Application of polyamine in preparation of product for treating radiation-induced oral mucositis

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of polyamine in preparation of a product for treating radiation-induced oral mucositis. The polyamine comprises any one or more of spermine, spermidine and putrescine. The invention finds that the oral basal cell death mode caused after radiation exposure is mainly ferroptosis, the polyamine synthetase ODC1 is activated in the early stage of radiation, the polyamine production is increased, and the polyamine aggregates in the nucleus to inhibit the activation of JUND transcription factors, thereby inhibiting the SAT1-ACSL4 ferroptosis pathway. Research results show that after radiation, polyamine is used as an important medium to inhibit ferroptosis of basal cells and support renewal of mucosal epithelium. The inhibition effect of polyamine on ferroptosis provides a potential strategy for clinical treatment of radiation-related complications.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and disease treatment, and specifically relates to the application of polyamines in preparing products for treating radiation oral mucositis. Background Art

[0002] Radiation-induced oral mucosal damage is called radiation oral mucositis, which is one of the most common adverse reactions to radiotherapy in patients with head and neck tumors. Severe oral mucositis may require feeding tubes to maintain nutrition, treat severe pain, or even stop radiotherapy prematurely. This can seriously affect the effect of tumor treatment, increase the risk of local and systemic infection, and significantly increase the use and cost of medical resources, and even cause treatment interruptions, resulting in a shortened patient survival period. Although supportive treatments such as mouthwashes and cooling devices are currently used clinically to relieve patients' pain, these interventions are not suitable for all types of radiation oral mucositis and are only used for symptom management. Currently, no drugs have been approved to reduce the duration, incidence, or severity of radiation oral mucositis in patients with solid tumors. Therefore, we urgently need to explore the mechanism of mucosal damage and intervene to inhibit the degree of radiation oral mucositis to relieve patients' pain.

[0003] As the first line of defense against various potentially harmful substances, the oral epithelium plays a vital role in protecting the underlying tissues. One of the characteristics of radiation-induced oral mucositis is epithelial ablation. The maintenance of epithelial tissue structure and function depends largely on the stem cell activity of epithelial basal cells. Basal cells can promote the proliferation and differentiation of the oral epithelium, but in radiation-induced oral mucositis, the basal layer cells are damaged and cannot continue to perform this function, and the homeostasis of the oral mucosa is destroyed.

[0004] Ionizing radiation with high linear energy density can cause direct and indirect damage to basal cells after being exposed to radiation. Fibroblasts in the oral mucosa are very likely to regulate the homeostasis of basal stem cells in radiation-induced oral mucositis. Metabolism, iron regulation, and ROS defense jointly control sensitivity to ferroptosis. Therefore, metabolic regulation is one of the key means to regulate the susceptibility of cells to ferroptosis. Radiation also activates the stem cell niche to resist metabolic disorders, which may be the key to inhibiting ferroptosis in basal cells. This key mechanism may guide the development of early intervention strategies for radiation-induced oral mucositis.

[0005] Polyamines are a class of organic compounds containing two or more amino groups. They are widely present in organisms and participate in various physiological processes, but the role of polyamines in radiation-induced oral mucositis is unclear. Summary of the invention

[0006] In order to solve the problem of unclear use of polyamines in radiation oral mucositis, the present invention provides a use of polyamines in preparing a product for treating radiation oral mucositis.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides the use of polyamines in preparing products for treating radiation oral mucositis, provides a new method for treating radiation oral mucositis, and solves the problem of unclear use of polyamines in radiation oral mucositis.

[0009] Wherein, the polyamine includes any one or more of spermine, spermidine and putrescine.

[0010] The present invention has found through research that supplementing polyamines can maintain the proliferation activity of basal cells after ionizing radiation, thereby increasing the thickness of oral mucosa and reducing mucosal damage. Wherein, the polyamine is any one of spermine and spermidine. Moreover, among the above polyamines, spermidine has the best effect, followed by spermine.

[0011] Preferably, the product comprises a medicament.

[0012] Preferably, the drug contains the polyamine as an active ingredient and is supplemented with pharmaceutically acceptable excipients or carriers.

[0013] Preferably, the drug comprises any one of a liquid, a patch, an injection and a solution. In a preferred embodiment of the present invention, the drug is a solution, because the solution can disperse the polyamine well and can be administered in a variety of ways.

[0014] According to the actual treatment purpose and treatment plan, the polyamine can be prepared into different types of drugs to achieve the effect of treating radiation oral mucositis.

[0015] Preferably, the auxiliary materials include any one or more of an antioxidant, a diluent and a coating material. The antioxidant includes any one or more of sodium bisulfite, cysteine, citric acid, ascorbic acid and disodium edetate.

[0016] The diluent includes any one or more of starch, powdered sugar, dextrin, lactose, microcrystalline cellulose and inorganic salts.

[0017] The coating material comprises any one or more of polyvinyl alcohol, blank pellet core, high osmotic pellet core, microcrystalline cellulose pellet core, sugar pellet and high strength starch pellet core.

[0018] Preferably, the carrier comprises any one or more of a hydrogel, a polymer carrier and a liposome.

[0019] Among them, hydrogel plays an important role as a carrier in drug delivery. Due to the biocompatibility, physicochemical properties and designed interaction with the living environment of hydrogel, it is one of the most reliable biomaterial groups. The diversity and versatility of hydrogel make it very outstanding not only in the field of targeted drug delivery but also in tissue engineering, contact lens manufacturing and wound dressing. Therefore, the drug provided by the present invention can be prepared using hydrogel as a carrier, thereby realizing its efficient function of delivering drugs.

[0020] Polymer carriers and high molecular weight nanocarriers, as carriers, can increase drug enrichment in diseased areas and reduce drug toxic side effects.

[0021] Liposomes can change the release properties of drugs in the body and thereby increase drug efficacy and reduce toxic side effects. Their characteristics include targeting and lymphatic orientation, prolonging drug blood circulation time, reducing drug toxicity, and improving stability.

[0022] Preferably, the drug is a solution.

[0023] The solution is prepared by compounding the polyamine with a solvent. After the polyamine is dissolved in the solvent, it can be better delivered in vivo.

[0024] Preferably, the solvent is any one of DMSO, water, ethanol and glycerol, wherein the solvent water can make the polyamine dissolve better.

[0025] Preferably, the concentration of the polyamine in the solution is 2 mg / kg to 10 mg / kg. The solution drug has a large dispersion, fast absorption, rapid action, and good bioavailability; the active ingredients are evenly dispersed and can be accurately measured and used, especially suitable for children and elderly patients; the dosage size is easy to adjust and control; it can reduce the local irritation of certain soluble drugs; and it can increase the stability and safety of certain drugs. Therefore, the dosage form of the above-mentioned drug provided by the present invention is a solution.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention provides the use of polyamines in the preparation of a method for treating radiation oral mucositis, provides a new method for treating radiation oral mucositis, and solves the problem of unclear use of polyamines in radiation oral mucositis.

[0028] 2. In the present invention, we used single-cell RNA sequencing and related experiments to verify the mechanism of obvious ferroptosis of oral epithelial basal stem cells after radiation-induced lipid metabolism changes. The fibroblasts in the stem cell niche are in close contact with the basal cells. We found that two endogenous polyamines, spermine and spermidine, provide metabolic support to maintain the stem cell proliferation activity of basal cells and inhibit their ferroptosis. This metabolism is particularly important in the early proliferation stress stage of RIOM radiation. Based on the crosstalk in polyamine metabolism mediated by fibroblasts and basal cells, it is shown that clinical supplementation of polyamines can be used as a potential treatment for preventing mucosal damage in clinical RIOM patients.

[0029] 3. The present invention aims to provide a method for preparing a product for treating radiation oral mucositis using polyamines, which can not only effectively improve the patient's symptoms and improve the treatment effect, but also ensure the safety and convenience of use. The present invention aims to overcome the deficiencies in the prior art and provide a new treatment option for clinical use by optimizing the use conditions of polyamines, including but not limited to concentration, formulation composition, and administration method. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the animal model of radiation oral mucositis (A) and mucosal damage (B) in the embodiment of the present invention.

[0031] Figure 2 Radiation induced significant ferroptosis in basal cells.

[0032] Figure 3 The staining (A) and statistics (B) of polyamine content in the late radiation period of the present invention.

[0033] Figure 4 The figure shows the effects of spermine (A), spermidine (B) and putrescine (C) on the activity of basal cells after radiation in the present invention.

[0034] Figure 5 The polyamines in the present invention can inhibit the level of ferroptotic lipid peroxidation in basal cells.

[0035] Figure 6 The polyamines in the present invention can inhibit the SAT1-ACSL4 ferroptosis signaling pathway.

[0036] Figure 7 This is the detection of the binding site between the SAT1 upstream transcription factor JUND and SAT1 in the present invention.

[0037] Figure 8 The staining (A) and statistics (B) of the JUND expression after supplementation of the ferroptosis inhibitor LIP-1 and spermidine after radiation in the present invention.

[0038] Fig. 9 The staining (A) and statistics (B) of basal cell proliferation after supplementation of spermine, spermidine and ferroptosis inhibitor in the present invention.

[0039] Fig.10 This is the effect of supplementing spermine, spermidine and ferroptosis inhibitor on the thickness of mucosal tissue in the present invention.

[0040] Fig.11 It is the time for injection treatment of spermine, spermidine and ferroptosis inhibitor in the present invention. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0042] Example 1

[0043] 1. Methods

[0044] 1. scRNA-seq sample preparation

[0045] Tongue tissues were freshly collected from non-irradiated mice, mice 3 days after irradiation, and mice 9 days after irradiation using surgical scissors. The mice were from the Experimental Animal Center of the Air Force Medical University. Non-irradiated mice refer to mice that were not exposed to X-rays. Irradiated mice refer to mice that were exposed to 18Gy X-rays.

[0046] Under sterile conditions, the collected tongue tissue was washed twice with pre-cooled RPMI 1640 medium with 0.04% BSA (MACS) added. After removing the muscle tissue on ice, the tongue tissue was fully minced into 0.5 mm pieces with surgical scissors. 3 The small pieces were placed in freshly prepared collagenase II and 0.5mg / ml hyaluronidase, and digested by enzymatic digestion for 30min in a constant temperature incubator at 37℃. The mixture was mixed by inversion every 5min to obtain a digested cell suspension. The digested cell suspension was filtered once with a 0.4mm cell sieve and centrifuged at 4℃, 300g for 5min. After centrifugation, the precipitate was collected and resuspended with DMEM medium. An equal volume of red blood cell lysis buffer (MACS) was added. After mixing, the precipitate was allowed to stand at 4℃ for 10min. The cell suspension was centrifuged at 300g for 5min and the supernatant was discarded. The precipitate was washed once with DMEM medium, centrifuged at 300g for 5min, and the supernatant was discarded. The cell precipitate was resuspended with 100μL DMEM medium to obtain a single cell suspension. The cell concentration and viability in the cell suspension were calculated using a Luna cell counter.

[0047] Among them, the Chinese name of BSA is bovine serum albumin.

[0048] RPMI 1640 medium: Gibco.

[0049] Collagenase II: Gibco.

[0050] Hyaluronidase: Sigma-Aldrich.

[0051] Cell screen: BD Bioscience.

[0052] Luna cell counter: Logos Biosystems.

[0053] 2. scRNA Sequencing

[0054] Single cell isolation and sequencing were performed at OE Biotech. According to the manufacturer's protocol (Shanghai, China), the cell concentration of freshly prepared single cell suspension was adjusted to 1000 cells / μl, and the ChromiumNext GEMSingle Cell 3′ReagentKits v3.1 (10×Genomics) instructions were followed for loading and library construction.

[0055] The constructed library was sequenced using the Illumina Nova 6000PE150 platform.

[0056] 3. Radiation oral mucositis model

[0057] We induced oral mucositis by single-dose 18 Gy cranial irradiation. -1 Lead shielding their bodies (except the head), we guided each mouse into a custom-made mouse mold with standard snout positioning and closed the end of the clamp. We monitored the irradiated mice every day after a single 18 Gy irradiation. We collected mouse tongue tissue on the ninth day after irradiation, fixed the tissue in paraformaldehyde for 24 h, embedded it in paraffin, and cut 5 μm sections for HE staining to analyze histological changes using mucosal thickness and cell composition.

[0058] 4. Lipid peroxidation assay

[0059] Basal cells were plated in triplicate in six-well plates 24 hours before X-ray irradiation. One of them was pretreated with nothing for 24 hours, the second was pretreated with 10 μM spermine for 24 hours, and the third was pretreated with 10 μM spermidine for 24 hours. After pretreatment, 6 Gy of X-ray irradiation was performed. After 24 hours of incubation of basal cells, fresh DMEM medium with 5 μM BODIPY 581 / 591C11 dye for lipid peroxidation measurement was added to each well. After 30 minutes of incubation at 37°C and 5% CO2 in a humidified incubator, the cells were washed with PBS, trypsinized to obtain cell suspension, and lipid peroxidation levels were analyzed using a flow cytometer.

[0060] Among them, PBS was purchased from Wuhan Punosai Biotechnology Co., Ltd. with a concentration of 1× and a pH of 7.3.

[0061] BODIPY 581 / 591C11 dye: Invitrogen, D3861.

[0062] Confocal microscope: Olympus, FV3000.

[0063] Trypsin: Gibco.

[0064] Flow cytometer: BD FACSCelesta.

[0065] 5. Histology

[0066] Mouse tongue tissue was fixed in 4% buffered paraformaldehyde overnight at room temperature and subsequently dehydrated through a series of isopropanol baths. Finally, the isopropanol was replaced with xylene and the tissue was infiltrated with paraffin before embedding. The tissue was cut with a microtome at a thickness of 4 μm.

[0067] Among them, overnight means time ≧12h.

[0068] Paraformaldehyde: Beyotime.

[0069] A series of isopropanol baths are: 80% by volume isopropanol, 90% by volume isopropanol, and 100% by volume isopropanol.

[0070] Microtome: Leica, RM2016.

[0071] For further use, sections were cleared with xylene and rehydrated. Hematoxylin-eosin staining and Masson staining were performed according to the instructions of the manufacturer of Beyotime. Stained sections were examined under a light microscope and analyzed using ImageJ software.

[0072] The hydration reagents are: EtOH with a volume fraction of 100%, EtOH with a volume fraction of 96%, and ddH2O.

[0073] Optical microscope: Leica Microsystems, Wetzlar, Germany.

[0074] ImageJ software: National Institutes of Health, Bethesda, MD.

[0075] For immunofluorescence (IF) staining, immunofluorescence was performed by heat-mediated antigen retrieval with Tris-EDTA buffer in a Retriever 2100 pressure cooker. The sections were blocked with 3% BSA (MACS) and incubated with primary antibodies overnight at 4°C. The day after antibody incubation, the sections were washed with PBS, and the IF samples were incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature in the dark, followed by DAPI as a nuclear counterstain mixture, anti-fluorescence quenching mounting medium, and observation under a confocal microscope. ImageJ software was used for analysis. For IF staining, the staining intensity of each channel was evaluated, and the relative fluorescent area was semi-quantitatively analyzed.

[0076] Among them, overnight refers to a time ≥12h.

[0077] Retriever 2100 Pressure Cooker: Electron Microscopy Sciences.

[0078] The formula composition of Tris-EDTA buffer is: 10mM Tris, 1mM EDTA, pH 9.0, and the solvent is water.

[0079] Primary antibodies: KRT14, JUND, SPM / SPD, PCNA.

[0080] DAPI: Invitrogen, D1306.

[0081] Mounting medium: Beyotime, P0126.

[0082] Confocal microscope: Olympus, FV3000.

[0083] ImageJ software: National Institutes of Health, Bethesda, MD.

[0084] 6. Western blot assay

[0085] The basal cells were washed with pre-cooled PBS, scraped in PBS, and the pellet was dissolved in lysis buffer supplemented with a mixture of protease / phosphatase inhibitors. The total protein level in the cell lysate was determined by BCA kit, and equal amounts of denatured lysate were resolved on 10% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked in 4% low-fat milk at 20°C for 1 hour and incubated with primary antibodies at 4°C overnight. HRP-conjugated secondary antibodies were blocked at room temperature for 1 hour at 1:10000, and proteins were visualized using ECL substrate.

[0086] Lysis buffer: NCM Biotech.

[0087] BCA Kit: Thermo Fisher Scientific.

[0088] PVDF membrane: Millipore.

[0089] Primary antibodies: Table S3.

[0090] Overnight refers to a time ≥12h.

[0091] Secondary antibody: Proteintech.

[0092] ECL Substrate: Thermo Scientific.

[0093] 7. Determination of cell viability and proliferation ability

[0094] In the determination of cell viability and proliferation ability, we seeded basal cells in 96-well plates at a density of 2000 cells per well, and added DMSO and spermine or spermidine at concentrations of 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, and 100nM; putrescine (MCE) at 20nM, 40nM, 60nM, 80nM, 100nM, 200nM, 300nM, 400nM, and 500nM to DMEM culture medium for pretreatment for 24h before irradiation. 10μL CCK8 solution was added 24h after irradiation, and the wavelength was detected using a microplate reader (Thermo Scientific) after further incubation in the cell culture incubator for 2h.

[0095] Among them, microplate reader: Thermo Scientific.

[0096] 8. Statistical analysis

[0097] Data from three or four experiments are shown as mean ± SD. GraphPad Prism 9.0 was used. All quantitative measurements were tested for normal distribution. Differences between groups were assessed using Student's t-test, one-way or two-way analysis of variance (ANOVA), followed by Holm- Multiple comparison test. To ensure the validity of the observed results, the quantitative experiments were repeated at least 3 times. In all cases, **p<0.01, *p<0.05, ns=not statistically significant (p>0.05).

[0098] Among them, GraphPad Prism 9.0: GraphPad Software, San Diego, CA.

[0099] 2. Results

[0100] 1. Changes in mucosa and basal cells caused by ionizing radiation Figure 1 shown.

[0101] Depend on Figure 1 (A) The method of establishing an animal model of radiation oral mucositis can be clearly seen. Figure 1 (B) Changes in mucosal thickness and basal cell number caused by ionizing radiation can be observed, indicating that ionizing radiation can lead to a significant thinning of the mucosa after a decrease in basal cells.

[0102] 2. Single-cell sequencing scoring to observe the scoring results of basal cell death caused by ionizing radiation Figure 2 shown.

[0103] Depend on Figure 2 It can be seen intuitively that among the scores of apoptosis, pyroptosis, ferroptosis and necrosis, ferroptosis has the highest score, so ferroptosis is the main mode of death of basal cells.

[0104] 3. Pass Figure 3 Immunofluorescence staining can be used to observe the location and content changes of polyamines. The expression of polyamines in basal cells decreased significantly in the late stage of radiation, so ionizing radiation caused a decrease in the content of polyamines in basal cells.

[0105] 4. The results of cell viability test after polyamine supplementation in basal cells are as follows Figure 4 shown.

[0106] Depend on Figure 4 It can be observed that a certain concentration of spermine and spermidine can maintain cell viability. The results of lipid peroxidation level detection after polyamine supplementation in basal cells are as follows Figure 5 As shown by Figure 5 It can be observed that the level of lipid peroxidation in basal cells decreased significantly after polyamine supplementation, so polyamine supplementation can maintain the activity of basal cells after ionizing radiation and inhibit ferroptosis.

[0107] 5. The results of changes in ferroptosis-related proteins in basal cells after polyamine supplementation are as follows Figure 6 As shown. Figure 6 It can be observed that the expression of SAT1 and ACSL4 in basal cells decreased significantly after polyamine supplementation. Figure 7 As shown. Figure 7 It can be seen that JUND can bind to SAT1. The immunofluorescence staining results of JUND after polyamine supplementation are as follows Figure 8 As shown in the figure, JUND expression decreased after polyamine supplementation. Therefore, the mechanism by which polyamine inhibits ferroptosis is to inhibit JUND transcription factors and then inhibit the SAT1-ACSL4 signaling pathway.

[0108] 6. What is the time to supplement polyamines and ferroptosis inhibitors? Fig.11 As shown in Figure 2, the proliferation of basal cells and mucosal thickness after supplementation are as follows Fig. 9 and Fig.10 As shown in the figure, the proliferation level of basal cells increased significantly and the mucosal thickness increased significantly after polyamine supplementation. Therefore, polyamine supplementation can maintain the proliferation activity of basal cells after ionizing radiation, thereby increasing the thickness of the oral mucosa and reducing mucosal damage.

[0109] From the above experimental results, it can be seen that polyamines can inhibit basal cell ferroptosis after ionizing radiation and are a potential strategy for treating radiation oral mucositis. The present invention provides the use of polyamines in the preparation of a novel method for treating radiation oral mucositis, and expands the research of polyamines in radiation oral mucositis.

[0110] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0111] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. The use of polyamines in the preparation of products for treating radiation oral mucositis, characterized in that: The polyamine includes any one or more of spermine, spermidine and putrescine.

2. The use according to claim 1, characterized in that: The polyamine is any one of spermine and spermidine.

3. The use according to claim 1, characterized in that: The products include pharmaceuticals.

4. The use according to claim 3, characterized in that: The drug uses the polyamine as an active ingredient and is supplemented with pharmaceutically acceptable excipients or carriers.

5. The use according to claim 4, characterized in that: The medicine includes any one of liquid, patch, injection and solution.

6. The use according to claim 4, characterized in that: The auxiliary materials include any one or more of antioxidants, diluents and coating materials.

7. The use according to claim 4, characterized in that: The carrier includes any one or more of a hydrogel, a polymer carrier and a liposome.

8. The use according to claim 5, characterized in that: The drug is a solution; The solution is prepared by compounding the polyamine and a solvent.

9. The use according to claim 8, characterized in that: The solvent is any one of DMSO, water, ethanol and glycerol.

10. The use according to claim 8, characterized in that: The concentration of the polyamine in the solution is 2 mg / kg to 10 mg / kg.

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