Application of indobufen in preparation of medicine for preventing and treating diseases induced by excessive ferroptosis

Abstract

The invention discloses application of indobufen in preparation of a medicine for preventing and treating diseases induced by excessive ferroptosis, and particularly relates to the technical field of medicines. The invention relates to an application of indobufen in preparation of drugs for preventing and treating diseases induced by excessive ferroptosis by inhibiting ferroptosis. The indobufen is a ferroptosis inhibitor. The invention provides a new application of indobufen alone or in combination with other drugs in promoting wound healing. Cell and animal level experiments prove that the indobufen can inhibit the local ferroptosis level of the wound surface, so that the wound surface healing is obviously accelerated, and the indobufen has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of indobufen in the preparation of medicines for preventing and treating diseases induced by excessive ferroptosis. Background Art

[0002] Indobufen is a nonsteroidal anti-inflammatory drug (NSAID) mainly used to prevent and treat thrombotic diseases. Its mechanism of action is mainly through inhibiting the activity of cyclooxygenase (COX) enzymes, reducing the synthesis of prostaglandins and thromboxanes, thereby inhibiting platelet aggregation and reducing thrombosis, thus playing an important role in the prevention and treatment of cardiovascular and cerebrovascular diseases. Indobufen is usually administered orally, and the dosage and onset time vary according to the specific conditions of the patient, with good safety and tolerability. In addition to cardiovascular and cerebrovascular diseases, indobufen also has certain application value in other diseases. For example, in diabetic patients, indobufen can reduce the occurrence of diabetic complications by inhibiting platelet aggregation. Studies have shown that indobufen can significantly reduce the incidence of cardiovascular events in diabetic patients and improve the quality of life of patients; Chinese patent CN101801373A, looking for and developing new drugs for the treatment of pancreatic diabetes. The invention is based on the development of more effective and less toxic drugs based on indole derivatives, which show anti-diabetic, hypolipidemic, hypoglycemic, cholesterol-lowering activities and insulin resistance improvement effects, and their structures are different from those of traditionally used compounds. The indole derivatives of the invention are also low-toxic and easy to tolerate. In tumor treatment, indobufen also shows certain potential. Studies have found that indobufen can reduce the proliferation and metastasis of tumor cells by inhibiting the expression of COX-2, thereby playing a role in the prevention and treatment of tumors. Chinese patent CN108822017A, the invention discloses a novel indole ketone compound shown in formula I compound and its physiologically acceptable salts, solvates and crystalline forms, pharmaceutical preparations containing the compound, and the anti-tumor effect of the compound.

[0003] However, there are no reports on the effect of indobufen in promoting wound healing. Summary of the invention

[0004] To this end, the present invention provides the use of indobufen in the preparation of a drug for preventing and treating diseases induced by excessive ferroptosis, so as to solve the above-mentioned problems.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: According to one aspect of the present invention, indobufen is provided for use in the preparation of a medicament for preventing and treating diseases induced by excessive ferroptosis by inhibiting ferroptosis, wherein the indobufen is a ferroptosis inhibitor.

[0006] The indobufen structural formula is: It can be used alone as an active ingredient of medicine, or it can be used as an active ingredient after being combined with a pharmaceutically acceptable carrier.

[0007] Furthermore, the disease induced by excessive ferroptosis is a neurodegenerative disease, an autoimmune disease, a wound or tuberculosis.

[0008] Among them, tuberculosis itself promotes ferroptosis, leading to inflammatory cell death during the infection process, thereby promoting the spread of pathogens and tissue pathological damage.

[0009] As an example, the present invention provides the use of indobufen in preparing a medicament for promoting wound healing.

[0010] Furthermore, the wound surface is any one of a chronic inflammatory wound surface, a pressure ulcer, a venous ulcer, a bedsore wound, a burn wound, and a surgical wound.

[0011] Furthermore, the chronic inflammatory wounds include diabetic wounds and diabetic foot ulcers.

[0012] Furthermore, the neurodegenerative disease includes one of Parkinson's disease, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis.

[0013] Furthermore, the autoimmune disease includes any one of rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, scleroderma, and autoimmune thyroiditis.

[0014] Furthermore, the anti-tuberculosis medicinal ingredients include, but are not limited to, one or more of antibiotics and drugs that promote epidermal growth.

[0015] Further, the dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. The liquid dosage form can be a solution (including a true solution and a colloidal solution), an emulsion (including an o / w type, a w / o type and a multiple emulsion), a suspension, an injection (including a water injection, a powder injection and an infusion), an eye drop, a nasal drop, a lotion and an liniment, etc.; the solid dosage form can be a tablet (including a common tablet, an enteric-coated tablet, a lozenge, a dispersible tablet, a chewable tablet, an effervescent tablet, an orally disintegrating tablet), a capsule (including a hard capsule, a soft capsule, an enteric-coated capsule), a granule, a powder, a micropill, a drop pill, a suppository, a film, a patch, an aerosol (powder) aerosol, a spray, etc.; the semisolid dosage form can be an ointment, a gel, a paste, etc. More preferably, the dosage form of the drug includes at least one of a suspension, a granule, a capsule, a powder, a tablet, a drop pill, an injection, a suppository, a spray, a drop pill, a gel, a patch and a semisolid preparation.

[0016] As an example, liquid dosage forms include solutions, suspensions and emulsions, for example, aqueous solutions or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as water-polyethylene glycol solutions.

[0017] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0018] Furthermore, the administration route may be enteral or parenteral; as an example, the administration route of the drug includes at least one of injection, oral administration, and transdermal administration. , such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0019] According to another aspect of the present invention, a wound healing promoting drug is provided, the drug comprising indobufen and a pharmaceutically acceptable carrier.

[0020] Furthermore, the pharmaceutically acceptable carrier is at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubility aid, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

[0021] As an example, a solid carrier can be one or more materials that also act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, adhesive, preservative, tablet disintegrating agent or encapsulation material. In a powder, the carrier is a subdivided solid that is mixed with the subdivided active ingredient. In a tablet, the active ingredient is mixed with a carrier with necessary adhesive properties in an appropriate ratio and compressed into a desired shape and size. Suitable carriers are magnesium carbonate, magnesium stearate, talcum powder, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, etc.

[0022] Preferably, the diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. The solubilizer or cosolvent may be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator may be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, when preparing lyophilized powder injection, mannitol, glucose, etc. may also be added as a support agent.

[0023] If necessary, colorants, preservatives, perfumes, flavoring agents or other additives may be added to the pharmaceutical preparations.

[0024] Indobufen of the present invention promotes wound healing mainly through the following mechanisms: Antiplatelet aggregation: In the early stage of wound healing, platelet aggregation will start the coagulation process, but excessive aggregation may lead to local microcirculation disorders. Indobufen can inhibit platelet aggregation and prevent thrombosis, thereby improving blood circulation in the wound, ensuring that nutrients and oxygen can be smoothly transported to the wound tissue, and providing a good blood supply for wound healing.

[0025] Anti-inflammatory Effects: Post-traumatic inflammatory response is an inevitable stage of wound healing, but excessive inflammation will have a negative impact on wound healing. Indobufen can reduce the production of inflammatory factors (such as interleukin-1β, tumor necrosis factor-α, etc.) and alleviate the inflammatory response. At the same time, inhibiting the inflammatory response can also help reduce cell damage caused by inflammation and protect cells around the wound.

[0026] Promotes cell proliferation and migration: Cell proliferation and migration are crucial for wound healing. Indobufen may promote the proliferation and migration of fibroblasts and keratinocytes by activating certain signaling pathways within cells. For example, it stimulates fibroblasts to produce collagen, increases the synthesis of extracellular matrix, and provides a scaffold for new tissue; it promotes the migration of keratinocytes and accelerates the epithelialization process of the wound.

[0027] Antioxidant Effect: Trauma can cause local tissues to produce a large amount of reactive oxygen species (ROS), causing oxidative stress and subsequently damaging cells. Indobufen has antioxidant properties that can remove ROS, reduce the damage of oxidative stress to cells, help maintain normal cell function, and promote wound healing.

[0028] The present invention has the following advantages: The present invention uses indobufen as a drug for promoting wound healing, which has a multi-target mechanism of action. It can simultaneously exert multiple effects such as anti-platelet aggregation, anti-inflammation, anti-oxidation and promotion of cell proliferation and migration. This multi-target characteristic enables it to intervene in the wound healing process from multiple aspects, and compared with drugs with a single mechanism of action, it can more comprehensively cope with the complex physiological and pathological changes in wound healing.

[0029] The present invention uses indobufen as a wound healing promoting agent with relatively high safety. Compared with some traditional wound healing promoting drugs, indobufen has relatively few adverse reactions. It has less irritation to organs such as the gastrointestinal tract and is not likely to cause complications such as severe bleeding when used within a reasonable dosage range, which provides better safety guarantee for long-term or local use.

[0030] The present invention uses indobufen as a wound healing agent to improve local blood circulation. By inhibiting platelet aggregation, indobufen can effectively improve local blood circulation in the wound. Good blood circulation can not only provide sufficient oxygen and nutrients for the wound, but also remove metabolic waste in time, which is very important for cell metabolism and tissue repair in the wound healing process.

[0031] The present invention uses indobufen as a wound healing agent to reduce inflammatory response, which is necessary in the early stage of wound healing, but excessive inflammation will hinder healing. Indobufen can effectively reduce inflammatory response, reduce the damage of inflammatory factors to wound tissue, and help create a microenvironment conducive to wound healing.

[0032] The present invention provides a new use of indobufen alone or in combination with other drugs in promoting wound healing. The present application proves through cell and animal level experiments that indobufen can inhibit the local ferroptosis level of wounds, thereby significantly accelerating wound healing, and has a good clinical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0034] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0035] Figure 1 A flow cytometry assay for detecting the effect of indobufen on cell function ferroptosis at the cell level provided in Example 1 of the present invention, wherein A-cell level detection of the effect of indobufen on cell death and oxidized lipid reactive oxygen species expression; B-cell level detection of the effect of indobufen on the expression of oxidized lipid reactive oxygen species; ****, p<0.0001.

[0036] Figure 2 This is a comparison chart of the skin wound results of mice treated with different doses of indobufen at different time points provided in Example 2 of the present invention, wherein A-skin appearance of mice treated with different doses of indobufen at different time points; B-statistics of skin wound healing of mice treated with different doses of indobufen; in the figure, empty control group, no treatment; PVA, polyvinyl alcohol glue (drug carrier). ***, p<0.001; ****, p<0.0001.

[0037] Figure 3 The cross-sectional hematoxylin-eosin staining and statistical graph of skin wounds of mice treated with different doses of indobufen for 8 days provided in Example 3 of the present invention; wherein, A-hematoxylin-eosin staining results of cross-sectional tissue of skin wounds of mice treated with different doses of indobufen for 8 days; B-statistics of the maximum diameter of skin wounds of mice treated with different doses of indobufen for 8 days. *, p<0.05; **, p<0.001; ****, p<0.0001.

[0038] Figure 4The present invention provides Example 4 of the present invention, which shows Prussian blue staining (marking the iron ion content in the tissue) of the cross-section of the skin wound tissue of mice treated with different doses of indobufen for 6 days and statistical graphs; wherein, A-Prussian blue staining results of the cross-section of the skin wound tissue of mice treated with different doses of indobufen for 6 days; B-statistics of relative iron concentration in the skin wound tissue of mice treated with different doses of indobufen for 6 days; ***, p<0.001; ****, p<0.0001.

[0039] Figure 5 The 4-HNE immunohistochemistry and statistical graphs of the cross-section of the skin wound tissue of mice treated with different doses of indobufen for 6 days provided in Example 5 of the present invention, wherein A-4-HNE immunohistochemistry results of the cross-section of the skin wound tissue of mice treated with different doses of indobufen for 6 days; B-4-HNE relative concentration statistics of the skin wound tissue of mice treated with different doses of indobufen for 6 days. ***, p<0.001; ****, p<0.0001. DETAILED DESCRIPTION

[0040] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In the present invention, indobufen as a ferroptosis inhibitor may play the following roles in the wound healing process: Reduce cell death: Wound healing requires the participation of multiple cells, such as fibroblasts, keratinocytes, and endothelial cells. In the wound microenvironment, oxidative stress may induce cell ferroptosis, thereby hindering wound healing. Indobufen can inhibit ferroptosis, reduce the death of these key cells, maintain cell numbers, and facilitate wound repair.

[0042] Reducing inflammatory response: Post-traumatic inflammatory response is the early stage of wound healing, but excessive inflammation will produce a large amount of reactive oxygen species (ROS), inducing cell ferroptosis. While indobufen inhibits ferroptosis, it may reduce the production of inflammatory factors, reduce inflammatory response, and create a good environment for wound healing.

[0043] Promoting angiogenesis: The ferroptosis inhibitor indobufen may promote the expression of angiogenesis-related factors such as vascular endothelial growth factor (VEGF) by inhibiting endothelial cell ferroptosis, thereby contributing to the formation of new blood vessels. New blood vessels can provide nutrients and oxygen to the wound surface, accelerating wound healing.

[0044] DMSO, dimethyl sulfoxide solvent; Indo, indobufen; Indo-R, indobufen dextrorotatory isomer; Indo-S, indobufen levorotatory isomer.

[0045] 4-HNE, 4-Hydroxynonenal, is a harmful metabolite produced when fatty acid peroxidation induces ferroptosis (indicating the level of ferroptosis occurring).

[0046] Example 1 The flow cytometry provided in this example detects the effect of indobufen on cell function ferroptosis at the cell level: 1. Detection of cell death level The degree of cell death is detected using propidium iodide (PI) to indicate the occurrence of overall cell death. The specific operation process is as follows: (1) RAW264.7 cells were seeded into 6-well plates covered with coverslips. After the cells adhered, 10 μM RSL3, an ferroptosis inducer, was added to treat the cells for 12 hours. Then, 10 μM indobufen, dextro-indobufen, or levo-indobufen combined treatment (DMSO solvent treatment group was used as control) was added to the cells and cultured for another 24 hours.

[0047] (2) The cells treated as above were digested with trypsin to prepare a cell suspension, and propidium iodide (PI) with a final concentration of 5 μg / mL was added and incubated with the cells for 20 min; the cells were washed three times with Hank's buffer.

[0048] (3) Resuspend the cells in Hank's buffer and analyze by flow cytometry (488 nm excitation light) to calculate the proportion of cells positive for PI labeling.

[0049] Experimental results: Treatment with the ferroptosis inducer RSL3 can cause about 15% of cells to die (DMSO control group), while treatment with indobufen (Indo), dextrorotatory indobufen (Indo-R) and levorotatory indobufen (Indo-S) can significantly inhibit cell death, and the proportion of dead cells decreased from 15% to 4-5% (such as Figure 1 A).

[0050] 2. Lipid peroxidation detection experiment The degree of lipid peroxidation is detected using BODIPY C11 fluorescent probe to indicate the degree of cell membrane peroxidation (ferroptosis indicator). The specific operation process is as follows: (1) RAW264.7 cells were seeded into 6-well plates covered with coverslips. After the cells adhered, 10 μM RSL3, an ferroptosis inducer, was added to treat the cells for 12 hours. Then, 10 μM indobufen, dextro-indobufen, or levo-indobufen combined treatment (DMSO solvent treatment group was used as control) was added to the cells and cultured for another 24 hours.

[0051] (2) The cells treated as above were digested with trypsin to prepare a cell suspension, and 1.5 μM BODIPY C11 fluorescent probe diluted with Hank's buffer was added to the cells and incubated for 20 min; the cells were washed three times with Hank's buffer.

[0052] (3) Resuspend the cells in Hank's buffer and analyze by flow cytometry (488 nm excitation light) to calculate the proportion of cells positive for probe labeling.

[0053] Experimental results: Oxidized lipid reactive oxygen species (ROS) is another important indicator of ferroptosis. The proportion of cells expressing oxidized lipid reactive oxygen species (ROS) in the DMSO group was about 30%, while the proportion of cells expressing oxidized lipid reactive oxygen species (ROS) in the Indo, Indo-R and Indo-S treatment groups was about 9-10% ( Figure 1 B).

[0054] The above two experimental results suggest that Indo, Indo-R and Indo-S can inhibit the occurrence of cell ferroptosis, and their ability to inhibit ferroptosis is basically the same.

[0055] Example 2 This example provides a comparison of the skin wound results of mice treated with different doses of indobufen at different time points: (1) Animal preparation: C57BL / 6 mice were adaptively raised for 7 days to ensure that they were in good health. The mice were fasted for 12 hours and deprived of water for 2 hours before the experiment.

[0056] (2) Anesthesia: Use sodium pentobarbital (50 mg / kg, intraperitoneal injection) to ensure that the mice are fully anesthetized.

[0057] (3) Depilation and disinfection: Depilate the back of the anesthetized mouse with an electric clipper and disinfect with 75% ethanol to avoid infection.

[0058] (4) Wound preparation: Use a circular punch or scalpel to create full-thickness skin defects (8 mm in diameter) on both sides of the mouse's back symmetrically. Keep the wound depth to the full thickness of the skin, but do not damage the muscle layer.

[0059] (5) Grouping: The mice were randomly divided into 5 groups (blank control group, no treatment control group; PVA, PVA gel treatment group; PVA+Indo (50 μg / mL) Gel, PVA gel treatment group containing indobufen, each 200 μL gel containing 10 μg indobufen; PVA+Indo (100 μg / mL) Gel, PVA gel treatment group containing indobufen, each 200 μL gel containing 20 μg indobufen; PVA+Indo (200 μg / mL) Gel, PVA gel treatment group containing indobufen, each 200 μL gel containing 40 μg indobufen); each group had 5 mice.

[0060] (6) Treatment cycle: Take photos of the wound surface at 0-8 days after drug treatment, using a ruler as a reference. Use image analysis software (such as ImageJ) to calculate wound changes and wound healing rate. Figure 2 As shown in A.

[0061] Experimental results: From the appearance of the wound and the statistical results, PVA as a drug carrier has no obvious effect on the wound healing process, while PVA mixed with indobufen can promote wound healing, and as the dosage of indobufen increases, the wound heals faster. For example, when the wound is treated with PVA+Indo (200μg / mL) gel drug, the wound can be basically healed within 6 days, while other treatment groups need more than 8 days to heal (such as Figure 2 B).

[0062] Example 3 This example provides hematoxylin-eosin staining and statistics of cross-sections of skin wounds of mice treated with different doses of indobufen for 8 days: (1) Tissue fixation: Complete skin tissue with intact wound surface was obtained from mice treated with the above drugs at different time points, and fixed with 10% formalin for 24 hours. Tissue sections were prepared using conventional paraffin embedding techniques with a thickness of about 4-5 microns.

[0063] (2) Dewaxing and hydration: Dewax the sections in xylene for 5 minutes each time, twice. Then, rinse the sections in 100%, 95%, 85%, and 75% ethanol for 2 minutes each, and finally rinse the sections with distilled water.

[0064] (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5-10 minutes and rinse with tap water until there is no excess dye.

[0065] (4) Bluing: Immerse the slices in an alkaline solution (such as 0.25% ammonia water) for a few seconds to blue the cell nuclei, and then rinse with tap water. After blueing, the cell nuclei will appear blue.

[0066] (5) Eosin staining: Immerse the sections in eosin staining solution for 1-3 minutes, and rinse with tap water until there is no excess dye.

[0067] (6) Dehydration and sealing: Dehydrate the sections in 75%, 85%, 95%, and 100% ethanol for 2 minutes each, and transparentize with xylene for 5 minutes each, twice in total. Seal the sections with neutral gum to ensure long-term storage of the sections.

[0068] (7) Wound size statistics: Use a ruler as a reference. Calculate the maximum wound diameter of the wound cross section using image analysis software (such as ImageJ). Figure 3 As shown in A.

[0069] Experimental results: HE staining and wound diameter statistics showed that PVA as a drug carrier had no significant effect on the wound healing process, while PVA mixed with indobufen promoted wound repair and healing, and with the increase in indobufen dosage, wound repair and healing were faster (e.g. Figure 3 B).

[0070] Example 4 This example provides Prussian blue staining and statistics of cross-sections of skin wound tissues of mice treated with different doses of indobufen for 6 days: (1) Tissue fixation and sectioning: Wound tissue was obtained and fixed in 10% formalin for 24 hours. Tissue sections were prepared using conventional paraffin embedding techniques with a thickness of approximately 4-5 microns.

[0071] (2) Dewaxing and hydration: Dewax the sections in xylene for 5 minutes each time, twice. Then, rinse the sections in 100%, 95%, 85%, and 75% ethanol for 2 minutes each, and finally rinse the sections with distilled water.

[0072] (3) Bruce blue staining: Mix equal volumes of 2% potassium ferric chloride and 2% hydrochloric acid. Immerse the sections in the mixed staining solution and stain for 10-30 minutes. Rinse the sections with distilled water to remove excess stain.

[0073] (4) Dehydration and sealing: Dehydrate in 75%, 85%, 95%, and 100% ethanol for 2 minutes each. Transparentize with xylene for 5 minutes each, twice. Seal the slides with neutral gum such as Figure 4 As shown in A.

[0074] Experimental results: Prussian blue staining results showed that the iron ion accumulation in the wound tissue of the group without indobufen treatment was obvious (relative iron ion density was about 17%), and as the dosage of indobufen treatment increased, the iron ion concentration in the wound tissue gradually decreased, and the relative iron ion density in the PVA+Indo (200μg / mL) treatment group dropped to about 3% (e.g. Figure 4 As shown in B). Since the accumulation of iron ions is an important factor in the occurrence of ferroptosis, this result suggests that indobufen can reduce the level of ferroptosis in wound tissue.

[0075] Example 5 This example provides 4-HNE immunohistochemistry and statistics of the cross-section of mouse skin wound tissue treated with different doses of indobufen for 6 days: (1) Tissue fixation and sectioning: The tissue was fixed with 10% formalin for 24 hours. After paraffin embedding, the section thickness was 4-5 μm.

[0076] (2) Dewaxing and hydration: Dewax the sections in xylene for 5 minutes each time, twice. Then, rinse the sections in 100%, 95%, 85%, and 75% ethanol for 2 minutes each, and finally rinse the sections with distilled water.

[0077] (3) Antigen retrieval: Heat retrieval (microwave or water bath heating) in citric acid buffer (pH 6.0) for about 10-20 minutes. After cooling to room temperature naturally, rinse with PBS.

[0078] (4) Blocking: Add 5-10% normal goat serum or bovine serum albumin (BSA) to the slices and incubate at room temperature for 30 minutes to block nonspecific binding sites.

[0079] (5) Primary antibody incubation: Add 4-HNE specific primary antibody and dilute it according to the recommended dilution ratio. Incubate overnight at 4°C or at room temperature for 1-2 hours.

[0080] (6) Washing: Wash the sections three times with PBS, 5 minutes each time.

[0081] (7) Secondary antibody incubation: Add appropriate biotinylated secondary antibody or enzyme-conjugated secondary antibody and incubate at room temperature for 30-60 minutes. Wash the sections three times with PBS, 5 minutes each time.

[0082] (8) Color development: Add DAB color developer and observe the color development reaction, usually for 1-5 minutes. Terminate the reaction with distilled water. Figure 5 As shown in A.

[0083] Experimental results: 4-HNE is a harmful metabolite produced by fatty acid peroxidation-induced ferroptosis, and its level can indicate the level of tissue ferroptosis. The results of 4-HNE immunohistochemistry showed that the relative density of 4-HNE in the wound tissue of the group without indobufen treatment was about 60%, and as the amount of indobufen treatment increased, the level of 4-HNE in the wound tissue gradually decreased, and the relative 4-HNE density in the PVA+Indo (200μg / mL) treatment group dropped to about 5% (e.g. Figure 5 The results show that indobufen can significantly inhibit the occurrence of ferroptosis in wound tissue.

[0084] Example 6 This embodiment provides a cream for promoting wound healing: Add 1.5g beeswax, 2.3g lanolin, 3.1g stearic acid, and 1.7g glyceryl monostearate into a dry 100mL beaker (referred to as beaker A), place the beaker in a glass water bath and heat it. Keep the water bath temperature constant at 70°C, and stir at 700r / min until all of them are melted. Take another 100 mL dry beaker (referred to as beaker B), add 5.3 g of glycerol, 15.77 g of distilled water, 1.2 g of 1,2-propylene glycol, 0.09 g of propylparaben, and 0.003 g of indobufen, and stir thoroughly; Place beaker B in a glass water bathtub, control the water bath temperature to 75°C, heat and stir for about 10 min, then add the mixture in beaker A to beaker B while stirring, mix thoroughly, and then emulsify at 75°C, 600 r / min for 10 min, stop heating, and continue stirring until cooled to room temperature to obtain indobufen cream.

[0085] Example 7 The cream obtained in Example 6 was applied to the SPF grade C57BL / 6 mouse skin wound model (model construction was the same as in Example 2). The experiment was divided into 4 groups (experimental group: cream in Example 6; positive control group: MEBO moist burn ointment, national medicine standard Z20000004; blank matrix group: blank matrix prepared from other matrices except indobufen in Example 6; blank control group: infiltration with physiological saline), and the wound changes were observed and the healing status was recorded on the 1st, 3rd, 5th and 7th days after modeling of each batch.

[0086] The grouping and medication of each batch of animals are shown in Table 1. Each batch of animals was treated with drugs 24 hours after wound formation. As the wound gradually shrank in the later stage, the dosage of the drug was gradually reduced, and the ointment was evenly applied to the wound with a thickness of 1mm to 2mm.

[0087] Table 1

[0088] The statistical results of wound diameters of mice in each group are shown in Table 2, and the complete healing time of wounds of mice in each group is shown in Table 3.

[0089] Table 2

[0090] Table 3

[0091] It can be seen from Tables 1 to 3 that the indobufen cream of the present invention can significantly accelerate the healing of skin wounds.

[0092] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Use of indobufen in the preparation of a drug for preventing and treating diseases induced by excessive ferroptosis by inhibiting ferroptosis, characterized in that: The indobufen is a ferroptosis inhibitor.

2. The use according to claim 1, characterized in that: The disease induced by excessive ferroptosis is a neurodegenerative disease, an autoimmune disease, a wound or tuberculosis.

3. The use according to claim 2, characterized in that: The wound surface is any one of a chronic inflammatory wound surface, a pressure ulcer, a venous ulcer, a bedsore wound, a burn wound, and a surgical wound.

4. The use according to claim 3, characterized in that: The chronic inflammatory wounds include diabetic wounds and diabetic foot ulcers.

5. The use according to claim 2, characterized in that: The neurodegenerative disease includes one of Parkinson's disease, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis.

6. The use according to claim 2, characterized in that: The autoimmune disease includes any one of rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, scleroderma, and autoimmune thyroiditis.

7. The use according to claim 1, characterized in that: The dosage form of the drug includes liquid dosage form, solid dosage form or semisolid dosage form.

8. The use according to claim 1, characterized in that: The administration route of the drug is enteral or parenteral administration.

9. A drug for promoting wound healing, characterized in that: The medicament comprises indobufen and a pharmaceutically acceptable carrier.

10. The wound healing promoting drug according to claim 9, characterized in that: The pharmaceutically acceptable carrier is at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

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