Hypoxia-inducible factor alpha inhibitor and application thereof

Abstract

The invention discloses a hypoxia-inducible factor alpha inhibitor and application thereof, it is found that ipriflavone serves as the hypoxia-inducible factor alpha inhibitor and plays an important role in diseases such as hypoxia gastrointestinal mucosal lesion, inflammation and tumors, the safety of ipriflavone serving as a clinical common medicine is widely verified, and the ipriflavone can be used as a hypoxia-inducible factor alpha inhibitor. The invention finds that ipriflavone can improve the hydroxylation effect of prolyl hydroxylase structural domain protein 2 on hypoxia-inducible factor-alpha so as to reduce the expression of hypoxia-inducible factor-alpha, can inhibit hypoxia-inducible factor 1 alpha and hypoxia-inducible factor 2 alpha at the same time, and provides a wide prospect for treating hypoxia-inducible factor alpha overexpression diseases. The ipriflavone can effectively inhibit the expression of the hypoxia-inducible factor alpha in vivo and in vitro, and can be used for treating hypoxia-inducible factor-alpha overexpression diseases, including hypoxia gastrointestinal mucosa injury, inflammation, tumor and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a hypoxia inducible factor alpha inhibitor and application thereof. Background Art

[0002] Hypoxia-inducible factors are the main regulators of cell perception and adaptation to hypoxic environment, including hypoxia-inducible factor-α (HIF-α; mainly including hypoxia-inducible factor 1α and hypoxia-inducible factor 2α) and hypoxia-inducible factor 1β (HIF-1β). Among them, the α subunit is the main regulator of the transcriptional activity of hypoxia-inducible factor (hypoxia-inducible factor α increases in hypoxia), while the constitutive expression of the hypoxia-inducible factor 1β subunit is independent of the intracellular oxygen content. In addition to hypoxia-inducible factor, another important cellular oxygen sensor is the prolyl hydroxylase domain protein (also known as prolyl hydroxylases, prolyl hydroxylase domain enzymes; Prolyl hydroxylase domain proteins, Prolyl hydroxylases, Prolyl hydroxylase domain enzymes; PHDs), including prolyl hydroxylase domain protein 1, prolyl hydroxylase domain protein 2 and prolyl hydroxylase domain protein 3, among which prolyl hydroxylase domain protein 2 plays a major role in regulating the expression of hypoxia-inducible factor-α. Prolyl hydroxylase domain proteins regulate hypoxia-inducible factor-α expression through the following pathway: prolyl hydroxylase domain proteins hydroxylate proline residues in the oxygen-dependent degradation domain of hypoxia-inducible factor-α subunits (hypoxia-inducible factor 1α: proline 402 and 564; hypoxia-inducible factor-2α: proline 405 and proline 531), and the hydroxylated hypoxia-inducible factor α subunits are recognized by the von Hippel-Lindau E3 ubiquitin ligase complex and rapidly degraded through the polyubiquitination / proteasome pathway.

[0003] Hypoxia-inducible factor α is also a major regulator of cellular basal metabolism, and participates in the occurrence and development of various tumors and inflammations in the hypoxic microenvironment of tumors and inflammation. Hypoxia-inducible factor α affects the energy metabolism, proliferation and apoptosis of tumor cells by participating in the transcriptional regulation of multiple target genes, causing cells and tissues to produce a series of reactions to adapt to the hypoxic environment, while promoting tumor angiogenesis, and increasing the invasiveness of the tumor itself and its resistance to radiotherapy and chemotherapy. Inhibition of hypoxia-inducible factor α may inhibit tumor proliferation, metabolic reprogramming, angiogenesis, invasion and metastasis. In addition, inhibition of hypoxia-inducible factor α can regulate the overactivation of certain types of immune cells in inflammation. The use of hypoxia-inducible factor α inhibitors alone or in combination can improve the efficacy of existing treatment options, thereby benefiting patients. It is particularly noteworthy that recent studies have found that hypoxia-inducible factor α can induce the expression of programmed cell death ligand 1 (PD-L1) in tumor cells. The combination of hypoxia-inducible factor α inhibitors with antibodies or inhibitors of programmed cell death protein 1 (PD-1) and its ligand 1 can enhance the therapeutic effect in a variety of tumors.

[0004] In plateau hypoxic gastrointestinal mucosal injury, hypoxia-inducible factor-α mediates ferroptosis of mucosal cells, and inhibition of hypoxia-inducible factor-α can significantly alleviate hypoxic gastrointestinal mucosal injury.

[0005] At present, a large number of preclinical studies have shown that inhibiting hypoxia-inducible factor α can treat inflammatory diseases such as sepsis, inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis. Hypoxia-inducible factor α is a key regulator of immune cell function. Immune cells gather at the site of inflammation and are in a rapidly hypoxic environment, which then induces immune cells to transcribe hypoxia-inducible factor α. Hypoxia-inducible factor α is activated during the immune response process, increasing the transcription of inflammatory target genes downstream of hypoxia-inducible factor α, causing high expression of cell molecules / chemokines, adhesion factors and pro-inflammatory response enzymes, and activating inflammasomes, leading to a high response to inflammation.

[0006] Ipriflavone is a plant isoflavone that is clinically used to treat osteoporosis with high safety and effectiveness. In addition to anti-osteoporosis, ipriflavone has also been reported to play a therapeutic role in Alzheimer's disease rat models due to its antioxidant effect. In addition, ipriflavone has also been shown to play an anti-esophageal squamous cell carcinoma and breast cancer bone metastasis role in animal models. Currently, ipriflavone has only been found to play a therapeutic role in environmental metal-induced neurobehavioral abnormalities and cognitive dysfunction and insulin resistance-induced brain tissue dysfunction. There is a lack of relevant research on ipriflavone in the treatment of other types of inflammation, especially as a hypoxia-inducible factor α inhibitor. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a hypoxia-inducible factor α inhibitor and its application. As a hypoxia-inducible factor α inhibitor, ipriflavone plays an important role in hypoxic gastrointestinal mucosal damage, inflammation, tumors and other diseases.

[0008] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0009] Application of ipriflavone as hypoxia inducible factor α inhibitor.

[0010] Application of ipriflavone as a hypoxia-inducible factor α inhibitor in the preparation of drugs for treating diseases with high expression of hypoxia-inducible factor α.

[0011] Furthermore, the diseases with high expression of hypoxia-inducible factor-α include hypoxic gastrointestinal mucosal damage, inflammation, and tumors.

[0012] Furthermore, the ipriflavone is used alone or in combination with other drugs.

[0013] Furthermore, the dosage form of the drug is tablets, powders, granules, hard capsules or pills.

[0014] Furthermore, the administration methods of the drug include oral administration, injection or external application, and the injection administration method includes intravenous injection, subcutaneous injection or intramuscular injection.

[0015] The reported hypoxia-inducible factor α inhibitors are hypoxia-inducible factor 1α or hypoxia-inducible factor 2α inhibitors, and only a few drugs have entered Phase II clinical trials, and their safety remains to be studied. As a commonly used clinical drug, ipriflavone has been widely verified for its safety and can inhibit both hypoxia-inducible factor 1α and hypoxia-inducible factor 2α, providing broad prospects for the treatment of diseases with overexpression of hypoxia-inducible factor α.

[0016] Hypoxia-inducible factor α inhibitors can also be used in combination with other drugs to enhance the therapeutic effect. The combination of hypoxia-inducible factor α inhibitors and immune checkpoint inhibitors can benefit patients by affecting the expression of immune checkpoints (such as PD-L1, PD-1, and HLA-G), and can also improve the response of immune cells in the tumor microenvironment. In addition to solid tumors, hypoxia-inducible factor α inhibitors may also play a therapeutic role in non-solid tumors such as leukemia.

[0017] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0018] The present invention finds that ipriflavone, as a hypoxia-inducible factor-α inhibitor, plays an important role in hypoxic gastrointestinal mucosal damage, inflammation, tumors and other diseases. On the first hand, it is proved that ipriflavone can increase the hydroxylation effect of prolyl hydroxylase domain protein 2 on hypoxia-inducible factor-α, thereby reducing the expression of hypoxia-inducible factor-α. On the second hand, it is proved that ipriflavone can effectively inhibit the expression of hypoxia-inducible factor-α both in vivo and in vitro, and can be used to treat diseases with overexpression of hypoxia-inducible factor-α, including hypoxic gastrointestinal mucosal damage, inflammation, tumors and the like.

[0019] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are part of this application and are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0021] Figure 1 Schematic diagram of restriction enzyme digestion-mass spectrometry analysis technology.

[0022] Figure 2 The Venn diagram of the number of proteins with different abundance and structure after ipriflavone intervention.

[0023] Figure 3 This is a QR code image of the structural change region of the prolyl hydroxylase domain protein 2 after ipriflavone intervention.

[0024] Figure 4 The binding mode of ipriflavone to prolyl hydroxylase domain protein 2 predicted by AutodockTools.

[0025] Figure 5 Western blotting was used to analyze the protein expression in normal human gastric epithelial cells and normal human small intestinal epithelial cells after intervention with ipriflavone under hypoxia.

[0026] Figure 6 Western blotting analysis of protein expression in normal human gastric epithelial cells and normal human small intestinal epithelial cells after intervention with ipriflavone and inhibition of prolyl hydroxylase domain-containing protein 2 under hypoxia.

[0027] Figure 7 Western blotting was used to detect endogenous von Hippel-Lindau immunoprecipitates after ipriflavone intervention, MG132 intervention, and inhibition of prolyl hydroxylase domain protein 2 under hypoxia.

[0028] Figure 8These are the crystal violet staining results and cell viability detection results of normal human gastric epithelial cells after overexpression of hypoxia-inducible factor-α under hypoxia.

[0029] Fig. 9 These are the crystal violet staining results and cell viability detection results of normal human small intestinal epithelial cells after overexpression of hypoxia-inducible factor-α under hypoxia.

[0030] Fig.10 This is a transmission electron microscopic image of normal human gastric epithelial cells treated with ipriflavone under hypoxia.

[0031] Fig.11 This is a transmission electron micrograph of normal human small intestinal epithelial cells treated with ipriflavone under hypoxia.

[0032] Fig.12 The bar graph shows the levels of reactive oxygen species and 4-hydroxynonenal in hypoxic normal human gastric epithelial cells and normal human small intestinal epithelial cells after ipriflavone intervention.

[0033] Fig.13 These are the crystal violet staining results and cell viability detection results of normal human gastric epithelial cells and normal human small intestinal epithelial cells after ipriflavone intervention and inhibition of prolyl hydroxylase domain protein 2 under hypoxia.

[0034] Fig.14 Western blotting was used to analyze the protein expression in the gastric and small intestinal mucosa of mice after oral administration of ipriflavone.

[0035] Fig.15 Representative images of hematoxylin-eosin staining of the stomach, duodenum, jejunum, and ileum of hypoxic mice after oral administration of ipriflavone.

[0036] Fig.16 The histopathological scores of the stomach, duodenum, jejunum, and ileum of hypoxic mice after oral administration of ipriflavone were obtained.

[0037] Fig.17 The bar graph is a graph of serum FITC-Dextran levels in hypoxic mice after oral administration of ipriflavone.

[0038] Fig.18 It is a bar graph of the levels of reactive oxygen species and 4-hydroxynonenal in the gastric mucosa of hypoxic mice after oral administration of ipriflavone.

[0039] Fig.19 This is a bar graph showing the levels of reactive oxygen species and 4-hydroxynonenal in the small intestinal mucosa of hypoxic mice after oral administration of ipriflavone.

[0040] Fig. 20 The cytokine levels after intervention with macrophage cell line Raw264.7 overexpressing hypoxia-inducible factor-1α, hypoxia-inducible factor-2α, lipopolysaccharide and ipriflavone.

[0041] Fig.21 This is the cell viability level after knocking down hypoxia-inducible factor-1α and intervening with ipriflavone in the colon cancer cell line HCT116.

[0042] Fig. 22 The cell viability level after intervention with breast cancer cell line MDA-MB-231 overexpressing hypoxia-inducible factor-1α, cisplatin and ipriflavone.

[0043] Fig.23 To investigate the expression of programmed death ligand 1 protein in colon cancer cell line MC38 overexpressing hypoxia-inducible factor-1α, hypoxia-inducible factor-2α and ipriflavone intervention.

[0044] Fig.24 Mice were inoculated with colon cancer cell line MC38 xenografts and then given control reagents, ipriflavone, cytotoxic T-lymphocyte-associated protein 4 monoclonal antibody, programmed death receptor 1 monoclonal antibody, ipriflavone + cytotoxic T-lymphocyte-associated protein 4 monoclonal antibody, and the changes in tumor size were measured every 3 days.

[0045] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0047] Example 1

[0048] (1) Restriction enzyme digestion-mass spectrometry analysis technology

[0049] Lysis buffer containing 10% protease inhibitors was added to the samples, and the supernatant was collected after centrifugation at 14100×g for 20 minutes. The protein concentration was determined by Bradford method. Each sample was divided into pure trypsin sample and restriction digestion sample. Proteinase K was added to the restriction digestion sample at an enzyme / substrate (E:S) ratio of 1:100 (weight / weight) and incubated at 25°C for 1 minute. All samples were heated at 100°C for 5 minutes to terminate the digestion of proteinase K, and then the samples were cooled to room temperature. SDC was added to all samples (trypsin group and restriction digestion group). To reduce cysteine ​​residues, TCEP was added to the sample, and then IAA was added and incubated at room temperature in the dark for 60 minutes. Lysis buffer was added at a ratio of m(sample protein): m(lysine) = 100:1, and incubated at 37°C for 4 hours; SDC in the sample was diluted to 1% and the pH was adjusted to 7-9. Trypsin was added at a ratio of m(sample protein):m(trypsin) = 100:1, and digested overnight at 37°C. After digestion, FA was added to adjust the pH of the system to <3. Centrifuge at 16,000×g for 10 minutes at 4°C to obtain the supernatant. The sample was then immediately analyzed using mass spectrometry.

[0050] Nano-LC-MS / MS analysis of tryptic peptides was performed on a quadrupole-Orbitrap mass spectrometer (Q Exactive HF-X, Thermo Fisher Scientific) and an EASYnLC1200 ultrahigh pressure system (Thermo Fisher Scientific) with a nano-electrospray ionization source. 500 ng of peptides were loaded onto a 25 cm column (150 μm inner diameter, packed with ReproSil-PurC18-AQ 1.5 μm silica beads). Peptide separation was performed using a gradient method from 8% B to 12% B in 8 min, from 12% B to 30% B in 55 min, to 40% B in 12 min, followed by a gradient wash of 95% B for 14 min at a flow rate of 600 nl per minute, with solvent A being 0.1% formic acid in water and solvent B being 80% ACN and 0.1% formic acid in water. The total run time was 90 min. The column temperature was maintained at 60 °C using an oven developed in-house. Briefly, the mass spectrometer was operated in “top-40” data-dependent mode, and MS spectra were collected in the Orbitrap mass analyzer (120,000 resolution, 350–1500 m / z range) with an automatic gain control (AGC) target of 3E6 and a maximum ion injection time of 80 ms. The most intense ions were isolated from the full scan with an isolation width of 1.6 m / z. MS / MS spectra were collected in the Orbitrap (15,000 resolution) after high-energy collision dissociation (HCD) at a normalized collision energy (NCE) of 27, with an AGC target of 5E4 and a maximum ion injection time of 45 ms. Dynamic exclusion of precursors was enabled and lasted for 16 s.

[0051] All raw files were analyzed using the Proteome Discoverer suite (version 2.4, Thermo Fisher Scientific). MS2 spectra were searched against the uniprot Mus musculus proteome database (55260 target sequences, downloaded on 2023-03-07). The Sequest HT search engine was used with the following parameters: semi-trypsin, a maximum of two missed cleavages, a minimum peptide length of 6, fixed carbamylation of cysteine ​​residues (+57.02146 Da), variable modification by oxidation of methionine residues (+15.99492 Da), a precursor mass tolerance of 15 ppm, and a fragment mass tolerance of 0.02 Da for MS2 spectra collected in the Orbitrap. Peptide spectral matches and peptides were filtered using Percolator to a false discovery rate of less than 1%. After spectral assignment, peptides were combined into proteins and further filtered based on the combined probability of their constituent peptides, with a final FDR of 1%. By default, the protein with the highest match or "master protein" is the protein with the highest number of unique peptides and the lowest percent peptide coverage value (i.e., the longest protein). Only unique peptides and razor peptides (i.e., fitted peptides) are considered for quantification.

[0052] GO and InterPro (IPR) analyses were performed against a non-redundant protein database using the interroscan-5 program, and protein families and pathways were analyzed using the COG and KEGG databases. Potential interacting proteins were predicted based on related species using the STRING-db server (http: / / string.embl.de / ).

[0053] See the flowchart Figure 1 The results are shown in Figure 2 and Figure 3 , Figure 2 It showed that there were 302 potential targets with structural differences, 40 potential targets with abundance differences, and 14 potential targets with both structural and abundance differences, among which prolyl hydroxylase domain protein 2 was one of the structural difference targets. Figure 3 It was shown that ipriflavone caused structural changes in the 140-152 amino acid sequence of prolyl hydroxylase domain protein 2.

[0054] (2) Molecular docking

[0055] The protein of prolyl hydroxylase domain protein 2 was obtained from Uniprot database. The structure of ipriflavone was downloaded from Pubchem database for docking. The prolyl hydroxylase domain protein 2 protein was docked with ipriflavone. Ipriflavone was designated as ligand and prolyl hydroxylase domain protein 2 as receptor. PyMOL (version 4.3.0) software (https: / / pymol.org / ) was used to separate the original ligand and protein structure, dehydrate and remove organic matter, AutodockTools (http: / / mgltools.scripps.edu / downloads) was used to add hydrogen, check the charge, designate the atom type as AD4 type, calculate gasteiger, and build the docking grid box of the protein structure. In addition, the chemical composition (small molecule ligand) should determine the root, and the torsional bond of the ligand should be selected in AutodockTools. Finally, in AutodockTools, the format of both protein structure and small molecule ligand should be converted from ".PDB" to ".PDBQT" for further docking. After docking using Vina, the scores of the docking combination of prolyl hydroxylase domain protein 2 protein and ipriflavone were calculated, and three-dimensional and two-dimensional force analysis and visualization were performed using Pymol and Discovery Studio software.

[0056] Results Figure 4 The three-dimensional graph shows that ipriflavone can be transported through two The hydrophobic force and 1 The hydrophobic forces of the prolyl hydroxylase domain protein 2 are all bound to the lysine (LYS) residue 146; through 1 The hydrophobic force binds to the 144th proline (PRO) amino acid residue of the prolyl hydroxylase domain protein 2; the two-dimensional graph shows that ipriflavone can bind to the 146th lysine (LYS) amino acid residue of the prolyl hydroxylase domain protein 2 through three hydrophobic binding forces, and can also bind to the 147th glutamic acid (GLU) amino acid residue through one hydrogen bond. In the three-dimensional graph, the gray dotted line represents the hydrophobic force; in the two-dimensional graph, the green dotted line represents the hydrogen bond, and the pink dotted line represents the hydrophobic force.

[0057] (3) Cell experiments

[0058] Human normal gastric epithelial cells and human normal small intestinal epithelial cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Corning, USA) and placed in a normoxic incubator at 37°C. In the non-transfection experiment, human normal small intestinal epithelial cells were cultured in an environment of 1% O2 and 5% CO2 (hypoxic incubator, NAWORDE, GC-C01, Beijing, China) for 24 hours, and then treated with 10 μM ipriflavone or control and then treated with hypoxia for 24 hours. In the transfection plus reagent treatment experiment, human normal gastric epithelial cells and human normal small intestinal epithelial cells were transfected with normal control and prolyl hydroxylase domain protein 2 siRNA under normoxic conditions for 24 hours, and then the cells were cultured under hypoxia (1% O2) conditions for 24 hours, and then co-cultured with dimethyl sulfoxide (control solvent), ipriflavone (10 μM), or MG132 (10 μM, S2619, Selleck) under hypoxia conditions for 24 hours.

[0059] (4) Cell protein extraction

[0060] S1. Discard the culture medium in the six-well plate and wash twice with 2 ml of PBS;

[0061] S2. Add 1 ml PBS to scrape the cells, blow evenly with a gun and then suck into a 1.5 ml EP tube;

[0062] S3. Centrifuge at 3000 rpm for 3 min to pellet the cells;

[0063] S4. Prepare protein lysis buffer. The formula is shown in the table below:

[0064]

[0065] S5. Add 100 μL of protein lysis buffer to each EP tube;

[0066] S6. Oscillation: When using a vortex oscillator, oscillate once every 10 minutes, 15 seconds each time, oscillate for half an hour, and oscillate 3 times in total;

[0067] S7. Centrifuge at 12000 rpm, 20 min, 4°C; prepare the material for BCA concentration measurement during the centrifugation process;

[0068] S8. Take the supernatant and transfer it to another EP tube by measurement. The supernatant is the cell protein extract;

[0069] S9. BCA method to detect tissue protein concentration

[0070] According to the BCA kit, the working solution was prepared with a ratio of 50:1 between solution A and solution B. 10uL 5ng / mL BCA was taken and diluted with 90μL NaCl to prepare 100μL 0.5mg / ml BCA solution. Prepare a 96-well plate and add 180μL BCA working solution to the standard wells and sample wells respectively; 0, 1, 2, 4, 8, 16, 20μL 0.5mg / mL BCA solution and 20, 19, 18, 16, 12, 4, 0μL deionized water were added to the standard wells in sequence, and 18μL deionized water and 2μL protein sample were added to the sample wells respectively. After fully mixing, the wells were placed in a 37℃ incubator for incubation for 30min. Use a spectrophotometer to record the optical density value of each well at 562nm, draw a standard curve with the optical density value of the standard well as the ordinate and the protein concentration as the abscissa, and calculate the protein concentration of the sample wells based on this standard curve.

[0071] S10. Protein concentration normalization

[0072] The sample protein concentration was adjusted to be consistent according to the calculation results. Taking the lowest concentration as the standard, the remaining samples were added with appropriate amounts of protein lysis buffer to make up, and then protein loading buffer was added to prepare the sample solution, which was mixed and stored in a -80℃ refrigerator.

[0073] (5) Western blotting

[0074] S1. Cell protein extraction

[0075] Proteins from human normal gastric epithelial cell line and human normal small intestinal epithelial cell line were extracted according to the method described in (4) Cell protein extraction.

[0076] S2.Western blot

[0077] S201. Preparation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0078] Align the two glass plates and place them on the clamping plate, then install them on the fixed frame. Prepare the lower glue mixture according to the reagent instructions: take one piece of glue as an example, the lower glue mixture contains 5 mL of liquid A and liquid B, add 100 μL of ammonium persulfate (APS), mix thoroughly, add the lower glue mixture to the glass plate sandwich, and then gently add 2 mL of anhydrous ethanol along the plate wall to press the glue. After the lower glue solidifies (about 40 minutes), pour out the anhydrous ethanol and absorb the residual anhydrous ethanol with absorbent paper. Prepare the upper glue: take one piece of glue as an example, the upper glue mixture contains 1 mL of liquid A and liquid B, add 20 μL of APS, mix thoroughly, add the upper glue mixture to the glass plate sandwich, and then insert a comb that has been cleaned and dried in advance. After the upper layer of gel solidifies (about 20 minutes), remove the glass plate, wipe it dry and insert it into the electrophoresis tank, add 1× electrophoresis solution (10× electrophoresis solution prepared with deionized water), then take out the comb and wait for sample loading.

[0079] S202. Electrophoresis

[0080] Add 2μL protein Maker and 18μL protein loading buffer to the Maker wells, and 20μL protein sample (previously boiled and cooled in a metal bath) to the sample wells, leaving the holes at both ends. After loading, connect the electrophoresis tank and power supply, 80V for the upper gel, 30-40min, 120V for the lower gel, 80-100min.

[0081] S203. Transfer

[0082] Add 4℃ pre-cooled transfer solution (10× transfer solution, methanol and deionized water in a ratio of 1:2:7) to the transfer tank and operation tank, place the transfer clip, sponge pad, and filter paper (from bottom to top) in the operation tank, gently pry open the glass plate along the gap with the cutting board, cut the gel according to the molecular weight of the protein Maker, place the cut gel strip on the filter paper of the black transfer clip, take out the pre-cut nitrocellulose filter membrane (Nitrocellulose filter membrane, NC membrane) and stick it on the gel strip, close the sponge pad and use a roller brush to gently drive out bubbles, close the transfer clip and transfer to the transfer tank. Transfer the membrane at 4℃, 100V, and 100min constant voltage (taking 10% SDS-PAGE as an example, the specific duration is adjusted according to the molecular weight).

[0083] S204. Ponceau Red Staining

[0084] Use tweezers to take out the NC membrane and place it in the incubation box. Add the pre-prepared Ponceau red dye solution and place it on a shaker. After 5 minutes, discard the Ponceau red dye solution and add distilled water to rinse repeatedly to reduce the background color.

[0085] S205. Film cutting

[0086] The membrane was cut according to the molecular weight of the target protein, the cut NC membrane was placed in an incubation box, and the residual Ponceau red dye was washed away with 1×TBST.

[0087] S206. Closed

[0088] Add 5% skim milk (prepared in 1×TBST) prepared in advance and block at room temperature for 2 h.

[0089] S207. Incubate with primary antibody

[0090] After blocking, carefully discard the milk. Add 1×TBST (10×TBST and deionized water) and rinse on a shaker for 10 minutes and discard. Repeat three times and add the pre-prepared primary antibody incubation solution (5% skim milk) and incubate on a shaker at 4°C overnight.

[0091] S208. Incubate with secondary antibody

[0092] After the primary antibody incubation is completed, carefully recover the primary antibody incubation solution and store it in a -20℃ refrigerator. Add 1×TBST and rinse on a shaker for 10 minutes and discard. Repeat three times and select the corresponding secondary antibody according to the species of the primary antibody. Prepare the secondary antibody incubation solution (the ratio of secondary antibody to 1×TBST is 1:10000), mix thoroughly and add to the corresponding incubation box, and incubate at room temperature for 2 hours.

[0093] S209. Chemiluminescence imaging

[0094] After the secondary antibody incubation is completed, discard it, add 1×TBST, place it on a shaker to rinse for 10 minutes and discard it, repeat three times for development. Place the membrane on the imaging system and drop the prepared enhanced chemiluminescence (ECL) developer (prepared in dark, A solution and B solution are 1:1), start exposure and save the image.

[0095] Results Figure 5 and Figure 6 , Figure 5 It was shown that under hypoxic conditions, ipriflavone downregulated hypoxia-induced hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in a concentration-dependent manner, so 10 μM (high dose) of ipriflavone was selected for subsequent experiments. Figure 6It was shown that ipriflavone decreased the expression of HIF-1α and HIF-2α, while increasing the expression of hydroxylated HIF-1α (there is no commercial hydroxylated HIF-2α antibody to detect hydroxylated HIF-2α expression). Inhibition of prolyl hydroxylase domain protein 2 using siRNA increased the expression of HIF-1α and HIF-2α, while decreasing the expression of hydroxylated HIF-1α. However, once prolyl hydroxylase domain protein 2 was inhibited, ipriflavone was unable to regulate the expression of HIF-1α, HIF-2α, and hydroxylated HIF-1α, indicating that ipriflavone inhibits HIF-α by targeting prolyl hydroxylase domain protein 2. ipriflavone may inhibit HIF-α expression by enhancing prolyl hydroxylase domain protein 2-mediated HIF-α hydroxylation.

[0096] (6) Co-immunoprecipitation

[0097] The cell lysate was incubated with 2 μg of anti-von Hippel-Lindau antibody (PA5-27322, Invitrogen) at 4°C overnight, and rabbit immunoglobulin (control group) was added. Then 30 μl of protein G Sepharose TM 4Fast Flow (17061802, Cytiva, Uppsala, Sweden) was added to the cell lysate and incubated at 4°C for 2 hours. The results were then obtained according to the above-mentioned cell protein extraction and protein immunoelectrophoresis methods.

[0098] Results Figure 7 , Figure 7 showed that ipriflavone increased the binding of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α to von Hippel-Lindau under conditions of proteasome inhibition using MG132 to ensure hypoxia-inducible factor-α stabilization, whereas this effect was abolished after inhibition of prolyl hydroxylase domain protein 2.

[0099] (7) Gene overexpression

[0100] Normal human gastric epithelial cells and normal human small intestinal epithelial cells were placed in a cell culture plate for culture, and the cells were transfected after they grew to 80%. Plasmids pCMV-hypoxia-inducible factor 1α (containing the full-length wild-type cDNA of human hypoxia-inducible factor 1α) and pCMV-hypoxia-inducible factor 2α (containing the full-length wild-type cDNA of human hypoxia-inducible factor 2α) were used to transiently transfect normal human gastric epithelial cells and normal human small intestinal epithelial cells with hypoxia-inducible factor 1α and hypoxia-inducible factor 2α, and the cells were cultured under hypoxia for 24 hours after 24 hours, and after replacing the cell culture medium, 10 μM ipriflavone or dimethyl sulfoxide (control solvent) was added to the culture plate and treated under hypoxia for 24 hours, and the cells were collected for crystal violet staining and CCK-8 detection.

[0101] (8) Crystal violet staining

[0102] Add 1 mL of 4% paraformaldehyde to the cell culture plate for fixation for 1 h, rinse with PBS three times, add 1 mL of crystal violet stain to each well for staining, rinse slowly with PBS after 30 min to remove excess crystal violet, and take pictures of each well after drying using a scanner.

[0103] (9) CCK-8 cell activity assay

[0104] Add 10 μL of CCK-8 solution to the cells in the 96-well plate after the experimental treatment, avoiding the formation of bubbles during the process, and then place the 96 wells in a CO2 constant temperature incubator and incubate in the dark for 2 hours, and use an enzyme reader to measure the absorbance at 450 nm.

[0105] Crystal violet staining and CCK-8 results are shown in Figure 8-9 , Figure 8-9 It was shown that in normal human gastric epithelial cells, overexpression of hypoxia-inducible factor 1α or hypoxia-inducible factor 2α aggravated hypoxia-induced cell death, while ipriflavone treatment significantly alleviated cell death induced by overexpression of hypoxia-inducible factor 1α or hypoxia-inducible factor 2α under hypoxic conditions. **P<0.01, ***P<0.001, ****P<0.0001; a indicates that the group is different from the normoxic control, and the difference is at least P<0.05; b indicates that the group is different from the hypoxic control, and the difference is at least P<0.05.

[0106] (10) Transmission electron microscopy observation

[0107] S1. Collection and fixation: For adherent cells, first discard the culture medium, add 2.5% glutaraldehyde, fix at 4°C for 15 minutes, scrape the cells, collect by centrifugation (retain the fixative), and store at 4°C for at least 4 hours.

[0108] S2. Osmium acid fixation: The cells fixed with glutaraldehyde were rinsed three times with 0.1 M phosphate buffer (pH 7.4) for 15 minutes each time, fixed with 1% osmium acid × 0.1 M phosphate buffer (pH 7.4) at room temperature (20°C) for 2 hours, and then rinsed three times with 0.1 M phosphate buffer (pH 7.2) for 15 minutes each time.

[0109] S3. Dehydration: The cells are dehydrated in an ascending gradient of 30%, 50%, 70%, 80%, 85%, 90%, and 100% (twice) alcohol, each time for 15-20 minutes (the dehydration time can be appropriately extended for samples with high water content and thick cell membrane).

[0110] S4. Penetration: The penetrants are acetone: epoxy resin (2:1), acetone: epoxy resin (1:1), and epoxy resin, in a 37-degree incubator, and each penetration lasts 8-12 hours.

[0111] S5. Embedding: Place the infiltrated sample into the embedding plate, add embedding agent epoxy resin, and polymerize in a 60-degree incubator for 48 hours.

[0112] S6. Sectioning: Ultrathin sectioning: 80-100nm.

[0113] S7. Double staining: uranium-lead double staining (2% saturated aqueous solution of uranyl acetate, lead citrate, staining at room temperature for 15 min, sections dried at room temperature overnight, and observed under an electron microscope.

[0114] Results Figure 10-11 , Fig.10 It was shown that hypoxia caused the mitochondria of normal human gastric epithelial cells to shrink, decrease in number, increase in membrane density, and loss of mitochondrial cristae, and ipriflavone treatment could alleviate the above hypoxic damage. Fig.11 It was shown that hypoxia caused the mitochondria of normal human small intestinal epithelial cells to shrink, decrease in number, increase in membrane density, and loss of mitochondrial cristae, and ipriflavone treatment could alleviate the above hypoxic damage.

[0115] (11) Detection of ferroptosis markers

[0116] S1. Cell protein extraction: See Example 1 (4) Cell protein extraction.

[0117] S2. Analyze the levels of reactive oxygen species and 4-hydroxynonenal in gastric mucosa and small intestinal mucosa according to the reagent instructions.

[0118] Results Fig.12 , Fig.12 The results showed that ipriflavone could reduce the levels of reactive oxygen species and 4-hydroxynonenal induced by hypoxia in normal human gastric epithelial cells and normal human small intestinal epithelial cells. *P<0.05, **P<0.01, ****P<0.0001.

[0119] (12) Gene knockdown

[0120] Human normal gastric epithelial cells and human normal small intestinal epithelial cells were transfected with normal control and prolyl hydroxylase domain protein 2 siRNA under normoxic conditions for 24 hours, and then the cells were cultured under hypoxic conditions (1% O2) for 24 hours, and then co-cultured with dimethyl sulfoxide (control solvent) or ipriflavone (10 μM) under hypoxic conditions for 24 hours.

[0121] (13) Crystal violet staining and CCK-8 detection

[0122] See Example 1 (8) crystal violet staining and (9) CCK-8 detection.

[0123] Results Fig.13 , Fig.13 The results showed that in normal human gastric epithelial cells and normal human small intestinal epithelial cells, ipriflavone treatment could significantly reduce hypoxia-induced cell death, and after knocking down prolyl hydroxylase domain protein 2, the protective effect of ipriflavone on cells disappeared, which once again indicated that the target of ipriflavone was prolyl hydroxylase domain protein 2. ****P<0.0001; a indicates that the group is different from the normoxic control, and the difference is at least P<0.05; b indicates that the group is different from the hypoxic control, and the difference is at least P<0.05.

[0124] Example 2

[0125] Experimental animals: C57BL / 6J mice (male, 6 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were raised in the specific pathogen free (SPF) animal room of Tsinghua University at room temperature of 20-24°C, with a 12h / 12h day and night cycle, free access to food and water, and mice were allowed to adapt to the environment for one week before the experiment. The experimental protocol was approved by the Animal Care and Use Committee of Tsinghua University (Protocol No. 23-CZJ1).

[0126] This example provides a study on the inhibition of hypoxia-inducible factor-α by ipriflavone in vivo, which specifically includes the following steps:

[0127] (1) Animal model construction

[0128] Mice were placed in a ventilated hypobaric oxygen chamber system to simulate an altitude of 5500 meters for 3 days to induce hypoxic gastrointestinal injury. The mice were randomly divided into five groups: ① normoxia + control group, exposed to room air (21% O2) for 3 days, and gavaged with 100 μL of control solution (10% dimethyl sulfoxide + 40% polyethylene glycol 300 + 5% Tween-80 + 45% PBS) on the first day; ② hypoxia + control group, placed in the hypoxia chamber for 3 days, and gavaged with 100 μL of control solution (10% dimethyl sulfoxide + 40% polyethylene glycol 300 + 5% Tween-80 + 45% PBS) on the first day; ③ hypoxia + ipriflavone treatment group, placed in the hypoxia chamber for 3 days, and gavaged with 100 μL of ipriflavone (15 mg / kg) on ​​the first day. The mice were euthanized on the third day and the gastric and small intestinal mucosa were collected for subsequent experiments.

[0129] (2) Western blot

[0130] S1. Tissue protein extraction: The protein lysis solution formula is shown in Example 1 step (4) S4; the extraction method is: collect about 1 cm of gastric and small intestinal mucosal tissue in a 1.5 mL EP tube, add 200 μL of protein lysis solution, cut the tissue on ice and grind it with a homogenizer, place it on ice, vortex once every 10 minutes, vortex three times and lyse it on ice for 30 minutes, then centrifuge at 12000 rpm and 4°C for 20 minutes, and transfer the supernatant to a new 1.5 mL EP tube.

[0131] S2. Perform Western blot detection according to the method described in step (5) of Example 1.

[0132] Results Fig.14 , Fig.14 It was shown that under hypoxic conditions, the expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in the gastric and small intestinal mucosa increased, while ipriflavone could reduce the expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α. The commonly used oral dose of ipriflavone for the treatment of osteoporosis in mice is 200 mg / kg. In this study, oral administration of 15 mg / kg of ipriflavone can reduce the expression of hypoxia-inducible factor-α in the gastric and small intestinal mucosa.

[0133] Example 3

[0134] Experimental animals: C57BL / 6J mice (male, 6 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were raised in the specific pathogen free (SPF) animal room of Tsinghua University at room temperature of 20-24°C, with a 12h / 12h day and night cycle, free access to food and water, and mice were allowed to adapt to the environment for one week before the experiment. The experimental protocol was approved by the Animal Care and Use Committee of Tsinghua University (Protocol No. 23-CZJ1).

[0135] This example provides a study on the therapeutic effect of ipriflavone on hypoxia-induced gastric and small intestinal mucosal damage, which specifically includes the following steps:

[0136] (1) Animal model construction

[0137] Refer to the animal model construction in Example 2. On the third day, the mice were euthanized and the entire gastrointestinal tract (including the esophagus, stomach, duodenum, jejunum, ileum and colon) was removed for H&E staining. Three H&E-stained specimens were taken from each group for histopathological scoring, and the gastric and small intestinal mucosa were collected for reactive oxygen detection.

[0138] The gavage method is as follows: fix the mouse with the left hand, hold the gavage device with the right hand (the gavage needle is installed and the control solution or ipriflavone has been sucked), insert the gavage needle into the mouth from the corner of the mouse's mouth, press the mouse's head to make the mouth and esophagus in a straight line, gently turn the needle to stimulate the mouse to swallow, slowly insert the gavage needle into the esophagus along the posterior wall of the pharynx, until the front end of the gavage needle reaches the level of the diaphragm, slowly push the syringe to complete the operation, and suck the control solution to clean the gavage needle before each gavage.

[0139] (2) H&E staining steps

[0140] Fresh mouse gastrointestinal tissue was fixed with 4% paraformaldehyde fixative for more than 24 hours. The fixed specimens were trimmed and then dehydrated step by step, embedded in paraffin, and the wax blocks were cut into 4 μm thick slides, and then the prepared sections were placed in a section box for storage.

[0141] The prepared sections were dewaxed and hydrated, then placed in hematoxylin for staining for 3-5 minutes, rinsed with distilled water, dried, and then placed in eosin for counterstaining. After transparent dehydration, the sections were sealed with neutral resin.

[0142] Results Fig.15 , Fig.15 Studies have shown that hypoxia can cause serious damage to the gastric and small intestinal mucosal barriers, including loss, shedding and death of gastric and small intestinal mucosal cells, as well as smaller villi, shallower crypts and thinner small intestinal mucosa. Taking ipriflavone can improve this mucosal damage.

[0143] (3) Scoring criteria for esophageal, gastric, and intestinal histopathology

[0144] Scoring criteria for esophageal and gastric histopathological examinations: mucosal injury (normal: 0 points; epithelial exfoliation: 1 point; superficial lamina propria or 1 / 3 epithelial exfoliation: 2 points; middle lamina propria or 2 / 3 epithelial exfoliation: 3 points; lower lamina propria or more than 2 / 3 epithelial exfoliation: 4 points); inflammatory cell infiltration (normal: 0 points; lamina propria infiltration: 1 point; muscularis mucosa infiltration: 2 points; extensive muscularis mucosa infiltration with edema: 3 points; submucosal infiltration: 4 points); and bleeding (normal: 0 points; bleeding area less than or equal to 10%: 1 point; bleeding area 11%-20%: 2 points; bleeding area 21%-30%: 3 points; greater than 30%: 4 points).

[0145] Intestinal histopathological scoring criteria included epithelial cell damage (normal: 0 points, mild goblet cell loss: 1 point, extensive goblet cell loss: 2 points, mild crypt loss and extensive goblet cell loss: 3 points, and extensive crypt loss: 4 points), inflammatory cell infiltration (normal: 0 points, infiltration around the crypt base: 1 point, infiltration of the muscularis mucosa: 2 points, extensive infiltration of the muscularis mucosae with edema: 3 points, and infiltration of the submucosal layer: 4 points).

[0146] Results Fig.16 , Fig.16 The results showed that hypoxia could increase the pathological scores of stomach and small intestine, while ipriflavone could reduce the pathological scores. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0147] (4) In vivo gastrointestinal permeability test

[0148] Mice were fasted for 4 hours before oral gavage. FITC-dextran was orally administered at a concentration of 0.6 mg / g body weight. Serum was collected four hours later and the fluorescence intensity was measured by fluorimetry.

[0149] Results Fig.17 , Fig.17 The results showed that hypoxia caused an increase in serum FITC-Dextran levels, while ipriflavone could significantly reduce serum FITC-Dextran levels, indicating that ipriflavone could significantly improve hypoxia-induced gastrointestinal mucosal barrier damage. **P<0.01, ***P<0.001.

[0150] (5) Detection of ferroptosis markers

[0151] S1. Tissue protein extraction: See Example 2 (2) Western blot detection of S1.

[0152] S2. Analyze the levels of reactive oxygen species and 4-hydroxynonenal in gastric mucosa and small intestinal mucosa according to the reagent instructions.

[0153] Results Fig.18 and Fig.19 , Fig.18 It was shown that ipriflavone could reduce the levels of reactive oxygen species and 4-hydroxynonenal induced by hypoxia in the gastric mucosa. Fig.19 The results showed that ipriflavone could reduce the levels of reactive oxygen species and 4-hydroxynonenal induced by hypoxia in the small intestinal mucosa. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0154] Example 4

[0155] (1) Cell experiments

[0156] Mouse macrophage cell line Raw264.7 was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Corning, USA) and placed in a 37°C normoxic incubator. After transfection with control plasmids PCMV-Vector, PCMV-hypoxia-inducible factor 1α, and PCMV-hypoxia-inducible factor 2α for 24 hours, they were co-cultured with lipopolysaccharide (100nM), dimethyl sulfoxide (control solvent), and ipriflavone (10μM) for 24 hours. The levels of inflammatory factors TNF-α, IFN-γ, and IL-6 in the culture medium were then detected by enzyme-linked immunosorbent assay. The results are shown in Fig. 20 , Fig. 20 The results showed that overexpression of hypoxia-inducible factor 1α or 2α could increase the level of inflammatory factors induced by lipopolysaccharide, while ipriflavone could downregulate the inflammatory factors increased by hypoxia-inducible factor 1α or 2α, indicating that ipriflavone reduced lipopolysaccharide-induced macrophage inflammation by inhibiting hypoxia-inducible factor 1α and 2α. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0157] Example 5

[0158] (1) Cell experiments

[0159] Human colon cancer HCT16 cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Corning, USA) and placed in a 37°C normoxic incubator. After transfection with control siRNA and si hypoxia-inducible factor 1α for 24 hours, they were co-cultured with dimethyl sulfoxide (control solvent) and ipriflavone (10 μM) for 24 hours. Cell viability was then detected using CCK-8. Results are shown in Fig.21 , Fig.21 The results showed that knocking down hypoxia-inducible factor 1α or ipriflavone intervention would downregulate the activity of HCT116, while the combination of si hypoxia-inducible factor 1α and ipriflavone had no further effect, which in turn confirmed that ipriflavone inhibited the growth of colon cancer HCT16 by inhibiting hypoxia-inducible factor 1α. ***P<0.001.

[0160] Example 6

[0161] (1) Cell experiments

[0162] The human breast cancer cell line MDA-MB-231 was cultured in L15 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Corning, USA) and placed in a 37°C normoxic incubator. After transfection with the control plasmids PCMV-Vector and PCMV-hypoxia-inducible factor-1α for 24 hours, they were co-cultured with dimethyl sulfoxide (control solvent), ipriflavone (10 μM), and cisplatin (30 μM) for 24 hours. Cell viability was then detected using CCK-8. The results are shown in Fig. 22 Cisplatin can inhibit the viability of MDA-MB-231 cells. Overexpression of hypoxia-inducible factor-1α can significantly interfere with the ability of cisplatin to inhibit the viability of MDA-MB-231 cells, while the addition of ipriflavone can restore the ability of cisplatin to inhibit the viability of MDA-MB-231 cells. This proves that ipriflavone can increase the killing of cisplatin on the breast cancer cell line MDA-MB-231 by inhibiting hypoxia-inducible factor-1α. **P<0.01,***P<0.001,****P<0.0001.

[0163] Example 7

[0164] (1) Cell experiments

[0165] Mouse colon cancer cell line MC38 Cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Corning, USA) and placed in a 37°C normoxic incubator. After transfection with the control plasmid PCMV-Vector (empty vector), PCMV-hypoxia-inducible factor 1α, and PCMV-hypoxia-inducible factor 2α for 24 hours, they were co-cultured with dimethyl sulfoxide (control solvent) and ipriflavone (10 μM) for 24 hours. After extracting cell proteins, Western blot was used to detect the expression of hypoxia-inducible factor 1α, hypoxia-inducible factor 2α, and programmed death ligand 1 proteins. The results are shown in Fig.23 Overexpression of hypoxia-inducible factors 1α and 2α can induce programmed death-ligand 1, while ipriflavone can inhibit the expression of programmed death-ligand 1 by inhibiting hypoxia-inducible factors 1α and 2α.

[0166] (2) Animal experiments

[0167] Experimental animals: C57BL / 6J mice (female, 4-5 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were raised in a specific pathogen free (SPF) animal room at Tsinghua University, with a room temperature of 20-24°C, a 12h / 12h day and night cycle, free access to food and water, and mice were allowed to adapt to the environment for one week before the experiment. The experimental protocol was approved by the Animal Care and Use Committee of Tsinghua University (Protocol No. 23-CZJ1).

[0168] MC38 cells were suspended in RPMI-1640 medium at a concentration of 1 × 10 6 The density of cells / 100 μL was injected into the flank of the recipient mice. The mice were divided into groups on the 6th day after transplantation to ensure that the initial average tumor volume between the experimental group and the control group was comparable. The formula V = ab 2 / 2 to calculate the tumor volume, where a is the longer diameter and b is the shorter diameter. Starting from the 6th day, mice were given oral administration of 80 mg / kg of ipriflavone or control solvent (20% dimethyl sulfoxide + 40% polyethylene glycol 300 + 5% Tween-80 + 35% PBS) every day. Every three days, mice were intraperitoneally injected with 0.2 mg of cytotoxic T lymphocyte-associated protein 4 antibody, programmed death receptor 1 antibody or control IgG antibody (monoclonal antibodies were purchased from R&D System). The groups were as follows: control group, which was given control solvent by gavage and control antibody by intraperitoneal injection; ipriflavone group, which was given ipriflavone by gavage and control antibody by intraperitoneal injection; cytotoxic T-lymphocyte-associated protein 4 antibody, which was given control solvent by gavage and cytotoxic T-lymphocyte-associated protein 4 antibody by intraperitoneal injection; programmed death receptor 1 antibody group, which was given control solvent by gavage and programmed death receptor 1 antibody by intraperitoneal injection; ipriflavone + cytotoxic T-lymphocyte-associated protein 4 antibody group, which was given ipriflavone by gavage and cytotoxic T-lymphocyte-associated protein 4 antibody by intraperitoneal injection; ipriflavone + programmed death receptor 1 antibody group, which was given ipriflavone by gavage and programmed death receptor 1 antibody by intraperitoneal injection. Results are shown in Table 1. Fig.24 , cytotoxic T lymphocyte-associated protein 4 antibody and programmed death receptor 1 antibody can significantly reduce tumor size; although ipriflavone has limited downregulation function, its combination with cytotoxic T lymphocyte-associated protein 4 antibody or programmed death receptor 1 antibody can significantly improve the therapeutic effect of single antibody. a is the difference between ipriflavone + cytotoxic T lymphocyte-associated protein 4 monoclonal antibody group and cytotoxic T lymphocyte-associated protein 4 monoclonal antibody group, the difference is at least P<0.05, b is the difference between ipriflavone + programmed death receptor 1 monoclonal antibody group and programmed death receptor 1 monoclonal antibody group, the difference is at least P<0.05.

[0169] Statistical analysis

[0170] The research data were analyzed using GraphPad Prism 9.5 software. The differences between the two groups were compared using t-test, and the research data of three or more groups were compared using one-way ANOVA. P < 0.05 was defined as a statistically significant difference, and the data were expressed as mean ± standard error.

[0171] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Application of ipriflavone as hypoxia inducible factor α inhibitor.

2. The application of ipriflavone as a hypoxia-inducible factor-α inhibitor in the preparation of drugs for the treatment of diseases with high expression of hypoxia-inducible factor-α.

3. The use according to claim 2, characterized in that: The diseases with high expression of hypoxia-inducible factor α include hypoxic gastrointestinal mucosal damage, inflammation, and tumors.

4. The use according to claim 2, characterized in that: The ipriflavone is used alone or in combination with other drugs.

5. The use according to claim 4, characterized in that: Combination regimens include but are not limited to: for example, in tumors, combination with cisplatin, cytotoxic T lymphocyte-associated protein 4 antibodies or inhibitors, programmed death receptor 1 and its ligand 1 antibodies or inhibitors and other drugs.

6. The use according to claim 2, characterized in that: The dosage form of the drug is liquid, gas, solid or semisolid.

7. The use according to claim 6, characterized in that: The dosage form of the medicine is tablet, powder, granule, hard capsule or pill.

8. The use according to claim 2, characterized in that: The administration methods of the drug include oral, sublingual, rectal, inhalation, epidermal or injection, and the injection administration method includes intravenous injection, subcutaneous injection or intramuscular injection.

Images