistical analysis method is independent sample T test; if it does not conform to normal distribution, it is expressed in the form of median (interquartile range IQR), and the statistical analysis method is non-parametric test. For data of count, the statistical analysis method is chi-square test. For analysis of multiple data samples, normal distribution data is analyzed by one way anova, and non-normal distribution data is analyzed by kruskal-wallis test. Since the clinical scores and weight changes of animal models are affected by the interaction of two factors, the statistical method is two way anova. P value ≤ 0.05 is considered to have significant difference.

[0113] By early intervention of the three metabolites TRP, KynA and XanA in CIA mouse model, it is found that the arthritis clinical score of KynA group is significantly lower than that of the solvent control group (inflammation control group), TRP and XanA groups, and the anti-type II collagen antibody titer of KynA group is significantly reduced, while the antibody titers of TRP and XanA groups have no significant difference with the solvent control group; the IL-6 concentration of KynA group is significantly lower than that of the solvent control group, as shown in Figure 1 , indicating that KynA can alleviate CIA arthritis inflammation.

[0114] By flow cytometry analysis of the changes of several major immune cell subsets related to the pathogenesis of RA in the solvent control group and the KynA group, we first analyzed the MHCII+CD11b+ cell population (macrophages) and found that the macrophages in the DLNs of KynA group mice were reduced compared with the solvent control group, while the macrophages in the MLNs had no significant difference between the two groups, as shown in Figure 6 A. In addition, we found that the proportion of CD4+IL-17+ cells (Th17) in the DLNs and MLNs of KynA group mice was lower than that of the solvent control group, as shown in Figure 6 B. The above results show that KynA alleviates arthritis inflammation by inhibiting Th17 cells.

[0115] Through F4 / 80 immunohistochemical staining of mouse joint sections, it is found that the expression of F4 / 80 in the solvent control group is significantly higher than that in the KynA group, as shown in Figure 3 , indicating that KynA alleviates joint local inflammation by affecting CIA synovial macrophages.

[0116] Therefore, the above animal experiment results show that KynA can alleviate collagen-induced arthritis in animals and reduce the production of inflammatory factors.

[0117] Example Two

[0118] On the basis of Example One, in vitro cell experiments were also carried out, and TRP, KynA and XanA were used to treat primary bone marrow-derived macrophages BMDM, and lipopolysaccharide (LPS) was used to activate macrophages to simulate an inflammatory environment. The inflammatory factors TNF-α, IL-6 and IL-1β secreted by macrophages pretreated with KynA decreased significantly, and the inhibitory ability of KynA on inflammatory factors increased with increasing concentration, as shown in Figure 4 .

[0119] The macrophage cell line Raw 264.7 was treated with TRP, KynA and XanA, respectively, and activated by LPS to simulate an inflammatory environment. Then, the secretion levels of inflammatory factors in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA), and the genes and pathways affecting macrophage inflammation were screened and enriched by transcriptome sequencing (RNA-seq).

[0120] The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in macrophages were analyzed using a Seahorse XF96 extracellular flux analyzer. Before starting the assay, the cells were washed and incubated in Seahorse assay medium supplemented with 10 mM glucose and 1 mM sodium pyruvate for 45 minutes at 37°C in a CO2-free incubator. Oligomycin (ATP enzyme inhibitor, 1 µM), FCCP (1 µM) and rotenone (0.5 µM) were injected at designated positions, and ECAR (mpH / min) and OCR (pMoles O2 / min) were measured in real time.

[0121] For the study of whether the changes in glycolysis and mitochondrial metabolism profiles in the macrophage cell line Raw 264.7 were caused by mitochondrial dysfunction, MitoTracker Green (mitochondria staining agent independent of Δψm) and MitoTracker Deep Red (mitochondria staining agent dependent on Δψm) were used to distinguish respiratory mitochondria and dysfunctional mitochondria.

[0122] Transcriptome sequencing analysis found that the differentially expressed genes in the KynA-treated macrophage group were mainly enriched in cytokine chemokine signaling pathways, G protein-coupled receptor internalization pathways, and oxidative reductase regulation pathways, as shown in A and B of Figure 5 . Among them, the pro-inflammatory cytokine and chemokine genes in the KynA-treated group were down-regulated, the anti-oxidative stress genes such as Nqo1 were significantly up-regulated, and the genes Nod1 and Nlrp3 in the NOD-like receptor signaling pathway were significantly down-regulated. Transcriptome analysis further found that KynA up-regulated IL-10 receptor beta (IL10rb), down-regulated key genes of the glycolysis pathway, such as glucose transporter gene Glut1 (Slc2a1), hexokinase-2 (Hk2) and lactate dehydrogenase A (Ldha), hypoxia-inducible factor-1α (HIF-1α), and down-regulated key genes of mitochondrial metabolism such as silent information regulator 3 (Sirt3) and FoxO transcription factor (Foxo1), as shown in B and C of Figure 5 . The above results show that KynA can down-regulate the glycolysis level through the HIF-1α / Glut1 pathway and down-regulate mitochondrial respiration through the Sirt3 / Foxo1 pathway, suggesting that KynA mediates the metabolic reprogramming of macrophages in an inflammatory environment.

[0123] Activated macrophages undergo metabolic reprogramming, including rapid upregulation of glycolysis, remodeling of the TCA cycle, and inhibition of mitochondrial respiration. For analysis of glycolysis and oxidative phosphorylation indicators using a cell energy metabolic detector, by Seahorse analysis of OCR, it was found that KynA restored mitochondrial oxidative function in LPS-stimulated macrophages by improving oxygen consumption, regulating mitochondrial respiration of macrophages, and the results are shown as A and B in FIG. 6. LPS-stimulated macrophage cell line Raw 264.7 was stained with MitoTracker Green (mitochondria staining agent independent of Δψm) and MitoTracker Deep Red (mitochondria staining agent dependent on Δψm), and it was found that LPS increased dysfunctional mitochondria, while KynA could reduce the proportion of dysfunctional mitochondria, and the results are shown as C and D in FIG. 6. The above results show that KynA can repair mitochondrial function in an inflammatory state. Figure 6 Figure 6

[0124] Therefore, the above cell experiments show that KynA can alleviate inflammation by mediating the metabolic reprogramming of macrophages in an inflammatory environment.

[0125] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered within the scope of the claims of the present application.​​

Claims

1. The use of kynurenic acid in the preparation of drugs for relieving and / or treating rheumatoid arthritis, characterized in that, The drug is used to relieve collagen-induced arthritis and reduce the production of inflammatory factors, and the drug has kynurenic acid as its active ingredient.

2. The application according to claim 1, characterized in that: The drug is also formulated into a dosage form with pharmaceutically acceptable excipients for use.

3. The application according to claim 2, characterized in that: The preparation is selected from one of the following: injection, tablet, powder, granule, capsule, oral liquid, pill, and sustained-release preparation.

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