Application method of targeted MTHFD2 in preparation of medicine for treating rheumatoid arthritis bone destruction

Abstract

The invention relates to the field of application of medicines for treating rheumatoid arthritis, and discloses an application method of targeted MTHFD2 in preparation of medicines for treating bone destruction of rheumatoid arthritis, (1) the level of MTHFD2 in knee joints of rheumatoid arthritis patients and mice is increased; (2) the expression of MTHFD2 is up-regulated in the process of differentiating osteoclast precursor cells from mice and humans into osteoclasts; (3) the MTHFD2 inhibitor can significantly inhibit differentiation of osteoclasts from mice and humans; (4) MTHFD2 gene silencing can inhibit differentiation of mouse-derived osteoclasts, MTHFD2 gene overexpression can promote differentiation of osteoclasts, and reliability of MTHFD2 as a therapeutic target is verified; and (5) the MTHFD2 inhibitor can improve the bone destruction of arthritis mice and increase the bone tissue density, which prompts that the targeted MTHFD2 is useful for the treatment or prevention of rheumatoid arthritis and other diseases.

Description

Technical Field

[0001] The present invention relates to the application field of rheumatic joint drugs, and specifically to an application method for preparing a drug for treating rheumatoid arthritis bone destruction by targeting MTHFD2. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by symmetrical synovial inflammation and imbalance of bone homeostasis. It is reported that more than 45% of RA patients experience bone erosion in the early stages. The maintenance of bone homeostasis is achieved by a dynamic balance between bone formation and bone resorption. Osteoclasts are the only cells in the human body that have bone resorption function. Under the stimulation of RANKL (Receptor Activator of Nuclear Factor-κBLigand), they gradually differentiate from monocytes / macrophages into mature osteoclasts. Osteoclasts play a vital role in promoting bone development, growth, repair and reconstruction. Abnormal activation of osteoclasts is an important pathological basis for the occurrence and development of RA. During the onset of RA, osteoclasts are generated in large numbers, leading to enhanced bone resorption and bone destruction, which ultimately leads to disability and loss of social function in patients. Therefore, inhibiting the excessive activation of osteoclasts is extremely important to prevent the occurrence of bone erosion diseases.

[0003] At present, the drugs used in clinical practice to improve bone absorption mainly include four major categories of drugs: estrogens, estrogen receptor modulators, calcitonin, and bisphosphonates. The clinical efficacy is acceptable and they are widely used in clinical practice for diseases such as postmenopausal osteoporosis. However, the therapeutic effects of the above four types of drugs are non-selective for cells, so they bring different degrees of toxic side effects to patients, including venous thrombosis, hypocalcemia, nephrotoxicity, and cardiotoxicity, etc., causing great physical and mental pain to patients. Finding new specific targets for regulating osteoclast differentiation and developing selective targeted therapeutic drugs has important clinical and social significance.

[0004] MTHFD2 (methylenetetrahydrofolate dehydrogenase 2) is a molecule specifically expressed in immune cells such as monocytes / macrophages and tumor cells, and is involved in the differentiation of T and B lymphocytes, immune regulation function, and the progression of tumors such as breast cancer. Since MTHFD2 is selectively expressed in immune cells, it is a potential target for the treatment of autoimmune diseases. For this reason, we proposed a method for using MTHFD2 in the preparation of drugs for the treatment of bone destruction in rheumatoid arthritis. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the deficiencies of the prior art, the present invention provides a method for using MTHFD2 in the preparation of a drug for treating bone destruction in rheumatoid arthritis, wherein: (1) the level of MTHFD2 in the knee joints of arthritis patients and mice is increased; (2) the expression of MTHFD2 is upregulated during the differentiation of mouse-derived and human-derived osteoclast precursor cells into osteoclasts; (3) an MTHFD2 inhibitor can significantly inhibit the differentiation of mouse-derived and human-derived osteoclasts; (4) MTHFD2 gene silencing can inhibit the differentiation of mouse-derived osteoclasts, while MTHFD2 gene overexpression promotes osteoclast differentiation, verifying the reliability of MTHFD2 as a therapeutic target; and (5) an MTHFD2 inhibitor can improve bone destruction in arthritic mice and increase bone tissue density, suggesting that targeting MTHFD2 is useful for the treatment or prevention of diseases such as rheumatoid arthritis.

[0007] (II) Technical solution

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a method for using MTHFD2 in preparing a drug for treating bone destruction in rheumatoid arthritis, comprising the following steps:

[0009] Step 1: Isolation and culture of mouse bone marrow-derived macrophages and human peripheral blood CD14+ monocytes;

[0010] Step 2: Collect mouse BMM and human CD14+ monocytes at different time points during the differentiation process, extract RNA and protein samples, and examine the MTHFD2 levels at different points;

[0011] Step 3: Reverse transcription-polymerase chain reaction (qRT-PCR) experiment;

[0012] Step 4: Western Blot detection;

[0013] Step 5: The crystal violet method was used to detect the effect of MTHFD2 inhibitor on cell viability;

[0014] Step 6: MTHFD2 inhibitor was added during osteoclast induction. TRAP staining, phalloidin staining, bone resorption experiment and expression level of osteoclast differentiation marker genes were performed after the formation of giant vesicular multinuclear cells. The above experiments were then repeated in osteoclasts derived from mouse BMM.

[0015] Step 7: Establish a collagen-induced arthritis mouse model and divide it into two groups: control and MTHFD2 inhibitor, and then perform bone mass related data detection and analysis. All data are expressed as mean ± standard deviation, with significant differences: *P<0.05, **P<0.01, ***P<0.001.

[0016] Preferably, the specific content of the first step is as follows:

[0017] The femur and tibia of mice were separated in a clean bench, the bone marrow cavity was repeatedly flushed, the flushing fluid containing bone marrow cells was collected, centrifuged at 1500 rpm for 5 minutes, the red blood cells were filtered and lysed for 5 minutes, the cells were resuspended with 12 ml of α-MEM cell culture medium, inoculated in a 6-well plate and cultured overnight, non-adherent cells were collected after 12 hours, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and α-MEM culture medium containing recombinant RANKL (75 ng / ml) and M-CSF (30 ng / ml) was added to continue culturing for 5 days to induce osteoclast differentiation, and the medium was changed every two days;

[0018] Peripheral blood was obtained from patients with rheumatoid arthritis, and the mononuclear cell buffy coat was separated after gradient centrifugation and lysis of red blood cells. CD14 magnetic beads were then added to sort CD14+ monocytes. The sorted cells were centrifuged at 1500rpm for 5min, the supernatant was discarded, and α-MEM medium containing recombinant RANKL (75ng / ml) and M-CSF (30ng / ml) was added and cultured for 10 days to induce osteoclast differentiation, with the medium changed every three days.

[0019] Preferably, the specific content of the second step is as follows:

[0020] Lyse the cells with 1 mL of Trizol, collect the cell lysate into an enzyme-free centrifuge tube, add 200 μL of chloroform, shake vigorously for 15 seconds, and place at room temperature for 10 minutes; then centrifuge at 4°C, 12,000 rpm, for 15 minutes;

[0021] Transfer the supernatant to another EP tube, add isopropanol of equal volume to the supernatant, flip the EP tube upside down to mix, and place at room temperature for 10 minutes; centrifuge at 4°C, 12000 rpm, 10 minutes;

[0022] The supernatant was discarded, and the cells were washed with 1 mL of 75% ethanol, followed by centrifugation at 4 °C, 12,000 rpm, for 5 min;

[0023] Discard the supernatant and dry in a fume hood;

[0024] Add 10 μL of sterile enzyme-free water to dissolve RNA, then reverse transcribe the RNA into cDNA, mix the cDNA with different gene primers and SYBR reagent, and measure the relative expression of the gene.

[0025] Preferably, the third step includes the following contents:

[0026] Remove the cell culture medium, wash the cells 3 times with PBS, add Western cell lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to each well, lyse on ice for 30 minutes, centrifuge at 12000rpm for 10 minutes, and collect the supernatant, which is the total cell protein. After the protein concentration of the sample is determined by the BCA method, 10 μg of protein is loaded per well according to the BCA protein content determination result, separated by 10% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, blocked with blocking solution, added corresponding primary antibody, and incubated overnight at 4°C;

[0027] After washing the membrane with TBST, the secondary antibody was incubated at room temperature for 1 h. After color development with the color developing solution, the gel was photographed in a gel imaging system. The protein bands were analyzed using Image J software, and β-actin was used as the internal reference protein to determine the relative expression level of the target protein.

[0028] Preferably, the fifth step includes the following contents:

[0029] Mouse BMM and human CD14+ monocytes in the logarithmic growth phase were taken, and MTHFD2 inhibitors were added at a final concentration of 0μM-20μM. Three replicate wells were set up for each concentration. After culturing for 3-9 days, the cell viability was determined by the crystal violet method, and the absorbance at 570nm was detected. When the MTHFD2 inhibitor was below 15μM, it had no toxic effect on mouse and human osteoclast precursor cells.

[0030] Preferably, the sixth step includes the following contents:

[0031] α-MEM medium containing recombinant RANKL (75 ng / ml) and M-CSF (30 ng / ml) was added to the mouse BMM culture system for 5 days to induce osteoclast differentiation, and the medium was changed every two days, and a control solvent or 10 μM MTHFD2 inhibitor was added at the same time; α-MEM medium containing recombinant RANKL (75 ng / ml) and M-CSF (30 ng / ml) was added to the human CD14+ monocyte culture system for 10 days to induce osteoclast differentiation, and the medium was changed every three days, and a control solvent or 7.5 μM MTHFD2 inhibitor was added at the same time. At the end of differentiation, tartrate-resistant acid phosphatase staining, phalloidin cytoskeleton staining, and osteoclast-specific molecular gene and protein levels were continued.

[0032] Preferably, the seventh step includes the following contents:

[0033] Use 2.5 ml of glacial acetic acid solution to dissolve 5 mg of chicken type II collagen to prepare a 2 mg / mL chicken type II collagen solution;

[0034] In an ice bath, 2 mg / mL CII solution was mixed and emulsified with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant using an ultrasonic disruptor. The primary immunization was performed on day 0, and the booster immunization was performed on day 21. The emulsified solution was injected subcutaneously at the base of the tail of 8-week-old DBA / 1 male mice.

[0035] From day 21 onwards, MTHFD2 inhibitor / control solvent was administered intragastrically every day, and arthritis scores were performed every 3 days;

[0036] The experimental mice were euthanized on day 42, and the lower limbs were removed for micro CT scanning and TRAP staining.

[0037] (III) Beneficial effects

[0038] Compared with the prior art, the present invention provides a method for using MTHFD2 in the preparation of a drug for treating bone destruction in rheumatoid arthritis, which has the following beneficial effects:

[0039] 1. The application method of MTHFD2 in the preparation of a drug for treating bone destruction in rheumatoid arthritis. Compared with the prior art, the MTHFD2 in the present invention is a molecule specifically expressed in immune cells such as monocytes / macrophages, and the MTHFD2 level increases in the disease state of rheumatoid arthritis and is continuously upregulated during osteoclast differentiation, which is different from the non-selective effect of traditional therapeutic drugs on cells.

[0040] 2. The use of MTHFD2 in the preparation of a drug for treating rheumatoid arthritis bone destruction. The present invention finds that the MTHFD2 inhibitor has low toxicity and is safe, and can well inhibit osteoclast differentiation and alleviate bone tissue destruction in arthritis mice. Therefore, the present invention provides a new drug target for preventing and treating bone metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the structural formula of MTHFD2 inhibitor;

[0042] Figure 2 Schematic diagram of the changes in MTHFD2 levels in arthritis and during osteoclast differentiation;

[0043] Figure 3 Schematic diagram showing that the MTHFD2 inhibitor has no significant effect on the activity of human and mouse osteoclast precursor cells;

[0044] Figure 4 Schematic diagram showing that MTHFD2 inhibitors can inhibit human osteoclast differentiation;

[0045] Figure 5 Schematic diagram showing that MTHFD2 inhibitors can inhibit osteoclast differentiation in mice;

[0046] Figure 6 Schematic diagram showing that MTHFD2 gene silencing can inhibit osteoclast differentiation in mice;

[0047] Figure 7 Schematic diagram showing that overexpression of the MTHFD2 gene can promote osteoclast differentiation in mice;

[0048] Figure 8 Schematic diagram of how MTHFD2 inhibitor inhibits bone damage and osteoclastogenesis in collagen-induced arthritis mice in vivo. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] See also Figure 1-8 The method for using MTHFD2 in preparing a drug for treating bone destruction in rheumatoid arthritis comprises the following contents:

[0051] (1) Isolation and culture of mouse bone marrow macrophages (BMM): 6-8 week old C57BL / 6 mice were euthanized and soaked in 75% alcohol for 10 min. The femur and tibia of the mice were then separated in a clean bench, and the muscle, fascia and other tissues were removed. The ends of the femur and tibia were cut open, and PBS buffer was drawn with a 5 ml syringe. The bone marrow cavity was repeatedly flushed, and the flushing fluid containing bone marrow cells was collected. The flushing fluid was centrifuged at 1500 rpm for 5 min, and the red blood cells were filtered and lysed for 5 minutes. The cells were resuspended in 12 ml of α-MEM cell culture medium and inoculated into a 6-well plate for overnight culture. After 12 hours, the non-adherent cells were collected, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and α-MEM culture medium containing recombinant RANKL (75 ng / ml) and M-CSF (30 ng / ml) was added for further culture for 5 days to induce osteoclast differentiation. The medium was changed every two days. Isolation and culture of human peripheral blood CD14+ monocytes: Peripheral blood from patients with rheumatoid arthritis was collected, and the mononuclear cell buffy coat was separated after gradient centrifugation and lysis of red blood cells, followed by the addition of CD14 magnetic beads for CD14+ monocyte sorting. The sorted cells were centrifuged at 1500rpm for 5min, the supernatant was discarded, and α-MEM medium containing recombinant RANKL (75ng / ml) and M-CSF (30ng / ml) was added for continued culture for 10 days to induce osteoclast differentiation, and the medium was changed every three days.

[0052] (2) Reverse transcription-polymerase chain reaction (qRT-PCR) experiment: 1 mL Trizol was used to lyse cells, and the cell lysate was collected into an enzyme-free centrifuge tube, 200 μL chloroform was added, and the mixture was shaken vigorously for 15 seconds and placed at room temperature for 10 minutes; then centrifuged at 4°C, 12,000 rpm, for 15 minutes; the supernatant was transferred to another EP tube, and an equal volume of isopropanol was added to the supernatant, and the EP tube was flipped up and down to mix, and placed at room temperature for 10 minutes; centrifuged at 4°C, 12,000 rpm, for 10 minutes; the supernatant was discarded, and 1 mL 75% ethanol was used for washing, and then centrifuged at 4°C, 12,000 rpm, for 5 minutes; the supernatant was discarded and dried in a fume hood; 10 μL sterile enzyme-free water was added to dissolve RNA, and then reverse transcribed to convert RNA into cDNA. The cDNA was mixed with different gene primers and SYBR reagent to measure the relative expression of the gene.

[0053] (3) Western Blot detection: Remove the cell culture medium, wash the cells three times with PBS, add Western cell lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to each well, lyse on ice for 30 minutes, centrifuge at 12000rpm for 10 minutes, and collect the supernatant (total cell protein). After determining the protein concentration of the sample by BCA method, prepare the sample. According to the results of BCA protein content determination, 10 μg of protein was loaded per well, separated by 10% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, blocked with blocking solution, added with corresponding primary antibody, and incubated at 4°C overnight. After washing the membrane with TBST, incubate the secondary antibody at room temperature for 1 hour, and take pictures in the gel imaging system after color development with color developing solution. Use Image J software to analyze the protein bands, and use β-actin as the internal reference protein to determine the relative expression level of the target protein.

[0054] (4) Collagen-induced arthritis model mice: 5 mg of chicken type II collagen was dissolved in 2.5 ml of glacial acetic acid solution to prepare a 2 mg / mL chicken type II collagen solution. Under ice bath conditions, 2 mg / mL CII solution was mixed and emulsified with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant using an ultrasonic disruptor. The primary immunization was performed on day 0, and the booster immunization was performed on day 21. The emulsifier was injected subcutaneously at the base of the tail of 8-week-old DBA / 1 male mice. From day 21 onwards, the MTHFD2 inhibitor / control solvent was gavaged daily, and arthritis scores were performed every 3 days. The experimental mice were euthanized on day 42, and the lower limbs were removed for microCT scanning and TRAP staining.

[0055] (5) Data analysis: Data were analyzed using Prism 10.0 software, and all data were expressed as mean ± standard deviation. Significant differences: *P < 0.05, **P < 0.01, ***P < 0.001.

[0056] Results: The present invention established the effects of MTHFD2 on osteoclast differentiation and function through in vitro models and animal experiments. The results showed that MTHFD2 plays an important role in osteoclast differentiation and may be a potential therapeutic target for bone destructive diseases such as rheumatoid arthritis.

[0057] (1) MTHFD2 expression in RA state and changes in MTHFD2 levels during osteoclast differentiation:

[0058] Synovial samples from RA patients and knee joint samples from arthritic mice were collected, and the MTHFD2 level was detected by immunohistochemistry. Figure 2 As shown in A. As shown in the figure, MTHFD2 was significantly increased in the inflammatory state compared with the control group. Mouse BMM and human CD14+ monocytes were collected at different time points during the differentiation process, RNA and protein samples were extracted, and the MTHFD2 levels at different points were examined. The results are shown in Figure 2 As shown in B and C, the mRNA and protein levels of MTHFD2 in mouse BMM and human CD14+ monocytes increased with the extension of RANKL stimulation.

[0059] (2) Crystal violet method to detect the effect of MTHFD2 inhibitors on cell viability:

[0060] Mouse BMM and human CD14+ monocytes in the logarithmic growth phase were taken, and the MTHFD2 inhibitor was added at a final concentration of 0μM-20μM. Three replicate wells were set for each concentration. After culturing for 3-9 days, the cell viability was determined by the crystal violet method, and the absorbance at 570nm was detected. The results were as follows: Figure 3 As shown in A and B. As can be seen from the figure, the MTHFD2 inhibitor has no toxic effect on mouse and human osteoclast precursor cells when it is below 15 μM.

[0061] (3) Effects of MTHFD2 inhibitors on osteoclast differentiation:

[0062] During the osteoclast induction process, MTHFD2 inhibitors were added, and TRAP staining, phalloidin staining, bone resorption experiments, and osteoclast differentiation marker gene expression levels were detected after giant vesicular multinuclear cells were formed. TRAP staining-positive areas are red. In the control group without MTHFD2 inhibitor intervention, a large number of irregular multinuclear giant cells, namely osteoclasts, were seen. After treatment with MTHFD2 inhibitors, osteoclasts derived from human CD14+ monocytes were significantly reduced, and the dark-stained areas were significantly reduced. Only a small amount of purple-red positive areas were seen. Figure 4 A. Similarly, the osteoclast cytoskeleton phalloidin fluorescence staining showed that the control group could clearly observe the well-differentiated F-actin ring, while the inhibitor group did not see obvious F-actin ring, see Figure 4B. The present invention further found that in the MTHFD2 inhibitor treatment group, the expression of osteoclast-specific genes and proteins, including matrix metalloproteinase 9 (MMP9), integrin β3, cathepsin K (CTSK) and nuclear factor of activated T cells 1 (NFATc1), were all significantly downregulated. Figure 4 C.

[0063] The present invention repeated the above experiment in mouse BMM-derived osteoclasts, and the results showed that the MTHFD2 inhibitor can also significantly inhibit the differentiation and function of mouse BMM-derived osteoclasts. Figure 5 In addition, the present invention shows that the addition of MTHFD2 inhibitors during osteoclast formation significantly inhibits osteoclast function, and the bone plate absorption experiment shows that multiple erosions of the bone plate can be seen in the control group without MTHFD2 inhibitor intervention, while no obvious erosion was seen in the MTHFD2 inhibitor group. Figure 5 Middle E.

[0064] (4) Effects of MTHFD2 gene silencing and overexpression on osteoclast differentiation:

[0065] In order to further clarify the reliability of MTHFD2 as a target for osteoclast differentiation, the present invention intervenes in MTHFD2 at the gene level to verify the specificity of the inhibitory effect of MTHFD2 inhibitors. After using adenovirus vectors to silence the gene of MTHFD2 in mouse BMM, it was found that both the RNA and protein expressions of MTHFD2 were reduced. Figure 6 At the same time, the TRAP-positive osteoclasts and F-actin ring-positive osteoclasts in the low MTHFD2 expression group were significantly reduced, as shown in Figure 6 B and C. Correspondingly, the expression of osteoclast-specific genes and proteins was significantly downregulated in D and E. In addition, the present invention used a lentiviral vector to perform a gene overexpression experiment on MTHFD2. Figure 7 A shows that the RNA and protein expressions of MTHFD2 were significantly upregulated. At the same time, the TRAP-positive osteoclasts and F-actin ring-positive osteoclasts in the high MTHFD2 expression group were significantly reduced, as shown in Figure 7 In B and C, the expression of osteoclast-specific genes and proteins was significantly downregulated. Figure 7 D.E.

[0066] (5) Therapeutic effect of MTHFD2 on bone damage in collagen-induced arthritis in mice:

[0067] The animals were randomly divided into 2 groups (n=8): solvent control group and MTHFD2 inhibitor group. The tibiae fixed with 4% paraformaldehyde were scanned and reconstructed by microCT, the knee joints were embedded after decalcification, and the sections were stained with TRAP. Figure 8As shown in A and B, after microCT bone scanning, bone parameters of the upper end of the tibia were analyzed to obtain bone density (BMD), trabecular bone volume (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp) data. Compared with the control mice, the inhibitor-intervention arthritis mice had increased bone mass and shortened trabecular spacing, indicating that the degree of bone destruction in arthritis was lower. In addition, the present invention shows that the percentage of osteoclast surface on the bone surface in TRAP staining in the inhibitor-intervention group (OcS / BS, %) was reduced, as shown in Figure 2. Figure 8 Middle C and D. In summary, administration of MTHFD2 inhibitors improved arthritic bone damage in mice, reduced bone resorption, and increased bone density.

[0068] See also Figure 2 : A. Immunohistochemistry detected increased expression of MTHFD2 in synovial samples of RA patients; B. MTHFD2 expression was increased in knee joint samples of mice with collagen-induced arthritis; C. MTHFD2 protein levels in mouse BMM and human CD14+ monocytes increased with prolonged RANKL stimulation; D. MTHFD2 mRNA levels in mouse BMM and human CD14+ monocytes increased with prolonged RANKL stimulation (n=3-4, *p<0.05, **p<0.01, ***p<0.001).

[0069] See also Figure 3 : A. MTHFD2 inhibitors had no toxic effect on human osteoclast precursor cells on the 6th and 9th days of culture when the concentration was below 15 μM; B. MTHFD2 inhibitors had no toxic effect on mouse osteoclast precursor cells on the 3rd day of culture when the concentration was below 15 μM (n=4, ns=not statistically significant).

[0070] See also Figure 4 : A. When MTHFD2 inhibitor was added during human osteoclast differentiation, the number of TRAP-positive osteoclasts decreased; B. When MTHFD2 inhibitor was added during human osteoclast differentiation, the number of F-actin ring-positive osteoclasts decreased; C. MTHFD2 inhibitor reduced the mRNA expression of human osteoclast-specific molecules (n=4, *p<0.05, **p<0.01, ***p<0.001).

[0071] See also Figure 5: A. When MTHFD2 inhibitors were added during osteoclast differentiation in mice, the number of TRAP-positive osteoclasts decreased; B. When MTHFD2 inhibitors were added during osteoclast differentiation, the number of F-actin ring-positive osteoclasts decreased; C. MTHFD2 inhibitors reduced the mRNA expression of osteoclast-specific molecules; D. MTHFD2 inhibitors reduced the protein expression of osteoclast-specific molecules; E. MTHFD2 inhibitors could reduce the number of bone resorption pits in bone plates, indicating that osteoclast function was weakened (n=4, *p<0.05, **p<0.01, ***p<0.001).

[0072] See also Figure 6 : A. MTHFD2 gene silencing during osteoclast differentiation in mice reduced the mRNA and protein levels of MTHFD2; B. The number of TRAP-positive osteoclasts differentiated from osteoclast precursor cells with low MTHFD2 expression decreased; C. The number of F-actin ring-positive osteoclasts differentiated from osteoclast precursor cells with low MTHFD2 expression decreased; D. Low MTHFD2 expression inhibited the mRNA expression of osteoclast-specific molecules; E. Low MTHFD2 expression inhibited the protein expression of osteoclast-specific molecules (n=4, *p<0.05, **p<0.01, ***p<0.001).

[0073] See also Figure 7 : A. MTHFD2 gene was overexpressed during osteoclast differentiation in mice, and the mRNA and protein levels of MTHFD2 increased; B. The number of TRAP-positive osteoclasts differentiated from osteoclast precursor cells with high MTHFD2 expression increased; C. The number of F-actin ring-positive osteoclasts differentiated from osteoclast precursor cells with high MTHFD2 expression increased; D. High MTHFD2 expression promoted the mRNA expression of osteoclast-specific molecules; E. High MTHFD2 expression promoted the protein expression of osteoclast-specific molecules (n=4, *p<0.05, **p<0.01, ***p<0.001).

[0074] See also Figure 8 :A, microCT showed that MTHFD2 inhibitor could improve osteopenia in mice with collagen-induced arthritis; B, analysis of bone-related indicators at the upper end of the tibia; CD, TRAP staining and relative quantitative analysis of osteoclast number (n=3-8, *p<0.05, **p<0.01, ***p<0.001).

[0075] The sources of consumables in this solution are as follows:

[0076] Main experimental consumables: 6-8 weeks old C57BL / 6 and DBA / 1 male mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.; MTHFD2 inhibitor (DS18561882, structural formula as Figure 1 As shown, purity ≥99%), purchased from MedChemExpress, USA; α-MEM medium and fetal bovine serum were purchased from Gibco, USA; mouse and human recombinant RANKL and M-CSF (Macrophage colony-stimulating factor) were purchased from PeproTech, USA; Trizol reagent, reverse transcription reagent and SYBR Premix Ex Taq reagent were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; Western Blot related reagents were purchased from Shanghai Yamei Company; tartrate-resistant acid phosphatase (TRAP) staining reagent was purchased from Wuhan Sewell Company; phalloidin fluorescent antibody was purchased from Beijing Solebao Company; chicken type II collagen, complete adjuvant and incomplete adjuvant were purchased from Chondrex Company.

[0077] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application method for preparing a drug for treating rheumatoid arthritis bone destruction by targeting MTHFD2, characterized in that: The following steps are involved: Step 1: Isolation and culture of mouse bone marrow-derived macrophages and human peripheral blood CD14+ monocytes; Step 2: Collect mouse BMM and human CD14+ monocytes at different time points during the differentiation process, extract RNA and protein samples, and examine the MTHFD2 levels at different differentiation points; Step 3: Reverse transcription-polymerase chain reaction (qRT-PCR) experiment; Step 4: Western Blot detection; Step 5: The crystal violet method was used to detect the effect of MTHFD2 inhibitor on cell viability; Step 6: MTHFD2 inhibitor was added during the differentiation of human osteoclasts. After the formation of giant vesicular multinuclear cells, tartrate-resistant acid phosphatase staining, phalloidin staining, bone resorption experiment and expression level of osteoclast differentiation marker genes were performed. The above experiments were then repeated in mouse BMM-derived osteoclasts. Step 7: Establish a collagen-induced arthritis mouse model and divide it into two groups: control group and MTHFD2 inhibitor group. Then perform bone mass related data detection and analysis. All data are expressed as mean ± standard deviation. Significant differences: *P<0.05, **P<0.01, ***P<0.

001.

2. The method for preparing a drug for treating rheumatoid arthritis bone destruction by targeting MTHFD2 according to claim 1, characterized in that: The femur and tibia of mice were separated in a clean bench, the bone marrow cavity was repeatedly flushed, the flushing fluid containing bone marrow cells was collected, centrifuged at 1500rpm for 5min, the red blood cells were filtered and lysed for 5 minutes, the cells were resuspended with 12ml α-MEM cell culture medium, inoculated in a 6-well plate and cultured overnight, non-adherent cells were collected after 12 hours, centrifuged at 1500rpm for 5min, the supernatant was discarded, and α-MEM culture medium containing recombinant RANKL (75ng / ml) and M-CSF (30ng / ml) was added and cultured for 5 days to induce osteoclast differentiation, and the medium was changed every two days.

3. The application method of targeting MTHFD2 to prepare a drug for treating rheumatoid arthritis bone destruction according to claim 1, characterized in that: Peripheral blood was obtained from patients with rheumatoid arthritis, and the mononuclear cell buffy coat was separated after gradient centrifugation and lysis of red blood cells. CD14 magnetic beads were then added to sort CD14+ monocytes. The sorted cells were centrifuged at 1500rpm for 5min, the supernatant was discarded, and α-MEM medium containing recombinant RANKL (75ng / ml) and M-CSF (30ng / ml) was added and cultured for 10 days to induce osteoclast differentiation, with the medium changed every three days.

4. The application method of targeting MTHFD2 to prepare a drug for treating rheumatoid arthritis bone destruction according to claim 1, characterized in that: The specific contents of the third step are as follows: Lyse the cells with 1 mL of Trizol, collect the cell lysate into an enzyme-free centrifuge tube, add 200 μL of chloroform, shake vigorously for 15 seconds, and place at room temperature for 10 minutes; then centrifuge at 4°C, 12,000 rpm, for 15 minutes; Transfer the supernatant to another EP tube, add isopropanol of equal volume to the supernatant, flip the EP tube upside down to mix, and place at room temperature for 10 minutes; centrifuge at 4°C, 12000 rpm, 10 minutes; The supernatant was discarded, and the cells were washed with 1 mL of 75% ethanol, followed by centrifugation at 4 °C, 12,000 rpm, for 5 min; Discard the supernatant and dry in a fume hood; Add 10 μL of sterile enzyme-free water to dissolve RNA, then reverse transcribe the RNA into cDNA, mix the cDNA with different gene primers and SYBR reagent, and measure the relative expression of the gene.

5. The application method of targeting MTHFD2 to prepare a drug for treating rheumatoid arthritis bone destruction according to claim 1, characterized in that: The fourth step includes the following contents: Remove the cell culture medium, wash the cells 3 times with PBS, add Western cell lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to each well, lyse on ice for 30 minutes, centrifuge at 12000rpm for 10 minutes, and collect the supernatant as total cell protein. The protein concentration of the sample was determined by BCA method. According to the results of BCA protein content determination, 10 μg of protein was loaded per well. After separation by 10% SDS-PAGE gel electrophoresis, the membrane was transferred to PVDF membrane, blocked with blocking solution, added with corresponding primary antibody, and incubated overnight at 4°C; After washing the membrane with TBST, the secondary antibody was incubated at room temperature for 1 h. After color development with the color developing solution, the gel was photographed in a gel imaging system. The protein bands were analyzed using Image J software, and β-actin was used as the internal reference protein to determine the relative expression level of the target protein.

6. The application method of targeting MTHFD2 to prepare a drug for treating rheumatoid arthritis bone destruction according to claim 1, characterized in that: The fifth step includes the following contents: Mouse BMM and human CD14+ monocytes in the logarithmic growth phase were taken, and MTHFD2 inhibitors were added at a final concentration of 0μM-20μM. Three replicate wells were set up for each concentration. After culturing for 3-9 days, the cell viability was determined by the crystal violet method, and the absorbance at 570nm was detected. When the MTHFD2 inhibitor was below 15μM, it had no toxic effect on mouse and human osteoclast precursor cells.

7. The application method of targeting MTHFD2 to prepare a drug for treating rheumatoid arthritis bone destruction according to claim 1, characterized in that: The sixth step includes the following: adding α-MEM medium containing recombinant RANKL (75ng / ml) and M-CSF (30ng / ml) to the mouse BMM culture system for 5 days to induce osteoclast differentiation, changing the medium every two days, and adding a control solvent or 10μM MTHFD2 inhibitor; adding α-MEM medium containing recombinant RANKL (75ng / ml) and M-CSF (30ng / ml) to the human CD14+ monocyte culture system for 10 days to induce osteoclast differentiation, changing the medium every three days, and adding a control solvent or 7.5μM MTHFD2 inhibitor. At the end of differentiation, tartrate-resistant acid phosphatase staining, phalloidin cytoskeleton staining, and osteoclast-specific molecular gene and protein level detection are continued.

8. The method for preparing a drug for treating rheumatoid arthritis bone destruction by targeting MTHFD2 according to claim 1, characterized in that: The seventh step includes the following contents: Use 2.5 ml of glacial acetic acid solution to dissolve 5 mg of chicken type II collagen to prepare a 2 mg / mL chicken type II collagen solution; In an ice bath, 2 mg / mL CII solution was mixed and emulsified with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant using an ultrasonic disruptor. The primary immunization was performed on day 0, and the booster immunization was performed on day 21. The emulsified solution was injected subcutaneously at the base of the tail of 8-week-old DBA / 1 male mice. From day 21 onwards, MTHFD2 inhibitor / control solvent was administered intragastrically every day, and arthritis scores were performed every 3 days; The experimental mice were euthanized on day 42, and the lower limbs were removed for micro CT scanning and TRAP staining.

Images