Application of biomarker for rheumatoid arthritis

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a biomarker for rheumatoid arthritis. The anti-lactylated histone autoantibody and various autoantibodies are found, the level of the anti-lactylated histone autoantibody and various autoantibodies in a serum sample of a rheumatoid arthritis patient is remarkably increased, and compared with a normal person, the anti-lactylated histone autoantibody and various autoantibodies can be used as a biomarker for diagnosis of rheumatoid arthritis. In addition, the level of H3K9la in synovial fibroblasts of rheumatoid arthritis patients is increased, and the H3K9la has the effect of promoting joint destruction. Therefore, the effect of relieving arthritis can be achieved by inhibiting the level of H3K9la, and the H3K9la can be used as a biomarker with the effect of treating rheumatoid arthritis. The invention provides an effective means for diagnosis and treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a biomarker for rheumatoid arthritis. Background Art

[0002] Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by the production of autoantibodies, synovitis, and bone and cartilage destruction, and is known as the "cancer that does not kill". The early manifestations are migratory joint pain and dysfunction. In the late stage, joint stiffness and severe deformation often occur, with a high disability rate, seriously endangering the physical health of the general public. "Early detection, early diagnosis, and early treatment" are the main principles for controlling the progression of RA. Therefore, early and accurate diagnosis of RA is of great significance for patients to receive early intervention and then delay the disease progression.

[0003] Currently, the clinical diagnosis of RA mainly relies on joint involvement, symptom duration, acute-phase reactants, and serum indicators such as serum rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA). However, due to the atypical and diverse early clinical symptoms of RA, joint involvement often occurs in the late stage, and combined with the non-specificity of acute-phase reactants, serological indicators are particularly important for the early and accurate diagnosis of RA. However, both RF and ACPA recommended by current guidelines have certain limitations. RF and ACPA can not only be detected in RA patients, but also widely exist in patients with autoimmune diseases such as osteoarthritis (OA) and systemic lupus erythematosus (SLE). Therefore, their sensitivity or specificity is relatively low, 69%, 85% and 67%, 95% respectively. The sensitivity and specificity of the combined diagnosis of RA are only 78% and 82%, with a misdiagnosis rate as high as 19%. In addition, their performance in early RA is also not satisfactory, with a positive rate of only 57%. Therefore, there is an urgent need in this field to find diagnostic indicators that can early and accurately diagnose RA patients and are simple and convenient to operate. This research is of great significance for early detection, early diagnosis, and early treatment of RA patients, so as to slow down the disease process, control clinical symptoms, and improve the quality of life.

[0004] Numerous studies have shown that joints of RA patients present a high-lactate environment, and the high level of intra-articular lactate further promotes the proliferation and migration of synovial fibroblasts, forming invasive pannus and damaging cartilage and bone tissues. In 2019, lactylation was first discovered as a brand-new post-translational modification of proteins, regulated by the concentration of substrate lactate and involved in the occurrence and development of various lactate-related diseases. At the same time, post-translational modification of proteins not only affects the immune response but may also have an important impact on the production of autoantibodies due to the change in its immunogenicity. For example, the citrullination of protein arginine leads to the production of anti-citrullinated protein antibodies (ACPA), which is an important serological diagnostic indicator for RA. Based on these, we discovered new autoantibodies against lactylated proteins, providing a new indicator for the diagnosis of RA. Summary of the Invention

[0005] The object of the present invention is to overcome the disadvantages and deficiencies existing in the prior art and provide an application of a biomarker for rheumatoid arthritis.

[0006] In the first aspect of the present invention, there is provided an application of a biomarker for rheumatoid arthritis in the preparation of a product for diagnosing rheumatoid arthritis, wherein the biomarker is an autoantibody against lactylated histone, an autoantibody against lactylated H2B, an autoantibody against lactylated H3, an autoantibody against lactylated H4, an autoantibody against H3K9la or H3K9la (lactylation modification of lysine at position 9 of histone H3).

[0007] Among them, the levels of the autoantibody against lactylated histone, the autoantibody against lactylated H2B, the autoantibody against lactylated H3, the autoantibody against lactylated H4, and the autoantibody against H3K9la are elevated in the sera of rheumatoid arthritis patients; the level of the biomarker H3K9la is elevated in synovial fibroblasts of rheumatoid arthritis patients.

[0008] In the second aspect of the present invention, there is provided a diagnostic product for rheumatoid arthritis, wherein the diagnostic product comprises a reagent for detecting the expression of an autoantibody against lactylated histone, an autoantibody against lactylated H2B, an autoantibody against lactylated H3, an autoantibody against lactylated H4, an autoantibody against H3K9la or H3K9la in a biological sample.

[0009] Among them, the biological sample is fresh serum or synovial fibroblasts, and the levels of the autoantibody against lactylated histone, the autoantibody against lactylated H2B, the autoantibody against lactylated H3, the autoantibody against lactylated H4, and the autoantibody against H3K9la are elevated in the sera of rheumatoid arthritis patients; the level of the biomarker H3K9la is elevated in synovial fibroblasts of rheumatoid arthritis patients.

[0010] The diagnostic tool for rheumatoid arthritis in the present invention is for quantitative detection of proteins, such as Western blot, Immunohistochemistry, Immunofluorescence, immunochromatography, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence detection (ECL), etc. Therefore, the diagnostic tool for rheumatoid arthritis in the present invention at least includes detection products based on the above methods, and their types can be reagent kits, chips, test strips, high-throughput sequencing platforms. Among them, the reagent kit also includes instructions for diagnosing or predicting early rheumatoid arthritis.

[0011] In the third aspect of the present invention, there is provided an application of a biomarker for rheumatoid arthritis in the preparation of a product for preventing or treating rheumatoid arthritis. The biomarker is H3K9la, and the product for preventing or treating rheumatoid arthritis contains an H3K9la inhibitor.

[0012] In the fourth aspect of the present invention, there is provided a drug for preventing or treating rheumatoid arthritis, which contains an H3K9la inhibitor.

[0013] Preferably, the H3K9la inhibitor is an LDHA inhibitor. Excessive production of lactic acid by synovial fibroblasts is the direct cause of the increase in H3K9la in them, and lactate dehydrogenase (LDHA) is the key enzyme for producing lactic acid. By inhibiting the expression of LDHA, the H3K9la level in synovial fibroblasts can be reduced.

[0014] The beneficial effects of the present invention are as follows: The present invention discloses biomarkers for the diagnosis of rheumatoid arthritis, which are anti-lactylated histone autoantibodies, anti-lactylated H2B autoantibodies, anti-lactylated H3 autoantibodies, anti-lactylated H4 autoantibodies, anti-H3K9la autoantibodies, and H3K9la. The present invention discovers that the levels of anti-lactylated histone autoantibodies, anti-lactylated H2B autoantibodies, anti-lactylated H3 autoantibodies, anti-lactylated H4 autoantibodies, and anti-H3K9la autoantibodies in the sera of rheumatoid arthritis patients are significantly different from those of normal people, and the level of H3K9la in synovial fibroblasts of rheumatoid arthritis patients is significantly different from that of normal people, which can be used as biomarkers for the diagnosis of rheumatoid arthritis. The present invention also discloses the application of the anti-H3K9la autoantibody, a biomarker for the diagnosis of rheumatoid arthritis, including its application in the preparation of detection reagents for the diagnosis of rheumatoid arthritis and the preparation of drugs for targeted treatment of rheumatoid arthritis. In addition, the present invention discovers that the level of H3K9la in synovial fibroblasts of rheumatoid arthritis patients is increased and has the effect of promoting joint destruction. Therefore, by inhibiting the level of H3K9la, the effect of relieving arthritis can be achieved, which can be used as a biomarker with the therapeutic effect on rheumatoid arthritis. It provides an effective means for the diagnosis and treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, obtaining other drawings based on these drawings still belongs to the scope of the present invention.

[0016] Figure 1 For ELISA detection of the levels of anti-lactylated histone autoantibodies (A), anti-lactylated H2B autoantibodies (B), anti-lactylated H3 autoantibodies (C), anti-lactylated H4 autoantibodies (D), and anti-H3K9la autoantibodies (E) in the sera of rheumatoid arthritis patients, healthy people, and psoriatic arthritis patients;

[0017] Figure 2 For correlation analysis of the level of anti-H3K9la autoantibodies in the sera of rheumatoid arthritis patients and the disease activity (DAS28) of the patients;

[0018] Figure 3 For dot blot hybridization to detect the levels of anti-lactylated histone autoantibodies, anti-lactylated H2B autoantibodies, anti-lactylated H3 autoantibodies, anti-lactylated H4 autoantibodies, and anti-H3K9la autoantibodies in the sera of rheumatoid arthritis patients, healthy people, and psoriatic arthritis patients;

[0019] Figure 4 Protein immunoblotting was used to detect the level of H3K9la in synovial fibroblasts of 10 patients with rheumatoid arthritis, 8 healthy people and 10 patients with gouty arthritis. The detection was carried out on both sides, and the comparison results of 5 cases of rheumatoid arthritis, 4 normal people and 5 cases of gouty arthritis in the upper figure and the comparison results of another 5 cases of rheumatoid arthritis, 4 normal people and 5 cases of gouty arthritis in the lower figure were obtained;

[0020] Figure 5 Protein immunoblotting was used to detect the level of H3K9la after LDHA inhibition and knockdown;

[0021] Figure 6 Transwell assay was used to detect the migration and invasion ability of synovial fibroblasts after reducing the level of H3K9la;

[0022] Figure 7 Wound healing assay was used to detect the migration ability of synovial fibroblasts after reducing the level of H3K9la;

[0023] Figure 8 The joint swelling degree of arthritic mice after reducing the level of H3K9la was measured;

[0024] Figure 9 The joint score of arthritic mice after reducing the level of H3K9la was measured;

[0025] Figure 10 H&E staining and safranin-O-fast green staining were used to detect the joint destruction of arthritic mice after reducing the level of H3K9la;

[0026] Figure 11 ELISA was used to detect the levels of serum inflammatory cytokines (TNF-α, IL-6, IL-1β and IL-10) in arthritic mice after reducing the level of H3K9la. Detailed implementation manners

[0027] Next, in combination with the accompanying drawings and specific implementation manners, the present invention will be further described. It should be noted that, on the premise of no conflict, the following described embodiments or technical features can be combined arbitrarily to form new embodiments.

[0028] Embodiment 1:

[0029] Blood samples were collected from 50 patients with rheumatoid arthritis, 50 healthy volunteers (abbreviated as HC), and 20 patients with psoriatic arthritis (abbreviated as PsA). All cases were newly diagnosed and untreated patients. The inclusion criteria for patients with rheumatoid arthritis met the 2010 ACR / EULAR classification criteria for rheumatoid arthritis. Patients with psoriatic arthritis met the classification criteria for psoriatic arthritis. This study was approved by the Ethics Committee of Wenzhou Medical University, and each patient was informed of the use of the samples and signed an informed consent form.

[0030] In this example, the levels of anti-lactylated histone autoantibodies, anti-lactylated H2B autoantibodies, anti-lactylated H3 autoantibodies, anti-lactylated H4 autoantibodies, and anti-H3K9la autoantibodies in clinical serum samples were detected by ELISA. The specific process is as follows:

[0031] 1. Serum separation: The blood samples were centrifuged at 3000g for 10 minutes at 4°C, and the upper-layer serum was collected.

[0032] 2. Peptide coating: The peptide was diluted to 5 μg / mL and added to a Costar ELISA plate, and incubated overnight at 4°C.

[0033] overnight.

[0034] 3. Blocking: 2% BSA was added to the well plate and blocked at room temperature for 1 hour, and the liquid was aspirated completely.

[0035] 4. Serum incubation: Human serum (diluted 100-fold) was added to the well plate and incubated at room temperature for 1 hour. The well plate was washed 3 times with PBST, 5 minutes each time. The liquid was aspirated completely.

[0036] 5. Secondary antibody incubation: Diluted HRP-conjugated goat anti-human IgG was added to the well plate and incubated at room temperature for 1 hour. The well plate was washed 3 times with PBST, 5 minutes each time. The liquid was aspirated completely.

[0037] 6. Substrate color development: 100 μL of chromogenic solution was added to each well and incubated at room temperature in the dark for 30 minutes.

[0038] 7. Detection: The OD value was measured at 405 nm using an enzyme-linked immunosorbent assay reader.

[0039] The results were as Figure 1 shown. The levels of anti-lactylated histone autoantibodies, anti-lactylated H2B autoantibodies, anti-lactylated H3 autoantibodies, anti-lactylated H4 autoantibodies, and anti-H3K9la autoantibodies in the sera of patients with rheumatoid arthritis were significantly higher than those of healthy subjects and patients with psoriatic arthritis.

[0040] Next, the correlation between the level of anti-H3K9la autoantibodies in the sera of patients with rheumatoid arthritis and the disease activity (DAS28) of the patients was analyzed. The results were as Figure 2The level of anti-H3K9la autoantibodies in the serum of rheumatoid arthritis patients shown is positively correlated with the disease activity (DAS28) of the patients, indicating that anti-H3K9la autoantibodies can reflect the disease status of rheumatoid arthritis patients.

[0041] Example 2:

[0042] In this example, the levels of anti-lactylated histone autoantibodies, anti-lactylated H2B autoantibodies, anti-lactylated H3 autoantibodies, anti-lactylated H4 autoantibodies, and anti-H3K9la autoantibodies in clinical serum samples were detected by dot blot hybridization. The clinical serum samples were the same as those in Example 1, and the specific procedure was as follows:

[0043] 1. Polypeptide dissolution: Take out the polypeptide dry powder from -20 °C, place it in the environment to return to room temperature, and dissolve it with the accompanying PBS.

[0044] 2. Spotting: Dilute the polypeptide to 1 μg / mL, and spot (1 μL / spot) on a nitrocellulose membrane (1 cm ×

[0045] 1 cm) with a narrow-mouth pipette tip, and dry it naturally.

[0046] 3. Blocking: Block with 10% BSA at room temperature for 2 hours, and wash the membrane 3 times with PBST, 5 minutes each time.

[0047] 4. Serum incubation: Incubate the membrane with 200 μL of serum sample overnight at 4 °C. The next day, wash the membrane 3 times with

[0048] PBST, 5 minutes each time.

[0049] 5. Secondary antibody incubation: Incubate the membrane with diluted HRP-conjugated goat anti-human IgG at room temperature for 1 hour. Wash the membrane 3 times with PBST, 5 minutes each time.

[0050] 6. Exposure: Use chemiluminescence method and an exposure instrument for image exposure.

[0051] The results are as Figure 3 shown. Anti-lactylated histone autoantibodies were present in the sera of 10 (100%) RA patients, while only 1 (10%) of the healthy control group had anti-lactylated histone autoantibodies in their sera. Meanwhile, 2 (20%), 8 (80%), and 4 (40%) of the RA patients had autoantibodies against lactylated H2B, lactylated H3, and lactylated H4, respectively. Further experimental results showed that anti-H3K9la autoantibodies were present in the sera of 7 (70%) of the 10 RA patients

[0052] Example 3

[0053] The synovium is the main pathological site in patients with rheumatoid arthritis, which consists of fibroblast-like synoviocytes (FLS). Its excessive proliferation causes the synovium to form invasive pannus, leading to cartilage destruction, which is an important cause of joint destruction in patients. Blood samples were collected from 10 patients with rheumatoid arthritis, 8 healthy volunteers, and 10 patients with gouty arthritis (abbreviated as GA). The inclusion criteria for patients with rheumatoid arthritis met the 2010 ACR / EULAR classification criteria for rheumatoid arthritis. Patients with gouty arthritis met the 2015 ACR / EULAR classification criteria for gout. This study was approved by the Ethics Committee of Wenzhou Medical University, and each patient was informed of the use of the samples and signed an informed consent form.

[0054] In this example, the level of H3K9la in synovial fibroblasts was detected by Western blotting:

[0055] 1. Isolation and culture of primary synovial FLS

[0056] 1) Turn on the ultraviolet lamp in the laminar flow hood in the cell room for 30 minutes for disinfection.

[0057] 2) Take out the fresh synovial tissue from the sterile surgical bag and place it in a sterile culture dish.

[0058] 3) Add sterile PBS buffer to the sterile culture dish, wash the synovial tissue to remove blood, etc., and repeat this step 2-3 times.

[0059] 4) Use a pipette to aspirate the excess PBS, and cut the synovial tissue into small pieces with sterile ophthalmic scissors, the smaller the better. Transfer it to a 50 mL centrifuge tube when it appears paste-like or mass-like.

[0060] 5) Centrifuge at 350 g for 5 minutes to remove the upper adipose tissue, and repeat step 4 until there is almost no adipose tissue and the synovial tissue is small enough.

[0061] 6) Prepare the digestion solution, add 10-15 mL to the centrifuge tube, and then add a 1:100 mixture of penicillin and streptomycin and type II collagenase at a concentration of 0.4%.

[0062] 7) Add the digestion solution to the precipitated tissue after the last centrifugation, mix well, and place it in an incubator at 37 °C to start digestion. Shake the centrifuge tube every 2 hours during the digestion process, and digest for about 6-8 hours.

[0063] 8) Place a 100 μm cell sieve on a 50 mL centrifuge tube, filter the digested digestion solution, and centrifuge the filtrate at 350 g for 5 minutes.

[0064] 9) Discard the supernatant, resuspend the cells with DMEM medium containing 10% fetal bovine serum, and transfer them to a cell culture flask for culture.

[0065] 10) After culturing for one day, the FLS will compete with impurity cells for adherent growth. The impurity cells will float, and the impurity cells can be removed by changing the culture medium. The adherent cells are the FLS.

[0066] 2. Western Blot

[0067] 1) Protein extraction: Wash the intervened FLS cells with PBS twice, then add 200 μL of RIPA protein lysate, pipette and mix well, lyse on ice for 30 minutes, centrifuge at 4°C and 12,000 g for 20 minutes, and take the supernatant for long-term storage at -80°C.

[0068] 2) Protein quantification: Dilute the 2 mg / mL BSA standard with PBS to a final concentration of 0.5 mg / mL; add the standard to the 96-well plate in volumes of 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL in sequence, and make up to 20 μL with PBS in each well; add 19 μL of PBS and 1 μL of protein sample to the sample wells; mix solution A and solution B at a ratio of 1:50, add 200 μL to each well, incubate in a 37°C incubator for 30 minutes, measure the OD value at 562 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the protein concentration in the sample according to the standard curve.

[0069] 3) Protein denaturation: Determine the loading volume according to the protein concentration calculated above, dilute all samples with PBS to the same concentration to ensure the same loading volume. Mix the diluted protein sample and 5× Loading Buffer at a volume ratio of 4:1, seal with a sealing film, and heat in a metal bath at 100°C for 10 minutes.

[0070] 4) Electrophoresis: Add the calculated protein and marker to the loading wells, perform electrophoresis at a constant voltage of 80 V for 30 minutes, then change to a constant voltage of 120 V until the bromophenol blue runs to the bottom of the gel, and then stop.

[0071] 5) Membrane transfer: Cut the gel strip of the target size, cover it with a PVDF membrane activated by methanol, and place it in the membrane transfer apparatus in the order of filter paper - gel - PVDF membrane - filter paper. Place the membrane transfer apparatus in a box filled with ice cubes, and transfer the membrane at a constant current of 250 mA.

[0072] 6) Blocking: Place the PVDF membrane in a 5% skim milk blocking solution and block it on a shaker at room temperature for 1 hour.

[0073] 7) Primary antibody incubation: Wash the blocked PVDF membrane to remove the blocking solution, transfer it to the antibody dilution solution containing the primary antibody, and incubate it on a shaker at 4°C overnight (12 - 16 hours).

[0074] 8) Washing: The next day, transfer the incubated PVDF membrane to TBST, wash it on a shaker at room temperature for 10 minutes each time, and perform three washes in total.

[0075] 9) Secondary antibody incubation: Prepare the corresponding secondary antibody, transfer the washed PVDF membrane to the antibody diluent containing the secondary antibody, and incubate it on a shaker at room temperature for 1 hour.

[0076] 10) Washing: Transfer the incubated PVDF membrane to TBST, wash it on a shaker at room temperature for 10 minutes each time, and perform three washes in total.

[0077] 11) Exposure: Prepare the exposure solution and perform exposure imaging on the exposure instrument.

[0078] 12) Grayscale analysis: Use ImageJ software to perform grayscale analysis on the images.

[0079] The results are as Figure 4 shown. The level of H3K9la in synovial fibroblasts of rheumatoid arthritis patients is significantly higher than that of normal people and gouty arthritis patients.

[0080] Example 4:

[0081] The excessive production of lactic acid by synovial fibroblasts is the direct cause of the increase in H3K9la, and lactate dehydrogenase (LDHA) is the key enzyme for lactic acid production. In this example, inhibiting and knocking down LDHA can reduce the level of H3K9la in synovial fibroblasts.

[0082] Transwell and scratch assays were used to detect the effect of H3K9la on the migration and invasion ability of synovial fibroblasts:

[0083] 1. Intervention of synovial fibroblasts

[0084] Treat synovial fibroblasts with the LDHA inhibitor (FX-11) or knockdown the expression of LDHA in cells using siRNA, collect the cells, and detect the change in the level of H3K9la by Western blot.

[0085] The results are as Figure 5 shown. After inhibiting and knocking down LDHA in synovial fibroblasts of rheumatoid arthritis patients, the level of H3K9la in the cells decreased.

[0086] 2. Transwell invasion assay

[0087] 1) Take out Matrigel matrix glue from -80 °C and thaw it at 4 °C the night before the experiment. Pre-cool the sterile pipette tips, transwell chambers, cell well plates, and ice boxes in advance.

[0088] 2) Place the transwell chamber in a 24-well plate. Mix the Matrigel and serum-free medium thoroughly at a ratio of 1:8. Take 60 μL of the diluted Matrigel and spread it evenly in the transwell chamber. Place the plate in the cell culture incubator for 3 hours to allow it to solidify.

[0089] 3) Inoculate the RA-FLS of different treatment groups that have been digested and counted into the transwell chamber (5×10 4 cells / well).

[0090] 4) Add 500 μL of DMEM medium containing 10% FBS to the lower chamber well plate and incubate at 37°C and 5% CO 2 for 24 hours.

[0091] 5) Take out the transwell chamber, aspirate all the medium, and gently wipe off the residual cells on the upper layer of the chamber with a dry cotton swab.

[0092] Fix with 4% paraformaldehyde for 10 minutes and wash 3 times with PBS.

[0093] 6) Subsequently, fix with methanol for 30 minutes, wash 3 times with PBS, stain with 0.1% crystal violet solution for 30 minutes, and wash 3 times with PBS.

[0094] 7) Take pictures under an inverted microscope and randomly select five fields of view for photographing and cell counting..

[0095] The results are as Figure 6 shown. After the level of H3K9la in synovial fibroblasts decreases, the number of cell migration and invasion significantly decreases. This indicates that H3K9la has a promoting effect on the migration and invasion ability of synovial fibroblasts.

[0096] 3. Scratch migration assay

[0097] 1) Inoculate the RA-FLS of different treatment groups that have been digested and counted into a six-well plate (1×10 5 cells / well).

[0098] 2) When the cells reach 95% confluence, scratch along the diameter of the well with the tip of a 10 μL pipette.

[0099] 3) Wash away the scratched cells 3 times with sterile PBS. After aspirating all the PBS solution, add serum-free DMEM solution.

[0100] 4) Culture in a 37°C incubator and take pictures under an inverted microscope at the same field of view at 0 h and 24 h respectively.

[0101] The results are as Figure 7As shown, after the level of H3K9la in synovial fibroblasts decreased, the cell migration ability was significantly weakened. This indicates that H3K9la has a promoting effect on the migration ability of synovial fibroblasts.

[0102] Example 5

[0103] In this example, the level of H3K9la was inhibited in an animal model of arthritis, and the disease condition of arthritis was observed and inflammatory indicators were detected. The specific process is as follows:

[0104] 1. Construction and sampling of CIA model

[0105] 1) Collagen preparation: Dissolve bovine type II collagen dry powder with 0.05 M acetic acid to prepare a solution with a concentration of 4 mg / mL, and fully dissolve it overnight at 4°C.

[0106] 2) Homogenization and emulsification: In a laminar flow hood, add complete Freund's adjuvant and collagen solution to the bottle in a volume ratio of 1:1 with a sterile syringe needle, and mix well. Place the bottle on ice, and vertically insert a high-speed homogenizer into the mixed solution in the bottle. Homogenize at 20,000 rpm for 2 minutes, stop for 5 minutes, and repeat this process two to three times until the emulsified droplets do not disperse in water for a long time, indicating successful emulsification.

[0107] 3) Primary immunization: Anesthetize DBA / 1 mice by intraperitoneal injection with 1% pentobarbital sodium. Use a 1 mL sterile disposable syringe to aspirate the emulsifier, insert the needle 3 cm from the base of the mouse tail, and subcutaneously inject 100 μL of the emulsion into each mouse.

[0108] 4) Secondary immunization: On the 21st day after primary immunization, replace complete Freund's adjuvant with incomplete Freund's adjuvant and mix it with collagen for emulsification (the emulsification step is the same as step 2). After the mice are anesthetized, insert the needle 2 cm from the base of the tail, avoiding the first immunization needle hole, and subcutaneously inject 100 μL of the emulsion into each mouse.

[0109] 5) Clinical scoring: Within 28 days after primary immunization, observe the degree of joint swelling in mice and count the score every three days. Each limb is scored from 0 to 4 points, with a total of 0 to 16 points. The scoring system is defined as 0 = no signs of erythema and swelling, 1 = erythema and mild swelling limited to the tarsus or ankle joint, 2 = erythema and mild swelling extending from the ankle joint to the tarsus, 3 = erythema and moderate swelling extending from the ankle joint to the metatarsophalangeal joint, 4 = erythema and severe swelling including the ankle joint, foot, fingers, or limb ankylosis. Repeated measures analysis of variance (ANOVA) was used to test for significance.

[0110] 6) Intervention: On the day of the second immunization, mice were intraperitoneally injected with FX-11 (2 mg / kg) or AZD-3965 (100 mg / kg) every day.

[0111] 7) Photographing mouse joints: Before sacrificing the mice, photograph the limbs of the mice and group and save the records.

[0112] 8) Obtaining joint samples: Sacrifice the mice, peel off the skin of the mouse legs, separate the mouse legs from the pelvic bones along the greater trochanter, separate the muscles on the mouse legs and fix them on sponge foam, soak the separated hind feet of the mice in 4% paraformaldehyde solution for fixation, and change to decalcifying solution after 24 hours, and update the decalcifying solution every 3 days.

[0113] The results are as Figure 8 shown. In CIA mice (arthritis mice), the joints were significantly swollen. Treatment with the lactate dehydrogenase inhibitor (FX-11) and the lactate transporter MCT1 inhibitor (AZD-3965) could reduce the level of H3K9la in synovial fibroblasts, thereby alleviating joint swelling in mice. And as Figure 9 shown, the joint clinical score showed that after using the inhibitor to reduce the level of H3K9la, the joint score could be decreased, indicating that the joint damage in mice was alleviated.

[0114] 2. H&E staining

[0115] 1) Paraffin sections of mouse joints: Place the sections in fresh xylene for dewaxing overnight, and then transfer them to 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, and distilled water for gradient hydration, 5 minutes for each step. Then start the staining steps.

[0116] 2) Nucleus staining: Slowly drip hematoxylin staining solution onto the tissue sample and stain for 10 minutes.

[0117] 3) Differentiation and blueing: Differentiate with 1% hydrochloric acid ethanol for 1 - 2 seconds, and rinse with tap water for 15 minutes for blueing.

[0118] 4) Cytoplasm staining: Blot the surrounding moisture with filter paper, and slowly drip eosin staining solution and stain for 5 minutes.

[0119] 5) Dehydration and mounting: Then transfer to 50% alcohol, 75% alcohol, 85% alcohol, 95% alcohol, 100% alcohol for gradient dehydration, 5 minutes for each step. Dry the sections and mount them with neutral balsam, cover with a coverslip, and avoid generating air bubbles.

[0120] 6) Photographing: Use an upright microscope for photographing.

[0121] 3. Safranin O-fast green staining

[0122] 1) Dewaxing and hydration: Place the sections in fresh xylene for dewaxing overnight, and then transfer them to 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, and distilled water for gradient hydration, 5 minutes for each step.

[0123] 2) Dry the moisture around the joint tissue, add freshly prepared Weigert solution and stain for 3 minutes, then wash with water.

[0124] 3) Add acidic differentiating solution and differentiate for 15 seconds, then wash with distilled water for 10 minutes.

[0125] 4) Immerse in solid green solution for 5 minutes, quickly wash the sections with weak acid solution for 10 seconds to remove residual solid green, and air dry.

[0126] 5) Immerse in safranin staining solution for 5 minutes.

[0127] 6) Dehydrate in 95% ethanol for 3 seconds, absolute ethanol for 3 seconds, and absolute ethanol for 1 minute.

[0128] 7) Dry the slides, add neutral resin for mounting.

[0129] 8) Use an upright microscope for photography.

[0130] As shown in the results Figure 10 H&E staining showed synovial tissue hyperplasia at the joints of CIA mice, infiltration of a large number of immune cells, and cartilage destruction. Safranin-O-fast green staining also showed joint cartilage destruction. Treatment with lactate dehydrogenase inhibitor (FX-11) and lactate transporter MCT1 inhibitor (AZD-3965) could relieve joint destruction and improve arthritis symptoms.

[0131] 4. ELISA detection of serum inflammatory cytokine levels

[0132] Mouse ELISA kits for IL-1β, IL-6, IL-10, and TNF-α from R&D Systems were used to detect the levels of IL-1β, IL-6, IL-10, and TNF-α in the sera of mice in each group according to the instructions. The specific operation steps are as follows:

[0133] 1) Dissolve the standard product and dilute it to the corresponding concentration according to the instructions.

[0134] 2) Add 50 μL of the standard product and the sample to be tested to the center of each well.

[0135] 3) Cover with a strip and incubate at room temperature for 2 hours.

[0136] 4) Aspirate each well with a pipette and add washing solution for washing. Repeat this process four times, for a total of five washes. After the last wash, aspirate any residual washing buffer with a pipette. Invert the ELISA plate and blot it dry with a clean tissue.

[0137] 5) Add 100 μL of the corresponding secondary antibody conjugate for mice to each well. Cover with a new strip. Incubate at room temperature for 2 hours.

[0138] 6) Repeat the washing step in step 4.

[0139] 7) Add 100 μL of substrate solution to each well. Incubate in the dark at room temperature for 30 minutes.

[0140] 8) Add 100 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.

[0141] 9) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the optical density (OD) value of each well at 450 nm within 30 minutes.

[0142] As shown in the results Figure 11 The levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in CIA mice increased and decreased after treatment with the inhibitor. The level of the anti-inflammatory cytokine (IL-10) in CIA mice decreased and was restored after treatment with the inhibitor. This indicates that reducing the level of H3K9la can improve the inflammatory state of arthritic mice.

[0143] The preferred embodiments of the present application have been described in detail above in conjunction with the accompanying drawings. However, the present application is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all fall within the protection scope of the present application.

[0144] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present application will not separately describe various possible combination methods.

[0145] Furthermore, any combination can be made between the various different embodiments of the present application as long as it does not violate the idea of the present application, and it should also be regarded as the content disclosed in the present application.

Claims

1. Use of a biomarker for rheumatoid arthritis in the preparation of a product for diagnosing rheumatoid arthritis, characterized in that: The biomarker is an anti-lactated histone autoantibody, an anti-lactated H2B autoantibody, an anti-lactated H3 autoantibody, an anti-lactated H4 autoantibody, an anti-H3K9la autoantibody or H3K9la.

2. The use according to claim 1, characterized in that: in, The levels of anti-lactated histone autoantibodies, anti-lactated H2B autoantibodies, anti-lactated H3 autoantibodies, anti-lactated H4 autoantibodies, and anti-H3K9la autoantibodies are increased in the serum of patients with rheumatoid arthritis; the marker H3K9la is increased in the synovial fibroblasts of patients with rheumatoid arthritis.

3. A diagnostic product for rheumatoid arthritis, characterized in that: The diagnostic product includes a reagent for detecting anti-lactated histone autoantibodies, anti-lactated H2B autoantibodies, anti-lactated H3 autoantibodies, anti-lactated H4 autoantibodies, anti-H3K9la autoantibodies or H3K9la expression in a biological sample.

4. The diagnostic product for rheumatoid arthritis according to claim 3, characterized in that: The biological sample is fresh serum or synovial fibroblasts, wherein the levels of anti-lactated histone autoantibodies, anti-lactated H2B autoantibodies, anti-lactated H3 autoantibodies, anti-lactated H4 autoantibodies, and anti-H3K9la autoantibodies are increased in the serum of patients with rheumatoid arthritis; and the level of marker H3K9la is increased in synovial fibroblasts of patients with rheumatoid arthritis.

5. The diagnostic product for rheumatoid arthritis according to claim 3, characterized in that: The product is a test kit, a chip, a test paper or a high-throughput sequencing platform.

6. Use of a biomarker for rheumatoid arthritis in the preparation of a product for preventing or treating rheumatoid arthritis, characterized in that: The biomarker is H3K9la, and the product for preventing or treating rheumatoid arthritis contains an H3K9la inhibitor.

7. A drug for preventing or treating rheumatoid arthritis, characterized in that: Contains an H3K9la inhibitor.

8. The drug for preventing or treating rheumatoid arthritis according to claim 7, characterized in that: The H3K9la inhibitor is a LDHA inhibitor.

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