ELF1-targeting small molecule drug and application thereof in intervertebral disc degeneration

Abstract

The invention discloses a micromolecular drug targeting ELF1 and application of the micromolecular drug in intervertebral disc degeneration, and belongs to the technical field of biological medicine, it is found for the first time that the micromolecular compound Mycopherolate Mofetin has a treatment effect on intervertebral disc degeneration, in-vivo and in-vitro verification experiments show that the micromolecular compound Mycopherolate Mofetin has a remarkable inhibition effect on senescence of nucleus pulposus cells, and the micromolecular drug can be used for treating intervertebral disc degeneration. According to the present invention, the application of the Mycopherate Mofetil in the treatment of intervertebral disc degeneration is provided, the progress of intervertebral disc degeneration is significantly inhibited, the new method and the new idea are provided for the clinical treatment of intervertebral disc degeneration, and the significant significance in the adjuvant treatment and prevention of intervertebral disc degeneration is provided, such that the small molecule compound Mycopherate Mofetil has the good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small molecule drug targeting ELF1 and an application thereof in intervertebral disc degeneration. Background Art

[0002] Low back pain is the leading cause of disability worldwide, and the annual medical insurance expenditure for the treatment of low back pain is as high as hundreds of billions of dollars, causing a heavy socioeconomic burden. In the past few years, many researchers around the world have paid attention to the fact that nucleus pulposus (NP) cell degeneration plays a major role in IVDD. NP cell degeneration is caused by a series of pathological processes, including oxidative stress, inflammatory response, apoptosis, abnormal proliferation, autophagy, etc. Interestingly, many studies have found that there is a close relationship between NP cell aging and the progression of NP degeneration, and the classic aging pathway has also been confirmed to be involved in the pathological process of IVDD. In addition, there are currently a variety of anti-aging drugs that treat IVDD by inhibiting NP cell aging, such as proanthocyanidins, resveratrol, and bone morphogenetic protein. Therefore, the current diagnosis and treatment ideas for IVDD should shift to precise treatment based on the molecular biological mechanism of nucleus pulposus cell aging, in order to delay the progression of IVDD from the root.

[0003] In recent years, small molecule drugs have shown significant efficacy in in vitro, in vivo and clinical studies of IVDD as alternative therapeutic agents, and can play an antioxidant, anti-inflammatory, anti-aging, anti-apoptotic and anti-catabolic role in the process of disc degeneration. Compared with macromolecule drugs, small molecule drugs have the following advantages: ① Small molecules play a significant therapeutic role at low doses; ② Small molecules are effective when the amount of their active drug ingredients is small, which is more cost-effective; ③ Small molecules are easily absorbed through the digestive tract and can be taken orally without injection; ④ Small molecules usually do not require the cold chain guarantee required by macromolecule drugs, thereby improving their shelf life and transportation process. Therefore, small molecule drugs have therapeutic potential in delaying disc degeneration and promoting tissue regeneration. Small molecule drugs can be used as the preferred tool to achieve targeted intervention in the aging of nucleus pulposus cells. With the increasing degree of dataization in the field of drug research and development, people use artificial intelligence (AI) technology to meet the challenges in drug discovery and improve the efficiency of drug development. The application of artificial intelligence technology in virtual screening can predict the pharmacological properties according to the structure of the compound, provide direction for subsequent experimental research, and is an important auxiliary means for new drug discovery. By utilizing molecular docking operations between small molecule compounds and drug targets, active compounds with drugability can be quickly selected from tens to millions of molecules, greatly reducing the number of experimental screening compounds, shortening the research cycle, and reducing drug development costs.

[0004] Epigenetic dysregulation is considered a core hallmark and driving force of aging, and repairing the integrity of the epigenome may be the key to reverse cellular aging with targeted drugs. Epigenomic changes include changes in transcription factor binding, histone marks, DNA methylation, N6 methyladenine (m6A) and non-coding RNA, and age-related changes in transcription factors and chromatin states are considered to be early drivers of aging and important hubs connecting many aging characteristics. Genetic regulation of a single transcription factor in an evolutionarily conserved manner is sufficient to affect the lifespan and healthspan of model organisms. Some transcription factors have been associated with human lifespan and age-related diseases. For example, the aging-related transcription factors p53 and MYC are both key cell cycle regulators that affect aging and tumorigenesis in a complex manner, have become targets for cancer treatment, and may delay aging itself. ELF1 (E74-like factor 1) is a transcription factor belonging to the ETS (erythroblast transformation-specific) family. Recent studies have shown that ELF1 is closely related to a variety of diseases and is involved in a variety of biological processes such as cell proliferation, differentiation, apoptosis, and immune response. At present, ELF1 has been found to play a role in various organs and tissues such as nerves, brain, kidneys, cardiovascular, and gastrointestinal tract; ELF1 plays an anti-proliferative role in squamous cell carcinoma cell lines; in breast cancer, ELF1 has been found to enhance the proliferation and invasion of breast cancer cells and is associated with lymph node metastasis of breast cancer; abnormal expression of ELF1 is also associated with the occurrence and prognosis of many other tumors such as colorectal cancer, prostate cancer, and lung cancer. However, small molecule drugs that target and regulate ELF1 protein expression and regulate the senescence of nucleus pulposus cells have not yet been reported.

[0005] Mycophenolate Mofetil (MMF) is a specific inhibitor of inosine monophosphate dehydrogenase involved in de novo purine synthesis and an inhibitor of T- and B-lymphocyte proliferation. Therefore, MMF is used to prevent acute, as well as chronic rejection after transplantation. Interestingly, MMF is an immunosuppressive drug known to have effects on cells other than the immune system, particularly antiproliferative effects on vascular smooth muscle cells. MMF has been shown to inhibit intimal smooth muscle cell proliferation in vivo after balloon injury. In addition, MMF has been reported to have an inhibitory effect on growth factor-induced vascular smooth muscle cell proliferation in vitro. MMF is the 2-morpholino ethyl ester of MPA (mycophenolic acid). The availability of MMF in healthy volunteers orally is 94.1%. After absorption, MMF is rapidly converted to the active metabolite MPA by esterases in plasma, liver, and kidney. MPA is inactivated in the liver by a first glucuronidation. A large portion of the phenolic glucuronide of the resulting MPA (MPAG) is secreted into the bile and recirculated to the liver via the enterohepatic pathway. This enterohepatic recycling process is believed to be an important contribution to maintaining serum MPA / MPAG levels. Studies have shown that mycophenolic acid can prolong the replication ability of yeast and thus prolong its lifespan, and mycophenolate mofetil can improve oxidative stress, so it is defined as an "anti-aging" drug, but whether it can inhibit the aging of nucleus pulposus cells and its target of action have not yet been clarified.

[0006] Through the above analysis, the problems and defects of the prior art are as follows:

[0007] (1) None of the current therapies successfully address the underlying biological problem—the symptomatic degenerative disc. Spinal fusion and total disc replacement devices both reduce spinal motion and are associated with adjacent segment disease. Therefore, there is an unmet need for novel and stage-adapted therapies to combat IVDD.

[0008] (2) Small molecule drugs can act as anti-inflammatory, anti-apoptotic, antioxidant, and anabolic agents, which can prevent further degeneration of intervertebral disc cells and enhance their regeneration. So far, several in vivo studies using small molecules to target intervertebral disc degeneration and related degenerative processes have been published. All of these in vivo studies have shown that different small molecules have good therapeutic effects on intervertebral disc degeneration; however, there are currently limited reports on the effects of small molecules on the aging process of nucleus pulposus cells.

[0009] (3) In IVDD-related diseases, the transcription factor ELF1 is upregulated in IVDD, but there is currently no small molecule drug targeting ELF1 during the aging process of nucleus pulposus cells to inhibit their aging and thus delay intervertebral disc degeneration. Summary of the invention

[0010] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a small molecule drug targeting ELF1 for use in the diagnosis and treatment of intervertebral disc degeneration based on research on the function and mechanism of ELF1 in intervertebral disc degeneration.

[0011] The technical solution of the present invention is as follows:

[0012] The present invention provides the use of a small molecule drug targeting ELF1 in the preparation of a drug for treating and / or preventing intervertebral disc degeneration. The small molecule drug is Mycophenolate Mofetil, and its structural formula is as follows:

[0013]

[0014] Furthermore, the small molecule drug inhibits the degeneration process of the intervertebral disc by inhibiting the aging of nucleus pulposus cells, and / or promoting the proliferation of nucleus pulposus cells, and / or promoting the production of extracellular matrix of nucleus pulposus cells.

[0015] Furthermore, the small molecule drug delays the occurrence and development of intervertebral disc degeneration by inhibiting the expression of senescence proteins and degeneration-promoting proteins.

[0016] Furthermore, the drug is composed of a therapeutically effective amount of a small molecule drug and a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutically acceptable carrier and / or excipient includes a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant: preferably, the small molecule drug is used at a concentration of 0nM-55nM.

[0017] The present invention also provides an application of a small molecule drug targeting ELF1 in the preparation of a reagent, wherein the small molecule drug is Mycophenolate Mofetil, and the structural formula is shown above.

[0018] Further, the reagent is used in any one or more of the following aspects:

[0019] (1) Inhibit the senescent phenotype of nucleus pulposus cells in vitro;

[0020] (2) promoting the proliferation phenotype of nucleus pulposus cells in vitro;

[0021] (3) Promote the increase of extracellular matrix of nucleus pulposus cells in vitro;

[0022] (4) Delaying intervertebral disc degeneration and inhibiting the aging of nucleus pulposus cells in vivo.

[0023] Furthermore, the nucleus pulposus cells include primary human nucleus pulposus cells.

[0024] The present invention also provides a pharmaceutical composition for preventing and / or treating intervertebral disc degeneration, wherein the pharmaceutical composition comprises the small molecule drug Mycophenolate Mofetil; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0025] The present invention also provides a method for screening candidate drugs for treating and / or preventing intervertebral disc degeneration, the method comprising the following steps:

[0026] Step 1: Using ELF1 as the drug target, select small molecule compounds through computer-aided design:

[0027] Step 2: Conduct verification experiments on the small molecule compounds screened in step 1, and screen again small molecule compounds that can inhibit nucleus pulposus cell aging, promote cell proliferation, and / or inhibit intervertebral disc degeneration as candidate drugs.

[0028] Furthermore, the verification experiments include cell proliferation experiments, cell senescence experiments, cell cycle experiments, and natural aging intervertebral disc degeneration animal model experiments.

[0029] Furthermore, the candidate drug for treating and / or preventing intervertebral disc degeneration screened in step 2 is Mycophenolate Mofetil.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The present invention discovers for the first time that the small molecule compound Mycophenolate Mofetil has a therapeutic effect on intervertebral disc degeneration. In vivo and in vitro verification experiments show that the small molecule compound Mycophenolate Mofetil has a significant inhibitory effect on the aging of nucleus pulposus cells and significantly inhibits the progression of intervertebral disc degeneration, providing a new method and new idea for the clinical treatment of intervertebral disc degeneration. It also has significant significance in the auxiliary treatment and prevention of intervertebral disc degeneration. Therefore, the small molecule compound Mycophenolate Mofetil has a very good clinical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A diagram showing the location of the AI-predicted binding pocket in the box used for virtual screening in the protein (the protein is in surface representation; the green part is the AI-predicted binding pocket);

[0033] Figure 2 is the box for the protein used for virtual screening (the protein is in the form of surface representation);

[0034] Figure 3 is the protein Ramachandran plot;

[0035] Figure 4 TOP 1 interaction diagram of protein ELF1; blue solid line: hydrogen bond; gray dashed line: hydrophobic interaction;

[0036] Figure 5 TOP 2 interaction diagram of protein ELF1; blue solid line: hydrogen bond; gray dashed line: hydrophobic interaction;

[0037] Figure 6 TOP 3 interaction diagram of protein ELF1; blue solid line: hydrogen bond; gray dashed line: hydrophobic interaction;

[0038] Figure 7 The molecular structure of the Mycophenolate Mofetil compound and its binding configuration with the protein ELF1.

[0039] Figure 8 The overall and detailed images of the surface binding configuration of protein ELF1 and small molecule drugs.

[0040] Fig. 9 This is a detailed diagram of the 2D interaction between protein ELF1 and Top1 small molecule.

[0041] Fig.10 This is the IC50 concentration test graph of Mycophenolate Mofetil compound in nucleus pulposus cells.

[0042] Fig.11 This is the result of Western blotting test on the anti-aging and type II collagen secretion promoting effects of Mycophenolate Mofetil.

[0043] Fig.12 This is the immunofluorescence detection result of the inhibitory effect of Mycophenolate Mofetil on ELF1 and aging protein p21.

[0044] Fig.13 This is the immunofluorescence detection result of Mycophenolate Mofetil's inhibitory effect on ELF1 and p16 and its promoting effect on type II collagen secretion.

[0045] Fig.14 This is the result of the galactosidase experiment on the anti-aging effect of Mycophenolate Mofetil after IL-1β induction.

[0046] Fig.15 This is the result of the galactosidase test of Mycophenolate Mofetil on the anti-aging of replicative aging nucleus pulposus cells.

[0047] Fig.16 Magnetic resonance imaging of the inhibitory effect of Mycophenolate Mofetil on intervertebral disc degeneration in naturally aged mice.

[0048] Fig.17 This is a grayscale value evaluation diagram of the inhibitory effect of Mycophenolate Mofetil on intervertebral disc degeneration detected by magnetic resonance imaging in naturally aged mice.

[0049] Fig.18 This is a graph showing the degeneration grading evaluation of the inhibitory effect of Mycophenolate Mofetil on intervertebral disc degeneration detected by magnetic resonance imaging in naturally aged mice.

[0050] Fig.19 This is a picture of the histological changes after HE and Safranin Fast Green staining detected the inhibitory effect of Mycophenolate Mofetil on intervertebral disc degeneration.

[0051] Fig. 20 This is a histological scoring diagram after HE and Safranin Fast Green staining to detect the inhibitory effect of Mycophenolate Mofetil on intervertebral disc degeneration.

[0052] Fig.21 This is a graph showing the changes in the expression levels of ELF1 and aging protein P21 after Mycophenolate Mofetil inhibited intervertebral disc degeneration using immunofluorescence detection. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific examples. All data analyses involved in the following examples were statistically analyzed using Graphpad Prism9 software, including t-test, variance analysis, etc. A P value of less than 0.05 indicates statistical significance.

[0054] Embodiment 1

[0055] In this example, various compounds that can theoretically inhibit the activity of ELF1 were designed by computer-aided design based on the ELF1 target.

[0056] 1. Virtual screening based on human ELF1 protein structure

[0057] (1) Virtual Screening (VS) is the selection of active compounds based on small molecule databases. By using molecular docking operations between small molecule compounds and drug targets, virtual screening can quickly screen out active compounds with drugability from tens to millions of molecules, greatly reducing the number of experimental screening compounds, shortening the research cycle, and reducing the cost of drug development. It is reported that the positive rate of virtual screening is 5%-30%. The number of cases in which virtual screening has successfully assisted drug design has increased year by year. Virtual screening has become the most promising drug development tool. Virtual screening methods are mainly divided into two types: structure-based virtual screening (SBVS) based on the structure of receptor biomacromolecules and ligand-based virtual screening (LBVS) based on small molecule ligands.

[0058] (2) Parameter description

[0059] In order to ensure the scientific nature of the results of virtual screening, the size of the docking box is usually determined based on the size of the binding pocket to ensure that the box can contain the pocket. The docking box has two important parameters, one is the coordinates of the box center and the other is the size of the box. The following is an explanation of the parameter settings and calculations. Figure 1 and Figure 2 Parameter settings: The center position of the box in xyz coordinates (-8.1, 2.4, -12.9) A: The dimensions of the box in the x, y, and z axes are 20A, 22A, and 20A respectively.

[0060] (3) Species information: Human

[0061] (4) Protein information: ELF1

[0062] (5) Compound library: Physical compound library (LCO020020), total: 11036 compounds

[0063] (6) Active site information: AI site prediction

[0064] (7) Virtual screening software: The software used is Autodockvinavina, adjust the parameters to allocate server resources, and start the program

[0065] (8) Structural evaluation:

[0066] from Figure 3 As can be seen, the number of amino acids outside the 0.002 curve is relatively small, accounting for 3.4%*. Generally, if this probability is less than 5, the structure is considered reasonable, so this structure can be used for subsequent calculations.

[0067] Note: The probability is calculated as: number of amino acids outside the 0.002 curve / total number of protein amino acids.

[0068] (9) Virtual screening: According to the conventional judgment method of calculation, we usually believe that the lower the score result, the stronger the binding affinity between the small molecule and the protein compound may have with the target. After manually checking whether the result structure overlaps or other abnormal phenomena occur, the top ten small molecule data are displayed as follows:

[0069]

[0070] Note: 1) Each molecular docking here obtains the top ten results ranked by the affinity of the molecular conformation after binding to the receptor; 2) The second to fifth columns in the table represent the compound number (Compound Index), affinity (Affinity (kcal / mol)), compound name, and CAS number respectively: Compound Index: the compound number corresponding to the compound in the virtual screening library; Affinity: represents the affinity after the molecular conformation binds to the receptor; Name: compound name; CAS: CAS number of the compound.

[0071] (10) Analysis of the top 10 interactions of protein ELF1 affinity ranking, such as Figure 4-6 shown.

[0072] (11) Detailed action analysis of the top 1 compound, such as Figure 7-9 shown.

[0073] like Fig. 9 As shown, the interaction type description:

[0074] van der Waals: Van der Waals forces, used to describe the interactions between molecules;

[0075] Pi-AlkyAlky: π-alkyl interaction, existing between the π ring and the center of mass of the alkyl group;

[0076] Carbon Hydrogen Bond: A carbon-hydrogen bond interaction between a hydrogen bond donor atom and a carbon atom. When the donor is a polarized carbon atom, the carbon-hydrogen bond interaction is considered to be a weaker hydrogen bond.

[0077] Conventional Hydrogen Bond: Conventional hydrogen bond interaction, which exists between hydrogen bond donor atoms and acceptor atoms;

[0078] Alkyl: Alkyl is hydrophobic and is an interaction between non-polar, non-π system alkyl groups.

[0079] In this implementation case, small molecule drugs for ELF1 were screened based on virtual screening of the human ELF1 protein structure, and the compound with the highest docking score (TOP 1) Mycophenolate Mofetil was screened out.

[0080] Embodiment 2

[0081] This example uses the small molecule drug Mycophenolate Mofetil to detect the proliferation and aging ability of nucleus pulposus cells in vitro.

[0082] The experimental method is as follows:

[0083] 1. IC50 detection of small molecule compounds

[0084] (1) Cell culture: Inoculate cells on a culture plate, ensuring that the cell density in each well is the same. Usually a 96-well plate is used. Incubate the cells in an incubator at 37°C and 5% CO2 for 24 hours to ensure that the cells adhere to the wall and grow stably.

[0085] (2) Treatment compound: Prepare a dilution series of the compound, usually with multiple dilutions (e.g., 2-fold, 5-fold dilutions). Each concentration of the compound needs to be tested in at least three replicate wells.

[0086] (3) Drug treatment: Add different concentrations of compounds to each well, and set up a control group (add the same volume of solvent, such as DMSO). Continue to culture the cells for 24-72 hours, the specific time is determined according to the cell type and the nature of the compound.

[0087] (4) Cell activity detection: Use the CCK-8 cell activity detection kit and perform the detection according to the instructions. Add the detection reagent to each well, incubate for an appropriate time, and measure the absorbance of each well (usually around 450 nm).

[0088] (5) Data analysis: The absorbance value of each well was normalized (relative to the control group) and the IC50 value was calculated using nonlinear regression analysis software (such as GraphPadPrism).

[0089] (6) Result report: Summarize and analyze the experimental results and draw a curve showing the relationship between compound concentration and cell activity.

[0090] 2. Western Blot

[0091] (1) Cell lysis and protein extraction

[0092] ① Collect cells: wash the cells with PBS and scrape the cells with a cell scraper.

[0093] ② Lyse cells: Add an appropriate amount of lysis buffer (containing protease inhibitors and phosphatase inhibitors) and lyse on ice for 30 minutes with interval shaking.

[0094] ③ Centrifugation: Centrifuge at high speed (such as 12,000g) for 15 minutes at 4°C. Collect the supernatant (containing total protein) and discard the precipitate.

[0095] ④ Determine protein concentration: Use Bradford or BCA protein concentration determination kit to determine the protein concentration.

[0096] (2) SDS-PAGE (polyacrylamide gel electrophoresis)

[0097] ①Prepare samples: Mix an appropriate amount of protein sample with 2x or 4x loading buffer and heat at 95°C for 5 minutes (to denature the protein).

[0098] ② Loading samples: Load the protein samples into the SDS-PAGE gel wells and load the prestained protein molecular weight standards at the same time.

[0099] ③Electrophoresis: Run electrophoresis at an appropriate voltage (usually 80V to 120V) until the dye front reaches the bottom of the gel.

[0100] (3) Transfer (protein transfer to PVDF or nitrocellulose membrane)

[0101] ① Activation of membrane: PVDF membrane: soak in methanol for a few seconds, then equilibrate in transfer buffer. Nitrocellulose membrane: equilibrate directly in transfer buffer.

[0102] ② Assemble the transfer clip: Soak the filter paper and sponge for transfer in the transfer buffer. Assemble the sandwich structure of sponge, filter paper, gel, membrane, filter paper, and sponge in sequence, making sure there are no bubbles.

[0103] ③ Transfer: Transfer the membrane at an appropriate voltage (usually 100V, 1 hour).

[0104] (4) Membrane sealing

[0105] Blocking membrane: Incubate the membrane in blocking solution for 1 hour at room temperature with gentle agitation.

[0106] (5) Primary antibody incubation

[0107] Incubate with primary antibody: Incubate the membrane in primary antibody dilution (primary antibody diluted in TBS-T), usually overnight at 4°C with gentle agitation.

[0108] (6) Wash the membrane: Wash the membrane three times with TBS-T, 10 minutes each time.

[0109] (7) Secondary antibody incubation: Incubate the membrane in secondary antibody diluent (secondary antibody diluted in TBS-T), usually for 1 hour at room temperature with gentle shaking.

[0110] (8) Wash the membrane: Wash the membrane three times with TBS-T, 10 min each time.

[0111] (9) Color development: Incubate the membrane with ECL substrate according to the manufacturer's instructions. Use an imaging system or X-ray film to detect the luminescent signal of the target protein.

[0112] 3. Cell Immunofluorescence

[0113] (1) Cell culture: Cultivate cells in appropriate culture medium until they reach an appropriate density.

[0114] The day before the experiment, seed cells onto coverslips or culture dishes and allow them to attach and reach appropriate density.

[0115] (2) Cell fixation: Remove the culture medium and gently wash the cells with PBS. Add pre-cooled 4% paraformaldehyde (PFA) solution and fix the cells for 10-20 minutes (room temperature or 4°C). After fixation, remove PFA and wash the cells 3 times with cold PBS, 5 minutes each time.

[0116] (3) Cell permeabilization: Treat cells with a permeabilizing agent (such as 0.1%-0.5% TritonX-100 in PBS) for 5-15 minutes. After permeabilization, wash cells with PBS three times, 5 minutes each time.

[0117] (4) Blocking: Add blocking solution (such as 5%-10% BSA or normal serum in PBS) and incubate at room temperature for 30-60 minutes to block nonspecific binding sites.

[0118] (5) Primary antibody incubation: Cover the cells with diluted primary antibody solution (diluted in blocking solution) and incubate at 4°C overnight or at room temperature for 1-2 hours. After incubation, wash the cells three times with PBS, 5 minutes each time.

[0119] (6) Secondary antibody incubation: Cover the cells with fluorescently labeled secondary antibody solution (diluted in blocking solution) and incubate at room temperature in the dark for 1 hour. After incubation, wash the cells three times with PBS, 5 minutes each time.

[0120] (7) Nuclear staining: Stain with DAPI (1 μg / mL in PBS) and incubate at room temperature in the dark for 5 minutes. After nuclear staining, wash the cells three times with PBS, 5 minutes each time.

[0121] (8) Mounting and sealing: Move the coverslip onto the slide and seal the slide with an anti-fluorescence quenching reagent (such as Fluoromount-G). Gently cover the coverslip to ensure there are no bubbles.

[0122] (9) Microscope observation: Use a fluorescence microscope or a confocal microscope to observe cells and record images.

[0123] 4.Senescence-Associated β-Galactosidase Staining (SA-β-Gal)

[0124] (1) Cell culture: Cultivate cells in appropriate culture medium until they reach an appropriate density.

[0125] The day before the experiment, seed cells onto culture dishes or coverslips and allow them to attach and reach the appropriate density.

[0126] (2) Cell fixation: Remove the culture medium and gently wash the cells with PBS. Add fixative (2%-4% formaldehyde or glutaraldehyde in PBS) and fix at room temperature for 5-10 minutes. After fixation, remove the fixative and wash the cells three times with PBS, 5 minutes each time.

[0127] (3) Prepare staining solution: Prepare SA-β-Gal staining buffer and make sure the pH is adjusted to 6.0. Add X-gal, K4Fe(CN)6 and K3Fe(CN)6 to the staining buffer and mix well.

[0128] (4) Staining: Add the staining solution to the fixed cells, ensuring that the cells are completely covered. Incubate the cells at 37°C (without CO2) overnight (usually 12-16 hours), away from light.

[0129] (5) Observation: After staining, wash the cells gently with PBS three times, 5 minutes each time. Observe the staining results under a microscope. Aged cells will appear blue.

[0130] In this embodiment, we first detected the IC50 concentration of the small molecule compound Mycophenolate Mofetil, which was 55.4nmol ( Fig.10 ). After the nucleus pulposus cells were treated with Mycophenolate Mofetil concentration gradient (20nmol and 40nmol), protein immunoblotting (Western Blot) was used to detect the expression levels of aging proteins and degenerative proteins. The experimental results showed that with the increase of Mycophenolate Mofetil concentration, the expression of aging protein P16 was significantly inhibited, and the expression level of degenerative protein CollagenⅡ was increased ( Fig.11). In addition, we further used IL-1β to construct a nucleus pulposus cell degeneration model and then used Mycophenolate Mofetil for treatment. Finally, using immunofluorescence staining, we found that the addition of IL-1β can significantly increase the expression of ELF1 protein, aging proteins P21 and P16, while reducing the expression level of degeneration protein CollagenⅡ. After adding IL-1β and using Mycophenolate Mofetil for treatment, the expression level of ELF1 protein, aging proteins P21 and P16, and the expression level of degeneration protein CollagenⅡ can be significantly inhibited ( Fig.12 , Fig.13 In addition, the results of the β-galactosidase aging staining experiment showed that Mycophenolate Mofetil can significantly reduce the aging state of nucleus pulposus cells ( Fig.14 , Fig.15 ). The above results indicate that the small molecule compound Mycophenolate Mofetil can reduce the expression level of senescence protein in nucleus pulposus cells by targeting and regulating the expression level of ELF1, thereby inhibiting the senescence of nucleus pulposus cells.

[0131] Embodiment 3

[0132] This example uses the small molecule drug Mycophenolate Mofetil to conduct in vivo experiments on aging mice to verify its effect on intervertebral disc degeneration.

[0133] 1. Observation of intervertebral disc changes in aged mice treated with Mycophenolate Mofetil

[0134] At the beginning of the experiment, we selected 6 young mice aged 2 months and 12 old mice aged 24 months for follow-up study. The 12 old mice were randomly divided into 6 groups. One group of old mice was selected for Mycophenolate Mofetil treatment. 30 mg kg was administered daily by gastric gavage. -1 ·day -1 of MMF, which was diluted in 200 μl of water. Since Mycophenolate Mofetil is insoluble in water, we prepared each dose individually and stirred the mixture vigorously to obtain a suspension, which was then administered to mice immediately before the precipitation of MMF. The young group and the non-drug treated group of old mice received the same volume of water daily by the same administration route.

[0135] 2. Small Animal MRI Lumbar Spine Imaging Experiment

[0136] (1) Anesthesia: According to the animal's weight and experimental requirements, select an appropriate anesthetic and perform anesthesia. Isoflurane inhalation anesthesia is generally used to ensure that the animal remains painless and still during the imaging process.

[0137] (2) Fixation: The anesthetized animal is fixed in a dedicated animal fixation device to ensure that the lumbar region is in the best imaging position. The fixation device usually contains a support board and restraints to prevent the animal from moving.

[0138] (3) Imaging coil: Select an appropriate imaging coil (such as a body coil or a surface coil) and place it above the animal's lumbar region.

[0139] (4) Temperature control system: Connect the temperature control system to ensure that the animal maintains normal body temperature (approximately 37°C) during imaging.

[0140] (5) MRI imaging: A quick pre-scan was performed to ensure that the animal's lumbar region was correctly positioned within the imaging area.

[0141] (6) Imaging parameter setting: Set appropriate imaging parameters according to experimental requirements. Commonly used imaging sequences include: T2-weighted imaging: used to display the structure of the intervertebral disc, spinal cord, and surrounding soft tissue. T1-weighted imaging: used to display bone structure and adipose tissue. Fat suppression sequence: used to improve soft tissue contrast.

[0142] (7) Scanning: Start the MRI scan, which usually takes 10-30 minutes, depending on the imaging sequence used and the resolution required.

[0143] (8) Data processing and image reconstruction: High-resolution images of the lumbar spine were generated using the image reconstruction software of the MRI scanner.

[0144] (9) Data analysis: Use image processing software (such as ImageJ, Analyze, etc.) to perform quantitative analysis and three-dimensional reconstruction of the lumbar spine and surrounding tissues.

[0145] 3. Tissue HE (Hematoxylin and Eosin) staining

[0146] (1) Sample fixation: The tissue samples are fixed in 10% neutral buffered formaldehyde solution, usually for 24 hours.

[0147] (2) Dehydration: The fixed tissue samples were placed in different concentrations of ethanol for dehydration: 70%, 95%, and 100% ethanol, each step for 30 minutes.

[0148] (3) Transparency: The dehydrated tissue sample is placed in xylene, usually twice, each time for 30 minutes, to make the tissue transparent.

[0149] (4) Wax immersion and embedding: The transparent tissue sample is immersed in molten paraffin, usually twice, each time for 1 hour. The tissue sample is embedded in a paraffin block.

[0150] (5) Sectioning: Use a microtome to cut the paraffin-embedded tissue into 3-5 μm thick slices. Spread the slices flat on a glass slide and bake them in a 60°C oven for 30 minutes to 1 hour to ensure that they adhere firmly.

[0151] (6) Dewaxing and hydration: Dewaxing the slides in xylene for 5 minutes each step, usually twice. Sequentially place the slides in 100%, 95%, 70%, and 50% ethanol for 2-3 minutes each step to rehydrate the tissue. Finally, rinse with distilled water.

[0152] (7) Hematoxylin staining: Immerse the slide in hematoxylin staining solution and stain for 5-10 minutes. Rinse with running water for several minutes until the excess stain is washed off.

[0153] (8) Blueing: Place the slide in ammonia or lithium carbonate solution for 1-2 minutes to turn the hematoxylin-stained cell nuclei blue. Rinse with running water for several minutes.

[0154] (9) Eosin staining: Immerse the slide in eosin staining solution and stain for 2-5 minutes. Rinse with running water for a few seconds until the excess stain is washed off.

[0155] (10) Dehydration and clearing: Place the stained slides in 50%, 70%, 95%, and 100% ethanol in turn for 2-3 minutes each for dehydration. Then place the slides in xylene for 5 minutes each step, usually twice, to make the tissue clear.

[0156] (11) Sealing: Add neutral gum sealing agent to the slide and carefully cover it with a coverslip to avoid bubbles. After sealing, dry it at room temperature.

[0157] 4. Safranin O / Fast Green Staining

[0158] (1) Sample fixation: The tissue samples are fixed in 10% neutral buffered formaldehyde solution, usually for 24 hours.

[0159] (2) Dehydration: The fixed tissue samples were placed in different concentrations of ethanol for dehydration: 70%, 95%, and 100% ethanol, each step for 30 minutes.

[0160] (3) Transparency: The dehydrated tissue sample is placed in xylene, usually twice, each time for 30 minutes, to make the tissue transparent.

[0161] (4) Wax immersion and embedding: The transparent tissue sample is immersed in molten paraffin, usually twice, each time for 1 hour. The tissue sample is embedded in a paraffin block.

[0162] (5) Sectioning: Use a microtome to cut the paraffin-embedded tissue into thin slices of 5-10 μm thickness. Spread the slices flat on a glass slide and bake them in a 60°C oven for 30 minutes to 1 hour to ensure that they adhere firmly.

[0163] (6) Dewaxing and hydration: Dewaxing the slides in xylene for 5 minutes each step, usually twice. Sequentially place the slides in 100%, 95%, 70%, and 50% ethanol for 2-3 minutes each step to rehydrate the tissue. Finally, rinse with distilled water.

[0164] (7) Safranin staining: Immerse the slide in 0.5% safranin staining solution and stain for 5-10 minutes. Rinse with distilled water for a few seconds until the excess stain is washed off.

[0165] (8) Acetic acid wash: Immerse the slide in 1% acetic acid for a few seconds to remove background staining. Rinse with distilled water for a few seconds.

[0166] (9) Fast green staining: Immerse the slide in 0.1% fast green staining solution and stain for 2-5 minutes. Wash quickly with 95% ethanol for a few seconds to remove background staining.

[0167] (10) Dehydration and clearing: Place the stained slides in 95% and 100% ethanol, respectively, for 2-3 minutes each, to dehydrate. Then place the slides in xylene, for 5 minutes each step, usually twice, to make the tissue clear.

[0168] (11) Sealing: Add neutral gum sealing agent to the slide and carefully cover it with a coverslip to avoid bubbles. After sealing, dry it at room temperature.

[0169] 5. Tissue Immunofluorescence Staining

[0170] (1) Sample fixation and embedding: The tissue samples are fixed in 10% neutral buffered formaldehyde solution, usually for 24 hours. After dehydration and transparency, the tissue samples are embedded in paraffin.

[0171] (2) Sectioning and slide preparation: Use a microtome to cut the paraffin-embedded tissue into thin slices of 5-10 μm thickness.

[0172] Spread the slice flat on a glass slide and bake it in a 60°C oven for 30 minutes to 1 hour to allow it to adhere firmly.

[0173] (3) Dewaxing and hydration: Dewaxing the slides in xylene for 5 minutes each step, usually twice. Sequentially place the slides in 100%, 95%, 70%, and 50% ethanol for 2-3 minutes each step to rehydrate the tissue. Finally, rinse with distilled water.

[0174] (4) Antigen retrieval: Immerse the slides in preheated antigen retrieval solution (such as citrate buffer, pH 6.0) and treat at high temperature in a microwave oven for 10-20 minutes. Allow the slides to cool at room temperature and then wash them three times with PBS for 5 minutes each.

[0175] (5) Blocking: Immerse the slide in blocking solution (such as 5% BSA or normal serum in PBS) and incubate at room temperature for 30-60 minutes to block nonspecific binding sites.

[0176] (6) Primary antibody incubation: Cover the tissue sections with diluted primary antibody solution (diluted in blocking solution) and incubate at 4°C overnight or at room temperature for 1-2 hours. After incubation, wash the slides three times with PBS, 5 minutes each time.

[0177] (7) Secondary antibody incubation: Cover the tissue sections with fluorescently labeled secondary antibody solution (diluted in blocking solution) and incubate at room temperature in the dark for 1 hour. After incubation, wash the slides three times with PBS, 5 minutes each time.

[0178] (8) Nuclear staining: Stain with DAPI (1 μg / mL in PBS) and incubate at room temperature in the dark for 5 minutes. After nuclear staining, wash the slides with PBS three times, 5 minutes each time.

[0179] (9) Sealing: Add anti-fluorescence quenching mounting agent (such as Fluoromount-G) on the slide and carefully cover it with a coverslip to avoid bubbles. After sealing, dry at room temperature.

[0180] (10) Microscope observation: Use a fluorescence microscope or a confocal microscope to observe tissue sections and record images.

[0181] In this implementation case, we further verified the role of Mycophenolate Mofetil in intervertebral disc degeneration through in vivo experiments. We selected 6 2-month-old young mice and 12 24-month-old old mice for follow-up research. The 12 old mice were randomly divided into 6 mice in each group. One group of old mice was selected for Mycophenolate Mofetil treatment. Magnetic resonance imaging results showed that the intervertebral disc degeneration of mice in the natural aging group was significantly aggravated, while taking Mycophenolate Mofetil treatment could significantly delay the process of intervertebral disc degeneration ( Figure 16-18In addition, HE staining and safranin fast green staining were used to further evaluate the severity of intervertebral disc degeneration. The results showed that the density of nucleus pulposus cells decreased in the intervertebral discs of naturally aged mice, and the number of myeloid cells (nuclear cells) in the nucleus pulposus gradually changed into fibroblast-like cells. However, treatment with Mycophenolate Mofetil significantly delayed the decrease in the density of nucleus pulposus cells and the number of nucleus pulposus cells ( Figure 19-20 At the same time, immunofluorescence results showed that the expression of ELF1 and P21 was significantly upregulated in the intervertebral disc of naturally aged mice, while treatment with Mycophenolate Mofetil significantly reduced the expression of ELF1 and P21 ( Fig.21 ). Therefore, taking Mycophenolate Mofetil can inhibit the aging of nucleus pulposus cells in mice and delay intervertebral disc degeneration.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a small molecule drug targeting ELF1 in the preparation of a drug for treating and / or preventing intervertebral disc degeneration, characterized in that: The small molecule drug is Mycophenolate Mofetil, and its structural formula is as follows:

2. The use according to claim 1, characterized in that: The small molecule drug inhibits the degeneration process of the intervertebral disc by inhibiting the aging of nucleus pulposus cells, and / or promoting the proliferation of nucleus pulposus cells, and / or promoting the generation of extracellular matrix of nucleus pulposus cells.

3. The use according to claim 1, characterized in that: The small molecule drug is used at a concentration of 0 nM-55 nM.

4. The use according to claim 3, characterized in that: The drug is composed of a therapeutically effective amount of a small molecule drug and a pharmaceutically acceptable carrier and / or excipient; The pharmaceutically acceptable carriers and / or excipients include diluents, adhesives, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

5. Use of a small molecule drug targeting ELF1 in the preparation of a reagent, characterized in that: The small molecule drug is Mycophenolate Mofetil, and the structural formula of the small molecule drug is as follows:

6. The use according to claim 5, characterized in that: The reagent is used in any one or more of the following aspects: (1) Inhibit the senescent phenotype of nucleus pulposus cells in vitro; (2) promoting the proliferation phenotype of nucleus pulposus cells in vitro; (3) Promote the increase of extracellular matrix of nucleus pulposus cells in vitro; (4) Delaying intervertebral disc degeneration and inhibiting the aging of nucleus pulposus cells in vivo.

7. The use according to claim 6, characterized in that: The nucleus pulposus cells include primary human nucleus pulposus cells.

8. A pharmaceutical composition for preventing and / or treating intervertebral disc degeneration, characterized in that: The pharmaceutical composition comprises the small molecule drug as described in claim 1.

9. A method for screening candidate drugs for treating and / or preventing intervertebral disc degeneration, characterized in that: The method comprises the following steps: Step 1: Using ELF1 as a drug target, screen small molecule compounds through computer-aided design: Step 2: Conduct verification experiments on the small molecule compounds selected in step 1, and screen again for small molecule compounds that can inhibit nucleus pulposus cell aging, promote cell proliferation, and / or inhibit intervertebral disc degeneration as candidate drugs.

10. The method according to claim 9, characterized in that The candidate drug for treating and / or preventing intervertebral disc degeneration screened in step 2 is Mycophenolate Mofetil, and the structural formula of the small molecule drug is as follows:

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