Application of ferroptosis inhibitor in preparation of medicine for resisting novel bunyavirus

Abstract

The invention discloses an application of a ferroptosis inhibitor in preparation of a novel bunyavirus resisting drug. In-vivo and in-vitro experiments find that the ferroptosis inhibitor Liproxstatin-1 or DFO can reduce ferroptosis, virus load and MDA (malondialdehyde) content caused by novel bunyavirus infection by improving iron overload, lipid peroxidation and cell death, so that multi-tissue organ injury caused by novel bunyavirus infection is relieved, and the novel bunyavirus is inhibited. Therefore, the ferroptosis inhibitor can be used for preparing the medicine for preventing and / or treating the novel bunyavirus disease.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a ferroptosis inhibitor in the preparation of a drug against a novel bunyavirus. Background Art

[0002] The new Bunyavirus disease (sever fever with thrombocytopnia syndrome) was first reported in 2009. Cases are mainly distributed in rural areas in mountainous and hilly areas. The cases are highly sporadic, but relatively concentrated in geographical distribution. The new Bunyavirus, sever fever with thrombocytopnia syndrome virus (SFTSV), is mainly transmitted through the bite of infected ticks (mainly long-horned ticks). The virus can also be transmitted through direct contact with the patient's blood or its contaminants, thereby causing infection to medical staff, visitors or funeral personnel. People are generally susceptible to this disease. People who participate in agricultural planting activities or tea picking, farmers living in rural hilly or forested areas, and people who travel to such areas are at high risk of infection with the new Bunyavirus, and the incidence rate shows an upward trend with increasing age.

[0003] The incubation period of the new Bunyavirus infecting humans is not very clear, but it may be 1-2 weeks. In cases of human-to-human transmission, the incubation period is mostly 6 to 9 days. Clinical symptoms include low platelets, low white blood cell count, fever, fatigue, general discomfort, muscle aches, poor appetite, nausea, vomiting, diarrhea and other digestive symptoms. Critically ill patients also have skin ecchymoses, involuntary movements of the mandible, limbs and other parts accompanied by increased muscle tension and neurological symptoms such as drowsiness, confusion or lethargy, combined with lung infection, bleeding in the digestive tract, lungs, uterus and other cavities, and even multiple organ failure leading to death.

[0004] In the mouse model infected with the new Bunyavirus, early histopathological changes mainly occurred in the spleen and bone marrow, with an increase in the number of monocytes and a decrease in the number of lymphocytes, which then gradually recovered after 2 weeks. In the later stages of infection, a large number of liver and kidney cells underwent degeneration and necrosis, showing pathological changes and damage. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of ferroptosis inhibitors in the preparation of anti-new bunyavirus drugs. In vitro and in vivo pharmacodynamics experiments have confirmed that ferroptosis inhibitors can effectively inhibit new bunyaviruses in both in vitro cell models and animal models, and have the potential to be used in the preparation of anti-new bunyavirus drugs.

[0006] In a first aspect, the present invention provides a new use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof.

[0007] The present invention provides the use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating a novel bunyavirus disease (or fever with thrombocytopenia syndrome).

[0008] The present invention also provides the use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in preventing and / or treating a novel bunyavirus disease (or fever with thrombocytopenia syndrome).

[0009] The present invention also provides the use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting a novel bunyavirus (or a fever with thrombocytopenia syndrome bunyavirus).

[0010] The present invention also provides the use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in inhibiting a novel bunyavirus (or a fever with thrombocytopenia syndrome bunyavirus).

[0011] The present invention also provides the use of the ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a novel bunyavirus inhibitor.

[0012] Any of the above products can be a drug or a drug preparation.

[0013] In the above application, the pharmaceutically acceptable salt of the ferroptosis inhibitor refers to a salt that is suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable effect / risk ratio within the scope of reliable medical judgment. The pharmaceutically acceptable salt of the ferroptosis inhibitor is well known in the art, including but not limited to sodium salt, potassium salt, calcium salt, hydrochloride, nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, oxalate, lactate, citrate, tartrate and maleate, etc.

[0014] In the above applications, when preparing drugs or pharmaceutical preparations, the ferroptosis inhibitor can be used as one of the effective ingredients or as the only effective ingredient.

[0015] In the above application, when preparing drugs or pharmaceutical preparations, the pharmaceutically acceptable salt of the ferroptosis inhibitor can be used as one of the active ingredients or as the only active ingredient.

[0016] In the above applications, carrier materials may also be added when preparing the medicine.

[0017] The carrier material includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. 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, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also 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. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, flavors, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal administration; respiratory tract administration, such as nasal cavity; mucosal administration.

[0018] In a second aspect, the present invention provides pharmaceutical compounds.

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

[0020] The pharmaceutical compound provided by the present invention can be used for preventing and / or treating novel bunyavirus disease.

[0021] The medicinal compound provided by the invention can be used to inhibit the novel Bunyavirus.

[0022] The pharmaceutical compound provided by the present invention can be a novel bunyavirus inhibitor.

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

[0024] When the ferroptosis inhibitor provided by the present invention or its pharmaceutically acceptable salt is used to prevent and / or treat diseases caused by novel bunyavirus infection (novel bunyavirus disease or fever with thrombocytopenia syndrome), an effective amount of the ferroptosis inhibitor or its pharmaceutically acceptable salt is administered to the subject's organism.

[0025] The dosage and method of use of the ferroptosis inhibitor or its pharmaceutically acceptable salt depend on many factors, including the patient's age, weight, gender, natural health status, nutritional status, activity strength of the compound, time of administration, metabolic rate, severity of the disease, and subjective judgment of the treating physician. The preferred dosage is between 0.01-100 mg / kg body weight / day, with the optimal dosage being 20 mg / kg body weight / day.

[0026] In the present invention, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0027] In the present invention, the term "subject" may refer to a patient or other animal that receives the composition of the present invention to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present invention, particularly mammals, such as humans, dogs, monkeys, cows, horses, etc.

[0028] Any of the above-mentioned ferroptosis inhibitors is selected from any one of Liproxstatin-1, Desferrioxanium 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 present invention, the ferroptosis inhibitor is desferrioxanium DFO.

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

[0034]

[0035] In the present invention, the ferroptosis inhibitor improves iron overload, lipid peroxidation and cell death, thereby reducing ferroptosis, viral load and lipid peroxide content caused by novel bunyavirus infection, thereby alleviating multi-tissue organ damage caused by novel bunyavirus infection and inhibiting novel bunyavirus, which is specifically embodied in any one of the following a1)-a7):

[0036] a1) Inhibit cell death caused by novel bunyavirus infection;

[0037] a2) Inhibit lipid peroxidation caused by novel bunyavirus infection;

[0038] a3) improving the survival rate of mice infected with the novel bunyavirus;

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

[0040] a5) Reduction of viral load in spleen of mice infected with novel bunyavirus;

[0041] a6) reducing the lipid peroxide (MDA) content in the spleen of mice infected with the novel bunyavirus;

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

[0043] Any of the novel Bunyaviruses described above may specifically be the novel Bunyavirus HBMC16 strain.

[0044] The present invention has found through in vitro and in vivo experiments that the ferroptosis inhibitor Liproxstatin-1 or desferric ammonium DFO can reduce the ferroptosis, viral load and lipid peroxide content caused by the novel bunyavirus infection by improving iron overload, lipid peroxidation and cell death, thereby alleviating the multi-tissue organ damage caused by the novel bunyavirus infection, thereby inhibiting the novel bunyavirus. Therefore, the ferroptosis inhibitor can be used to prepare a drug for preventing and / or treating the novel bunyavirus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Liproxstatin-1 inhibits cell death induced by novel bunyavirus infection in a dose-dependent manner.

[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 the new Bunyavirus.

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

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

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

[0051] Figure 7 Liproxstatin-1 can alleviate multiple organ damage in mice infected with the new Bunyavirus.

[0052] Figure 8 Desferric ammonium (DFO) can inhibit cell death caused by novel bunyavirus infection.

[0053] Fig. 9 Desferric ammonium (DFO) can inhibit lipid peroxidation caused by novel bunyavirus infection. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0057]

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

[0059]

[0060] The novel Bunyavirus HBMC16 strain in the following example is recorded in the document “Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality”, isolated from the Wuhan Institute of Virology, Chinese Academy of Sciences (Wuhan, Hubei), and its virus titer was measured on Vero cells by plaque assay.

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

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

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

[0064] Example 1: The ferroptosis inhibitor Liproxstatin-1 can inhibit cell death caused by novel bunyavirus infection in a dose-dependent manner

[0065] 1. Select 6-week-old C57BL / 6 mice, extract bone marrow cells from the lower limbs (femur + tibia) of the mice, and use 50ng / mL macrophage colony stimulating factor (M-CSF, Peprotech, cat.315-02) to induce in vitro for 6 days to form bone marrow-derived macrophages (BMDMs). The specific induction method refers to 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 50ng / mL M-CSF.

[0066] 2. Use the new Bunyavirus to infect BMDMs cells, and collect the cells for detection three days later. According to the different doses of 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: DMEM medium containing the novel bunyavirus HBMC16 strain (MOI=5) was added to the BMDM cell culture system (DMEM medium containing BMDM cells).

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

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

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

[0072] The cell culture systems of each group were cultured at 5% CO2 and 37°C for 3 days, and the cells of each group were collected for detection after 3 days.

[0073] 3. Assessment of cell death using LDH release

[0074] Using CytoTox Non-Radioactive Cytotoxicity Assay (Promega, Cat. No. G1780) was used to evaluate the cell death of each group obtained in step 2. The specific steps are as follows: ① Rapidly thaw the Assay Buffer in a 37°C water bath, quickly return to room temperature, transfer 12mL of Assay Buffer to dissolve a bottle of Substrate Mix, configure it into CytoTox 96 Reagent, and store it in aliquots away from light. ② Each experiment needs to set up "pure culture medium group", "LDH maximum release group", "Mock group" and "experimental group". In this study, 5% serum concentration was used to prevent excessive LDH in the serum from causing excessive background. ③ After adding treatment, keep the solution volume in each well between 100 and 150 μL. Incubate at 37°C until the detectable time. ④ Before adding CytoTox 96 Reagent, add 10 μL of 10X lysis buffer to the "LDH maximum release group" (add 10 μL of 10X lysis buffer to every 100 μL), gently blow, mix thoroughly, and lyse for 45 minutes. The cell morphology can be observed under a microscope to determine whether the lysis solution is effective. ⑤ Take out the dissolved and packaged CytoTox 96 Reagent and add it to the sample tank, making sure to avoid light. ⑥ Transfer 50μL of the sample supernatant to a new translucent plate. ⑦ Use a spray gun to add 50μL of the dissolved CytoTox 96 Reagent to each well, gently tap the periphery of the translucent plate to mix the sample with the CytoTox 96 Reagent. Place at room temperature away from light for 15 minutes. ⑧ After 15 minutes, add 50μL of stop solution to each well. Avoid generating bubbles, and record the absorbance at 490nm within one hour. Cytotoxicity measurement: LDH release in the experimental group (OD490) / maximum LDH release (OD490)*100.

[0075] The results are as follows Figure 1As shown, the results showed that after the novel Bunyavirus infected BMDM cells, the level of cell death (assessed by the LDH release method) increased, but when Liproxstatin-1 was used to treat the novel Bunyavirus-infected BMDM cells, the level of cell death was significantly reduced in a dose-dependent manner.

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

[0077] Using BODIPY TM 581 / 591C11 (lipid peroxidation sensor, catalog number D3861) was used to detect Lipid ROS in each group of cells obtained in step 2 of Example 1. The specific steps are as follows: ① 1 mg BODIPY TM 581 / 591C11 was dissolved in 198.2μL DMSO to prepare a 10mM storage solution, which was divided into 10μL per tube and stored in a -80℃ refrigerator away from light to avoid repeated freezing and thawing. ② Collect the cell sample to be tested, prepare a single cell suspension, and centrifuge at 600g for 5min. ③ Discard the supernatant, keep the cell pellet, wash once with PBS, and centrifuge at 600g for 5min. ④ Discard the supernatant, keep the cell pellet. ⑤ Add 10mM BODIPY TM 581 / 591C11 was dissolved in PBS at a ratio of 1:1000 to prepare 10 μM BODIPY TM 581 / 591 working solution, add 100μL to each sample. Vortex to mix, place in a 37℃ incubator away from light for 30min. ⑥ After 30min, add 800μL PBS to each sample to wash away the dye, centrifuge at 600g for 5min. ⑦ Discard the supernatant, keep the cell pellet, add 100μL PBS to resuspend the cells, and store away from light. ⑧ Detect the fluorescence intensity on a flow cytometer.

[0078] The results are as follows Figure 2 As shown, the results showed that after the new Bunyavirus infected BMDM cells, the level of cellular lipid peroxidation increased, but when Liproxstatin-1 was used to treat BMDM cells infected with the new Bunyavirus, the lipid peroxidation phenomenon was significantly reduced in a dose-dependent manner.

[0079] Example 3: The ferroptosis inhibitor Liproxstatin-1 can improve the survival rate of mice infected with the new bunyavirus

[0080] 1. 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 with temperature and humidity controlled at 22±2°C and 50±10%. Mice were intraperitoneally injected with 300 μg IFNAR1 antibody (Bio X Cell, Cat. BE0241) one day in advance and 2×10 4 A new Bunyavirus-lethal mouse model was constructed by using PFU units of the new Bunyavirus HBMC16 strain.

[0082] 2. Effect of 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 intraperitoneally injected with 20 mg / kg dose of Liproxstatin-1 (the solvent consisted 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 every day after injection. When the weight of the mice decreased by 25%, they were judged dead and the survival curve was calculated. At the same time, the same amount of solvent was injected as the solvent treatment group (denoted as Vehicle).

[0084] The results are as follows Figure 3 As shown, the results showed that after the novel bunyavirus infected mice, the mortality rate of the lipoxstatin-1 treatment group was significantly reduced compared with the solvent treatment group (8.3%, 1 / 12 vs. 50%, 5 / 10), indicating that lipoxstatin-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 the new bunyavirus

[0086] 1. 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 with temperature and humidity controlled at 22±2°C and 50±10%. Mice were intraperitoneally injected with 300 μg IFNAR1 antibody (Bio X Cell, Cat. BE0241) one day in advance and 2×10 4 The novel Bunyavirus HBMC16 strain was injected with PFU units to construct a novel Bunyavirus-lethal mouse model (referred to as SFTSV). Meanwhile, mice not injected with the novel Bunyavirus were used as a model control group (referred to as Mock).

[0088] 2. Effect of ferroptosis inhibitor Liproxstatin-1 on the anti-lipid peroxidation capacity of tissues of mice infected with novel bunyavirus

[0089] Two hours after infection with the novel bunyavirus, mice were intraperitoneally injected with 20 mg / kg dose of Liproxstatin-1 (solvent consists 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 obtained 5 days after infection with the new Bunyavirus, and total RNA was extracted using the Trizol method. The expression level of the Gpx4 gene was detected by qPCR. The expression level of Gpx4 is related to lipid peroxidation. Increasing the expression level of Gpx4 can improve the ability of tissues to resist lipid peroxidation. The 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 showed that after the new Bunyavirus infected mice, the expression level of Gpx4 was downregulated and the tissue's ability to resist lipid peroxidation was reduced, but the ferroptosis inhibitor Liproxstatin-1 was able to improve the decrease in Gpx4 expression level caused by the new Bunyavirus infection and enhance the tissue's ability to resist lipid peroxidation.

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

[0095] The spleen tissue samples of mice injected with the novel Bunyavirus on the fifth day in Example 4 were taken, and the 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 showed that the viral load of the new Bunyavirus in the spleen of mice treated with the ferroptosis inhibitor Liproxstatin-1 (20 mg / kg) was significantly downregulated, and the ferroptosis inhibitor 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 peroxide (MDA) in the spleen of mice infected with the new bunyavirus

[0101] Take the spleen tissue sample of the mouse injected with the new Bunyavirus on the fifth day in Example 4, weigh and record the sample weight, and use the MDA detection kit (Biyuntian, item number S0131M) to detect the MDA content in the tissue. The specific steps are as follows: ① Use 120μL Western and IP cell lysis solution (item number P0013) to lyse the cell sample. Centrifuge at 10000g for 10min and take 100μL supernatant and transfer it to a new EP tube. ② Use TBA configuration solution to dissolve TBA and prepare a 0.37% TBA storage solution. When dissolving, it can be placed on a 70°C shaker and mixed in the dark. ③ Use distilled water to dilute the standard to prepare 1, 2, 5, 10, 20, and 50μM concentration gradient MDA standards for preparing standard curves. ④ Equipped with MDA detection working solution, the detection working solution of each sample contains 150μL TBA diluent, 50μL TBA storage solution and 3μL antioxidant. The MDA detection working solution is not easy to dissolve. It can be placed on a 70℃ shaker and mixed and dissolved in the dark. ⑤ Add 200μL MDA detection working solution to each sample and standard, mix well and place in a 100℃ metal bath for heating. Fasten the metal bath heating cover to prevent the liquid in the EP tube from overheating and boiling and splashing. ⑥ After 15 minutes, take the EP tube out of the metal bath and place it in a 4℃ refrigerator for a while to cool it to room temperature. ⑦ Centrifuge at 1000g for 10 minutes, take 200μL of supernatant and add it to the light-transmitting plate. ⑧ Use an enzyme reader to detect the absorbance at 532nm. ⑨ Calculate the MDA content of each sample according to the standard curve.

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

[0103] Example 7: The ferroptosis inhibitor Liproxstatin-1 can alleviate multiple organ damage in mice infected with the novel bunyavirus

[0104] The lung, liver and spleen tissues of the mice injected with the novel bunyavirus in Example 4 were taken on the fifth day, and the tissue lesions were observed using conventional HE staining.

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

[0106] Example 8: Ferroptosis inhibitor desferrioxamine (DFO) inhibits cell death caused by novel bunyavirus infection in vitro

[0107] 1. Infect BMDMs with the new Bunyavirus and collect the cells for detection three days later. According to the addition of ferroptosis inhibitor desferroxine DFO, 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): Desferrifen-DFO solution (solvent: DMSO) was added to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of desferrifen-DFO in the BMDM cell culture system was 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 desferrifen-DFO solution (solvent: DMSO) were added to the BMDM cell culture system (DMEM medium containing BMDM cells). The concentration of desferrifen-DFO in the BMDM cell culture system was 20 μM.

[0112] 2. The cell culture systems of each group were cultured at 5% CO2 and 37°C for 3 days. After 3 days, the cells of each group were collected for detection.

[0113] 3. Use LDH release to evaluate cell death, the detection method is the same as Example 1.

[0114] The results are as follows Figure 8 As shown, the results showed that after the new bunyavirus infected BMDM cells, the level of cell death (assessed by the LDH release method) increased, but the level of cell death was significantly reduced when the BMDM cells infected with the new bunyavirus were treated with the ferroptosis inhibitor desferric ammonium DFO.

[0115] Example 9: Ferroptosis inhibitor desferrioxamine (DFO) inhibits lipid peroxidation caused by novel bunyavirus infection in vitro

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

[0117] The results are as follows Fig. 9 As shown, the results showed that after the new Bunyavirus infected BMDM cells, the level of cellular lipid peroxidation increased, but when the BMDM cells infected with the new Bunyavirus were treated with the ferroptosis inhibitor desferric ammonium DFO, the lipid peroxidation phenomenon was significantly reduced.

[0118] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating a novel bunyavirus disease; Or, use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in preventing and / or treating novel bunyavirus disease.

2. Use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting a novel bunyavirus; Or, use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in inhibiting a novel bunyavirus.

3. Use of a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a novel bunyavirus inhibitor.

4. The use according to any one of claims 1 to 3, characterized in that: The ferroptosis inhibitor is Liproxstatin-1 or desferrioxamine DFO.

5. The use according to any one of claims 1 to 4, characterized in that: The product is a drug or a drug preparation.

6. A pharmaceutical compound, the active ingredient of which is a ferroptosis inhibitor or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical compound according to claim 6, characterized in that: The ferroptosis inhibitor is Liproxstatin-1 or desferrioxamine DFO.

8. The pharmaceutical compound according to claim 6 or 7, characterized in that: The medicinal compound is used for preventing and / or treating novel bunyavirus disease.

9. The pharmaceutical compound according to claim 6 or 7, characterized in that: The pharmaceutical compound is used for inhibiting novel bunyavirus.

10. The pharmaceutical compound according to any one of claims 6 to 9, characterized in that: The medicinal compound is a novel bunyavirus inhibitor.

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