A preparation method of 6-hydroxy-flavin adenine dinucleotide and its application as ferroptosis inhibitor

Abstract

The present invention relates to a preparation method of 6-hydroxy-flavin adenine dinucleotide and its application as an inhibitor of ferroptosis. The preparation method includes: (1) constructing a recombinant plasmid pET28a-Sumo-hFSP1; (2) constructing a recombinant bacterium hFSP1; (3) culturing the recombinant bacterium hFSP1, and obtaining hFSP1 protein after induction, lysis, centrifugation and purification; (4) denaturing and purifying the hFSP1 protein to obtain 6-hydroxy-flavin adenine dinucleotide. The present invention also finds that 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) has the function of inhibiting cell ferroptosis. Cell experiments show that 6-hydroxy-FAD can significantly inhibit ferroptosis caused by RSL3, and can be used to prepare ferroptosis inhibitors, prepare drugs for inhibiting cell ferroptosis, and prepare drugs for treating diseases caused by ferroptosis, which provides a new option for the research and development of efficient ferroptosis inhibitor-related drugs.

Description

Technical Field

[0001] The invention relates to a preparation method of 6-hydroxy-flavin adenine dinucleotide and application of the same as a ferroptosis inhibitor, belonging to the technical field of medicine. Background Art

[0002] Ferroptosis is a new type of iron-dependent programmed cell death, characterized by iron accumulation and lipid peroxidation in the cell membrane. The peroxide products destroy the integrity of the membrane, leading to cell death. At present, the ferroptosis defense system mainly includes glutathione peroxidase 4 (GPX4)-glutathione (GSH) system, ferroptosis inhibitory protein 1 (FSP1)-coenzyme Q10 (CoQ10) system, dihydroorotate dehydrogenase (DHODH)-CoQ10 system and guanosine triphosphate cyclohydrolase (GCH1)-tetrahydrobiopterin (BH4) system.

[0003] Glutathione peroxidase 4 (GPX4) can use glutathione (GSH) to reduce peroxidized lipids to non-toxic lipid alcohols, thereby protecting cells from ferroptosis. The GPX4-GSH signaling pathway is one of the most classic defense mechanisms against ferroptosis in cells. The FSP1-CoQ10 signaling pathway is a newly discovered independent anti-ferroptosis system that is parallel to the GSH-GPX4 signaling pathway and has been confirmed to be one of the main pathways against ferroptosis. FSP1 reduces ubiquinone on the cell membrane to ubiquinol, which can capture lipid peroxyl radicals that mediate lipid peroxidation, prevent lipid peroxidation and inhibit the occurrence of ferroptosis. Lipid peroxidation and iron accumulation are characteristics of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, Pelvic-Merzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis and Friedreich's ataxia. A large number of studies have shown that ferroptosis is an important pathogenic factor in neurodegenerative diseases. Many ferroptosis inhibitors have been shown to delay the development of neurodegenerative diseases under different disease conditions, and have the potential and research value to be clinical therapeutic drugs for neurodegenerative diseases. In addition, ferroptosis has been shown to be involved in a large number of animal models or cell models of ischemia-reperfusion injury in the myocardium, kidney, liver, intestine and brain, and ferroptosis inhibitors are expected to be a new drug to prevent and treat this type of ischemia-reperfusion injury.

[0004] However, the currently known ferroptosis inhibitors are mainly used for cell experiments in vitro, but their stability and effectiveness in animals are insufficient. Typical ferroptosis inhibitors such as Ferrostatin-1 (Fer-1) can inhibit ferroptosis of HT-1080 cells induced by RSL3 or Erastin, but Fer-1 is metabolically unstable in vivo and has poor inhibitory effects. Although the ferroptosis inhibitor Liproxstatin-1 (Lip-1) has stronger stability, its safety and effectiveness are still far from the clinical application standards. Therefore, the screening and development of ferroptosis inhibitors has become one of the hot spots and difficulties in the field of ferroptosis research. 6-Hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) is an organic compound with a molecular formula of C 27 H 33 N9O 16 P2 is a special flavin adenine dinucleotide modification, that is, the hydrogen at the 6th position of flavin adenine dinucleotide is replaced by a hydroxyl group. 6-Hydroxy-flavin adenine dinucleotide mainly exists occasionally as a cofactor in some flavoprotein oxidoreductases. It is generally bound to the enzyme in a non-covalent bond form and has a very low abundance, accounting for only a small part of the total flavin content bound by the enzyme. In many cases, the binding of 6-hydroxy-flavin adenine dinucleotide may lead to a decrease or even loss of protease catalytic activity compared with proteases that bind to flavin adenine dinucleotide. Unlike other flavoproteins, FSP1 is one of the few proteases that binds 6-hydroxy-FAD in a stoichiometric manner. FSP1 obtained by bacterial expression system contains a higher abundance of 6-hydroxy-FAD and has higher enzyme activity. At present, in addition to being an enzyme cofactor, other functions of 6-hydroxy-flavin adenine dinucleotide are rarely mentioned. So far, there has been no literature record or research report on the inhibitory effect or activity of 6-hydroxy-flavin adenine dinucleotide on cell ferroptosis. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing 6-hydroxy-flavin adenine dinucleotide and its application as a ferroptosis inhibitor.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing 6-hydroxy-flavin adenine dinucleotide comprises the following steps:

[0008] (1) PCR amplification was performed using the human ferroptosis inhibitory protein 1 (hFSP1) gene as a template to obtain the hFSP1 gene sequence; the hFSP1 gene sequence was then inserted into the vector plasmid pET28a-SUMO to obtain the recombinant plasmid pET28a-Sumo-hFSP1;

[0009] (2) Transforming the recombinant plasmid pET28a-Sumo-hFSP1 into competent Escherichia coli cells, verifying and selecting positive recombinants, and obtaining recombinant bacteria hFSP1;

[0010] (3) inoculating the recombinant bacterium hFSP1 into LB liquid culture medium for cultivation, obtaining the recombinant bacterium hFSP1 supernatant after induction, lysis and centrifugation; purifying the recombinant bacterium hFSP1 supernatant to obtain hFSP1 protein;

[0011] (4) The hFSP1 protein is denatured at 80-90° C. for 8-12 min, and then purified by centrifugation, ion exchange column, and chromatography column to obtain 6-hydroxy-flavin adenine dinucleotide.

[0012] Preferably according to the present invention, in step (1), the NCBI number of the human ferroptosis suppressor protein 1 (hFSP1) gene is Q8BUE4.1.

[0013] According to the preferred embodiment of the present invention, in step (3), the culture and induction are specifically as follows: first, the recombinant bacterium hFSP1 is inoculated into LB liquid culture medium, and cultured overnight at 37°C and 200rpm with shaking, and then the overnight cultured bacterial solution is inoculated into LB liquid culture medium at a volume ratio of 1:100, and cultured at 37°C until OD600 is 0.6-0.9, and then cooled to 18°C, IPTG with a final concentration of 0.1mM is added, and cultured overnight at 18°C ​​and 200rpm with shaking. The required amount of E. coli is 20L.

[0014] Preferably, in step (3), the purification is as follows: 20 L of overnight cultured cells are collected by centrifugation at 4000 rpm for 20 min at 4°C, and then the cells are resuspended in 1 L of resuspension buffer, the resuspended cells are lysed by a micro ultra-high pressure cell disruptor (JNBIO), and the lysate is centrifuged at 12000 rpm for 60 min at 4°C to obtain the supernatant of hFSP1; the hFSP1 supernatant is fixed on 10 mL of Ni-bound gel resin, and then impurities are washed away with a cleaning solution, and finally eluted with an eluent to obtain hFSP1 protein.

[0015] Further preferably, the resuspension buffer is a phosphate buffer containing 150mM NaCl, 5% glycerol, 5mM β-mercaptoethanol, pH 7.4; the cleaning solution is a phosphate buffer containing 150mM NaCl, 5% glycerol, 5mM β-mercaptoethanol, 5mM imidazole, pH 7.4; the elution solution is a phosphate buffer containing 150mM NaCl, 5% glycerol, 5mM β-mercaptoethanol, 250mM imidazole, pH 7.4.

[0016] Preferably according to the present invention, in step (4), the denaturation reaction conditions are: denaturation temperature 85° C., denaturation time 10 min.

[0017] According to the preferred embodiment of the present invention, in step (4), the ion exchange column is an anion exchange column, and the chromatographic column is a supelclean column. TM LC-18SPE.

[0018] The present invention also provides 6-hydroxy-flavin adenine dinucleotide prepared according to the above preparation method.

[0019] On the other hand, the present invention also provides the use of the above-mentioned 6-hydroxy-flavin adenine dinucleotide in inhibiting ferroptosis, in preparing ferroptosis inhibitors, as a ferroptosis inhibitor, in preparing drugs for inhibiting cell ferroptosis, and in preparing drugs for treating diseases caused by ferroptosis.

[0020] Preferably according to the present invention, the cell is a human fibrosarcoma cell HT1080.

[0021] Preferably according to the present invention, the disease caused by ferroptosis is a neurodegenerative disease or kidney / liver / heart ischemia-reperfusion.

[0022] Where the present invention is not exhaustive, prior art may be used.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention discovered for the first time that 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) has the function of inhibiting cell ferroptosis. Cell experiments showed that 6-hydroxy-FAD can significantly inhibit ferroptosis caused by RSL3, providing a new option for the research and development of efficient ferroptosis inhibitor-related drugs.

[0025] 2. The present invention provides a method for heterologously expressing hFSP1 protein, then heating and denaturing the hFSP1 protein, and then passing it through an anion exchange column and supelclean TM The invention discloses a method for preparing 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) by LC-18 SPE column purification. The method does not require additional raw materials, reduces production costs, and has simple steps, mild conditions, and environmentally friendly processes, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS-PAGE gel electrophoresis diagram of the positive recombinant bacterium hFSP1.

[0027] Figure 2This is a diagram showing the HPLC-MS / MS identification results of 6-hydroxy-FAD prepared in the present invention.

[0028] Figure 3 The 6-hydroxy-FAD prepared by the present invention 1 H NMR identification results.

[0029] Figure 4 The 6-hydroxy-FAD prepared by the present invention inhibits RSL3-induced HT1080 hFSP1 KO Comparison of cell ferroptosis effects.

[0030] Figure 5 This is a comparison chart of the effect of 6-hydroxy-FAD prepared by the present invention on inhibiting RSL3-induced ferroptosis in HT1080 cells. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. The experimental methods and reagents without specific conditions in the examples are all based on conventional conditions in the art. The "%" not specifically specified in the present invention are all mass percentages.

[0032] The vector plasmid pET28a-SUMO is available from Shanghai Beino Biotechnology Co., Ltd. The competent E. coli cell BL21 (DE3), product number 200131, is available from Agilent Technologies. The human fibrosarcoma cell HT1080 is from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0033] The chemical structural formula of the 6-hydroxy-FAD is shown below:

[0034]

[0035] Example 1: Construction of recombinant plasmid pET28a-Sumo-hFSP1

[0036] The human ferroptosis suppressor protein 1 (hFSP1) gene was artificially synthesized by Shanghai Jierui Bioengineering Co., Ltd., and then PCR amplification was performed using the hFSP1 gene as a template to obtain the hFSP1 gene sequence. The PCR primer sequences are as follows:

[0037] Upstream primer: 5'-CAGGATCCATGGGTAGCCAGGTTAGCGT-3' (containing BamHI restriction site),

[0038] Downstream primer: 5'-GACTCGAGTCATGGTGGGCTTTGAC-3' (containing XhoI restriction site);

[0039] PCR amplification program: pre-denaturation, 95°C for 5 min; denaturation, 95°C for 30 sec; annealing, 60°C for 40 sec; extension, 72°C for 30 sec (30 cycles); termination of extension, 72°C for 10 min; and finally incubation at 4°C.

[0040] By conventional recombinant plasmid construction method, the vector plasmid pET28a-SUMO and hFSP1 gene sequence were double-digested with BamHI and XhoI respectively, and then connected with ligase to obtain pET28a-Sumo-hFSP1 plasmid.

[0041] The NCBI number of the ferroptosis suppressor protein 1 (hFSP1) gene is Q8BUE4.1.

[0042] Example 2: Construction of hFSP1 recombinant bacteria and protein expression

[0043] 1. Conversion

[0044] Take 100 μL of BL21 (DE3) Escherichia coli competent cells, place them in a 1.5 mL tube, quickly place the tube on ice, bury it in ice except for the tube cover, and gently tap the tube wall after melting to resuspend the cells. Add the pET28a-Sumo-FSP1 plasmid constructed in Example 1 with a volume of 1 μL and a concentration of 100 ng / μL to the competent cells, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and ice bath again for 2 minutes. Then add 400 μL of LB liquid culture medium, mix well, and culture at 37°C and 200 rpm for 40 to 60 minutes. Take 100 μL of culture solution, evenly spread it on LB solid culture medium, and invert and culture at 37°C for 12 to 16 hours to obtain recombinant bacteria hFSP1.

[0045] 2. Positive clone verification and protein expression

[0046] The recombinant bacteria hFSP1 was picked from the above LB solid culture medium and inoculated into LB liquid culture medium containing kanamycin (final concentration of kanamycin: 50 μg / mL), and cultured at 37°C and 200 rpm with shaking overnight. Then, the overnight cultured bacterial solution was inoculated into 1000 mL of fresh LB liquid culture medium containing kanamycin (final concentration of kanamycin: 50 μg / mL) at a volume ratio of 1:100, and cultured at 37°C until OD600 was 0.6-0.9, cooled to 18°C, IPTG was added at a final concentration of 0.1 mM, and cultured at 18°C ​​and 200 rpm with shaking overnight to obtain 20 L of recombinant bacteria hFSP1 bacterial solution. The overnight cultured cells were collected by centrifugation at 4000 rpm for 20 min at 4°C, and then resuspended in 1 L of resuspension buffer. The resuspended cells were lysed using a micro ultra-high pressure cell disruptor (JNBIO), and the lysate was centrifuged at 12000 rpm for 60 min at 4°C to obtain the supernatant of hFSP1.

[0047] The resuspension buffer is a phosphate buffer containing 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, and pH 7.4.

[0048] The supernatant of recombinant hFSP1 was used as a sample for SDS-PAGE electrophoresis to detect the size of the target protein. The results are as follows: Figure 1 shown.

[0049] Depend on Figure 1 It can be seen that the ferroptosis inhibitory protein 1 (hFSP1) expressed by the recombinant bacterium hFSP1 is about 46.8 kDa in size, indicating that the colony transformation and heterologous expression are successful.

[0050] Example 3. Preparation of 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD)

[0051] A method for preparing 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) comprises the following steps:

[0052] (1) The supernatant of the recombinant bacterium hFSP1 obtained in Example 2 was fixed on 10 mL of Ni-bound gel resin, and impurities were first washed away with a cleaning solution, and then eluted with an eluent to obtain hFSP1 protein;

[0053] The cleaning solution is a phosphate buffer containing 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 5 mM imidazole, and pH 7.4; the elution solution contains 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 250 mM imidazole, and pH 7.4 phosphate buffer;

[0054] (2) The hFSP1 protein was heated at 85°C for 10 min for denaturation reaction, and then the heat-denatured hFSP1 protein was centrifuged at 12000 rpm for 20 min, and the supernatant was collected to obtain the crude 6-hydroxy-FAD product;

[0055] (3) The crude 6-hydroxy-FAD product was acidified to pH 4.0 with acetic acid, and then filtered with a glass filter (Jin Teng Technology) and a cellulose filter membrane (pore size 0.45 μm) to further remove precipitated impurities, and then loaded onto an anion exchange column HiTrap Q HP; the anion exchange column HiTrap Q HP needs to be installed in advance on the FPLC purifier system (GE Healthcare) and pre-equilibrated with ultrapure water acidified with acetic acid (pH = 4.0), and after loading, eluted with a gradient from 0% AQ-B buffer to 50% AQ-B buffer (AQ-B buffer, 500mM, NaCl, pH = 4.0); according to the recorded conductivity elution curve, the main peak corresponding to 6-hydroxy-FAD was collected; the collected 6-hydroxy-FAD samples were mixed, and methanol and 1% acetic acid were added at a final concentration of 5%, and then loaded onto a chromatographic column supelclean pre-equilibrated with washbuffer (5% methanol and 1% acetic acid). TM After loading, the LC-18SPE (Sigma-Aldrich) column was first rinsed with 200 mL of washbuffer, then rinsed with 50 mL of water, and finally eluted with 100% methanol to obtain 6-hydroxy-FAD, which was fractionated and freeze-dried in a vacuum freeze dryer (Heto-Holten, Denmark) to obtain 1 mg of dark green powdered 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD).

[0056] The 6-hydroxy-FAD prepared in this example was analyzed by HPLC-MS / MS and 1 H NMR characterization results are shown in Figure 2 and Figure 3 shown.

[0057] Depend on Figure 2 It can be seen that the chemical formula of 6-hydroxy-FAD prepared in this example is C 27 H 33 N9O 16 P2, molecular weight is 801.5491. The [M+H] + The measured value of 802.1603 is the same as [M+H] +The exact mass of 6-hydroxy-FAD is consistent with the theoretical value of 802.1599. The secondary mass spectrum of the parent ion ([M+H]+=802.16) confirmed that 6-hydroxy-FAD is a derivative in which the 6-hydrogen of FAD is replaced by a hydroxyl group, which is consistent with the structural formula.

[0058] Depend on Figure 3 It can be seen that the molecular structure of 6-hydroxy-FAD prepared in this example is consistent with the structural formula, and is a derivative in which the hydrogen at position 6 of FAD is replaced by a hydroxyl group. At the same time, the 6-hydroxy-FAD prepared in this example may contain an isomer with a keto form at position 6.

[0059] Example 4: Verification of 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) activity

[0060] 1. The hFSP1 gene of human fibrosarcoma cell HT1080 was knocked out by CRISPR-Cas9 method to obtain HT1080hFSP1 KO The CRISPR-Cas9 method is a prior art. Then HT1080 and HT1080 hFSP1 KO The cells were inoculated in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. When culturing HT1080 cells, glucose and glutamine were added at 4.5 g / L.

[0061] 2. The cultured HT1080 and HT1080 hFSP1 KO The cells were 6×10 4 100 μL / well were inoculated on a 96-well plate and grown for 12 to 24 hours. KO The cells were then divided into two groups: one group was treated with RSL3 agonist in HT1080 hFSP1 cells. KO 10 μM 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD), flavin adenine dinucleotide (FAD) and 1% DMSO were added to the cell wells, and three replicate wells were set for each drug concentration. HT1080 cells that were only added with RSL3 agonist were used as the control group (WT, corresponding to Figure 4); the other group was to add 10, 20, 50 μM 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) to the HT1080 cells that had been added with RSL3 agonist, and 3 replicate wells were set for each drug concentration, and the HT1080 cells that were only added with RSL3 agonist were used as the control group (WT, corresponding to Figure 5 After 24 hours of continuous culture, 10 μL of Cell Counting Kit-8 (Dojin Chemical Research Institute, Japan) was added to each well and incubated for 30 minutes. The absorbance at 450 nm was measured with an ELISA reader, and a comparison chart of ferroptosis effects was drawn. The results are shown in the figure. Figure 4 and Figure 5 shown.

[0062] Depend on Figure 4 It can be seen that the addition of 6-hydroxy-FAD (hFSP1 KO HT1080 hFSP1 after +6-hydroxy-FAD) KO The cell survival rate was significantly higher than that of the control group after addition of FAD (hFSP1 KO +FAD) and DMSO(hFSP1 KO +DMSO) after HT1080hFSP1 KO The cell survival rate was close to that of normal HT1080 cells containing the hFSP1 gene. This indicates that 6-hydroxy-FAD can significantly inhibit ferroptosis caused by RSL3.

[0063] Depend on Figure 5 It can be seen that the addition of 6-hydroxy-FAD can increase the survival rate of HT1080 wild-type cells, and this effect becomes more obvious with the increase of the concentration of 6-hydroxy-FAD added. This further shows that 6-hydroxy-FAD has a significant inhibitory effect on ferroptosis caused by RSL3.

[0064] In summary, the present invention found that 6-hydroxy-flavin adenine dinucleotide (6-hydroxy-FAD) has the function of inhibiting cell ferroptosis. 6-hydroxy-FAD can significantly inhibit ferroptosis caused by RSL3, providing a new option for the research and development of efficient ferroptosis inhibitor-related drugs.

Claims

1. A method for preparing 6-hydroxy-flavin adenine dinucleotide, characterized in that: The steps include: (1) PCR amplification was performed using the human ferroptosis inhibitory protein 1 gene as a template to obtain the hFSP1 gene sequence; the hFSP1 gene sequence was then inserted into the vector plasmid pET28a-SUMO to obtain the recombinant plasmid pET28a-Sumo-hFSP1; The NCBI number of the human ferroptosis inhibitory protein 1 gene is Q8BUE4.1; (2) Transform the recombinant plasmid pET28a-Sumo-hFSP1 into competent E. coli cells, verify and select positive recombinants, and obtain recombinant bacteria hFSP1; (3) The recombinant bacterium hFSP1 is inoculated into LB liquid culture medium for cultivation. After induction, lysis and centrifugation, the recombinant bacterium hFSP1 supernatant is obtained; after purifying the recombinant bacterium hFSP1 supernatant, the hFSP1 protein is obtained; (4) The hFSP1 protein was denatured at 80-90°C for 8-12 minutes, and then purified by centrifugation, ion exchange column and chromatography column to obtain 6-hydroxy-flavin adenine dinucleotide.

2. The method for preparing 6-hydroxy-flavin adenine dinucleotide according to claim 1, characterized in that: In step (3), the culture and induction are specifically as follows: first, the recombinant bacterium hFSP1 is inoculated into LB liquid culture medium, and cultured at 37°C and 200 rpm with shaking overnight, and then the overnight cultured bacterial solution is inoculated into LB liquid culture medium at a volume ratio of 1:100, and cultured at 37°C until OD600 is 0.6-0.9, then cooled to 18°C, IPTG is added at a final concentration of 0.1 mM, and cultured at 18°C ​​and 200 rpm with shaking overnight.

3. The method for preparing 6-hydroxy-flavin adenine dinucleotide according to claim 1, characterized in that: In step (3), the purification is specifically as follows: 20 L of overnight cultured cells are collected by centrifugation at 4000 rpm for 20 min at 4°C, and then the cells are resuspended in 1 L of resuspension buffer, the resuspended cells are lysed by a micro-ultra-high pressure cell disruptor, and the lysate is centrifuged at 12000 rpm for 60 min at 4°C to obtain the hFSP1 supernatant; the hFSP1 supernatant is fixed on 10 mL of Ni-bound gel resin, and then impurities are washed away with a cleaning solution, and finally eluted with an elution solution to obtain the hFSP1 protein; The resuspension buffer is a phosphate buffer containing 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH 7.4; the cleaning solution is a phosphate buffer containing 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 5 mM imidazole, pH 7.4; the elution solution is a phosphate buffer containing 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 250 mM imidazole, pH 7.

4.

4. The method for preparing 6-hydroxy-flavin adenine dinucleotide according to claim 1, characterized in that: In step (4), the denaturation reaction conditions are: denaturation temperature 85°C, denaturation time 10 min.

5. The method for preparing 6-hydroxy-flavin adenine dinucleotide according to claim 1, characterized in that: In step (4), the ion exchange column is an anion exchange column, and the chromatographic column is Supelclean™ LC-18 SPE.

6. Use of 6-hydroxy-flavin adenine dinucleotide prepared by the method for preparing 6-hydroxy-flavin adenine dinucleotide according to any one of claims 1 to 5 in preparing drugs for inhibiting cell ferroptosis and in preparing drugs for treating diseases caused by ferroptosis; The cells are human fibrosarcoma cells HT1080; the disease caused by ferroptosis is a neurodegenerative disease or kidney / liver / heart ischemia-reperfusion.

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