CN117503935A - Application of hexokinase 2 inhibitor 3-BP in wound repair - Google Patents

Application of hexokinase 2 inhibitor 3-BP in wound repair Download PDF

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CN117503935A
CN117503935A CN202311518625.2A CN202311518625A CN117503935A CN 117503935 A CN117503935 A CN 117503935A CN 202311518625 A CN202311518625 A CN 202311518625A CN 117503935 A CN117503935 A CN 117503935A
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protein
inhibitor
ionizing radiation
radiation
hexokinase
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袁增强
王树坤
袁方
弓玄伟
王丽彬
王景泽
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention belongs to the field of biological medicine, and in particular relates to an application of an inhibitor 3-BP of hexokinase 2 in wound repair. In particular, the invention provides an application of hexokinase 2 inhibitor 3-BP in preparing a medicament for promoting wound healing and preventing radiation injury. The function of reducing apoptosis and accelerating wound healing speed caused by ionizing radiation can be achieved by inhibiting hexokinase 2.

Description

Application of hexokinase 2 inhibitor 3-BP in wound repair
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an application of an inhibitor 3-BP of hexokinase 2 in wound repair.
Background
Wound healing is a complex and orderly process involving hematopoietic cell responses, repair cell responses, extracellular matrix and growth factor responses, etc., where cell responses are key elements in the overall repair process. The pathological process of obviously delaying wound healing under the action of large-dose ionizing radiation is mainly characterized by: the hematopoiesis is restrained, the inflammatory reaction is weakened, especially inflammatory cells such as macrophages and neutrophils which are locally infiltrated by the wound are obviously reduced, and the wound starting process is delayed; vascular damage, endothelial cell degeneration, necrosis and obvious bleeding; the formation and maturation of granulation tissue are obviously slowed down, and the number and the function of fibroblast are damaged; and the epithelialization process is delayed, and the healing time is prolonged. In recent years, research from the molecular level further deepens the understanding of the mechanism of retarding wound healing by ionizing radiation. Studies have shown that reduced hematopoietic cell sources and increased radiation-induced apoptosis are important causes of reduced local inflammatory cell numbers in wounds, while radiation-induced cell proliferation inhibition and increased apoptosis are important causes of reduced fibroblast numbers. Proliferative nuclear antigen (proliferate cell nuclear antigen, PCNA), cyclin E (cyclin E), cyclin-dependent kinase 4 (CDK 4) and other molecules closely related to cell proliferation are reduced in expression, and cell cycle G is inhibited 1 The transition from phase S to phase S is an important mechanism responsible for the inhibition of cell proliferation; and the increase of expression of pro-apoptosis genes such as bax and the like and the decrease of expression of anti-apoptosis genes such as bcl-2 and the like are important mechanisms for causing the increase of apoptosis. Further studies have also found that cell counts are removedIn addition to the reduced amount, the cell functions are also obviously affected, and the synthesis and secretion of extracellular matrix molecules such as collagen, fibronectin and the like and growth factors such as basic fibroblast growth factors, transforming growth factor beta and the like are obviously reduced.
Hexokinase 2 (Hexokinase 2, hk 2) is a key metabolic enzyme that catalyzes the first step in the glycolytic pathway, phosphorylating glucose to glucose-6-phosphate, primarily localized on the outer mitochondrial membrane. HK2 exhibits high expression in many tumors and plays an important role in promoting the production of the Warburg effect. No research has reported that HK2 can promote wound healing after ionizing radiation.
Disclosure of Invention
According to the invention, by constructing an ionizing radiation model, the expression level of Hexokinase 2 (Hexokinase 2, HK 2) in human epidermal stem cells is increased after ionizing radiation, and the function of reducing the apoptosis degree caused by ionizing radiation of the inhibitor of the HK2 is further verified; moreover, animal model experiments prove that the application of 3-BP (3-bromopyruvate, 3-Bromopyruvic acid) which is an inhibitor of Hexokinase 2 (Hexokunase 2, HK 2) in advance can promote the healing of wound surfaces caused by ionizing radiation.
Based on the above purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides an application of an inhibitor of hexokinase 2 in preparing a medicament for promoting wound healing and preventing radiation injury.
Further, the wound surface includes a wound surface caused by ionizing radiation.
Further, the radiation damage is damage caused by ionizing radiation, including but not limited to skin damage caused by ionizing radiation and other radioactive diseases. "ionizing radiation" as used in the present invention includes, in particular, radiation caused by the use of radioactive rays in radiotherapy (which may be simply referred to as radiotherapy). The types of radiation used in radiation therapy include alpha rays, beta rays, X rays, and gamma rays; wherein, the alpha rays and the beta rays are particle beams, and the X rays and the gamma rays are electromagnetic waves.
Further, the ionizing radiation refers to the use of 60 CoγRadiation from the radiation source.
In an embodiment of the present invention, the intensity of the ionizing radiation is 1 to 20Gy, preferably 5 to 8Gy, specifically including 1Gy, 2Gy, 3Gy, 4Gy, 5Gy, 6Gy, 7Gy, 8Gy, 9Gy, 10Gy, 11Gy, 12Gy, 13Gy, 14Gy, 15Gy, 16Gy, 17Gy, 18Gy, 19Gy, 20Gy. In a specific embodiment of the invention, the intensity of the ionizing radiation is 5Gy, 8Gy.
The abbreviation of Gy, namely "Gray", is a standard unit of the physical quantity "ionizing radiation energy absorbing dose". Gray may be interpreted as "Gray", "Golay", "Gray", and sometimes abbreviated as "Golay", and the concepts described above are intended to be used interchangeably herein.
Wound healing (Wound healing) refers to a series of pathophysiological processes in which local tissues are repaired by regeneration, repair and reconstruction after the tissue is lost due to the action of a Wound factor.
In the present invention, the term "inhibitor" refers to a molecule or substance or compound or composition or agent or any combination that is capable of inhibiting and/or reducing the activity or expression level of the target molecule.
Further, the inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules.
Further, the protein binding molecules include compounds.
Further, the protein binding agents also include, for example, receptors for proteins, lectins that bind proteins, antibodies to proteins, peptide antibodies (peptide bodies) to proteins, bispecific dual binding agents, or bispecific antibody formats.
Specifically, the inhibitor competitively or non-competitively binds with Hexokinase 2 (Hexokinase 2, hk 2), thereby achieving the function of inhibiting the biological activity of Hexokinase 2.
Further, the inhibitor is 3-BP or a stereoisomer, a pharmaceutically acceptable salt, a solvate or a hydrate thereof. The 3-BP is 3-bromopyruvate (3-Bromopyruvic acid). Further, the medicament also comprises a pharmaceutically acceptable carrier.
As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent.
Further, the pharmaceutically acceptable carrier includes diluents, buffers, suspensions, emulsions, granules, encapsulates, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorption carriers.
In another aspect, the invention provides the use of a reagent for detecting the amount of hexokinase 2 expressed in a sample from a subject in the manufacture of a product for diagnosing radiation damage, for diagnosing a radiation disease or for determining the severity of radiation damage to the skin.
Preferably, the subject is a mammal. Such as bovine, equine, ovine, porcine, canine, feline, rodent, primate, specifically such as human.
The term "sample" is defined as any material to be tested in the analytical or experimental methods described herein. In some embodiments, the sample is obtained via a non-invasive method (e.g., a non-invasive sample). Exemplary non-invasive methods include, but are not limited to, passive collection of bodily fluids or atraumatic scraping of tissue (e.g., of the epidermis or mouth) accessible from the external environment. Exemplary non-invasive samples include, but are not limited to, saliva, sputum, mucus, sweat, urine, stool, semen, cervical vaginal secretions, breast milk, inflammatory secretions, tears, or cheek epithelial swabs. In some embodiments, the sample is obtained via a micro-invasive method. Exemplary micro-invasive methods include, but are not limited to, capillary collection, venipuncture, thoracocentesis, amniocentesis, needle aspiration, or gastric lavage. Exemplary micro-invasive samples include, but are not limited to, blood or blood fractions (e.g., plasma or PBMC preparations), interstitial fluid, bile, gastric fluid, and amniotic fluid. In some embodiments, the sample is obtained via biopsy. Exemplary biopsy samples include, but are not limited to, skin biopsy samples (e.g., obtained by perforation, shaving, disk surgery, wedging, cutting, or resecting biopsy), bone marrow samples (e.g., obtained by aspiration biopsy), lymph node or breast biopsies (e.g., obtained by fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or image guided biopsy), surgical biopsy samples (e.g., samples of internal organs obtained by resecting or resecting biopsy), or mouth, gastrointestinal tract, lung, bladder, or urinary tract biopsy samples (e.g., obtained by endoscopic examination).
Preferably, the sample comprises an epidermal cell; more specifically, epidermal stem cells.
Further, the radiation damage is damage caused by ionizing radiation.
Further, the radioactive disease is a disease caused by ionizing radiation.
Further, the diseases caused by the ionizing radiation comprise malignant tumors caused by the radiation and degenerative diseases caused by the radiation.
In the present invention, the malignant tumors caused by the irradiation include, but are not limited to, leukemia, skin cancer caused by external irradiation, thyroid cancer, bone malignant tumor, lung cancer caused by internal irradiation, bone malignant tumor, malignant tumor of liver and biliary tract system, and thyroid cancer.
In the present invention, the degenerative diseases caused by the radiation include, but are not limited to, cataract, aplastic anemia, osteonecrosis, osteoporosis, fibrosis of other body parts.
Further, the reagent comprises a reagent for detecting the expression level of hexokinase 2 by a digital imaging technology, a protein immunization technology, a dye technology, a nucleic acid sequencing technology, a nucleic acid hybridization technology, a chromatography technology and a mass spectrometry technology.
Further, the expression amount includes a protein expression amount and an mRNA expression amount, and preferably, a protein expression amount.
Further, the reagent comprises a primer, a probe and a protein binding agent.
Unless otherwise indicated, the term "primer" refers to an oligonucleotide that hybridizes to a sequence in a target nucleic acid ("primer binding site") and is capable of serving as a point to initiate synthesis along the complementary strand of the nucleic acid under conditions suitable for such synthesis.
Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, the probe is able to bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probes may be labeled directly or indirectly.
Unless otherwise indicated, the term "protein binding agent" is for example a receptor for a protein, a lectin that binds a protein, an antibody directed against a protein, a peptide antibody (peptide body) directed against a protein, a bispecific dual binding agent or a bispecific antibody format.
Further, the product comprises a chip, a kit or a nucleic acid membrane strip. In the present invention, a "chip" is also referred to as an "array" and refers to a solid support comprising attached nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using mechanical synthesis methods or light-guided synthesis methods that combine a combination of photolithographic methods and solid-phase synthesis methods. The array may comprise a planar surface or may be a bead, gel, polymer surface, fiber such as optical fiber, glass or any other suitable nucleic acid or peptide on a substrate. The array may be packaged in a manner that allows for diagnosis or other manipulation of the fully functional device. The "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The substrate may be a solid substrate, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid substrate, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any arrangement thereof.
Further, the kit comprises a gene detection kit and a protein detection kit.
Further, the kit may further comprise one or more of the following group: a container, instructions for use, positive control, negative control, buffer, adjuvant, or solvent.
In another aspect, the present invention also provides a method for detecting whether an epidermal stem cell is subjected to ionizing radiation, which comprises detecting the expression level of hexokinase 2 in the epidermal stem cell. Also, it may be referred to as a method of detecting whether a subject has been subjected to ionizing radiation, the epidermal stem cells being taken from the subject.
Further, the epidermal stem cell is an isolated epidermal stem cell or a commercial cell line.
Further, the method is performed in vitro or for non-diagnostic purposes. Specifically, in the method, an increase in the expression level of hexokinase 2 is indicative of exposure to ionizing radiation, more specifically, of 60 And (5) Co gamma ray irradiation.
In another aspect, the present invention also provides a method for inhibiting apoptosis of epidermal stem cells, which comprises inhibiting the expression level of hexokinase 2 in the epidermal stem cells.
Further, the method is performed in vitro or is not therapeutic.
Further, the inhibiting the expression level of hexokinase 2 in the epidermal stem cell comprises treating the epidermal stem cell with an inhibitor of hexokinase 2.
In another aspect, the invention also provides a method of promoting wound healing, preventing radiation damage, comprising administering to a subject an inhibitor of hexokinase 2. Further, the hexokinase 2 inhibitor is applied by application to the skin.
Further, the inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules.
Further, the protein binding molecules include compounds.
Further, the protein binding agents also include, for example, receptors for proteins, lectins that bind proteins, antibodies to proteins, peptide antibodies (peptide bodies) to proteins, bispecific dual binding agents, or bispecific antibody formats.
Further, the inhibitor is 3-BP or a stereoisomer, a pharmaceutically acceptable salt, a solvate or a hydrate thereof.
More specifically, the 3-BP of the present invention is formulated using 3% methylcellulose as a solvent.
The invention has the beneficial effects that:
the invention discloses that the protein level of hexokinase 2 in human epidermal stem cells is obviously increased 48 hours after ionizing radiation treatment for the first time. The invention further proves that the HK2 inhibitor can reduce apoptosis caused by ionizing radiation through a cell experiment, and animal experiments prove that the HK2 inhibitor can promote wound healing. The invention provides a new method for promoting wound healing and has good clinical application prospect.
Drawings
FIG. 1 is a graph showing the results of immunoblotting for detecting protein levels of γH2A in human epidermal stem cells after ionizing radiation,
FIG. 2 is a graph showing the results of reactive oxygen species detection of human epidermal stem cells after ionizing radiation by ROS,
FIG. 3 is a graph showing the results of Tunel detection of apoptosis levels of human epidermal stem cells after ionizing radiation,
FIG. 4 shows the protein level of HK2 in human epidermal stem cells after detection of ionizing radiation by immunoblotting,
FIG. 5 is a graph showing the results of Tunel detection of apoptosis levels of human epidermal stem cells after 3-BP treatment,
FIG. 6 is a graph showing the results of 3-BP promoting wound healing after ionizing radiation.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 role of Hexokinase 2 (Hexokinase 2, HK 2) in wound repair
1. Experimental materials
1. Human epidermal stem cell line, stored in the laboratory (originally purchased from company);
2. ionizing radiation source: military medical institute 60 A Co gamma-ray source;
3. mice: for the C57BL/6J background, the culture was kept at room temperature (23 ℃) for 12 hours in a day-night period and allowed free ingestion and drinking water, purchased from Si Bei Fu (Beijing) Biotechnology Co., ltd;
4. inhibitors of HK2 3-BP (small compound) were purchased from selselect company;
5. ROS assay kit and apoptosis assay kit were purchased from Biyundian corporation.
2. Experimental method
1. Modeling and evaluation of ionizing radiation (Ionizing radiation, IR) damaged cells
Establishing an ionization injury cell model by using human epidermal stem cells, preparing single-cell suspension from the epidermal stem cells after digestion by pancreatin, and spreading the single-cell suspension in a cell culture plate; the next day cells were attached to the wall and the cell plates were placed 60 Irradiating under Co gamma rays, wherein the dose is 5Gy, and the irradiation dose is 79cGy/min; the irradiation time is about 6min, and then the sample is placed in an incubator, and samples are collected at different time points of 12h, D2 and the like for subsequent detection.
The acute phase DNA is damaged after ionizing radiation, while γh2ax is a DNA double strand break closely related, and is a marker of DNA damage, indicating whether the ionizing radiation cell model is successful.
2. ROS detection, apoptosis detection
The intracellular reactive oxygen species and apoptosis were analyzed using ROS kit and Tunel kit (bi yun) respectively, with reference to kit instructions.
3. Mouse ionizing radiation damage model
Male C57BL/6J mice were randomly divided into 4 groups, ctrl, ctrl+3-BP,After anesthesia with 2% tribromoethanol (intraperitoneal administration, 10 ul/g), the back hair was shaved off, and a round wound with a diameter of about 1cm was punched with a punch, and the results were obtained in the case of IR and IR+3-BP mice 60 And (3) carrying out local irradiation under Co gamma rays, wherein the dose is 8Gy, and a small compound 3-BP (prepared by using 3% methyl cellulose as a solvent) is smeared before irradiation, so as to record the wound healing condition.
3. Experimental results
1. Successfully build ionizing radiation model of human epidermal stem cells
As shown in fig. 1, human epidermal stem cells (EpSC) showed a significant increase in protein level of γh2a 0.5H after ionizing radiation (5 Gy), indicating DNA damage to the cells, which was successful in the ionizing radiation cell model.
2. Active oxygen in human epidermal stem cells increases after ionizing radiation, and cells undergo apoptosis
ROS detection results show that the human epidermal stem cells have significantly increased intracellular reactive oxygen species content and cell damage 2 days after ionizing radiation treatment. (as shown in FIG. 2)
Tunel test results show that the number of human epidermal stem cells is relatively obviously increased 3 days after ionizing radiation treatment, which indicates that cells undergo apoptosis after IR. (as shown in FIG. 3)
3. Increased expression of HK2 in epidermal stem cells in the acute phase after ionizing radiation
As shown in fig. 4, WB detection results show that 48 hours after ionizing radiation treatment, protein levels of HK2, a key limiting enzyme for sugar metabolism in cells, are significantly increased, suggesting that the cell sugar metabolism is disturbed after IR, and HK2, a key enzyme for metabolism, is abnormally increased, possibly involved in cell damage after IR.
4. The addition of the HK2 inhibitor 3-BP can reduce apoptosis of the epidermal stem cells
As shown in FIG. 5, the Tunel test results show that the number of human epidermal stem cells after the ionizing radiation treatment is relatively significantly increased, and the apoptosis is reduced after the treatment with the HK2 inhibitor 3-BP.
5. Application of HK2 inhibitor 3-BP in advance promotes wound healing after ionizing radiation
Establishing a mouse ionizing radiation skin injury model, smearing an HK2 inhibitor for 3-bp before IR, detecting wound healing conditions, and displaying the results: as shown in fig. 6, the IR group mice wound did not heal yet after 12 days of irradiation, and the ctrl and ir+ inhibitor groups wound healed better. This shows that HK2 plays an important role in the repair of ionizing radiation wound, and the use of its inhibitors can promote wound healing.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of the details are possible in light of the above teachings, and such variations are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. Application of hexokinase 2 inhibitor in preparing medicine for promoting wound healing and preventing radiation injury;
preferably, the wound surface comprises a wound surface caused by ionizing radiation;
preferably, the radiation damage is damage caused by ionizing radiation; more preferably, skin lesions;
preferably, the ionizing radiation includes radiation caused by alpha rays, beta rays, X rays and gamma rays;
preferably, the ionizing radiation is 60 Radiation by Co gamma rays;
preferably, the intensity of the ionizing radiation is 1-20Gy or 5Gy-8 Gy;
preferably, the intensity of the ionizing radiation is 5Gy, 8Gy.
2. The use according to claim 1, wherein the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, a proteolytic enzyme or a protein binding molecule;
preferably, the protein binding molecule comprises a compound;
preferably, the protein binding molecules further comprise a receptor for a protein, a lectin that binds a protein, an antibody directed against a protein, a peptide directed against a protein;
preferably, the inhibitor is 3-BP or a stereoisomer, pharmaceutically acceptable salt, solvate or hydrate thereof.
3. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier;
preferably, the pharmaceutically acceptable carrier includes diluents, buffers, suspensions, emulsions, granules, encapsulates, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorption carriers.
4. Application of reagent for detecting hexokinase 2 expression in a subject sample in preparing products for diagnosing radiation injury, diagnosing radioactive diseases or judging skin radiation injury severity;
preferably, the expression amount includes a protein expression amount and an mRNA expression amount;
preferably, the radiation damage is damage caused by ionizing radiation;
preferably, the radioactive disease is a disease caused by ionizing radiation.
5. The use according to claim 4, wherein the reagent comprises a reagent for detecting the expression level of hexokinase 2 by digital imaging technique, protein immunization technique, dye technique, nucleic acid sequencing technique, nucleic acid hybridization technique, chromatography technique, mass spectrometry technique.
6. The method of claim 4, wherein the reagent comprises a primer, a probe, and a protein binding agent.
7. The use according to claim 4, wherein the product comprises a chip, a kit or a nucleic acid membrane strip.
8. A method for detecting whether an epidermal stem cell is subjected to ionizing radiation, comprising detecting the expression level of hexokinase 2 in the epidermal stem cell.
9. A method for inhibiting apoptosis of epidermal stem cells, comprising inhibiting expression of hexokinase 2 in the epidermal stem cells.
10. The method of claim 9, wherein the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, a proteolytic enzyme, or a protein binding molecule;
preferably, the protein binding molecule comprises a compound;
preferably, the protein binding molecules further comprise a receptor for a protein, a lectin that binds a protein, an antibody directed against a protein, a peptide directed against a protein;
preferably, the inhibitor is 3-BP or a stereoisomer, pharmaceutically acceptable salt, solvate or hydrate thereof.
CN202311518625.2A 2023-05-30 2023-11-15 Application of hexokinase 2 inhibitor 3-BP in wound repair Pending CN117503935A (en)

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Citations (2)

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WO2022006184A2 (en) * 2020-06-29 2022-01-06 Kodiscovery, Llc Cellular energy inhibitor formulations for the treatment of pathogenic infections and associated methods

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