Application of inhibiting substance of purine nucleotidase in preparation of medicine for treating rheumatoid arthritis

Abstract

According to the application of the purine nucleotidase inhibiting substance in preparation of the medicine for treating rheumatoid arthritis, a high-efficiency inhibitor Forodesine of PNP is selected to be subjected to preventive intragastric administration on CIA mice, and after administration, arthritis morbidity is relieved, the disease course is delayed, the clinical score is reduced, and the morbidity is reduced.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to the application of a purine nucleotidase inhibitor in the preparation of a drug for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease with erosive arthritis as the main clinical manifestation. It can occur at any age, mainly in young and middle-aged women, with an average age of onset of about 45 years old. Its pathogenesis is still unclear, and there is no complete cure with current treatment methods. The basic pathological manifestations are synovitis, pannus formation, and gradual destruction of articular cartilage and bone, which eventually lead to joint deformity and loss of function.

[0003] One of the pathogenesis of RA is that abnormal activation of T cells infiltrates the synovium of the joints, which causes an autoimmune response and causes joint damage. In severe cases, it leads to joint deformity, quadriplegia, and even death. Currently, in the study of T cell metabolism in RA, few people pay attention to abnormalities in the purine metabolic pathway.

[0004] Previous studies have found that the level of 2'-Deoxyinosine (2'-Di) in the serum of RA patients is reduced, and 2'-Di treatment is effective in arthritis mouse models. Studies on multiple sclerosis have found that the level of 2'-Deoxyinosine in the serum of patients is also reduced. After the establishment of its animal model, experimental autoimmune encephalomyelitis (EAE) mice, the severity of the disease was significantly reduced after oral administration of 2'-Deoxyinosine. At the same time, we conducted experiments on the systemic lupus erythematosus nephritis mouse model, and 2'-Deoxyinosine treatment had no effect. However, due to the toxic side effects of 2'-Deoxyinosine treatment in the later stage, we considered its related enzymes.

[0005] Purine Nucleoside Phosphorylase (PNP) is one of the key enzymes in the purine salvage synthesis pathway. In human cells, its main function is to catalyze the conversion of (deoxy)guanosine and (deoxy)inosine into hypoxanthine, which is then converted into uric acid (UA) by xanthine oxidase (XO). In human blood, T cells lack the de novo synthesis of purines and rely on the salvage synthesis pathway of purines. Increased activity and content of PNP can lead to abnormalities in T cell immune function, T cell lymphoma, lymphocytic leukemia, etc. Enzyme deficiency can lead to bacterial and viral infections, systemic lupus erythematosus, autoimmune hemolytic anemia and other diseases.

[0006] Studies have shown that methotrexate (MTX), the first-line anti-inflammatory drug for RA, can effectively reduce the activities of adenosine deaminase (ADA), 5'-nucleotidase (5'-NT) and PNP enzyme in the purine metabolic pathway, among which the effect on PNP is the most significant. We speculate that one of the mechanisms of MTX in treating RA is to reduce PNP activity. Summary of the invention

[0007] The purpose of the present invention is to provide an application of a purine nucleotidase inhibitor in the preparation of a drug for treating rheumatoid arthritis.

[0008] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of a purine nucleotidase inhibitor in the preparation of a drug for treating rheumatoid arthritis.

[0009] The preferred technical solution is: the inhibitory substance is 7-[(2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-pyrrolidinyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one.

[0010] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a pharmaceutical composition for treating rheumatoid arthritis, comprising 7-[(2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-pyrrolidinyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one and pharmaceutically acceptable excipients.

[0011] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0012] Forodesine, a highly effective inhibitor of PNP, was selected and administered to CIA mice by oral gavage. After administration, the onset of arthritis was alleviated, the course of the disease was delayed, the clinical score and the incidence rate were reduced; there was no difference in liver and kidney pathological H&E staining and transaminase; the degree of spleen enlargement was reduced, and the spleen was weighed and the weight decreased; flow cytometry analysis of splenic lymphocytes showed that the proportions of lymphocytes, T cells, CD8+T cells, and CD8+Ki67+T cells decreased, and the proportion of Treg cells increased. Among the CD4+T cell subsets, the proportion of Th2 cells increased, the proportion of Th17 cells decreased, the proportion of Treg / Th17 cells increased, and the proportion of Th1 / Th2 cells decreased; qPCR of splenic lymphocytes showed that the expression of TNF-α and IL-17 decreased after administration, and the expression of IL-4 and Foxp3 increased; Western blot of splenic lymphocytes showed that NF-κB phosphorylation was inhibited and the phosphorylation of AMPK pathway increased. Flow cytometry analysis of peripheral blood and lymph node lymphocytes showed that the proportion of Treg cells increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The purine metabolic pathway is abnormal in RA patients.

[0014] Figure 2 Abnormalities of PNP enzyme in RA patients. (A) Schematic diagram of purine salvage biosynthesis pathway. (B) Electrophoresis of PNP protein in peripheral blood lymphocytes of RA vs healthy controls. (C) PNP enzyme activity in peripheral blood lymphocytes of RA vs healthy controls. (D) ADA enzyme activity in peripheral blood lymphocytes of RA vs healthy controls. (E) 5-NT enzyme activity in peripheral blood lymphocytes of RA vs healthy controls.

[0015] Figure 3 Schematic diagram of abnormal purine salvage synthesis pathway in RA patients.

[0016] Figure 4 The flowchart for the CIA model induction is shown in Figure 1.

[0017] Figure 5 Flowchart of animal experiments.

[0018] Figure 6 FDS alleviates arthritis symptoms in mice and delays disease progression. A) Clinical scores of CIA mice in the blank control group, model control group, low, medium and high dose groups. (B) Incidence of CIA mice. (C) Joints and mammographic targets of CIA mice in the blank control group, model control group, low, medium and high dose groups.

[0019] Figure 7 The degree of spleen enlargement was reduced after FDS administration. (A) Gross appearance of spleen of CIA mice in blank control group, model control group, low, medium and high dose groups. (B) Spleen weight of mice in each group.

[0020] Figure 8 No obvious toxic side effects were observed after FDS administration.

[0021] Fig. 9 Administration inhibits the secretion of inflammatory factors.

[0022] Fig.10 Administration inhibits the phosphorylation of NF-κB protein.

[0023] Fig.11 Administration promotes activation of the AMPK pathway.

[0024] Fig.12 qPCR was used to detect the inhibitory effect of FDS on PNP gene expression.

[0025] Fig.13 FDS inhibits the secretion of TNF-α in vitro.

[0026] Fig.14 FDS induces apoptosis of CD8+T cells. Left: apoptosis rates of CD4+T cells in three groups. Right: apoptosis rates of CD8+T cells in three groups. DETAILED DESCRIPTION

[0027] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in the embodiments.

[0028] See also Figure 1-14 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance, and any structural modification, change in proportional relationship or adjustment of size. The following examples are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0029] Example: Application of purine nucleotidase inhibitor in the preparation of rheumatoid arthritis treatment drugs

[0030] RA patients vs healthy controls

[0031] According to the medical record system of the Second Affiliated Hospital of Soochow University, 10 RA patients from the Department of Rheumatology and 10 healthy controls were selected. After the patients signed the informed consent form, serum, urine, and peripheral blood were collected from the patients. The serum and urine samples were processed and frozen at -80°C, and the peripheral blood was immediately isolated for mononuclear cells. The diagnostic criteria for RA patients refer to the 2010 version of the ACR diagnostic criteria. The inclusion criteria for the healthy control group: 1. The subjects who underwent a healthy physical examination during the same period must match the age and gender of the RA group. The healthy physical examination confirmed that they were in a healthy range and did not suffer from any serious organic or functional diseases; 2. No other immune system abnormalities; 3. No antibiotics, probiotic preparations, etc. were taken in the past month; 4. Voluntarily participate in this trial study and sign the informed consent form. Approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University, approval number: JD-LC2024008-I01.

[0032] Reagent Name Manufacturer:

[0033] PBS buffer solution - White Shark Biotechnology; human lymphocyte separation solution - Dakowei; L-2-chlorophenylalanine (mixed internal standard) - Shanghai Hengchuang Biotechnology; succinic acid-d4 (mixed internal standard) - SIGMA; L-valine-d8 (mixed internal standard) - Shanghai Haoyuan Biotechnology; bile acid-D4 (mixed internal standard) - Shanghai Yuanye Biotechnology; BCA kit - Biyuntian; RIPA (strong) lysis buffer - Biyuntian; PMSF (100×) - Biyuntian; phosphatase inhibitor cocktail (100×) - Yazyme; 5× Loading Buffer - Biyuntian; Tris / glycine / SDS electrophoresis buffer (10×) - Yazyme; Omni-Flash TM Ice-free fast transfer buffer (10×)-Yazyme; TBS / Tween buffer (10×)-Yazyme; Universal antibody diluent-Yazyme; PAGE gel rapid preparation kit (12.5%)-Yazyme; β-Actin Rabbit mAb (High Dilution)-Aibotek; Nucleosidephosphorylase Polyclonal antibody-Wuhan Mitsubishi Corporation; HRP-conjugated Affinipure GoatAnti-Rabbit IgG (H+L)-Wuhan Mitsubishi Corporation; PVDF membrane-Libo; Omni-ECL TM Ultra-sensitive chemiluminescence detection kit-Yazyme; protease inhibitor cocktail-MCE; purine nucleoside phosphorylase activity detection kit (colorimetric method)-SIGMA; adenosine deaminase (ADA) colorimetric kit-Elabscience; 5'-nucleotidase (5'-NT) colorimetric kit-Elabscience.

[0034] Experimental methods

[0035] 2.1 Processing of serum samples

[0036] (1) Blood is collected from the median cubital vein using a coagulant tube. The blood should be kept upright and still to avoid violent shaking.

[0037] (2) Let the serum stand at room temperature for 30 min to allow it to precipitate naturally;

[0038] (3) Eliminate hemolyzed samples and select specimens with good serum precipitation effect and basically consistent blood sample volume in the tube;

[0039] (4) After checking that the blood coagulation is complete (the supernatant is clearly separated), centrifuge at room temperature (3000 rpm, 10 min), transfer the upper serum layer into a new 1.5 mL sterile EP tube (400 μL / specimen), mark the date serial number, and freeze at -80°C for future use.

[0040] 2.2 Processing of urine samples

[0041] (1) Morning midstream urine is directly dispensed into centrifuge tubes, 1 mL per tube;

[0042] (2) Centrifuge at 4°C (10,000 rpm, 5 min), transfer the supernatant to a new 1.5 mL sterile EP tube (400 μL / specimen), mark the date, and freeze at -80°C for later use.

[0043] 2.3 Isolation of PBMCs from peripheral blood

[0044] (1) Take out the human lymphocyte separation solution and PBS buffer from the refrigerator in advance, let them stand at room temperature for equilibrium, and mix them by inversion.

[0045] (2) Peripheral blood was collected from the median cubital vein into a heparin anticoagulant tube and PBMCs were immediately isolated.

[0046] (3) Mix and dilute the peripheral blood with an equal volume of PBS in a 1:1 ratio in a clean bench. Add an equal volume of human lymphocyte separation solution to the peripheral blood in another centrifuge tube, and use a sterile pipette to slowly spread the blood dilution solution onto the separation solution along the tube wall. The volume of separation solution: blood: PBS is 1:1:1.

[0047] (4) Centrifuge at room temperature (400g, 30min), and change the speed up and down to 3 up and 1 down. According to the different densities of the components in the blood, after centrifugation, the yellow plasma layer, the white film layer, the transparent separation liquid layer, and the red blood cell layer at the bottom can be seen from top to bottom in the centrifuge tube. The white film layer is the required lymphocyte layer.

[0048] (5) Gently discard the upper yellowish plasma layer with a pipette, transfer the buffy coat cells to a new 15 mL centrifuge tube, add 10 mL PBS and mix by pipetting, centrifuge at room temperature (400 g, 5 min), and wash twice.

[0049] (6) After centrifugation, discard the supernatant, add 4 mL of complete culture medium, and gently pipette to mix. Transfer the cells to a 6-well plate, 2 mL per well, and incubate in a 37°C carbon dioxide incubator for 2 h to allow the macrophages to adhere to the wall so that pure lymphocytes can be obtained.

[0050] (7) After 2 hours, use a pipette to aspirate the suspended cells in the culture well, which are the peripheral blood lymphocytes. Centrifuge at room temperature (1000 rpm, 5 min), discard the supernatant, and adjust the cell concentration to 1×106 / mL with complete culture medium for later use.

[0051] 2.4LC-MS

[0052] 2.4.1 Pre-processing

[0053] (1) Transfer 100 μL of sample to a 1.5 mL EP tube, add 200 μL of methanol-acetonitrile (V:V = 2:1, containing mixed internal standard, 4 μg / mL), and vortex for 60 s;

[0054] (2) Ultrasonic extraction at 4°C for 10 min and then incubation at -40°C overnight;

[0055] (3) Centrifuge at 4°C (12,000 rpm, 10 min), collect 350 μL of supernatant into a sample vial, and air-dry in a safety cabinet;

[0056] (4) Redissolve with 300 μL methanol-water (V:V = 1:4), vortex for 30 s, sonicate at 4 °C for 3 min, and stand at -40 °C for 2 h;

[0057] (5) Centrifuge at 4°C (12000 rpm, 10 min), draw 150 μL of supernatant with a syringe, and use a 0.22 μm

[0058] The organic phase was filtered through a pinhole filter and transferred to an LC injection vial and stored at -80 °C until LC-MS analysis.

[0059] On-machine analysis.

[0060] (6) QC samples were prepared by mixing equal volumes of extracts from all samples.

[0061] 2.4.2 Liquid chromatography-mass spectrometry analysis conditions

[0062] The analytical instrument is a liquid chromatography-mass spectrometry system consisting of a Waters ACQUITY UPLC I-Class plus / Thermo QE HF ultra-high performance liquid phase tandem high-resolution mass spectrometer.

[0063] 2.4.3 Chromatographic conditions

[0064] Chromatographic column: ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.8 um); column temperature: 45°C; mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile; flow rate: 0.35 mL / min; injection volume: 3 μL.

[0065] 2.4.4 The elution gradient is as follows:

[0066] Time(s) A％ B％ 0 95 5 2 95 5 4 70 30 8 50 50 10 20 80 14 0 100 15 0 100 15.1 95 5

[0067] 2.4.5 Mass spectrometry conditions are as follows

[0068]

[0069] 2.5 Western blotting

[0070] 2.5.1 Specimen preparation

[0071] (1) Lysis buffer preparation: Add 1 μL PMSF (100 mM) and 1 μL phosphatase inhibitor (100×) to 100 μL RIPA lysis buffer.

[0072] (2) Specimen lysis: Count 1×106 peripheral blood lymphocytes, suspend in PBS buffer, centrifuge at room temperature (400g, 5 min), add 100 μL of lysis buffer to the cell pellet, repeatedly blow and beat, ultrasonically disrupt at 4°C for 30 min, centrifuge at 4°C (12000g, 10 min), and take the supernatant as the protein lysis buffer.

[0073] 2.5.2 BCA protein concentration determination

[0074] (1) Preparation of protein standards

[0075] a. Dissolve 30 mg of protein standard in 1.2 mL of protein standard preparation solution to prepare a protein standard solution with a final concentration of 25 mg / mL;

[0076] b. Dilute 20 μL of protein standard solution with 980 μL of PBS buffer solution to prepare a protein standard with a final concentration of 0.5 mg / mL.

[0077] (2) Preparation of BCA working solution

[0078] BCA A solution: B solution = 50:1, prepared according to the number of samples, 200 μL per well.

[0079] (3) Protein concentration determination

[0080] a. Add the standard and diluent into the standard wells according to the proportions shown in the table below.

[0081]

[0082] b. Add samples to the sample wells of the 96-well plate.

[0083] c. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min.

[0084] d. Use an ELISA reader to measure the absorbance of each well at 562 nm.

[0085] e. Calculate the protein concentration and sample volume based on the standard curve.

[0086] 2.5.3 Protein sample denaturation

[0087] Add lysis buffer to sample 1 / 4 volume of 5× Loading Buffer, denature at 100°C in a metal bath for 10 min, and freeze at -80°C for later use.

[0088] 2.5.4 Preparation of protein electrophoresis reagents

[0089] Electrophoresis solution: electrophoresis buffer (10×): pure water = 1:9;

[0090] Transfer solution: ice-free rapid transfer solution: anhydrous ethanol: pure water = 1:1:8;

[0091] TBST: TBS / Tween buffer (10×): pure water = 1:9;

[0092] Blocking solution (5% skim milk powder): 5 g skim milk powder dissolved in 100 mL TBST;

[0093] Primary antibody / secondary antibody diluent: dilute with the above blocking solution according to the ratio recommended in the literature;

[0094] 2.5.5 PAGE gel preparation

[0095] (1) 12.5% ​​PAGE gel configuration (1 mm gel as an example):

[0096] a. Lower gel: 2.7 mL each of lower gel solution and buffer, 60 μL of coagulant, mix thoroughly.

[0097] b. Upper gel: 750 μL each of lower gel solution and buffer solution, 15 μL of coagulant, mix thoroughly.

[0098] c. Slowly inject the lower glue and upper glue mixture into the gap between the glass plates, insert the comb teeth, and let it stand at room temperature for at least 15 minutes.

[0099] 2.5.6 Protein electrophoresis

[0100] (1) Clamp the completely solidified PAGE gel to the electrophoresis clamp, inject the electrophoresis solution, remove the comb teeth, and add

[0101] Protein maker, sample, room temperature, 150V, constant voltage electrophoresis for 50 minutes.

[0102] (2) Transfer

[0103] a. Cut the PVDF membrane according to the area of ​​the PAGE gel after electrophoresis and activate it with anhydrous ethanol for 1 minute;

[0104] b. Soak the transfer sponge pad, and place PAGE gel, PVDF membrane, and the soaked sponge pad on it in sequence.

[0105] Form a wet transfer "sandwich" structure, clamp the splint, and insert it into the transfer tank;

[0106] c. Pour transfer solution into the membrane transfer clip, and transfer the membrane at a constant current of 400 mA at room temperature for 35 minutes.

[0107] (3) Closed

[0108] Transfer the intact PVDF membrane and shake the blocking solution at room temperature for 1 hour.

[0109] (4) Primary antibody incubation

[0110] The membrane was washed with TBST for 6 min × 5 times and then discarded. The dilution solution of β-actin and PNP primary antibodies was added, mixed on a shaker at room temperature, and incubated at 4°C overnight.

[0111] (5) Secondary antibody incubation

[0112] The membrane was washed with TBST for 6 min × 5 times and then discarded. HRP secondary antibody dilution was added and shaken at room temperature for 1 h.

[0113] (6) Membrane washing

[0114] Wash the membrane with TBST for 6 min × 5 times.

[0115] (7) Development

[0116] Mix 1 mL of ECL chemiluminescent solution A and 1 mL of ECL chemiluminescent solution B in equal volumes and protect from light; immerse the membrane in the chemiluminescent solution for 1 min, place it flat in a developer, expose it, take pictures, and save the image.

[0117] (8) Results Analysis

[0118] Image J was used to analyze the grayscale value of the development results and compare the relative expression levels of the target protein and the internal reference protein in the same sample.

[0119] 2.6 Enzyme activity determination

[0120] 2.6.1 PNP enzyme activity (colorimetric method)

[0121] (1) Reagent preparation:

[0122] a.Assay Buffer: Assay Buffer (10×): pure water = 1:9;

[0123] Inosine substrate: aliquot for use, store at -20℃

[0124] Hypoxanthine standard (10mM): 10μL standard + 90μL Assay Buffer dilution to obtain a final concentration of 1mM hypoxanthine standard.

[0125] (2) Specimen processing:

[0126] a. After lymphocytes were separated from peripheral blood, the cells were washed by centrifugation with pre-ice-cold PBS;

[0127] b. Resuspend in 150-300 μL Assay Buffer (containing protease inhibitor cocktail) and pipette thoroughly;

[0128] c. 4℃, stirring with electric homogenizer for 15min;

[0129] d. Centrifuge at 4°C (12,000 g, 15 min) to remove insoluble matter, collect the supernatant and test immediately or store at -80°C.

[0130] (3) Sampling and detection

[0131] a. Standard hole preparation, as shown in the following table.

[0132]

[0133] Two replicate wells were set up for each dilution concentration.

[0134] b. Add sample

[0135] Standard wells: 50 μL standard dilution;

[0136] Sample well: 2-50 μL sample, fill in less than 50 μL with Assay buffer;

[0137] Positive control: 2μL PNP positive control + 48μL Assay buffer.

[0138] Background control well: same as this well.

[0139] System preparation: XμL ingredients × (number of reactions + 1), as shown in the following table:

[0140]

[0141] c. Detection

[0142] In the kinetic mode, the absorbance was measured at OD = 293 nm, once every 2-3 minutes, and continuously monitored for 30 minutes at room temperature. Two time points T1 and T2 were selected within the linear range, and the OD values ​​were A1 and A2, and the PNP activity was calculated.

[0143] 2.6.2 ADA enzyme activity detection (colorimetric method)

[0144] (1) Sample processing: Same as the sample processing for PNP enzyme activity assay.

[0145] (2) Sampling and detection

[0146] a. Standard hole preparation, as shown in the following table:

[0147]

[0148] Two replicate wells were set up for each dilution concentration.

[0149] b. Add sample

[0150] Standard wells: 10 μL standard dilution solution;

[0151] Sample well: 10 μL sample;

[0152] Add 180 μL of working solution and 90 μL of color developing agent in sequence.

[0153] c. Detection

[0154] Incubate at 37℃ for 7min, the first absorbance at 550nm is A1, and then continue to incubate for 10min.

[0155] The light intensity is A2, and the ADA activity is calculated.

[0156] 2.6.3 5'-NT enzyme activity detection (colorimetric method)

[0157] (1) Sample processing: Same as the sample processing for PNP enzyme activity assay.

[0158] (2) Sampling and detection

[0159] a. Standard hole preparation, as shown in the following table:

[0160]

[0161] Two replicate wells were set up for each dilution concentration.

[0162] b. Add sample

[0163] Standard wells: 10 μL standard dilution solution;

[0164] Sample well: 10 μL sample;

[0165] Add 180 μL of working solution, incubate at 37°C for 5 min, and then add 90 μL of color developer.

[0166] c. Detection

[0167] Incubate at 37℃ for 10min, the absorbance at 550nm is A1 for the first time, and then continue to incubate for 10min.

[0168] The absorbance is A2, and the 5'-NT activity is calculated.

[0169] Experimental results:

[0170] 3.1 Abnormal purine metabolism pathway in RA patients

[0171] LC-MS results showed that RA patients had abnormal metabolism, and the metabolomics of serum and urine showed significant differences. Among the differential metabolites, we noticed that the level of deoxyinosine (2-Deoxyinosine, 2-Di) in the T cell salvage synthesis pathway was decreased in both serum and urine of patients, such as Figure 1 shown.

[0172] 3.2 Abnormalities of PNP enzyme in RA patients

[0173] Through the analysis of serum and urine metabolomics data, we focused on the salvage synthesis pathway of the purine metabolic pathway ( Figure 2 The 2-Di of A) decreased, which was considered to be related to the abnormality of its upstream and downstream enzymes. First, we considered the downstream enzyme PNP, collected peripheral blood lymphocytes from RA patients and healthy controls, lysed them, and extracted cell proteins. The results of protein electrophoresis showed that there was no significant difference in the expression of PNP in lymphocytes of RA patients and healthy controls ( Figure 2 B), we also measured the activity of PNP enzyme, and also measured the activity of adenosine deaminase (ADA) and 5'-nucleotidase (5'-NT) upstream of 2-Di. The results showed that PNP activity increased, while the activity changes of ADA and 5-NT were not statistically significant ( Figure 2 C. Figure 2 D. Figure 2 E).

[0174] We first collected serum and urine from RA patients and healthy controls for metabolomics analysis, and found that RA patients had obvious metabolomics abnormalities, among which differential metabolites were significantly enriched in the purine metabolic pathway. Among them, we focused on a metabolite that was reduced in both serum and urine of patients: 2-Di, which is a product of the purine salvage synthesis pathway. It can be converted by PNP enzyme to hypoxanthine and then to uric acid by xanthine oxidase. It is considered to be related to increased downstream enzyme activity or decreased upstream enzyme activity. Purine nucleoside phosphorylase (PNP) is one of the key enzymes in the purine salvage synthesis pathway. In human blood, T cells lack de novo purine synthesis and rely on the purine salvage synthesis pathway. The activity of PNP is increased, T cell proliferation is accelerated, the activity is enhanced, and lymphocytic leukemia occurs. Since the level of deoxyinosine upstream of PNP decreased, in order to investigate the cause, peripheral blood was collected from RA patients and healthy controls for lymphocyte separation. First, a protein blotting experiment of PNP enzyme was performed, and no abnormal PNP protein level was found in RA patients. Then the activity of adenosine deaminase (ADA) and 5-nucleotidase (5-NT) upstream of deoxyinosine, as well as downstream PNP enzyme was measured. It was found that the decrease in 2-Di level was caused by the increase in the activity of downstream PNP enzyme. Studies have shown that methotrexate (MTX), a first-line anti-inflammatory drug for RA, can effectively reduce the activity of adenosine deaminase (ADA), 5'-nucleotidase (5'-NT) and PNP enzyme in the purine metabolic pathway, among which the effect on PNP is the most significant. We speculate that one of the mechanisms of MTX in treating RA is to reduce PNP activity. In order to verify this hypothesis, we selected a known inhibitor of PNP with high activity for subsequent in vivo and in vitro experiments to verify the role of PNP in the pathogenesis of RA.

[0175] Forodesine (FDS) is a highly active PNP inhibitor. We consider that PNP abnormality can be used as a new target for the treatment of RA. Inhibiting the activity of PNP enzyme can inhibit the abnormal proliferation and activation of lymphocytes, reduce the secretion of inflammatory factors, and thus alleviate clinical symptoms. In this chapter, we established an animal model of rheumatoid arthritis, a collagen-induced arthritis (CIA) mouse model. By giving mice FDS for preventive treatment, the incidence of the model control group and the treatment groups of each dose, as well as the proportion of T cell subsets in the spleen and lymph node tissues, was compared to determine the effect of FDS on the course of the disease, in order to conduct a more in-depth exploration of RA.

[0176]

[0177]

[0178] 1.3 Primer sequences The primer sequences used in this example are as follows.

[0179]

[0180] 1.4 Experimental animals

[0181] SPF male DBA1 / J mice, 6-8 weeks old, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and housed in the clean animal experimental center of the Second Affiliated Hospital of Soochow University. The mice were kept at a constant temperature of 22-25°C, with a 12h / 12h light-dark cycle, free access to water and food, and the experiment began after one week of adaptive feeding. Animal experiments were carried out in accordance with the guidelines of the Animal Ethics and Use Committee and approved by the Animal Ethics Committee of Soochow University.

[0182] 2 Experimental methods

[0183] 2.1 CIA modeling, animal medication, and grouping

[0184] (1) Reagent preparation

[0185] a.0.5M glacial acetic acid solution: Use a pipette to pipette 28.6μL of analytical grade glacial acetic acid (17.5M) and 10mL of sterile distilled water into a test tube, mix well and store at 4℃ for later use.

[0186] b. CII collagen solution: Dissolve 10 mg of bovine type II collagen in 5 mL of 0.5 M glacial acetic acid solution and stir overnight at 4°C using a magnetic stirrer to obtain a final concentration of 2 mg / mL.

[0187] c. Collagen-complete Freund's adjuvant (CFA) mixed emulsion: Mix CII collagen solution with CFA in a ratio of 1:1 to a final concentration of 1 mg / mL. Use a three-way tube to fully push and pull the emulsified mixture until it becomes white and milky. If a drop is dropped into water and does not disperse, the emulsification is considered complete. To prevent collagen denaturation, the entire operation must be performed on ice.

[0188] d. Collagen-Freund's incomplete adjuvant (IFA) mixed emulsion: Mix the CII collagen solution with IFA in a ratio of 1:1, and emulsify in the same manner as above.

[0189] e. Tribromoethanol solution: 25 g 2,2,2-tribromoethanol was dissolved in 15.5 mL tert-amyl alcohol and stirred with a magnetic stirrer.

[0190] 12h, store in dark place, and prepare a stock solution with a final concentration of 1600mg / mL; 0.5mL of stock solution is diluted with 39.5mL of raw

[0191] The final concentration of the working solution was 20 mg / mL.

[0192] Make liquid.

[0193] (2) Establishment of CIA model

[0194] a. Primary immunization: According to the weight of the mouse, 0.2mL / 10g of tribromoethanol solution was injected intraperitoneally to anesthetize the mouse. The hair from the back to the base of the tail was shaved and the skin was prepared. The needle was inserted obliquely upward parallel to the tail at 1-1.5cm from the base of the tail. After aspiration without blood, the collagen-complete adjuvant mixture was slowly and subcutaneously injected at multiple points, 100μL / mouse. The primary immunization was day 0.

[0195] Reinforced immunization: On d21, the same method as the initial immunization was used, with injection of a collagen-incomplete adjuvant mixture, 100 μL / mouse. The model induction process was as follows: Figure 4 Hint.

[0196] (3) Body weight and clinical score records: The first immunization was regarded as day 0. The mice were closely observed starting from day 21, and the data were recorded every other day. The clinical symptoms of the mice were scored according to the table below.

[0197]

[0198] The limbs of the mice were scored, and the sum of the scores was the arthritis index of the mouse.

[0199] (4) Mouse grouping: The animals were randomly divided into 5 groups, with 10 mice in each group, namely blank control group, model control group, low-dose group (10 mg / kg), medium-dose group (20 mg / kg), and high-dose group (40 mg / kg). The blank control group was normal mice without arthritis induction; the model control group and the low-, medium-, and high-dose groups were all CIA model mice. The three drug-treated groups began to receive preventive oral administration of Forodesine on the 19th and 24th days after modeling, and the control group and model group were given an equal amount of PBS. Around the 35th day, the model control group entered a mild stage of disease, and several groups of animals were treated. The animal experiment process is as follows: Figure 5 .

[0200] (5) Preparation of Forodesine solution (10, 20, 40 mg / kg, based on 20 g / mouse body weight, prepare the solution

[0201] Concentrations: 1, 2, 4 mg / mL)

[0202] 50 mg Forodesine was dissolved in 12.5 mL PBS solution to obtain 4 mg / mL Forodesine solution. The solution was diluted with equal volumes of PBS solution in proportion to obtain Forodesine solutions of 3 different concentrations.

[0203] 2.2 Tissue HE staining

[0204] 2.2.1 Preparation of paraffin sections

[0205] (1) Fixation: Fresh intact claw joints, livers, and kidneys were rinsed twice in PBS to remove blood stains and then fixed in 4% paraformaldehyde.

[0206] (2) Decalcification (claw): bone tissue was fixed in 10% formaldehyde solution for 24 hours and then a bone slice of about 0.5 cm thick was sawed. It was fixed in 10% formaldehyde solution for 2 days. The tissue was placed in a decalcifying agent and the solution was replaced daily until the tissue softened. It was rinsed with running water for 24 hours (deacidification).

[0207] (3) Dehydration: After fixation, rinse with running water three times, then soak in 70%, 80%, 95%, 95% ethanol, and anhydrous ethanol twice, each for 1 hour, for dehydration.

[0208] (4) Transparency: Soak in anhydrous ethanol and xylene twice, each time for 10 minutes, until transparent.

[0209] (5) Wax immersion: The tissue block was immersed in paraffin melted at 60°C for 1 h.

[0210] (6) Embedding: Place the tissue on the embedding mold, adjust the position, add paraffin wax, and place in the freezer overnight after the paraffin wax solidifies.

[0211] (7) Sectioning: The embedded wax block is placed in a microtome and sectioned to a thickness of 3 μm.

[0212] 2.2.2 Hematoxylin-eosin (H&E) staining

[0213] (1) Dewaxing and hydration: Dry the sheets in an oven at 60°C for 30 min, and place them in the following solutions in sequence: xylene for 10 min twice; anhydrous ethanol, 95%, 85%, and 75% ethanol for 3 min each; and distilled water for 2 min.

[0214] (2) Hematoxylin staining: hematoxylin for 5 min, then wash with distilled water; differentiate solution for 10 s, then wash twice with distilled water, 5 min each time.

[0215] (3) Eosin staining: Eosin for 1 min.

[0216] (4) Dehydration, transparency and sealing: 75%, 85%, 95% and anhydrous ethanol twice, each for 2 seconds; xylene twice, each for 1 minute, and then seal the slides with neutral gum.

[0217] (5) Microscopic examination

[0218] Three mice were observed in each group, three sections in different regions were observed for each mouse, and three fields of view (40×) were observed for each section.

[0219] 2.3 Mouse serum collection

[0220] a. The mouse is placed in a supine position, and the hair on the chest area is trimmed. After disinfecting the skin with alcohol, the 3rd to 4th intercostal space on the left side is touched and the point where the heart beats the strongest is selected for puncture. A successful puncture will show blood returning from the front end of the syringe. The amount of blood collected each time is 0.5 to 0.6 mL.

[0221] b. The blood was allowed to stand at room temperature, and after stratification, it was centrifuged at room temperature (10,000 rpm, 10 min).

[0222] 2.4 Isolation of mouse lymph node lymphocytes

[0223] a. Remove the inguinal lymph nodes on both sides in a clean bench;

[0224] b. Place a sterile nylon filter on the lymph node, grind it with a 5mL syringe core, add RPMI1640 culture medium, blow and mix, and filter the suspension through the filter to remove large pieces of tissue;

[0225] c. Centrifuge at room temperature (400g, 5min), collect the precipitate, wash twice with PBS, and the precipitate obtained is the pure lymph node lymphocytes.

[0226] 2.5 Isolation and culture of mouse spleen lymphocytes

[0227] (1) Spleen isolation: Remove the spleen in a clean bench and place it in a sterile culture dish.

[0228] (2) Lymphocyte separation:

[0229] a. Grinding: Same method as above;

[0230] b. Centrifuge at room temperature (400 g, 5 min), and resuspend the cell pellet in 4 mL of mouse lymphocyte separation medium;

[0231] c. Slowly add 1 mL of RPMI1640 on top of the cell suspension; centrifuge at room temperature (800 g, 30 min), up 3 times and down 1 times;

[0232] d. After centrifugation, aspirate the lymphocytes in the middle buffy coat layer, wash twice with PBS, collect the cell pellet, suspend it in R10 complete medium, and adjust the concentration to 1×106 / mL.

[0233] (3) Lymphocyte stimulation

[0234] a. The lymphocytes separated above were suspended in R10 at a concentration of 1×106 / mL. 2 μL of cocktail was added to each mL of cell suspension.

[0235] b. Incubate the cells in a 37°C, 5% CO2 incubator for 5 hours.

[0236] c. Collect the stimulated cells, centrifuge at room temperature (300g, 5min), and discard the supernatant.

[0237] d. Perform subsequent staining.

[0238] 2.6 Flow cytometric analysis

[0239] (1) Flow cytometer compensation adjustment

[0240] a. Take 10 flow cytometry tubes, add 100 μL PBS to each tube, and add one drop of mixed negative control compensation beads and anti-rat / hamster Igκ compensation beads to each tube, and vortex to mix.

[0241] b. Add 1 μL of mouse / hamster anti-mouse FITC, PE, APC, APC-cy7, AF-700, Percp-cy5.5, BV421, BV605, V500, and PE-cy7 labeled antibodies into the tubes respectively, label them, and incubate at 4°C in the dark for 15 minutes.

[0242] c. Add 2 mL of PBS to terminate the reaction, centrifuge at room temperature (200 g, 10 min), discard the supernatant, resuspend the pellet in 500 μL of PBS, and mix well.

[0243] d. Start the machine and run each test tube separately. After the capture is completed, the instrument will automatically make compensation adjustments.

[0244] (2) Preparation of membrane permeabilization fixative

[0245] a.1×Fix / Perm Buffer: 4×Fix / Perm Buffer: Diluent Buffer=1:3, 1mL / test.

[0246] b.1×Perm / Wash Buffer:5×Perm / Wash Buffer:pure water=1:4, 2mL / test.

[0247] (3) Treg subpopulation detection

[0248] a. Take 100 μL of spleen and lymph node cell suspension respectively into flow cytometry tubes, wash with 2.5 mL of PBS to fully remove fetal bovine serum.

[0249] b. Add PBS containing ghost dye (1:1000 dilution) to the tube, 300 μL / tube, mix well, incubate at 4℃ in dark for 20 min, add 1 mL PBS to terminate staining, centrifuge and discard the supernatant.

[0250] c. Add RPMI1640 containing CD3-FITC, CD4-AF700, and CD8-Percp cy5.5 antibodies (1 μL / test each) into the tube, 100 μL / tube, mix well, incubate at 4°C in the dark for 15 min, add 1 mL PBS to terminate staining, centrifuge and discard the supernatant.

[0251] d. Add 1 mL of freshly prepared 1×Fix / Perm working solution into the tube, mix well by pipetting, and fix at room temperature in the dark for 40 min.

[0252] e. Add 1 mL 1×Perm / Wash to terminate the reaction, centrifuge and discard the supernatant.

[0253] f. Add 1×Perm / Wash containing APC-Foxp3 and BV421-Ki67 antibodies (1 μL / test each) into the tube, 100 μL / tube, mix well, and incubate at room temperature in the dark for 40 min.

[0254] g. Add 1 mL 1× Perm / Wash to terminate staining, centrifuge and discard the supernatant, resuspend the cell pellet in 200 μL PBS, and detect on the microscope.

[0255] (4) Th1 / Th2 / Th17 subset detection

[0256] a. Collect spleen lymphocytes after 6 hours of stimulation by centrifugation, wash twice with PBS, and remove fetal bovine serum.

[0257] b. Ghost, CD3, CD4, CD8 staining and fixation and membrane permeabilization operations are the same as above

[0258] c. Add 1×Perm / Wash containing IL17-PE, IL4-BV605, and IFN-γ-Pecy-7 antibodies (1 μL / test each) into the tube, and set up isotype controls by replacing the corresponding antibodies with 100 μL / tube of the isotype antibodies of the antibodies (1 μL / test each), mix well, and incubate at room temperature in the dark for 40 min;

[0259] d. Add 1 mL 1× Perm / Wash to terminate the staining, centrifuge and discard the supernatant. Resuspend the cell pellet in 200 μL PBS and test on the microscope.

[0260] 2.7 Western Blotting

[0261] The specimen processing is the same as in the first part, and the antibody dilution concentrations are as follows:

[0262]

[0263] 2.8 Real-time fluorescence quantitative PCR

[0264] (1) Specimen processing: Take the remaining lymphocytes after the above separation and flow cytometry operation, wash them twice with PBS, discard the supernatant, add 1 mL of Trizol and pipette thoroughly to fully lyse the cells.

[0265] (2) RNA extraction

[0266] a. Add one fifth of the volume of chloroform to the above lysate, mix thoroughly by inversion, and let stand at room temperature for 10 min to separate layers;

[0267] b. Centrifugation at 4°C (12000 g, 15 min);

[0268] c. Transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, mix by inversion, and store at -20°C overnight; d. Centrifuge at 4°C (12000g, 15min);

[0269] e. Discard the supernatant. The white precipitate at the bottom of the tube is RNA. Add 1 mL of 75% anhydrous ethanol (prepared with DEPC water) and wash twice.

[0270] f. Centrifugation at 4°C (12000 g, 15 min);

[0271] g. Discard the supernatant, air-dry in a safety cabinet, dissolve the precipitate with 20-50 μL DEPC water, and detect and record the RNA concentration and purity using Nanodrop2000.

[0272] (3) Reverse transcription

[0273] RT-PCR reaction was performed according to the standard operating procedure of 5×All-in-one RT MasterMix reverse transcription kit.

[0274] The steps are as follows:

[0275] a. Prepare the following system for gDNA removal and subsequent reverse transcription on ice, as shown in the table below.

[0276]

[0277] Incubate in a metal bath at 42°C for 2 minutes or at room temperature for 5 minutes. Then add 2 μL of reaction stopper and mix well.

[0278] b. The reverse transcription reaction system configuration is as shown in the following table.

[0279]

[0280] Vortex to mix.

[0281] c. RT-PCR reaction, as shown in the following table.

[0282]

[0283] The reverse transcription product needs to be diluted 3 times before q-PCR and stored at -20°C when not in use for a long time.

[0284] (4) Real-time fluorescence quantitative PCR

[0285] According to TB Premix Ex Taq TM II (Tli RNaseH Plus) real-time fluorescence quantitative kit standard operating procedures for qPCR. The reaction steps are as follows:

[0286] a. Reaction system configuration, as shown in the following table:

[0287]

[0288] b.qPCR, as shown in the following table.

[0289]

[0290] Experimental Results

[0291] 3.1FDS alleviates arthritis symptoms in mice and delays disease progression

[0292] In order to verify the relationship between increased PNP activity and the onset of rheumatoid arthritis, three doses of FDS were given to the treatment group on day 19 and day 24. Since FDS is a known PNP inhibitor with high oral activity, oral administration was chosen. At the same time, the blank control group and the model control group were given an equal amount of PBS buffer. The results showed that after administration, the arthritis symptoms of CIA mice were alleviated and the course of the disease was delayed. The clinical symptoms of the low-dose group were alleviated, and the medium-dose and high-dose groups effectively inhibited the occurrence of arthritis ( Figure 5 The incidence rate (n incidence / n total) of the drug-treated group decreased, and the difference was statistically significant ( Figure 5 At the same time, we performed molybdenum target examination on the CIA joints and found that after administration of FDS, the degree of bone and joint deformity was alleviated and the degree of soft tissue redness and swelling was significantly reduced ( Figure 5 C).

[0293] 3.2 Splenomegaly was reduced after FDS administration

[0294] The spleen is an important central immune organ, a place where mature lymphocytes reside, and has immune response functions, of which T cells account for about 40%. In the CIA mouse model, due to the increase in the number of lymphocytes and enhanced activation after collagen induction, the spleen is infiltrated, and the autoimmune response is enhanced, resulting in splenomegaly. After the experimental observation, we treated the mice and found that the spleen in the model group was significantly enlarged. FDS administration can reduce splenomegaly. Among them, the low-dose group and the medium-dose group inhibited splenomegaly, and the opposite trend was shown as the concentration increased ( Figure 6 A. Figure 6 B).

[0295] 3.3 No obvious toxic side effects were observed after FDS administration

[0296] The liver and kidney of each group of experimental animals were stained with HE and serum transaminase was detected. The results showed that there was no significant change in liver and kidney pathology after administration ( Figure 7 A), transaminase results showed that FDS administration did not cause alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and obvious toxic side effects ( Figure 7 BD).

[0297] 3.4FDS administration regulates the proportion of T lymphocyte subsets

[0298] 3.4.1 After administration, the proportion of spleen T lymphocytes decreased, the proportion of CD8+T cells decreased, and the proliferation capacity decreased. Lymphocyte subset analysis is an important indicator of the body's immune function, which can reflect the body's current immune function, state and balance level, and is of great significance for observing efficacy and detecting prognosis. The spleen is a gathering place for lymphocytes, among which T lymphocytes are the main immune response form of cellular immunity. We separated spleen mononuclear cells, and the flow cytometry results showed that the proportion of lymphocytes and T cells infiltrating the spleen decreased after administration, and the effect of the medium-dose group (20 mg / kg) was obvious. T cells are divided into two major categories: CD4+T cells and CD8+T cells. The cells of each subpopulation are in a dynamic equilibrium state. Among them, CD4+T cells are also called helper T cells (Helper T cell, Th), and CD8+T cells are also called cytotoxic T lymphocytes (CTL cells). They are key components of the adaptive immune system. CD8+T cell dysfunction triggers excessive immune response, thereby leading to immune-mediated body damage or pathological response. There was no statistically significant change in the proportion of CD4+T cells after administration, and the proportion of CD8+T cells decreased. Ki67 is a nuclear proliferation antigen that reflects the proliferation ability of cells. We found that administration had no significant effect on the proliferation ratio of CD4+T cells; it inhibited the expression of CD8+Ki67+T cells, indicating that administration inhibited the proliferation of CD8+T cells.

[0299] In summary, we believe that the mechanism by which PNP inhibitors alleviate RA inflammation is by reducing the proportion of T cells and inhibiting the proliferation of CD8+T cells.

[0300] 3.4.2 Increased Treg ratio after drug administration

[0301] Regulatory T cells (Treg) are key regulators of inflammation and play an important role in regulating immune balance [20,21]. The absence or functional defects of Treg cells can lead to autoimmune diseases. Treg cells are an important component of peripheral immune tolerance. In RA, Treg cells act as negative regulatory cells and have anti-inflammatory effects. Correcting Treg defects can alleviate the occurrence and development of RA. The transcription factor forkhead box protein 3 (Foxp3) is the main marker of Treg cells and dominates the function of Treg cells. We separated lymphocytes for flow cytometry analysis and gated them using Foxp3 as a marker of Treg cells. We found that the proportion of Treg cells increased after drug administration. At the same time, the spleen tissue was lysed by RIPA, and the protein was extracted after ultrasonic disruption. Western blotting experiments were performed to explore the differences in Foxp3 protein expression levels. The expression level of Foxp3 protein increased after drug administration, which was consistent with the trend of flow cytometry results.

[0302] Lymph nodes are where mature T lymphocytes and B lymphocytes reside, among which T lymphocytes account for a larger proportion. The occurrence and development of RA is related to the abnormal activation of immune cells in lymph nodes. Lymph nodes, as the central site of peripheral immune tolerance, play an important role in initiating and regulating autoimmune responses (including the production of antibodies, such as RF and ACPA). We separated lymphocytes from lymph node tissues, and after drug administration, Treg cells

[0303] We also separated the peripheral blood of mice for flow cytometry and found that the proportion of Treg cells also increased after administration. In summary, we believe that inhibiting PNP can alleviate the onset of RA and is related to the increase in the proportion of Treg cells.

[0304] 3.4.3 Changes in the proportion of CD4+ T cell subsets after drug administration

[0305] According to the results of previous experiments, we found that drug administration had no significant effect on the proportion of CD4+T cells, so we continued to detect CD4+T cell subsets. After isolating spleen lymphocytes from each group of mice, T cells were stimulated with PMA+ionomycin in vitro for 5 hours, and then Th1, 2, and 17 cells were detected.

[0306] Th1 cells are a type of CD4+T cells that produce pro-inflammatory cytokines. Their main effector function is to mediate cellular immunity and inflammatory responses, including activation of other immune cells such as macrophages, B cells, and CD8+T lymphocytes for cytolysis and other effector functions. IFN-γ is a cytokine secreted during the differentiation and maturation of Th1 cells. It can also act on dendritic cells and macrophages. After Th1 cells mature, they will secrete IFN-γ, thereby inducing the differentiation of more Th1 cells.

[0307] IL-4 is a specific transcription factor for Th2 cells. We found that after administration, the proportion of Th2 cells in the CD4+T cell subset increased and the Th1 / Th2 ratio decreased, indicating that administration can correct the Th1 / Th2 imbalance in RA. Abnormalities in Th17 cells can disrupt the immune balance of Th1 / Th2 in RA and its animal model CIA mice. Cytokines produced by Th17 cells, including IL-17, TNF-α, etc., have proinflammatory effects and play a role in the immune pathogenesis of RA. We used IL-17 as a marker of Th17 cells and detected Th17 cells. It was found that the proportion of Th17 cells decreased after administration. In summary, we believe that PNP inhibitors can correct the imbalance between Th1 / Th2 and Treg / Th17 cells and reduce the onset of the disease.

[0308] 3.5 Medication inhibits the secretion of inflammatory factors

[0309] Tumor necrosis factor (TNF-α) is a proinflammatory factor in RA, and TNF-α inhibitors can be combined with methotrexate as a treatment for RA. The RNA of spleen lymphocytes from each group of mice was extracted for reverse transcription, and the product was used for real-time fluorescence quantitative PCR. The gene expression of inflammatory factors TNF-α, IL-17a, IL-4 and Foxp3 was measured. Administration of TNF-α inhibited the secretion of proinflammatory factors TNF-α and IL-17a, promoted the gene expression of anti-inflammatory factor IL-4 and Treg cell transcription factor Foxp3, and the trend was consistent with the flow cytometry results.

[0310] 3.6 Administration of drugs to inhibit phosphorylation of NF-κB protein

[0311] Nuclear factor-κB (NF-κB) is a transcription factor that usually forms homo / heterodimers with p65 and p50, which can regulate the expression of genes that control cell proliferation and apoptosis, and is related to the occurrence of inflammation and immune status in the body. In RA, abnormal activation of the NF-κB pathway can promote the development of inflammation, and inhibiting this pathway can inhibit the onset of RA. We lysed the isolated mouse spleen lymphocytes, extracted cell proteins, and performed western blot. We found that there was no significant difference in total p65 protein after administration, and the phosphorylated pp65 decreased. The pp65 / p65 ratio reflects the phosphorylation level of NF-κB pathway proteins. Administration inhibited the phosphorylation of NF-κB protein.

[0312] 3.7 Administration promotes activation of the AMPK pathway and exerts anti-inflammatory effects

[0313] AMPK (Adenosine 5'-monophosphate (AMP)-activated protein kinase) is an AMP-dependent protein kinase, which is a key factor in regulating biological metabolism. In RA, the metabolic state is abnormal, and activation of the AMPK pathway can correct the abnormality, increase AMPK pathway phosphorylation, and reduce inflammation. The isolated mouse spleen lymphocytes were lysed, cell proteins were extracted, and protein immunoblotting experiments were performed. It was found that there was no significant difference in the total AMPK protein after administration, and the phosphorylated AMPK (p-AMPK) increased. The p-AMPK / AMPK ratio reflects the phosphorylation level of AMPK pathway proteins. Administration promotes the phosphorylation of AMPK protein.

[0314] CIA mice are the most widely used induced animal model for studying rheumatoid arthritis. This model uses a mixed emulsion of heterologous II collagen and Freund's adjuvant (BCG and liquid paraffin) as a foreign antigen, and subcutaneously injects autoantibodies in patients with rheumatoid arthritis to induce autoimmune reactions in mice, produce autoantibodies targeting type II collagen in articular cartilage, and manifest as multiple peripheral arthritis. This model develops arthritis symptoms such as joint swelling and bone and joint deformities around 24 days. In this section, we found that FDS, as a highly efficient oral inhibitor of PNP, alleviated the clinical symptoms of CIA mice after oral administration, delayed the course of the disease, reduced the incidence rate, and reduced the degree of bone and joint deformities and redness and swelling of joint soft tissues. The results of liver and kidney H&E staining showed that there was no significant change in liver and kidney pathology after administration, no abnormal changes in transaminase, and no obvious toxic side effects were caused by administration. The gross observation of the spleen showed that the degree of splenomegaly was reduced, the mass decreased, and the lymphocyte infiltration decreased after administration. Combining the above results with the results of metabolomics of RA patients and increased activity of PNP enzyme in the first part, we believe that abnormalities in PNP are related to the occurrence of arthritis, and inhibiting PNP can correct this abnormality and have a therapeutic effect.

[0315] Lymphocyte subset analysis is an important indicator of the body's immune function. It can reflect the body's current immune function, state and balance level, and is of great significance for observing therapeutic effects and detecting prognosis. The spleen is a gathering place for lymphocytes, among which T lymphocytes are the main immune response form of cellular immunity. After administration, the proportion of spleen lymphocytes decreased, and the proportion of T cells decreased. T cells are divided into two subclasses, CD4 and CD8, among which the proportion of CD8+T cells decreased. We believe that this is related to the reduced proliferation ability of CD8+T cells. The proportion of CD8+T cells in RA increases, the proliferation ability is enhanced, cytotoxic mediators are expressed, and the killing of target cells is mediated, leading to joint inflammation and damage to synovial tissue. Experimental results show that inhibiting PNP can inhibit the abnormal proliferation of CD8+T cells and play a therapeutic role.

[0316] In addition, it is known that the occurrence of RA is related to the imbalance in CD4+T cell subsets, including Th1, Th2, Th17, Treg, etc. Current studies believe that the increase in the Th1 / Th2 ratio and the decrease in the Treg / Th17 ratio are related, and the imbalance between the two groups of cells will lead to the occurrence of autoimmune diseases. After inhibiting PNP, the Th1 / Th2 ratio decreased, the Th2 cell ratio increased, and the IL-4 expression increased, exerting an anti-inflammatory effect. Regulatory T cells (Treg) play an anti-inflammatory role in RA and regulate the abnormal activities of immune cells. After administration, the proportion of Treg cells in the spleen, lymph nodes, and peripheral blood increased, while the proportion of Th17 cells in the spleen decreased, indicating that after administration, the ratio between Treg / Th17 cells increased, exerting an anti-inflammatory effect. At the same time, Th17 cells secrete anti-inflammatory factors such as TNF-α and IL-17. Common treatments for RA include disease-modifying antirheumatic drugs (DMARDs) combined with TNF-α inhibitors (TNF-αi). After inhibiting PNP, the proportion of Th17 cells decreases, and the expression of two pro-inflammatory factors, TNF-α and IL-17, also decreases. NF-κB is a transcription factor that regulates the expression of genes that control cell proliferation and apoptosis, as well as genes that respond to inflammation and immune responses. Abnormal activation of the pathway promotes the occurrence and development of inflammation, and inhibiting this pathway can inhibit the onset of RA [39,40]. NF-κB inhibitors are known to be used to treat inflammation and joint destruction in RA. After administration, PNP is inhibited. Since PNP is a key enzyme in the synthesis of purine salvage in T cells, the proliferation and activation of T cells is inhibited, the NF-κB factor is inhibited, and inflammation is reduced. Since immune status is also closely related to metabolic abnormalities, AMPK (Adenosine 5'-monophosphate (AMP)-activated protein kinase), an AMP-dependent protein kinase, is a key factor in regulating biological metabolism. Studies have shown that inhibition of the AMPK pathway can lead to abnormal immune status and autoimmune diseases. As a metabolic sensor, AMPK increases the number and function of Treg cells in CIA, regulates abnormalities in the Th1 / Th2 and Treg / Th17 axes, and exerts an anti-inflammatory effect

[48] . Administration of drugs will increase the activation of the AMPK pathway and alleviate RA symptoms.

[0317] Forodesine inhibits T cell proliferation and reduces inflammation in vitro.

[0318]

[0319] The usage sequence is shown in the following table.

[0320]

[0321] 1.4 Experimental animals

[0322] SPF male Balb / c mice, 6-8 weeks old, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and kept in the clean animal experimental center of the Second Affiliated Hospital of Soochow University. The temperature was constant at 22-25℃, with a 12h / 12h light-dark cycle, free access to water and food, and the experiment began after one week of adaptive feeding. Animal experiments were approved by the Animal Ethics Committee of Soochow University in accordance with the guidelines of the Animal Ethics and Use Committee.

[0323] 2 Experimental methods

[0324] 2.1MACS cell sorting

[0325] (1) Isolation of mouse lymphocytes

[0326] a. The mice were anesthetized and killed by myelotomy, disinfected with alcohol, and the spleen was removed in a clean bench and placed in a sterile culture dish;

[0327] b. Grinding: Place a sterile nylon filter on the spleen, grind with a 5 mL syringe core, add RPMI1640 medium, pipette and mix, then filter the suspension through a filter to remove large pieces of tissue;

[0328] c. Centrifuge and discard the supernatant. Resuspend the precipitate in 2 mL ACK Lysing Buffer to lyse the red blood cells. After standing at room temperature for 3 minutes, add 2 mL RPMI1640 to terminate the reaction.

[0329] d. Centrifuge and discard the supernatant. The precipitate is the mononuclear cells. Resuspend it in 2 mL of complete culture medium and count it.

[0330] (2) Magnetic bead sorting (positive selection, taking CD4+T cells as an example, CD8+T cells have the same steps except that magnetic beads are used)

[0331] a. Resuspend every 107 cells in 90 μL buffer;

[0332] b. Add 10 μL of magnetic beads per 107 cells;

[0333] c. Mix well and incubate at 4°C for at least 10 min;

[0334] d. Add 2 mL of buffer, centrifuge and discard the supernatant, then resuspend with 500 μL of buffer;

[0335] e. Install the column on the magnetic frame with the mark facing outwards when installing the separator and the bayonet facing outwards when installing the separation column;

[0336] f. Rinse the column with an appropriate amount of buffer, 500 μL for MS;

[0337] g. Add the cell suspension to the column, collect the unlabeled cells that flow down, and the labeled CD4+T cells on the column;

[0338] h. Wash the column with an appropriate amount of buffer to continue collecting the unlabeled cells that flow through, and wash the MS with 500 μL three times;

[0339] i. Remove the column and place it on a centrifuge tube, add 1 mL of buffer (MS column), and quickly push the plunger into the column to obtain CD4+ T cells.

[0340] 2.2 Reagent preparation

[0341] (1) Preparation of Forodesine (7-[(2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-pyrrolidinyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one) stock solution: FDS molecular weight 302.71, solubility in H2O: 100 mg / mL (330.34 mM; ultrasonic dissolution). Dissolve 5 mg of powder in 16.517 mL of pure water to obtain a 1 mM stock solution, which was stored at -80°C.

[0342] (2) CD3 and CD28 dual signal stimulation: dilute the antibody in complete medium, the final concentration of CD3 is 1 μg / mL,

[0343] The final concentration of CD28 was 2 μg / mL.

[0344] 2.3 Cell culture

[0345] A control group and an experimental group were set up, and cells and culture medium supernatant were collected after culturing in a 37°C, 5% CO2 incubator for 48 hours for subsequent treatment.

[0346] 2.4 Real-time fluorescence quantitative PCR

[0347] Same specimen processing and operation as above.

[0348] 2.5 Cell apoptosis detection

[0349] (1) Specimen processing: After collecting cells by centrifugation, wash twice with pre-cold PBS and centrifuge at 4°C (2000 rpm, 5 min);

[0350] (2) Cell staining: Resuspend the cells in 100 μL Binding buffer to obtain a single-cell suspension; add 5 μL each of Annexin V-FITC and PI Staining Solution, mix well, incubate at room temperature in the dark for at least 15 min, then add 400 μL Binding buffer, mix well, and detect on an analyzer.

[0351] 2.6 Cytokine assay

[0352] (1) Supernatant collection: Collect cell culture supernatant and store at -20°C.

[0353] (2) CBA method for measuring cytokines:

[0354] a. Preparation for startup: clean the instrument and test it with quality control microspheres to ensure that the PE and APC channels are normal.

[0355] b. Standard: Dilute the standard with 2 mL of Assay Diluent, mix gently, and let stand for 15 minutes. Dilute the standard with Diluent in the following ratio.

[0356] c. Preparation of capture microspheres: Take 10 μL / test of each capture microsphere and mix (shake each microsphere vigorously for 20 seconds before adding the sample).

[0357] Centrifuge at room temperature (300 g, 5 min), discard the supernatant, and add serum enhancer at a 1:1 ratio.

[0358] d. Specimen processing: standard or sample: microsphere mixture: PE detection antibody = 1:1:1, 50 μL each, incubate at room temperature in the dark for 3 hours.

[0359] e. Detection and data analysis: 1mL / test, buffer to terminate the reaction, centrifuge, discard the supernatant. Resuspend in 300μL / test buffer. Detect on the machine, export the file. Use FCAP Array software to draw a standard curve and calculate the concentration.

[0360] Experimental Results

[0361] 3.1qPCR detection of the inhibitory effect of FDS on PNP gene expression and selection of appropriate concentration

[0362] Total RNA was extracted from mouse spleen lymphocytes, reverse transcribed and then qPCR was performed to compare the inhibitory effects of 10, 20, and 30 μM FDS on PNP gene expression. The results showed that 20 μM had the strongest inhibitory effect, so we chose 20 μM concentration for subsequent experiments.

[0363] 3.2 FDS inhibits TNF-α secretion in vitro

[0364] A concentration of 20 μM was selected as the FDS administration concentration, and CD3+CD28 dual signals were used to stimulate T cell activation. A blank control group, a stimulator control group, and a stimulator+FDS group were set up. After culturing at 37°C and 5% CO2 for 48 hours, the culture medium supernatant was collected and the TNF-α level was detected (flow CBA method). It was found that FDS administration inhibited the secretion of TNF-α and had an anti-inflammatory effect.

[0365] 3.3FDS induces CD8+ T cell apoptosis

[0366] The results of in vivo experiments showed that FDS has an effect on the proportion of CD4+T cell subsets and CD8+T cells, but due to the existence of immune homeostasis in the body, the interactions between multiple cells are balanced. In order to explore the effect of FDS on CD4+T cell subsets and CD8+T cell functions, CD4+T cells and CD8+T cells in normal mouse spleen lymphocytes were sorted by MACS method and cultured for 48 hours respectively. T cell activation was stimulated by CD3+CD28 dual signals, and blank control group, stimulator control group, and stimulator+FDS group were set up to verify the effect of FDS on CD4+T cell and CD8+T cell apoptosis. The results showed that after in vitro culture, FDS had no significant effect on CD4+T cell apoptosis, but induced CD8+T cell apoptosis.

[0367] 4. Discussion

[0368] Forodesine is currently known to act on T-cell lymphoma and induce apoptosis of leukemia cells. The results of this section show that inhibiting PNP can induce apoptosis of CD8+T cells, and Forodesine has therapeutic potential in autoimmune diseases. At the same time, the results of in vitro experiments also show that Forodesine administration can inhibit the secretion of TNF-α, which is consistent with the results of in vivo experiments.

[0369] Combined with the results of the in vivo experiments in the second part, we believe that Forodesine can exert its effect by inhibiting the NF-κB pathway and promoting the activation of the AMPK pathway: inhibiting the proliferation of CD8+T cells and inducing apoptosis; increasing the proportion of Treg cells and promoting the expression of Foxp3; increasing the proportion of IL-4 and Th2, correcting the imbalance between Th1 / Th2; inhibiting the secretion of IL-17 and TNF-α, correcting the imbalance between Treg / Th17 cells to exert a therapeutic effect on RA.

[0370] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. Application of purine nucleotidase inhibitors in the preparation of drugs for the treatment of rheumatoid arthritis.

2. The use of the purine nucleotidase inhibitor according to claim 1 in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The inhibitory substance is 7-[(2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-pyrrolidinyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one.

3. A pharmaceutical composition for treating rheumatoid arthritis, characterized in that: The invention comprises 7-[(2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-pyrrolidinyl]-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one and pharmaceutically acceptable excipients.

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