Use of ferroptosis inhibitors in the preparation of drugs against novel bunyaviruses

Abstract

The application discloses application of an iron death inhibitor in preparation of a medicine for resisting a novel bunyavirus. The application finds through in-vivo and in-vitro experiments that the iron death inhibitor Liproxstatin-1 or deferoxamine DFO can reduce iron death, virus load and MDA content caused by infection of the novel bunyavirus by improving iron overload, lipid peroxidation and cell death, and then relieve multi-organ damage caused by infection of the novel bunyavirus, so as to inhibit the novel bunyavirus. Therefore, the iron death inhibitor can be used for preparing a medicine for preventing and / or treating a novel bunyavirus disease.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of ferroptosis inhibitors in the preparation of drugs against novel Bunyaviruses. Background Technology

[0002] Novel Bunyavirus disease (severe fever with thrombocytopenia syndrome) was first reported in 2009. Cases are mainly distributed in rural areas of mountainous and hilly regions, with a high degree of sporadic occurrence but relatively concentrated geographical distribution. The primary route of infection for SFTSV is through the bite of infected ticks (mainly Haemaphysalis longicornis). The virus can also be transmitted through direct contact with the blood of infected individuals or their contaminated objects, leading to infection among healthcare workers, visitors, and those involved in mortuary services. The general population is susceptible to the disease. High-risk groups for infection include those involved in agricultural activities, tea picking, farmers living in rural hilly or mountainous areas, and tourists visiting such areas. The incidence rate increases with age.

[0003] The incubation period for novel Bunyavirus infection in humans is not fully understood, but it is likely 1-2 weeks. In cases of human-to-human transmission, the incubation period is usually 6-9 days. Clinical symptoms include decreased platelet count, decreased white blood cell count, fever, fatigue, general malaise, muscle aches, and gastrointestinal symptoms such as loss of appetite, nausea, vomiting, and diarrhea. Critically ill patients may also experience skin ecchymosis, involuntary movements of the jaw and limbs with increased muscle tone, and neurological symptoms such as drowsiness, confusion, or stupor. Complications may include lung infection, bleeding in the gastrointestinal tract, lungs, uterus, etc., and even multiple organ failure leading to death.

[0004] In a mouse model of novel Bunyavirus infection, early histopathological changes mainly occurred in the spleen and bone marrow, with an increase in the number of monocytes and an initial decrease in the number of lymphocytes, which gradually recovered after 2 weeks. In the later stages of infection, liver and kidney cells showed extensive degeneration and necrosis, exhibiting pathological changes and damage. Summary of the Invention

[0005] The purpose of this invention is to provide a novel use of ferroptosis inhibitors in the preparation of drugs against novel Bunyavirus. In vitro and in vivo pharmacodynamic studies have confirmed that ferroptosis inhibitors effectively inhibit novel Bunyavirus in both in vitro cell and animal models, demonstrating their potential for use in the preparation of drugs against novel Bunyavirus.

[0006] In a first aspect, the present invention provides novel uses for ferroptosis inhibitors or pharmaceutically acceptable salts thereof.

[0007] This invention provides the use of ferroptosis inhibitors or pharmaceutically acceptable salts thereof in the preparation of products for the prevention and / or treatment of novel Bunyavirus disease (or fever with thrombocytopenia syndrome).

[0008] The present invention also provides the use of ferroptosis inhibitors or pharmaceutically acceptable salts thereof in the prevention and / or treatment of novel Bunyavirus disease (or fever with thrombocytopenia syndrome).

[0009] The present invention also provides the use of ferroptosis inhibitors or pharmaceutically acceptable salts thereof in the preparation of products for inhibiting novel Bunyavirus (or Bunyavirus with fever and thrombocytopenia syndrome).

[0010] The present invention also provides the use of ferroptosis inhibitors or pharmaceutically acceptable salts thereof in the inhibition of novel Bunyavirus (or Bunyavirus with fever and thrombocytopenia syndrome).

[0011] The present invention also provides the use of ferroptosis inhibitors or pharmaceutically acceptable salts thereof in the preparation of novel Bunyavirus inhibitors.

[0012] Any of the products mentioned above may be drugs or drug preparations.

[0013] In the above applications, the pharmaceutically acceptable salts of ferroptosis inhibitors refer to salts that, within a reliable medical judgment range, are suitable for contact with human and lower animal tissues without causing excessive toxicity, irritation, allergic reactions, etc., and that are commensurate with a reasonable effect / risk ratio. The pharmaceutically acceptable salts of ferroptosis inhibitors are well known in the art and include, but are not limited to, sodium salts, potassium salts, calcium salts, hydrochloride salts, nitrates, sulfates, bisulfates, phosphates, hydrogen phosphates, acetates, oxalates, lactates, citrates, tartrates, and maleates.

[0014] In the above applications, when preparing drugs or drug formulations, the ferroptosis inhibitor can be one of the active ingredients or the sole active ingredient.

[0015] In the above applications, when preparing drugs or pharmaceutical formulations, the pharmaceutically acceptable salt of the ferroptosis inhibitor may be used as one of the active ingredients or as the sole active ingredient.

[0016] In the above applications, a carrier material may also be added during drug preparation.

[0017] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.In addition, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; via cavities, such as rectal and vaginal; via the respiratory tract, such as nasal; and via mucosal administration.

[0018] Secondly, the present invention provides pharmaceutical compounds.

[0019] The active ingredient of the pharmaceutical compound provided by this invention is a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof.

[0020] The pharmaceutical compounds provided by this invention can be used for the prevention and / or treatment of novel Bunyavirus diseases.

[0021] The pharmaceutical compounds provided by this invention can be used to inhibit novel Bunyaviruses.

[0022] The pharmaceutical compound provided by this invention can be a novel Bunyavirus inhibitor.

[0023] The pharmaceutical compounds provided by this invention can be formulated into dosage forms such as solutions, tablets, granules, suspensions, capsules, and injections using conventional methods known to those skilled in the art.

[0024] When using the ferroptosis inhibitor or a pharmaceutically acceptable salt thereof provided by the present invention for the prevention and / or treatment of diseases caused by novel Bunyavirus infection (novel Bunyavirus disease or fever with thrombocytopenia syndrome), an effective amount of the ferroptosis inhibitor or a pharmaceutically acceptable salt thereof is administered to the subject.

[0025] The dosage and method of administration of the ferroptosis inhibitor or its pharmaceutically acceptable salt depend on a number of factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the timing of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. Preferred dosages range from 0.01 to 100 mg / kg body weight / day, with the optimal dosage being 20 mg / kg body weight / day.

[0026] In this invention, the term "effective dose" refers to a dose that can achieve treatment, prevention, reduction, and / or relief of the disease or condition described in this invention in a subject.

[0027] In this invention, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the composition of this invention to treat, prevent, reduce and / or alleviate the disease or condition described in this invention.

[0028] The ferroptosis inhibitor described above is selected from any one of Liproxstatin-1, deferric ammonium DFO, Ferrostatin-1, Acetylcysteine, and UAMC-3203.

[0029] In one embodiment of the present invention, the ferroptosis inhibitor is Liproxstatin-1.

[0030] The structural formula of Liproxstatin-1 is shown in formula (Ⅰ).

[0031]

[0032] In another embodiment of the invention, the ferroptosis inhibitor is deferric ammonium ferrophosphate (DFO).

[0033] The structural formula of the deferric ammonium DFO is shown in formula (II).

[0034]

[0035] In this invention, the ferroptosis inhibitor reduces ferroptosis, viral load, and lipid peroxide content induced by novel Bunyavirus infection by improving iron overload, lipid peroxidation, and cell death, thereby alleviating multi-organ damage caused by novel Bunyavirus infection and inhibiting novel Bunyavirus, specifically manifested as any one of the following a1)-a7):

[0036] a1) Inhibits cell death induced by novel Bunyavirus infection;

[0037] a2) Inhibits lipid peroxidation induced by novel Bunyavirus infection;

[0038] a3) Improve the survival rate of mice infected with novel Bunyavirus;

[0039] a4) Increased the expression level of Gpx4 in the spleen of mice infected with novel Bunyavirus;

[0040] a5) Reduces viral load in the spleen of mice infected with novel Bunyavirus;

[0041] a6) Reduces the content of lipid peroxides (MDA) in the spleen of mice infected with novel Bunyavirus;

[0042] a7) Alleviates damage to tissues and organs (such as lungs, liver, and spleen) in mice infected with the novel Bunyavirus.

[0043] The novel Bunyavirus mentioned above can specifically be the novel Bunyavirus HBMC16 strain.

[0044] This invention, through in vitro and in vivo experiments, reveals that ferroptosis inhibitors Liproxstatin-1 or deferoxammonium DFO can reduce ferroptosis, viral load, and lipid peroxide levels induced by novel Bunyavirus infection by improving iron overload, lipid peroxidation, and cell death, thereby alleviating multi-organ damage caused by novel Bunyavirus infection and inhibiting the novel Bunyavirus. Therefore, ferroptosis inhibitors can be used to prepare drugs for the prevention and / or treatment of novel Bunyavirus disease. Attached Figure Description

[0045] Figure 1 Liproxstatin-1 was used to dose-dependently inhibit cell death induced by novel Bunyavirus infection.

[0046] Figure 2 Liproxstatin-1 can inhibit lipid peroxidation caused by novel Bunyavirus infection.

[0047] Figure 3 Liproxstatin-1 can improve the survival rate of mice infected with a novel Bunyavirus.

[0048] Figure 4 Liproxstatin-1 can increase the expression level of the Gpx4 gene in the spleen of mice infected with novel Bunyavirus.

[0049] Figure 5 Liproxstatin-1 can reduce the viral load in the spleen of mice infected with novel Bunyavirus.

[0050] Figure 6 Liproxstatin-1 can reduce the content of lipid peroxide MDA in the spleen of mice infected with novel Bunyavirus.

[0051] Figure 7 Liproxstatin-1 can alleviate multi-organ damage in mice infected with a novel Bunyavirus.

[0052] Figure 8 Deferric ammonium DFO can inhibit cell death induced by novel Bunyavirus infection.

[0053] Figure 9 Deferric ammonium DFO can inhibit lipid peroxidation caused by novel Bunyavirus infection. Detailed Implementation

[0054] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0056] The ferroptosis inhibitor Liproxstatin-1 in the following examples is a product of Selleck, catalog number S7699, CAS number 950455-15-9; its structural formula is shown in formula (I).

[0057]

[0058] The ferroptosis inhibitor Deferoxamine mesylate in the following examples is a product of MCE, catalog number Cat.HY-B0988, CAS number 138-14-7; its structural formula is shown in formula (II).

[0059]

[0060] The novel Bunyavirus HBMC16 strain described in the following examples is described in the literature "Calcium channelblockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality". It was isolated from the Wuhan Institute of Virology, Chinese Academy of Sciences (Wuhan, Hubei Province), and its viral titer was determined on Vero cells by plaque assay.

[0061] The autophagy inhibitor 3-Methyladenine (3-MA) ​​in the following examples is a product of Selleck, catalog number S2767, CAS number 5142-23-4.

[0062] The wild-type C57BL / 6 mice used in the following examples are products of Shanghai Southern Model Biotechnology Development Co., Ltd.

[0063] The Vero cells used in the following examples are ATCC products (cat.CCL-81) and were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco).

[0064] Example 1: The ferroptosis inhibitor Liproxstatin-1 was able to inhibit cell death induced by novel Bunyavirus infection in a dose-dependent manner.

[0065] 1. Six-week-old C57BL / 6 mice were selected, and bone marrow cells were extracted from the bilateral hind limbs (femur + tibia). These cells were induced in vitro for 6 days with 50 ng / mL macrophage colony-stimulating factor (M-CSF, Peprotech, cat.315-02) to form bone marrow-derived macrophages (BMDMs). The specific induction method followed the steps in the literature "CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus." BMDMs were cultured in DMEM medium containing 10% fetal bovine serum (FBS, CLARK) and 50 ng / mL M-CSF.

[0066] 2. BMDMs cells were infected with a novel Bunyavirus, and cells were collected three days later for analysis. Based on the dosage of the ferroptosis inhibitor Liproxstatin-1, the cells were divided into the following groups:

[0067] Group 1: BMDM cell culture system (DMEM medium containing BMDM cells).

[0068] Group 2: Add DMEM medium containing the novel Bunyavirus HBMC16 strain (MOI=5) to the BMDM cell culture system (DMEM medium containing BMDM cells).

[0069] Group 3: Add DMEM medium (MOI=5) containing the novel Bunyavirus HBMC16 strain and ferroptosis inhibitor Liproxstatin-1 solution (solvent: DMSO) to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of ferroptosis inhibitor Liproxstatin-1 in the BMDM cell culture system is 1 μM.

[0070] Group 4: Add DMEM medium (MOI=5) containing the novel Bunyavirus HBMC16 strain and ferroptosis inhibitor Liproxstatin-1 solution (solvent: DMSO) to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of ferroptosis inhibitor Liproxstatin-1 in the BMDM cell culture system is 4 μM.

[0071] Group 5: Add DMEM medium (MOI=5) containing the novel Bunyavirus HBMC16 strain and ferroptosis inhibitor Liproxstatin-1 solution (solvent: DMSO) to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of ferroptosis inhibitor Liproxstatin-1 in the BMDM cell culture system is 8 μM.

[0072] Each cell culture system was cultured at 5% CO2 and 37°C for 3 days. After 3 days, the cells were collected from each group for testing.

[0073] 3. Assess cell death using LDH release.

[0074] Using CytoTox The Non-Radioactive Cytotoxicity Assay (Promega, catalog number G1780) assessed cell death in each group obtained in step 2. The specific steps are as follows: ① Thaw the Assay Buffer rapidly in a 37°C water bath, quickly return to room temperature, and transfer 12 mL of Assay Buffer to dissolve one bottle of Substrate Mix to prepare CytoTox 96Reagent. Aliquot and store in the dark. ② Each experiment requires setting up a "pure culture medium group," "LDH maximum release group," "Mock group," and "experimental group." In this study, a 5% serum concentration was used to prevent excessive LDH in the serum from causing high background. ③ After adding the treatment, maintain the solution volume in each well between 100 and 150 μL. Incubate at 37°C until detectable. ④ Before adding CytoTox 96Reagent, add 10 μL of 10X lysis buffer to the "LDH maximum release group" (add 10 μL of 10X lysis buffer per 100 μL), gently pipette to mix thoroughly, and lyse for 45 min. Cell morphology can be observed under a microscope to determine the effectiveness of the lysis buffer. ⑤ Add the dissolved and aliquoted CytoTox 96Reagent to the sample well, protecting it from light. ⑥ Transfer 50 μL of sample supernatant to a new translucent plate. ⑦ Using a multi-channel pipette, add 50 μL of dissolved CytoTox 96Reagent to each well, gently tapping the sides of the translucent plate to mix the sample with the CytoTox 96Reagent. Incubate at room temperature for 15 min, protecting from light. ⑧ After 15 min, add 50 μL of stop solution to each well. Avoid generating air bubbles and record the absorbance at 490 nm within one hour. Cytotoxicity measurement: Experimental group LDH release (OD490) / Maximum LDH release (OD490) * 100.

[0075] The results are as follows Figure 1As shown, the results indicate that infection of BMDM cells with the novel Bunyavirus caused an increase in cell death levels (assessed by the LDH release method), but treatment of BMDM cells infected with the novel Bunyavirus with Liproxstatin-1 significantly reduced cell death levels in a dose-dependent manner.

[0076] Example 2: The ferroptosis inhibitor Liproxstatin-1 can inhibit lipid peroxidation induced by novel Bunyavirus infection in a dose-dependent manner.

[0077] Use BODIPY TM The 581 / 591C11 (lipid peroxidation sensor, catalog number D3861) was used to perform Lipid ROS detection on the cells obtained in step 2 of Example 1. The specific steps are as follows: ① 1 mg of BODIPY was added to the cells. TM ① Dissolve 581 / 591C11 in 198.2 μL of DMSO to prepare a 10 mM stock solution. Aliquot into 10 μL vials and store in the dark at -80°C, avoiding repeated freeze-thaw cycles. ② Collect the cell samples to be tested, prepare a single-cell suspension, and centrifuge at 600g for 5 min. ③ Discard the supernatant, retain the cell pellet, wash once with PBS, and centrifuge at 600g for 5 min. ④ Discard the supernatant, retain the cell pellet. ⑤ Add 10 mM Body Powder to the solution. TM 581 / 591C11 was dissolved in PBS at a ratio of 1:1000 to prepare 10 μM Body TM Add 100 μL of 581 / 591 working solution to each sample. Vortex to mix, then incubate at 37°C for 30 min in the dark. ⑥ After 30 min, add 800 μL of PBS to each sample to wash away the dye, and centrifuge at 600g for 5 min. ⑦ Discard the supernatant, retain the cell pellet, resuspend the cells in 100 μL of PBS, and store in the dark. ⑧ Detect the fluorescence intensity using flow cytometry.

[0078] The results are as follows Figure 2 As shown, the results indicate that infection of BMDM cells with the novel Bunyavirus caused an increase in cellular lipid peroxidation. However, treatment of BMDM cells infected with the novel Bunyavirus with Liproxstatin-1 significantly reduced lipid peroxidation in a dose-dependent manner.

[0079] Example 3: The ferroptosis inhibitor Liproxstatin-1 can improve the survival rate of mice infected with a novel Bunyavirus.

[0080] I. Construction of a Novel Bunyavirus-Lethal Mouse Model

[0081] Six-week-old, sex-matched C57BL / 6 mice were selected and housed in a specific pathogen-free (SPF) facility under controlled temperature and humidity conditions of 22±2℃ and 50±10%. Mice were intraperitoneally injected with 300 μg of IFNAR1 antibody (Bio X Cell, Cat.BE0241) one day prior to IFNAR1 administration, followed by an injection of 2×10⁻⁶ IFNAR1 antibody one day later. 4 A novel Bunyavirus-lethal mouse model was constructed using the novel Bunyavirus HBMC16 strain with PFU units.

[0082] II. Effects of the ferroptosis inhibitor Liproxstatin-1 on the survival rate of mice infected with novel Bunyavirus

[0083] Two hours after infection with the novel Bunyavirus, mice were treated with an intraperitoneal injection of 20 mg / kg Liproxstatin-1 (a solvent composed of 5% DMSO, 40% PEG300, 5% Tween 80, and 50% ddH2O) as the Liproxstatin-1 treatment group (denoted as Lip-1). The mice were observed daily after injection, and were considered dead when their body weight decreased by 25%. Survival curves were calculated. Simultaneously, mice were treated with an equal volume of the solvent as the solvent treatment group (denoted as Vehicle).

[0084] The results are as follows Figure 3 As shown, the results indicated that, compared with the solvent treatment group, the mortality rate of the Liproxstatin-1 treatment group was significantly lower in mice infected with the novel Bunyavirus (8.3%, 1 / 12 vs. 50%, 5 / 10). This demonstrates that Liproxstatin-1 can significantly improve the survival rate of mice infected with the novel Bunyavirus.

[0085] Example 4: The ferroptosis inhibitor Liproxstatin-1 can increase the expression level of Gpx4 in the spleen of mice infected with novel Bunyavirus.

[0086] I. Construction of a Novel Bunyavirus-Lethal Mouse Model

[0087] Six-week-old, sex-matched C57BL / 6 mice were selected and housed in a specific pathogen-free (SPF) facility under controlled temperature and humidity conditions of 22±2℃ and 50±10%. Mice were intraperitoneally injected with 300 μg of IFNAR1 antibody (Bio X Cell, Cat.BE0241) one day prior to IFNAR1 administration, followed by an injection of 2×10⁻⁶ IFNAR1 antibody one day later. 4 A novel Bunyavirus HBMC16 strain (PFU units) was used to construct a novel Bunyavirus-lethal mouse model (SFTSV). Mice that were not injected with the novel Bunyavirus served as the model control group (Mock).

[0088] II. Effects of the ferroptosis inhibitor Liproxstatin-1 on the anti-lipid peroxidation capacity of tissues in mice infected with novel Bunyavirus

[0089] Two hours after infection with the novel Bunyavirus, mice were treated with an intraperitoneal injection of 20 mg / kg of Liproxstatin-1 (solvent composed of 5% DMSO, 40% PEG300, 5% Tween 80 and 50% ddH2O) as the Liproxstatin-1 treatment group (denoted as SF+Lip-1).

[0090] Spleen tissue (10 mg) was collected 5 days after infection with a novel Bunyavirus. Total RNA was extracted using the Trizol method, and the expression level of the Gpx4 gene was detected by qPCR. Gpx4 expression level is associated with lipid peroxidation; increasing Gpx4 expression level can enhance the tissue's resistance to lipid peroxidation. Primer sequences are as follows:

[0091] Gpx4 Forward: 5'-CCTCCTGCTGCAAGAGCCTCCC-3';

[0092] Gpx4 Reverse: 5'-CTTATCCAGGCAGACATGTGC-3'.

[0093] The results are as follows Figure 4 As shown, the results indicate that novel Bunyavirus infection in mice leads to downregulation of GPx4 expression and reduced tissue resistance to lipid peroxidation. However, the ferroptosis inhibitor Liproxstatin-1 can improve the decrease in GPx4 expression caused by novel Bunyavirus infection and enhance the tissue resistance to lipid peroxidation.

[0094] Example 5: The ferroptosis inhibitor Liproxstatin-1 can reduce the viral load in the spleen of mice infected with novel Bunyavirus.

[0095] Spleen tissue samples were taken from mice injected with the novel Bunyavirus five days after the treatment in Example 4. Total RNA was extracted using the Trizol method, and the copy number of the novel Bunyavirus was detected by qPCR. The primer sequences are as follows:

[0096] SFTSV Forward: 5'-TTCACAGCAGCATGGAGAGG-3'.

[0097] SFTSV Reverse: 5'-GATGCCTTCACCAAGACTATCAATG-3'.

[0098] SFTSV Probe: 5'-AACTTCTGTCTTGCTGGCTCCGC-3'.

[0099] The results are as follows Figure 5 As shown, the results indicated that the viral load of novel Bunyavirus was significantly downregulated in the spleen of mice treated with the ferroptosis inhibitor Liproxstatin-1 (20 mg / kg), demonstrating that Liproxstatin-1 has an inhibitory effect on the virus in vivo.

[0100] Example 6: The ferroptosis inhibitor Liproxstatin-1 can reduce the content of lipid peroxides (MDA) in the spleen of mice infected with novel Bunyavirus.

[0101] Spleen tissue samples were collected from mice injected with the novel Bunyavirus five days after Example 4. The sample weight was recorded. The MDA content in the tissue was detected using an MDA detection kit (Beyotime, catalog number S0131M). The specific steps are as follows: ① Lyse the cell samples using 120 μL of Western blotting and IP cell lysis buffer (catalog number P0013). Centrifuge at 10000g for 10 min and collect 100 μL of supernatant, transferring it to a new EP tube. ② Dissolve TBA in TBA preparation buffer to prepare a 0.37% TBA stock solution. Dissolve the TBA in a shaker at 70℃, protecting it from light. ③ Dilute the standards with distilled water to prepare MDA standards at concentration gradients of 1, 2, 5, 10, 20, and 50 μM for preparing a standard curve. ④ Prepare MDA detection working solutions. Each sample's working solution contains 150 μL of TBA diluent, 50 μL of TBA stock solution, and 3 μL of antioxidant. MDA detection working solution is not easily dissolved; it can be dissolved by shaking at 70℃ in the dark. ⑤ Add 200 μL of MDA detection working solution to each sample and standard, mix well, and then heat in a 100℃ metal bath. Tightly seal the metal bath lid to prevent overheating and splashing of the liquid in the EP tube. ⑥ After 15 minutes, remove the EP tube from the metal bath and place it in a 4℃ refrigerator for a short time to cool to room temperature. ⑦ Centrifuge at 1000g for 10 minutes, and add 200 μL of the supernatant to a translucent plate. ⑧ Detect the absorbance at 532 nm using a microplate reader. ⑨ Calculate the MDA content of each sample based on the standard curve.

[0102] The results are as follows Figure 6 As shown, the results indicated that the lipid peroxidation MDA induced by novel Bunyavirus infection was significantly reduced in the spleen of mice treated with the ferroptosis inhibitor Liproxstatin-1 (20 mg / kg), and that the ferroptosis inhibitor Liproxstatin-1 had the effect of alleviating lipid peroxidation induced by viral infection in vivo.

[0103] Example 7: The ferroptosis inhibitor Liproxstatin-1 can alleviate multi-organ damage in mice infected with a novel Bunyavirus.

[0104] Lung, liver, and spleen tissues from mice injected with the novel Bunyavirus on the fifth day in Example 4 were collected, and the tissue lesions were observed using routine HE staining.

[0105] The results are as follows Figure 7 As shown, the results indicated that after infection with the novel Bunyavirus, the alveolar morphology in the lungs was significantly abnormal, manifested as alveolar shrinkage. The ferroptosis inhibitor Liproxstatin-1 could alleviate the degree of alveolar shrinkage, bringing it closer to the control group. After infection with the novel Bunyavirus, the hepatic cord structure in the liver was significantly lost, and a large number of vacuoles appeared in the liver tissue. The ferroptosis inhibitor Liproxstatin-1 could inhibit the formation of vacuoles. After infection with the novel Bunyavirus, the red and white pulp boundary in the spleen disappeared. The ferroptosis inhibitor Liproxstatin-1 was beneficial in maintaining the red and white pulp structure. All of these results indicate that inhibiting ferroptosis can significantly improve multi-organ damage caused by the novel Bunyavirus and help reduce the mortality rate caused by the novel Bunyavirus.

[0106] Example 8: In vitro inhibition of cell death induced by novel Bunyavirus infection by the ferroptosis inhibitor deferoxammonium DFO.

[0107] 1. BMDMs cells were infected with a novel Bunyavirus, and cells were collected for analysis three days later. Based on the different amounts of the ferroptosis inhibitor deferric ammonium disulfide (DFO) added, the cells were divided into the following groups:

[0108] Group 1 (Mock): BMDM cell culture system (DMEM medium containing BMDM cells).

[0109] Group 2 (DFO): Add deferric ammonium DFO solution (solvent DMSO) to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of deferric ammonium DFO in the BMDM cell culture system is 20 μM.

[0110] Group 3 (SFTFV): DMEM medium containing the novel Bunyavirus HBMC16 strain (MOI=5) was added to the BMDM cell culture system (DMEM medium containing BMDM cells).

[0111] Group 4 (SF+DFO): DMEM medium containing the novel Bunyavirus HBMC16 strain (MOI=5) and deferric ammonium DFO solution (solvent DMSO) were added to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of deferric ammonium DFO in the BMDM cell culture system was 20 μM.

[0112] 2. Each group of cell culture systems was cultured at 37°C with 5% CO2 for 3 days. After 3 days, the cells from each group were collected for testing.

[0113] 3. Cell death was assessed using LDH release, and the detection method was the same as in Example 1.

[0114] The results are as follows Figure 8 As shown, the results indicate that infection of BMDM cells with the novel Bunyavirus caused an increase in cell death levels (assessed by the LDH release method), but treatment of BMDM cells infected with the novel Bunyavirus with the ferroptosis inhibitor deferric ammonium DFO significantly reduced the cell death levels.

[0115] Example 9: In vitro inhibition of lipid peroxidation induced by novel Bunyavirus infection by the ferroptosis inhibitor deferoxammonium DFO.

[0116] Use BODIPY TM The 581 / 591C11 (lipid peroxidation sensor, catalog number D3861) was used to perform Lipid ROS detection on the cells obtained in step 2 of Example 8, using the same detection method as in Example 2.

[0117] The results are as follows Figure 9 As shown, the results indicate that infection of BMDM cells with the novel Bunyavirus caused an increase in cellular lipid peroxidation levels, but treatment of BMDM cells infected with the novel Bunyavirus using the ferroptosis inhibitor deferric ammonium FO significantly reduced lipid peroxidation.

[0118] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a product for the prevention and / or treatment of fever with thrombocytopenia syndrome and Bunyavirus disease; wherein the ferroptosis inhibitor is Liproxstatin-1.

2. The use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting Bunyavirus of fever with thrombocytopenia syndrome; wherein the ferroptosis inhibitor is Liproxstatin-1.

3. The use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a Bunyavirus inhibitor for fever with thrombocytopenia syndrome; wherein the ferroptosis inhibitor is Liproxstatin-1.

4. The application according to claim 1 or 2, characterized in that: The product in question is a medicine.

Images