Use of indobufen in the preparation of a medicament for preventing and treating diseases induced by excessive ferroptosis

Abstract

The present invention discloses the use of indobufen in the preparation of a drug for preventing and treating diseases induced by excessive ferroptosis, specifically relating to the field of pharmaceutical technology. The use of indobufen in the preparation of a drug for preventing and treating diseases induced by excessive ferroptosis by inhibiting ferroptosis, wherein the indobufen is a ferroptosis inhibitor. The present invention provides a new use of indobufen alone or in combination with other drugs in promoting wound healing. Through experiments at the cellular and animal levels, this application proves that indobufen can inhibit the local ferroptosis level of wounds, thereby significantly accelerating wound healing and having good clinical application prospects.

Description

Technical Field

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

[0002] Indobufen is a non-steroidal anti-inflammatory drug (NSAID), mainly used for the prevention and treatment of thrombotic diseases. Its mechanism of action mainly inhibits the activity of cyclooxygenase (COX) enzyme, reduces the synthesis of prostaglandins and thromboxanes, thereby inhibiting platelet aggregation, reducing thrombus formation, and 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 patients, with good safety and tolerance. In addition to cardiovascular and cerebrovascular diseases, indobufen also has certain application values in other diseases. For example, in diabetic patients, indobufen can reduce the occurrence of diabetic complications by inhibiting platelet aggregation. Research shows that indobufen can significantly reduce the incidence of cardiovascular events in diabetic patients and improve the quality of life of patients; Chinese Patent CN101801373A, searching for and developing new drugs for the treatment of pancreatic diabetes. The invention is based on the development of more effective and less toxic indole derivative-based drugs, and the indole derivatives exhibit anti-diabetic, lipid-lowering, hypoglycemic, cholesterol-lowering activities and improve insulin tolerance, and their structures are different from those of the traditionally used compounds. The indole derivatives of the invention are also low-toxic and easily tolerated. In tumor treatment, indobufen also shows certain potential. Research finds that indobufen can play a role in the prevention and treatment of tumors by inhibiting the expression of COX-2, reducing the proliferation and metastasis of tumor cells. Chinese Patent CN108822017A, the invention discloses novel indolinone compounds represented by formula I compounds, physiologically acceptable salts, solvates and crystalline forms thereof, pharmaceutical preparations containing the compounds, and the anti-tumor effects of the compounds.

[0003] However, there is no report on the promotion of wound healing by indobufen. Summary of the Invention

[0004] Therefore, the present invention provides the application of indobufen in the preparation of drugs for preventing and treating diseases induced by excessive ferroptosis to solve the above problems.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The application of indobufen provided by one aspect of the present invention in the preparation of drugs for preventing and treating diseases induced by excessive ferroptosis by inhibiting ferroptosis, wherein the indobufen is a ferroptosis inhibitor.

[0007] The structural formula of indobufen is as follows:

[0008] ; It can be used alone as the active pharmaceutical ingredient or in combination with a pharmaceutically acceptable carrier as the active ingredient.

[0009] Furthermore, the diseases induced by excessive ferroptosis are neurodegenerative diseases, autoimmune diseases, wounds or tuberculosis.

[0010] Among them, tuberculosis itself promotes ferroptosis, leading to inflammatory cell death during infection, and further promoting the spread of pathogenic bacteria and tissue pathological damage.

[0011] As an example, the present invention provides the use of indobufen in the preparation of a drug for promoting wound healing.

[0012] Furthermore, the wound is any one of chronic inflammatory wounds, pressure ulcers, venous ulcers, bedsore wounds, burn wounds, and surgical wounds.

[0013] Furthermore, the chronic inflammatory wounds include diabetic wounds and diabetic foot ulcers.

[0014] Furthermore, the neurodegenerative diseases include one of Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis.

[0015] Furthermore, the autoimmune diseases include any one of rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, and autoimmune thyroiditis.

[0016] Furthermore, the medicinal ingredients for anti-tuberculosis include, but are not limited to, one or more of antibiotics and drugs promoting epidermal growth.

[0017] Furthermore, the dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including aqueous injection, powder injection and infusion), an eye drop, a nasal drop, a lotion and a liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a dropping pill, a suppository, a film, a patch, an aerosol (powder aerosol), a spray, etc.; the semi-solid 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 dropping pill, an injection, a suppository, a spray, a drop, a gel, a patch and a semi-solid preparation.

[0018] As an example, liquid dosage forms include solutions, suspensions, and emulsions, such as aqueous solutions or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions of water-polyethylene glycol.

[0019] 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.

[0020] Furthermore, the administration route can be enteral or parenteral; as an example, the administration routes of the drug include at least one of injection administration, oral administration, and transdermal administration, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0021] According to another aspect of the present invention, a drug for promoting wound healing is provided, and the drug comprises indobufen and a pharmaceutically acceptable carrier.

[0022] 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 solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, or a buffer.

[0023] As an example, the solid carrier can be one or more substances that also act as a diluent, a flavoring agent, a solubilizer, a lubricant, a suspending agent, a binder, a preservative, a tablet disintegrant, or a encapsulating material. In powders, the carrier is a finely divided solid, which is mixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in an appropriate proportion and compressed into the desired shape and size. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, waxes with low melting points, cocoa butter, etc.

[0024] 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 paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin mucilage, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crospovidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; the lubricant and glidant can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing freeze-dried injection, mannitol, glucose, etc. can also be added as a bulking agent.

[0025] If necessary, colorants, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparation.

[0026] Indobufen of the present invention promotes wound healing mainly through the following mechanisms:

[0027] Antiplatelet aggregation:

[0028] In the initial stage of wound healing, platelet aggregation initiates the blood coagulation process, but excessive aggregation may lead to local microcirculation disorders. Indobufen can inhibit platelet aggregation, prevent thrombus formation, thereby improving the blood circulation of the wound surface, ensuring that nutrients and oxygen can be smoothly transported to the wound tissue, and providing good blood supply for wound healing.

[0029] Anti-inflammatory effect:

[0030] The inflammatory reaction after trauma 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 reaction. At the same time, inhibiting the inflammatory reaction also helps to reduce cell damage caused by inflammation and protect the cells around the wound.

[0031] Promote cell proliferation and migration:

[0032] For wound healing, the proliferation and migration of cells are crucial. 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, increasing the synthesis of the extracellular matrix and providing a scaffold for new tissue; it promotes the migration of keratinocytes, accelerating the epithelialization process of the wound.

[0033] Antioxidant effect:

[0034] Trauma can lead to the production of a large amount of reactive oxygen species (ROS) in local tissues, causing oxidative stress and then damaging cells. Indobufen has antioxidant properties, can scavenge ROS, reduce the damage of oxidative stress to cells, is beneficial to maintaining the normal function of cells, and promotes wound healing.

[0035] The present invention has the following advantages:

[0036] The present invention uses indobufen as a drug for promoting wound healing, which has a multi-target action mechanism. It can simultaneously exert multiple effects such as antiplatelet aggregation, anti-inflammatory, antioxidant, and promoting cell proliferation and migration. This multi-target characteristic enables it to intervene in the wound healing process from multiple aspects. Compared with drugs with a single action mechanism, it can more comprehensively respond to the complex physiological and pathological changes in wound healing.

[0037] The present invention uses indobufen as a drug for promoting wound healing, and it has relatively high safety. Compared with some traditional drugs for promoting wound healing, indobufen has relatively fewer adverse reactions. It has less irritation to organs such as the gastrointestinal tract, and when used within a reasonable dose range, it is not easy to cause serious complications such as bleeding, which provides better safety guarantee for long-term or local use.

[0038] The present invention uses indobufen as a drug for promoting wound healing, which can improve local blood circulation. By inhibiting platelet aggregation, indobufen can effectively improve the local blood circulation of the wound. Good blood circulation can not only provide sufficient oxygen and nutrients for the wound, but also timely remove metabolic wastes, which is very important for cell metabolism and tissue repair during the wound healing process.

[0039] The present invention uses indobufen as a drug for promoting wound healing, which can reduce the inflammatory response. The inflammatory response is necessary in the initial stage of wound healing, but excessive inflammation will hinder healing. Indobufen can effectively reduce the inflammatory response, reduce the damage of inflammatory factors to the wound tissue, and contribute to creating a microenvironment conducive to wound healing.

[0040] The present invention provides a new use of indobufen alone or in combination with other drugs in promoting wound healing. Through cell and animal level experiments, this application proves that indobufen can inhibit the level of ferroptosis in the local wound, and then significantly accelerate wound healing, having good clinical application prospects. Brief Description of the Drawings

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary. For those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained based on the provided drawings.

[0042] The structures, ratios, sizes, etc. shown in this specification are only used to cooperate with the content disclosed in the specification for those who are familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the effects that the present invention can produce and the purposes that can be achieved, should still fall within the scope that can be covered by the technical content disclosed in the present invention.

[0043] Figure 1 It is a diagram for detecting the effect of indobufen on ferroptosis of cell function at the cell level by flow cytometry provided in Example 1 of the present invention. Among them, A - detecting the effect of indobufen on cell death and the expression of oxidized lipid reactive oxygen species at the cell level; B - detecting the effect of indobufen on the expression of oxidized lipid reactive oxygen species at the cell level; ****, p < 0.0001.

[0044] Figure 2 It is a comparison diagram of the results of treating mouse skin wounds at different time points with different doses of indobufen provided in Example 2 of the present invention. Among them, A - the skin appearance after treating mouse skin wounds at different time points with different doses of indobufen; B - the statistics of the healing of mouse skin wounds treated with different doses of indobufen; in the figure, the blank control group, no treatment; PVA, polyvinyl alcohol glue (drug carrier). ***, p < 0.001; ****, p < 0.0001.

[0045] Figure 3 It is a hematoxylin-eosin staining and statistical chart of the cross-section of mouse skin wounds treated with different doses of indobufen for 8 days provided in Example 3 of the present invention; among them, A - the hematoxylin-eosin staining results of the cross-section of mouse skin wound tissues treated with different doses of indobufen for 8 days; B - the statistics of the maximum diameter of mouse skin wounds treated with different doses of indobufen for 8 days. *, p < 0.05; **, p < 0.001; ****, p < 0.0001.

[0046] Figure 4Cross-sectional Prussian blue staining (marking the iron ion content in the tissue) and statistical chart of the skin wound tissues of mice treated with different doses of indobufen for 6 days provided in Example 4 of the present invention; wherein, A - Cross-sectional Prussian Blue staining results of the skin wound tissues of mice treated with different doses of indobufen for 6 days; B - Statistical chart of the relative iron concentration in the skin wound tissues of mice treated with different doses of indobufen for 6 days; ***, p<0.001; ****, p<0.0001.

[0047] Figure 5 Cross-sectional 4-HNE immunohistochemistry and statistical chart of the skin wound tissues of mice treated with different doses of indobufen for 6 days provided in Example 5 of the present invention, wherein, A - Cross-sectional 4-HNE immunohistochemistry results of the skin wound tissues of mice treated with different doses of indobufen for 6 days; B - Statistical chart of the relative 4-HNE concentration in the skin wounds of mice treated with different doses of indobufen for 6 days. ***, p<0.001; ****, p<0.0001. Detailed implementation manners

[0048] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0049] In the present invention, during the wound healing process, indobufen, as an inhibitor of ferroptosis, may play the following roles:

[0050] Reduce cell death: Wound healing requires the participation of various cells, such as fibroblasts, keratinocytes, and endothelial cells. In the wound microenvironment, oxidative stress may trigger ferroptosis of cells, thereby hindering wound healing. Indobufen can inhibit ferroptosis, reduce the death of these key cells, maintain the cell number, and is beneficial to wound repair.

[0051] Alleviate the inflammatory response: The inflammatory response after trauma is an early stage of wound healing, but excessive inflammation will produce a large amount of reactive oxygen species (ROS), inducing ferroptosis of cells. While inhibiting ferroptosis, indobufen may reduce the production of inflammatory factors and alleviate the inflammatory response, creating a good environment for wound healing.

[0052] Promote angiogenesis: The ferroptosis inhibitor indobufen may promote the expression of angiogenesis-related factors such as vascular endothelial growth factor (VEGF) by inhibiting ferroptosis of endothelial cells, contributing to the formation of new blood vessels. New blood vessels can provide nutrients and oxygen for the wound, accelerating wound healing.

[0053] DMSO, dimethyl sulfoxide solvent; Indo, indobufen; Indo-R, dextrorotatory indobufen; Indo-S, levorotatory indobufen.

[0054] 4-HNE, 4-Hydroxynonenal, is a harmful metabolite produced during ferroptosis induced by fatty acid peroxidation (indicating the level of ferroptosis occurrence).

[0055] Example 1

[0056] This example provides the detection of the effect of indobufen on ferroptosis of cell function at the cell level by flow cytometry:

[0057] 1. Detection of cell death level

[0058] The degree of cell death was detected using propidium iodide (PI) to indicate the occurrence of overall cell death. The specific operation process is as follows:

[0059] (1) RAW264.7 cells were seeded into a 6-well plate coated with cover slips. After the cells adhered, the ferroptosis inducer 10 μmol RSL3 was added to treat the cells for 12 hours, and then 10 μmol of indobufen, dextrorotatory indobufen or levorotatory indobufen was added simultaneously for combined treatment (the DMSO solvent treatment group was used as a control), and the cells were cultured for another 24 hours.

[0060] (2) The cells treated above were digested with trypsin to make cell suspensions, and incubated with propidium iodide (PI) at a final concentration of 5 μg / mL for 20 min; the cells were washed 3 times with Hank's buffer.

[0061] (3) The cells were resuspended with Hank's buffer and analyzed by flow cytometry (488 nm excitation light), and the proportion of PI-labeled positive cells was calculated.

[0062] Experimental results: Treatment with the ferroptosis inducer RSL3 could cause about 15% of the cells to die (DMSO control group), while treatment with indobufen (Indo), dextrorotatory indobufen (Indo-R) and levorotatory indobufen (Indo-S) could significantly inhibit cell death, and the proportion of dead cells decreased from 15% to 4 - 5% (as shown in Figure 1 A).

[0063] 2. Lipid peroxidation detection experiment

[0064] The degree of lipid peroxidation was detected using the BODIPY C11 fluorescent probe to indicate the degree of cell membrane peroxidation (ferroptosis index). The specific operation process is as follows:

[0065] (1) Seed RAW264.7 cells into a 6-well plate with coverslips. After the cells adhered to the surface, add 10 μmol of the ferroptosis inducer RSL3 to treat the cells for 12 hours, and then add 10 μmol of indobufen, dextrorotatory indobufen or levorotatory indobufen simultaneously for combined treatment (the DMSO solvent treatment group serves as a control), and continue culturing for 24 hours.

[0066] (2) Trypsinize the cells treated above to make cell suspensions respectively, add 1.5 μM BODIPY C11 fluorescent probe diluted with Hank's buffer to co-incubate with the cells for 20 min; wash the cells 3 times with Hank's buffer.

[0067] (3) Resuspend the cells with Hank's buffer and perform flow analysis (excitation light at 488 nm), and calculate the proportion of cells with positive probe labeling.

[0068] Experimental results: Oxidized lipid reactive oxygen species (ROS) is another important indicator for the occurrence of ferroptosis. The proportion of cells expressing oxidized lipid ROS in the DMSO group is about 30%, while the proportion of cells expressing oxidized lipid ROS in the Indo, Indo-R and Indo-S treatment groups is about 9 - 10% ( Figure 1 as shown in Figure B).

[0069] The above two experimental results suggest that Indo, Indo-R and Indo-S can inhibit the occurrence of ferroptosis in cells, and their abilities to inhibit ferroptosis are basically the same.

[0070] Example 2

[0071] This example provides a comparison of the results of treating mouse skin wounds at different time points with different doses of indobufen:

[0072] (1) Animal preparation: Adaptively raise C57BL / 6 mice for 7 days to ensure their good health. Fast the mice for 12 hours and withhold water for 2 hours before the experiment.

[0073] (2) Anesthesia treatment: Use sodium pentobarbital (50 mg / kg, intraperitoneal injection) to ensure that the mice are completely anesthetized.

[0074] (3) Hair removal and disinfection: Remove the hair on the back of the anesthetized mice with an electric hair clipper and disinfect with 75% ethanol to avoid infection.

[0075] (4) Wound creation: Use a round punch or scalpel to prepare full-thickness skin defects (8 mm in diameter) at symmetric positions on both sides of the back of the mice. Keep the wound depth to the full thickness of the skin without damaging the muscle layer.

[0076] (5)Grouping: The mice were randomly divided into 5 groups (blank control group, no-treatment control group; PVA, PVA gel alone treatment group; PVA+Indo (50 μg / mL) Gel, PVA gel containing indobufen treatment group, 10 μg indobufen in every 200 μL gel; PVA+Indo (100 μg / mL) Gel, PVA gel containing indobufen treatment group, 20 μg indobufen in every 200 μL gel; PVA+Indo (200 μg / mL) Gel, PVA gel containing indobufen treatment group, 40 μg indobufen in every 200 μL gel; each group had 5 mice).

[0077] (6)Treatment period: Wound surface photos were taken at 0-8 days after drug treatment, using a scale as a reference. The wound surface changes were calculated by image analysis software (such as ImageJ), and the wound healing rate was calculated as Figure 2 shown in A.

[0078] Experimental results: From the appearance of the wound surface and the statistical results, PVA as a drug carrier had no obvious effect on the wound healing process, while the mixture of PVA and the drug indobufen promoted wound healing, and with the increase of indobufen dosage, the wound healed faster. When the wound surface was treated with the gel drug of PVA+Indo (200 μg / mL), the wound surface could basically heal within 6 days, while other treatment groups needed more than 8 days to heal (as Figure 2 shown in B).

[0079] Example 3

[0080] This example provides hematoxylin-eosin staining and statistics of the cross-sections of the skin wounds of mice treated with different doses of indobufen for 8 days:

[0081] (1)Tissue fixation: Take the intact skin tissues with complete wound surfaces from the mice at different time points after the above drug treatment, and fix them with 10% formalin for 24 hours. Tissue sections were prepared using conventional paraffin embedding techniques, with a thickness of about 4-5 microns.

[0082] (2)Deparaffinization and hydration: The sections were placed in xylene for deparaffinization, 5 minutes each time, for a total of two times. They were sequentially passed through 100%, 95%, 85%, 75% ethanol for 2 minutes each, and finally the sections were rinsed with distilled water.

[0083] (3)Hematoxylin staining: The sections were immersed in hematoxylin staining solution for 5-10 minutes, and rinsed with tap water until there was no excess dye.

[0084] (4)Blueing: The sections were immersed in an alkaline solution (such as 0.25% ammonia water) for a few seconds to blue the cell nuclei, rinsed with tap water, and the cell nuclei showed blue after blueing.

[0085] (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.

[0086] (6) Dehydration and mounting: Perform dehydration treatment by passing through 75%, 85%, 95%, and 100% ethanol for 2 minutes each in sequence. Transparify with xylene for 5 minutes each time, twice in total. Mount with neutral balsam to ensure long-term preservation of the sections.

[0087] (7) Wound size statistics: Use a scale as a reference. Calculate the maximum wound diameter of the wound cross-section through image analysis software (such as ImageJ) as Figure 3 shown in A.

[0088] Experimental results: The results of HE staining and wound diameter statistics show that PVA as a drug carrier has no obvious effect on the wound healing process, while the mixture of PVA and the drug indobufen can promote wound repair and healing, and with the increase in the dosage of indobufen, the wound repair and healing are faster (as Figure 3 shown in B).

[0089] Example 4

[0090] This example provides Prussian blue staining and statistics of the cross-section of mouse skin wound tissue treated with different doses of indobufen for 6 days:

[0091] (1) Tissue fixation and sectioning: Take the wound tissue and fix it with 10% formalin for 24 hours. Prepare tissue sections using conventional paraffin embedding techniques, with a thickness of about 4 - 5 microns.

[0092] (2) Deparaffinization and hydration: Place the sections in xylene for deparaffinization, 5 minutes each time, twice in total. Pass through 100%, 95%, 85%, and 75% ethanol for 2 minutes each in sequence, and finally rinse the sections with distilled water.

[0093] (3) Prussian blue staining: Mix equal volumes of 2% ferric potassium 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 the excess stain.

[0094] (4) Dehydration and mounting: Dehydrate by passing through 75%, 85%, 95%, and 100% ethanol for 2 minutes each in sequence. Transparify with xylene, 5 minutes each time, twice in total. Mount with neutral balsam as Figure 4 shown in A.

[0095] Experimental results: The Prussian blue staining results show that there is obvious iron ion accumulation in the wound tissue of the group without indobufen treatment (the relative iron ion density is about 17%), while with the increase in the dosage of indobufen treatment, the iron ion concentration in the wound tissue gradually decreases, and the relative iron ion density in the PVA + Indo (200 μg / mL) treatment group drops to about 3% (as Figure 4As shown in Figure 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 tissues.

[0096] Example 5

[0097] This example provides immunohistochemistry and statistics of 4-HNE in cross-sections of skin wound tissues of mice treated with different doses of indobufen for 6 days:

[0098] (1) Tissue fixation and sectioning: Fix the tissue with 10% formalin for 24 hours. After paraffin embedding, the section thickness is 4-5 μm.

[0099] (2) Deparaffinization and hydration: Place the sections in xylene for deparaffinization, 5 minutes each time, for a total of two times. Pass through 100%, 95%, 85%, and 75% ethanol for 2 minutes each in turn, and finally rinse the sections with distilled water.

[0100] (3) Antigen retrieval: Heat retrieval (microwave or water bath heating) in citrate buffer (pH 6.0) for about 10-20 minutes. After natural cooling to room temperature, rinse with PBS.

[0101] (4) Blocking: Drop 5-10% normal goat serum or bovine serum albumin (BSA) on the sections and incubate at room temperature for 30 minutes to block non-specific binding sites.

[0102] (5) Primary antibody incubation: Add a specific primary antibody against 4-HNE and dilute it according to the recommended dilution ratio. Incubate overnight at 4°C or for 1-2 hours at room temperature.

[0103] (6) Washing: Wash the sections with PBS 3 times, 5 minutes each time.

[0104] (7) Secondary antibody incubation: Add an appropriate biotinylated secondary antibody or enzyme-conjugated secondary antibody and incubate at room temperature for 30-60 minutes. Wash the sections with PBS 3 times, 5 minutes each time.

[0105] (8) Color development: Add DAB color developer and observe the color reaction, usually for 1-5 minutes. Terminate the reaction with distilled water as Figure 5 shown in Figure A.

[0106] Experimental results: 4-HNE is a harmful metabolite produced during ferroptosis induced by fatty acid peroxidation, and its level can indicate the level of ferroptosis in tissues. The immunohistochemistry results of 4-HNE showed that the relative density of 4-HNE in the wound tissues of the group without indobufen treatment was about 60%. With the increase in the dosage of indobufen treatment, the level of 4-HNE in the wound tissues gradually decreased. The relative density of 4-HNE in the PVA+Indo (200 μg / mL) treatment group decreased to about 5% (as Figure 5 shown in Figure B), and this result suggests that indobufen can significantly inhibit the occurrence of ferroptosis in wound tissues.

[0107] Example 6

[0108] This example provides a cream for promoting wound healing:

[0109] Add 1.5 g of beeswax, 2.3 g of lanolin, 3.1 g of stearic acid, and 1.7 g of glycerol monostearate to a dry 100 mL beaker (denoted as beaker A). Place the beaker in a glass water bath and heat it, controlling the water bath temperature to be constantly 70 °C. Stir evenly at a speed of 700 r / min until completely melted;

[0110] Take another 100 mL dry beaker (denoted as beaker B), add 5.3 g of glycerol, 15.77 g of distilled water, 1.2 g of 1,2 - propanediol, 0.09 g of propylparaben, and 0.003 g of indobufen to it, and stir well;

[0111] Place beaker B in a glass water bath, control the water bath temperature to be 75 °C, heat and stir for about 10 min, then add the mixture in beaker A to beaker B while stirring, mix well, and then emulsify at 75 °C and 600 r / min for 10 min. Then stop heating and continue stirring until cooled to room temperature to obtain indobufen cream.

[0112] Example 7

[0113] Apply the cream obtained in Example 6 to the skin wound model of SPF - level C57BL / 6 mice (the model construction is the same as in Example 2). The experiment is divided into 4 groups (experimental group: the cream of Example 6; positive control group: MEBO, national drug approval number Z20000004; blank matrix group: the blank matrix made of other matrices in Example 6 except indobufen; blank control group: soaked with normal saline). After each batch of model construction, observe the changes of the wound and record the healing situation on the 1st, 3rd, 5th, and 7th days respectively.

[0114] The grouping and drug administration of each batch of animals are shown in Table 1. Each batch of animals is given drug treatment 24 h after the wound is formed. As the wound gradually shrinks in the later stage, the drug dosage also gradually decreases, and it is evenly applied to the wound with a thickness between 1 mm and 2 mm.

[0115] Table 1

[0116]

[0117] The statistical results of the wound diameters of mice in each group are shown in Table 2, and the complete healing times of the wounds of mice in each group are shown in Table 3.

[0118] Table 2

[0119]

[0120] Table 3

[0121]

[0122] As can be seen from Tables 1 - 3, the indobufen cream of the present invention can significantly accelerate the healing of skin wounds.

[0123] Although the present invention has been described in detail above with general descriptions and specific examples, some modifications or improvements can be made to it based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Use of indobufen in the preparation of a drug for promoting wound healing, characterized in that, The wound healing is a surgical wound, and the administration route of the drug is topical administration.

2. The application according to claim 1, wherein The dosage form of the drug includes liquid dosage form, solid dosage form or semi-solid dosage form.

3. The application according to claim 2, characterized in that, The liquid dosage form is solution, suspension or emulsion.

4. The application according to claim 2, wherein The semi-solid dosage form is ointment, gel or paste.

5. The application according to claim 1, characterized in that, The drug includes indobufen and a pharmaceutically acceptable carrier.

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