Application of 2'-deoxyinosine in the treatment of autoimmune encephalitis

Abstract

The invention discloses an application of 2'-deoxyinosine in treating autoimmune encephalomyelitis. The structural formula of 2'-deoxyinosine is as follows: The invention proves that 2'-deoxyinosine (DI) inhibits lymphocyte proliferation and the expression of inflammation-related factors.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of 2'-deoxyinosine in treating autoimmune encephalitis. Background Art

[0002] Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its main pathological features are multiple myelin loss, axonal degeneration and neuronal damage in the CNS. MS is more common in women aged 20-40 years. Its repeated attacks and gradual progression lead to progressive damage to neurological function, causing physical disability and cognitive decline in patients, seriously affecting their quality of life. Experimental autoimmune encephalomyelitis (EAE) is a classic animal model of MS. It is similar to the clinical and pathological characteristics of human MS and is widely used in basic research on MS. Although the etiology and pathogenesis of MS have not yet been clarified, abnormalities in cellular and humoral immune responses are believed to play an important role in it. At present, the treatment of MS remission is mainly based on disease modifying therapies (DMTs). However, existing DMTs are only effective for some patients and may have serious side effects. Therefore, actively exploring new immunomodulatory methods for MS has important scientific research value. Summary of the invention

[0003] The purpose of the present invention is to provide an application of 2'-deoxyinosine in treating autoimmune encephalomyelitis.

[0004] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: use of 2'-deoxyinosine in treating autoimmune encephalomyelitis.

[0005] The preferred technical solution is: the structural formula of 2′-deoxyinosine is as follows:

[0006]

[0007] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a pharmaceutical composition for treating autoimmune encephalomyelitis: comprising 2′-deoxyinosine and pharmaceutically acceptable materials.

[0008] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0009] The present invention proves that 2'-deoxyinosine (DI) inhibits lymphocyte proliferation and the expression of inflammation-related factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Flow chart for the establishment of EAE mouse model.

[0011] Figure 2 Schematic diagram of the method for isolating spinal cord and brain mononuclear cells.

[0012] Figure 3 Figure 3 Body weight and clinical scores of EAE mice.

[0013] Figure 4 Comparison of spinal cord infiltrating lymphocytes in mice.

[0014] Figure 5 Comparison of lymphocyte subsets in the mouse spinal cord.

[0015] Figure 6 Comparison of mouse spinal cord lymphocyte activation and proliferation.

[0016] Figure 7 Changes in Th1 / Th17 / Treg gene expression in mouse spinal cord (Q-PCR).

[0017] Figure 8 Comparison of brain-infiltrating lymphocytes in mice.

[0018] Fig. 9 Comparison of lymphocyte activation and proliferation in mouse brain.

[0019] Fig.10 Comparison of splenic lymphocyte subsets.

[0020] Fig.11 Comparison of splenic lymphocyte activation.

[0021] Fig.12 Changes in gene expression in mouse spleen lymphocytes (Q-PCR).

[0022] Fig.13 Comparison of mouse peripheral blood lymphocytes and monocytes.

[0023] Fig.14 Comparison of mouse peripheral blood T lymphocytes.

[0024] Fig.15 Comparison of CD4+ / CD8+ / Treg lymphocytes in mouse peripheral blood.

[0025] Fig.16 Gross picture of the effect of DI on the proliferation of mouse spleen cells in vitro.

[0026] Fig.17 Fluorescence detection of the effect of DI on the proliferation of mouse spleen cells in vitro.

[0027] Fig.18 Effect of DI on cytokine secretion of mouse spleen cells (QPCR / ck). DETAILED DESCRIPTION

[0028] 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.

[0029] See also Figure 1-18 . 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 technical substantive 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.

[0030] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0031] Example 1: Use of 2′-deoxyinosine in the treatment of autoimmune encephalomyelitis

[0032] 1. The steps to establish the EAE model are as follows (such as Figure 1 shown):

[0033] (1) Antigen injection: The mice were anesthetized by intraperitoneal injection of chloral hydrate at a volume ratio of 0.1 ml:10 g. After the mice were anesthetized, the backs were shaved and 200 ul MOG was injected subcutaneously. 35-55 The mixed solution was injected at four locations, 50ul at each location.

[0034] (2) PTX injection: 200 ng PTX was injected intraperitoneally, and 200 ng PTX was injected intraperitoneally again 48 hours later.

[0035] (3) Body weight and score records: The last injection of PTX was regarded as day 0. The mice were closely observed from day 1, and their body weight was recorded every day. The clinical symptoms of the mice were scored according to the table below.

[0036]

[0037]

[0038] All mice that were modeled were counted as model mice (0-5).

[0039] (4) Mouse grouping: The animals were randomly divided into three groups, with 6 mice in each group, namely the control group, the model group, and the 2′-deoxyinosine (hereinafter referred to as DI) administration group. The control group was normal mice without EAE induction; the model group and the DI administration group were both EAE model mice. The DI administration group was given DI by gavage starting from the first day after modeling, 1 mg / kg, twice a day, and the control group and the model group were given the same amount of sterile water. The animals were killed around the 20th day.

[0040] like Figure 3 As shown, the weight loss of EAE model mice after DI treatment was low (e.g. Figure 3 A), low clinical symptom scores (such as Figure 3 B), the cumulative score of mice in the group is low ((such as Figure 3 C).

[0041] 2. Preparation of paraffin sections:

[0042] (1) Fixation: The fresh whole spinal cord was quickly washed in PBS to remove blood stains and fixed in 4% paraformaldehyde.

[0043] (2) Dehydration: After fixation, rinse three times with running water, and then dehydrate by soaking in 70% ethanol, 80% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II for 1 hour each.

[0044] (3) Transparency: Soak in anhydrous ethanol, xylene I, and xylene II for 10 min each.

[0045] (4) Wax immersion: Soak in melted paraffin at 60°C for 1 hour.

[0046] (5) Embedding: Cut the lumbar enlarged segment of the spinal cord and place it on the embedding mold. Position it properly and add paraffin. After the paraffin surface solidifies, place it in the freezer overnight. The wax block can be stored at 4°C for a long time.

[0047] (6) Slicing: The embedded spinal cord is placed in a slicer and sliced ​​to a thickness of 3 μm.

[0048] 3. Hematoxylin-eosin (HE) staining:

[0049] (1) Dewaxing and hydration: Dry the sheet in an oven at 60°C for 30 min, and then place it in the following solutions for treatment: xylene I for 10 min; xylene II for 10 min; anhydrous ethanol for 3 min; 95% ethanol for 3 min; 85% ethanol for 3 min; 75% ethanol for 3 min; and distilled water for 2 min.

[0050] (2) Hematoxylin staining: Stain with hematoxylin for 5 min, rinse with distilled water; differentiate with differentiation solution for 10 s, and rinse twice with distilled water, 5 min each time.

[0051] (3) Eosin staining: Eosin staining for 1 min.

[0052] (4) Dehydration, transparency and sealing: The sections were treated in the following order: 75% ethanol for 2 seconds; 85% ethanol for 2 seconds; 95% ethanol for 2 seconds; anhydrous ethanol I for 2 seconds; anhydrous ethanol II for 1 minute; xylene I for 1 minute; xylene II for 1 minute; and sealing with neutral gum.

[0053] (5) Microscopic examination: Assess the degree of spinal cord inflammatory infiltration according to the table below.

[0054] Points symptom 0 No inflammatory cell infiltration 1 Very few inflammatory cells, only infiltrating blood vessels and around the capsule 2 1-10 inflammatory cells infiltrate per field 3 10-100 inflammatory cells infiltrate per field 4 100-1000 inflammatory cells infiltrate per field of view

[0055] like Figure 4 As shown in the figure, EAE mice had mild spinal cord pathological changes after DI treatment, and the number of inflammatory cells infiltrating the spinal cord and brain was small. One group of model group mice was treated with DI, and the other group was treated with PBS. It can also be directly called the model group.

[0056] 4. Isolation and culture of mouse CNS mononuclear cells:

[0057] (1) Separation of the spinal cord and brain: Use scissors to cut the skin on the back of the mouse and remove the spinal column. Use a syringe to draw 5 ml of RPMI1640 and insert the needle from the tail end. Push the spinal cord out of the spinal cavity with force and place it in a six-well plate with the separated mouse brain.

[0058] (2) Mononuclear cell extraction:

[0059] a. Cut the spinal cord and brain into small pieces with scissors, add 1.5 ml RPMI1640, 100 mg / ml collagenase IV and 100 mg / ml DNAase I, and digest at 37°C for 45 min.

[0060] b. Add 2% FBS RPMI1640 to terminate digestion, grind thoroughly and filter through 100-mesh nylon mesh, centrifuge at 500 g for 5 min; resuspend the cell pellet in 7 ml RPMI1640, add 3 ml 100% Percoll, quickly invert and mix to obtain 30% Percoll.

[0061] c. Use a sterile pipette to draw up 2 ml of 70% Percoll, lift up the 30% Percoll from bottom to top, centrifuge at 20℃, 500g for 30 min, and increase by 5 and decrease by 0.

[0062] d. After centrifugation, discard the upper lipid layer, aspirate the mononuclear cell layer and transfer it to a new 15ml centrifuge tube, wash twice with RPMI1640, and suspend in 300ul complete culture medium.

[0063] (3) Lymphocyte stimulation: The method for stimulating brain and spinal cord lymphocytes is the same as that for the spleen.

[0064] V. Flow cytometry analysis:

[0065] (1) Compensation adjustment of flow cytometer

[0066] a. Take 10 flow tubes, add 100 μl of PBS to each tube, and add one drop of the mixed BD negative control compensation microspheres and anti-rat / hamster Igκ compensation microspheres to each tube, then mix well.

[0067] b. Add 1 μl of mouse / hamster-derived anti-mouse FITC, PE, APC, APC cy7, AF700, Percpcy5.5, BV421, BV605, BV500, PE CY7-labeled antibodies to the tubes respectively, make good marks, and incubate at 4 °C in the dark for 15 min.

[0068] c. Add 2 ml of PBS to terminate the binding, centrifuge at 200 g for 10 min, carefully aspirate and discard the supernatant, and resuspend and mix the microsphere precipitate with 500 μl of PBS.

[0069] d. Run each test tube on the flow cytometer respectively. After the capture is completed, the instrument automatically adjusts the compensation. After the adjustment, subsequent specimen detection can be carried out.

[0070] (2) Detection of Treg subsets

[0071] a. Take 100 μl of peripheral blood, spleen, spinal cord, and brain cell suspensions into flow tubes respectively, wash with 2.5 ml of PBS to fully remove fetal bovine serum.

[0072] b. Add PBS containing ghost (1:1000) to the tubes, 300 μl / tube, pipette and mix well, stain at 4 °C in the dark for 20 min, and centrifuge.

[0073] c. Add PBS containing FITC-CD3 antibody (1:100), AF700-CD4 antibody (1:100), and Percpcy5.5-CD8 antibody (1:100) to the tubes, 100 μl / tube, pipette and mix well, incubate at 4 °C in the dark for 15 min, and centrifuge.

[0074] d. Add 1 ml of freshly prepared 1x Fix / Perm working solution to the tubes, pipette and mix well, and fix at 4 °C for 40 min.

[0075] e. Add 2 ml of 1x Perm / Wash working solution to terminate the reaction, centrifuge and discard the supernatant, add 1x Perm / Wash working solution containing APC-Foxp3 antibody (1:100) and BV421-Ki67 antibody (1:100), 100 μl / tube, mix well, and incubate at 4 °C for 40 min.

[0076] f. Add 2 ml 1x Perm / Wash working solution again to terminate staining, centrifuge, discard the supernatant, suspend the cell pellet with 200 ul PBS, and detect on the machine.

[0077] (3) TH1 / Th2 / TH17 subpopulation analysis

[0078] a. Collect stimulated spleen, spinal cord and brain lymphocytes, centrifuge, wash with PBS and remove fetal bovine serum.

[0079] b. Except that the intracellular molecules were replaced with BV605-IL4 antibody (1:100), PE-IL17A antibody (1:100) and PE CY7-IFNγ (1:100) antibodies, the rest of the staining steps were the same as the above Treg subsets.

[0080] like Figure 5 As shown, after treatment of EAE mice, the numbers of CD3+T cells, CD4+T cells and Th17 cells in the spinal cord decreased; the numbers of CD8+T cells, Th2 cells and Treg cells increased.

[0081] like Figure 6 As shown, the number of proliferating and activated T cells in the spinal cord was reduced after treatment of EAE mice.

[0082] 6. Real-time fluorescence quantitative PCR:

[0083] (1) Sample preparation

[0084] The remaining spinal cord cells were collected, washed twice with PBS, and then lysed with Trizol, and RNA was extracted for real-time fluorescence quantitative PCR.

[0085] (2) Cell RNA extraction

[0086] a. Add 200 μL of chloroform to the fresh cell lysate, mix by inversion, and let stand at room temperature for 10 min.

[0087] b. Centrifuge at 12000g for 15 min at 4°C.

[0088] c. Carefully transfer the upper aqueous phase to a new enzyme-free EP tube, add an equal volume of isopropanol, invert to mix, and let stand at -20℃ for 15 minutes.

[0089] d. Centrifuge at 12000g for 10 min at 4°C.

[0090] e. Use a pipette to discard the liquid in the tube, retain the white RNA precipitate at the bottom of the tube, and add 1 ml of 75% anhydrous ethanol for washing (75% anhydrous ethanol is prepared with DEPC water).

[0091] f. Centrifuge at 12000g for 5 min at 4°C

[0092] g. Discard the supernatant, dissolve the precipitate in 20 μL DEPC water, and detect and record the RNA concentration and purity.

[0093] (3) Synthesis of first-strand cDNA

[0094] RNA was reverse transcribed according to the standard operating procedure of HiScript III 1st Strand cDNA Synthesis Kit.

[0095] (4) Real-time fluorescence quantitative PCR

[0096] Real-time fluorescence quantitative experiments were performed according to the standard operating procedures of the Taq Pro Universal SYBR qPCR Master Mix Fluorescence Quantification Kit.

[0097] a. Reaction system configuration

[0098]

[0099] b. QPCR reaction

[0100]

[0101] like Figure 7 As shown, after EAE mice were treated, lymphocytes in the spinal cord expressed higher levels of Foxp3 and lower levels of Il-17 and IFN-R, which was consistent with the results of flow cytometry.

[0102] like Figure 8 As shown, after EAE mice were treated, the CD3 + T cells, CD4 + The number of T cells and Th17 cells decreased; the number of Treg cells increased.

[0103] like Fig. 9 As shown, the number of proliferating and activated T cells in the brain was reduced after treatment of EAE mice.

[0104] VII. Isolation and Culture of Mouse Splenic Lymphocytes

[0105] (1) Spleen isolation: After blood collection, the mice were killed by myelotomy and soaked in 75% alcohol for 1 min. The spleen was removed on a clean bench, placed in a sterile culture dish, and weighed and recorded on a balance.

[0106] (2) Lymphocyte extraction:

[0107] a. Place a sterile 100-mesh nylon mesh on the spleen, add serum-free RPMI 1640 medium, grind with a 10 ml syringe core, and filter the ground suspension through a 100-mesh nylon mesh to remove large pieces of tissue.

[0108] b. Centrifuge at 200 g for 5 min, collect the supernatant and store at -20°C for cytokine determination, resuspend the cell pellet in 7 ml RPMI1640 medium and mix by pipetting.

[0109] c. Use a sterile pipette to draw up 2.5 ml of 60% Percoll, lift up the mixed cell suspension from bottom to top, and centrifuge at 20°C, 500 g for 30 min, with a 5% increase and a 0% decrease.

[0110] d. After centrifugation, aspirate the cells in the middle white film layer, wash twice with RPMI 1640, collect the cell pellet and suspend it in complete culture medium, and adjust the concentration to 1*10 6 / ml.

[0111] Flow cytometry was the same as that of spinal cord cells.

[0112] like Fig.10 As shown, after EAE mice were treated, CD3 + The number of T cells decreased; the number of Treg cells increased.

[0113] like Fig.11 As shown, the number of activated T cells in the spleen was reduced after treatment of EAE mice.

[0114] like Fig.12 As shown, after EAE mice were treated, lymphocytes in the spleen highly expressed inhibition-related indicators Foxp3, LAG-3, and IL-4; and lowly expressed inflammation-related indicators CXCR3 and IL-17.

[0115] 8. Mouse Peripheral Blood Isolation

[0116] (1) Blood collection from mice: The mice were captured and anesthetized with chloral hydrate. The heart of the mice was located and the needle was slowly inserted into the tube with a 1 ml sterile syringe. When blood returned to the syringe, the needle was stopped and blood was slowly drawn. The drawn blood was divided into two portions. One portion was injected into a vacuum blood collection tube for immune cell assay and the other portion was injected into 400 μl of EDTA-Na2 anticoagulant for flow cytometry.

[0117] (2) Peripheral blood immune cell detection: The lymphocyte ratio and absolute count in the peripheral blood of each group of mice were measured using a blood routine instrument.

[0118] (3) Flow cytometry: EDTA-Na2 anticoagulated peripheral blood was lysed with 2 ml red blood cell lysis buffer for 2 min, centrifuged, supernatant discarded, washed once with PBS, centrifuged again, and the cell concentration was adjusted to 2*10 with PBS. 6 / ml.

[0119] Flow cytometry was the same as that of spinal cord cells.

[0120] like Fig.13 , 14 As shown, EAE mice have a decrease in peripheral blood lymphocytes.

[0121] like Fig.15 As shown in Figure 2, after EAE mice were treated, the number of monocytes in the peripheral blood increased, while the number of lymphocytes decreased, with CD4 + T cell reduction is the main problem.

[0122] IX. Isolation of peripheral blood lymphocytes from MS patients

[0123] (1) Remove the Ficoll separation solution half an hour in advance, mix thoroughly by inversion, and return to room temperature.

[0124] (2) Collect 15 ml of fresh sterile peripheral blood from MS patients into heparin anticoagulant tubes and immediately isolate PBMCs.

[0125] (3) Transfer the collected peripheral blood to a 50ml centrifuge tube in a clean bench and add an equal volume of PBS for dilution. Take another 50ml centrifuge tube, add 15ml of Ficoll separation solution, and then use a sterile pipette to slowly add the above blood diluent along the tube wall to the 45ml mark. Be careful not to let the separation solution level fluctuate.

[0126] (4) Centrifuge at 500g for 30 min at room temperature (18-20°C), with the value increasing by 5 and decreasing by 0. According to the different densities of the components in the blood, after centrifugation, the contents of the 50ml tube are, from top to bottom, a pale yellow plasma layer, a mononuclear cell buffy coat layer, a transparent separation fluid layer, and the red blood cell layer at the bottom.

[0127] (5) Use a pipette to gently remove the upper plasma layer, transfer the buffy coat layer to a new 15 ml centrifuge tube, gently blow and mix, and add 10 ml of RPMI1640 to wash twice, centrifuging at 200 g for 5 min each time.

[0128] (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, add 2 ml per well, and incubate in a 37°C incubator for 2 h to allow the macrophages to adhere to the wall.

[0129] (7) After 2 hours, use a pipette to aspirate the suspended cells in the culture well, which are peripheral blood lymphocytes. Centrifuge at 200g for 5 minutes, discard the supernatant, and adjust the cell concentration to 1*106 / ml, for future use.

[0130] 10. Lymphocyte proliferation capacity assay

[0131] (1) CFTR staining: Take the above lymphocyte suspension, centrifuge and discard the supernatant, resuspend in PBS, add CTFR dye to a final concentration of 1 μM, stain at 37°C in the dark for 15 min, terminate staining with complete medium, centrifuge at 200 g for 5 min; resuspend the centrifuged cell pellet in complete medium, add 2 μg / ml anti-CD28 antibody, and adjust the cell concentration to 1*10 6 / ml.

[0132] (2) Stimulate cell proliferation: Aspirate the suspension in the OKT3-coated plate with a pipette and wash twice with sterile PBS to remove unbound antibodies. After washing, add 100 μl of lymphocyte suspension to each well, and add equal volumes of DI at different concentrations to make the final concentrations of DI 0 mM, 0.25 mM, 0.5 mM, and 1 mM, respectively. Incubate at 37°C for 48 h.

[0133] (3) Flow cytometry: Collect cells in the wells into flow cytometry tubes, centrifuge at 1000 rpm for 5 min, suspend the cells in PBS, and detect cell proliferation using a flow cytometer.

[0134] XI. Cytokine Assay

[0135] (1) Cytokine collection: Except for lymphocyte staining, the rest of the operation is the same as 2.3. Collect the supernatant after 48 hours for cytokine determination.

[0136] (2) Flow cytometry CBA method for cytokine determination:

[0137] a. Preparation of standard products: Redissolve the freeze-dried microspheres of cytokine standard products, and perform serial dilutions in the flow cytometry according to 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512, and set up blank tubes.

[0138] b. Preparation of capture microspheres: Calculate the required volume of various capture microspheres based on the number of samples and the number of standards, vortex the capture microspheres, centrifuge and resuspend in serum enhancement buffer for later use;

[0139] c. Preparation of detection antibody: Calculate the required volume of PE detection antibody and dilute it

[0140] d. Sample reaction: Pipette 50 μL of capture microspheres, PE detection reagent, and sample / standard into corresponding flow tubes and incubate at room temperature in the dark for 3 hours.

[0141] e. Detection and analysis: 1 mL of washing buffer was used to terminate the binding. After centrifugation to discard the supernatant, the microspheres were resuspended in 300 μL of washing buffer and detected for the PE fluorescence intensity on the machine. A standard curve was plotted with FCAP Array software to calculate the sample concentration.

[0142] The QPCR method was the same as that for spinal cord cells.

[0143] As Fig.18 shown, DI inhibited the proliferation of lymphocytes and the expression of inflammation-related factors.

[0144] The above are only preferred embodiments for explaining the present invention and are not intended to limit the present invention in any form. Therefore, any modification or change to the present invention made in the same inventive spirit should still be included within the scope intended to be protected by the present invention.

Claims

1. Application of 2'-deoxyinosine in the preparation of drugs for treating autoimmune encephalomyelitis; Features: The structural formula of 2'-deoxyinosine is as follows:

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