Novel ferroptosis inhibitor and application thereof in preparation of medicine for preventing and / or treating sepsis

Abstract

The invention discloses a novel ferroptosis inhibitor and application thereof in preparation of drugs for preventing and / or treating sepsis, and belongs to the technical field of pharmaceutical preparations and biological medicines. The invention discloses a preparation method of a novel ferroptosis inhibitor, which can be used for developing a high-efficiency and low-toxicity ferroptosis inhibitor. The medicament can be used for preparing a medicament for preventing organ injury related to sepsis, is a potential medicament for treating sepsis, and can inhibit ferroptosis in kidney, liver, lung and cardiomyopathy induced by sepsis and relieve inflammatory response and organ injury. Meanwhile, the invention has the outstanding advantages of strong specificity, high bioavailability, obvious effect and stable preparation method, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations and biomedicine technology, and relates to a novel ferroptosis inhibitor and its application in preparing a drug for preventing and / or treating sepsis. Background Art

[0002] Ferroptosis is an iron-dependent cell death process characterized biochemically by the excessive accumulation of reactive oxygen species and lipid peroxides, as well as decreased glutathione peroxidase 4 activity. This leads to intracellular redox imbalance and cell membrane disruption, ultimately leading to cell death. Ferroptosis is morphologically characterized by cell membrane fragmentation, reduced mitochondrial size, increased membrane density, decreased or absent cristae, and outer membrane rupture. Iron is a vital trace element and is involved in key metabolic processes in many pathogenic microorganisms. The virulence of some bacteria depends on iron, which can enhance bacterial virulence and promote proliferation. Current research indicates that ferroptosis plays an increasingly important role in organ dysfunction and has a significant impact on disease treatment and prognosis.

[0003] Sepsis is a systemic disease that severely impairs health. It is triggered by an excessive immune inflammatory response to infection, surgery, or trauma, leading to multi-organ dysfunction. Sepsis can originate in community settings or be caused by hospitalization or other medical institutions. Approximately 80% of hospital-treated sepsis cases occur in the community. When pathogens invade, macrophages recognize receptors on the pathogen's surface and activate other immune cells, releasing inflammatory cytokines that trigger an intracellular inflammatory cytokine storm and excessive inflammatory response, ultimately leading to immunosuppression. Sepsis can disrupt intracellular iron metabolism, including increased intracellular iron transport and uptake, decreased iron export, and intracellular iron overload. Cellular iron death can release damage-related pattern molecules or lipid oxides, thereby activating inflammatory signaling pathways, further promoting the inflammatory cytokine storm and exacerbating organ damage.

[0004] In recent years, the incidence of sepsis has increased at a rate of 8% to 11%, and the overall mortality rate is close to 30%. It is a major disease that threatens life safety worldwide. Therefore, exploring the therapeutic targets of sepsis is of great significance in reducing the mortality rate of sepsis. At present, the progress in the treatment of sepsis is limited to symptomatic treatment, including organ support and fluid resuscitation. The main methods are to eliminate pathogens, remove inflammatory mediators, and restore cell function. There is still a gap in the development of effective therapeutic drugs in sepsis research. It is crucial to explore new treatment directions and potential therapeutic targets. Intervening in the progression of sepsis from the perspective of trace elements is expected to become a form of precision treatment.

[0005] Therefore, exploring the application of ferroptosis inhibitors in the prevention and treatment of sepsis may provide new ideas and targets for the treatment of sepsis.

[0006] Currently, ferroptosis inhibitors mainly include antioxidants, iron chelators, acyl-CoA synthetase long-chain family member 4 inhibitors, and lipoxygenase inhibitors. However, these agents suffer from poor bioavailability, high toxicity, and low specificity. Therefore, the research and development of novel ferroptosis inhibitors is urgently needed. Summary of the Invention

[0007] The present invention aims to solve existing technical problems and provide novel ferroptosis inhibitors EfeB and HemH and their use in the treatment of sepsis.

[0008] To achieve the above object, the technical solution of the present invention is as follows: First, through bioinformatics analysis, it was predicted that EfeB can bind to free iron ions and has the function of ion transport, and HemH can bind to free iron ions and insert them into porphyrins in cells, thereby more efficiently reducing the intracellular iron ion level and inhibiting ferroptosis.

[0009] On the other hand, bioinformatics analysis predicted that EfeB and HemH could be exocytized.

[0010] On the other hand, the molecular cloning method was successfully used to construct efeB and hemH A prokaryotic expression system was developed to express and purify the recombinant proteins rEfeB ​​and rHemH.

[0011] That is, a ferroptosis inhibitor, which is a protein described in a) or b) below: a) a protein consisting of the amino acid sequence shown in SEQ ID No. 1 and / or SEQ ID No. 2; or b) A protein derived from a) which has the same ferroptosis inhibitory activity as the amino acid sequence defined in a) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence defined in a).

[0012] An isolated polynucleotide encoding the above-mentioned protein.

[0013] Specifically, the polynucleotide preferably has the following nucleotide sequence: 1) the nucleotide sequence shown by SEQ ID No. 3 or SEQ ID No. 4; 2) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence defined in 1); or 3) A nucleotide sequence that has at least 90% homology to the nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No. 4, and encodes a protein that has the same ferroptosis inhibitory activity as the protein composed of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2.

[0014] The present invention also provides the use of the ferroptosis inhibitor and the polynucleotide in preparing a drug for inhibiting cell ferroptosis.

[0015] Preferably, the cells are macrophages. Specifically, the effects include reducing the concentration of iron ions in the cells, reducing the production of ferroptosis-related molecules ROS and lipid peroxides, and promoting the expression of GPX4, a key negative regulatory protein for ferroptosis, thereby inhibiting the occurrence of ferroptosis.

[0016] The present invention also provides use of the ferroptosis inhibitor and the polynucleotide in preparing or screening drugs for preventing and / or treating sepsis.

[0017] Preferably, when used, the drug can inhibit sepsis-induced ferroptosis, reduce inflammatory response and / or alleviate organ damage.

[0018] In some embodiments of the invention, the organ is a kidney or a liver.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: 1. The ferroptosis inhibitor provided by the present invention has high purity, strong specificity and low cytotoxicity.

[0020] 2. The ferroptosis inhibitor provided by the present invention has a functional site for binding to iron ions in its molecular domain and has the function of iron ion transport, which can bind to and transport free iron in cells. Compared with other ferroptosis inhibitors, the inhibition efficiency of the present invention is higher, and the concentration of iron ions in cells can be efficiently reduced. Compared with other ferroptosis inhibitors, the production of ferroptosis-related molecular reactive oxygen species and lipid peroxides can be reduced, and the expression of glutathione peroxidase 4 can be promoted, thereby inhibiting the occurrence of ferroptosis.

[0021] 3. The ferroptosis inhibitor provided by the present invention can inhibit sepsis-induced ferroptosis, reduce inflammation and organ damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Expression and purification of EfeB and HemH. (A) The expected size of EfeB and HemH was amplified from DNA of strain CFT073. efeB (1272 bp) and hemH (963 bp) gene fragment. Note: M: DNA marker; 1: hemHGene amplification band; 2: efeB Gene amplification bands. (BC) Construction of TA cloning and subcloning of target gene fragments: DNA fragments of the expected size and position were obtained by double enzyme digestion and 1.5% agarose gel electrophoresis. Note: M in B: DNA marker; 1: pMD19-T hemH Depend on Nde I and Xho PMD19-T after I digestion and hemH ;2:pMD19-T efeB Depend on Nde I and Xho PMD19-T after I digestion and efeB C in M: DNA marker; 1: pET-42a hemH Depend on Nde I and Xho pET-42a after I digestion and hemH ; 2: pET-42a efeB Depend on Nde I and Xho pET-42a after I digestion and efeB (D) IPTG-induced prokaryotic expression system efficiently expressed recombinant proteins rEfeB ​​and rHemH: SDS-PAGE gel electrophoresis showed the expected size of recombinant proteins rEfeB ​​(46.5 kDa) and rHemH (35.2 kDa). M: Protein Marker; 1: E. coli BL21 (DE3) pET42a Total protein expressed; 2: E. coli BL21 (DE3) pET42a-hemH Total protein expressed; 3: E. coli BL21 (DE3) pET42a-efeB Total expressed protein. (EG) Successful purification of the recombinant protein: Automated protein purification results show a single peak, and SDS-PAGE results show a single bright band. Note: M: protein marker; 1: purified rHemH protein; 2: purified rEfeB ​​protein. (H) Detoxi-Gel column chromatography to remove bacterial endotoxin (LPS) from rEfeB ​​and rHemH. Discrete dots represent parallel samples.

[0023] Figure 2EfeB and HemH promote cell survival and GPX4 expression levels. (A) CCK8 assay for THP-1 cell viability. (B) MTT assay for THP-1 cell viability. Compared with the control group, rEfeB ​​and rHemH had no significant effect on THP-1 cell viability, suggesting that EfeB and HemH have little cytotoxicity; compared with the RSL3-induced ferroptosis-positive group, rEfeB ​​and rHemH promoted THP-1 cell survival. (C) WB assay for the effects of rEfeB ​​and rHemH on GPX4 expression. (D) ImageJ software performed statistical analysis of the grayscale of GPX4 bands in three experiments. Compared with the RSL3 group, rEfeB ​​and rHemH promoted the expression level of GPX4 in THP-1 cells. The discrete circles are all parallel samples; *: p <0.05,**: p <0.01, ns: p >0.05.

[0024] Figure 3 EfeB and HemH inhibit ROS levels in macrophages. (A) Flow cytometry analysis of intracellular ROS levels. (B) ImageJ software statistical analysis of mean fluorescence intensity of ROS in the experiment (A). Discrete circles represent parallel samples. (C) Fluorescence microscopy observation of ROS levels. Compared with the RSL3 group, rEfeB ​​and rHemH inhibited ROS levels in THP-1 cells. **: p <0.01,***: p <0.001, ns: p >0.05.

[0025] Figure 4 EfeB and HemH inhibit Fe 2+ and lipid peroxide levels. (A) FerroOrange fluorescent probe detects intracellular Fe 2+ The blue fluorescence is the color of DAPI, and the orange fluorescence is the color of FerroOrange after binding to free ferrous ions in the cells. (B) ImageJ software analyzes the Fe 2+ Statistical analysis of mean fluorescence intensity, where the discrete circles are parallel samples. The results showed that rEfeB ​​and rHemH could inhibit the expression of Fe in THP-1 cells compared with the control group. 2+(C) BDP 581 / 591 C11 probe was used to detect intracellular lipid peroxide levels. Red fluorescence indicates the color of the BDP 581 / 591 C11 probe, and green fluorescence indicates the color of the BDP 581 / 591 C11 probe after binding to lipid peroxides. The results showed that rEfeB ​​and rHemH inhibited the level of lipid peroxides in THP-1 cells compared with the RSL3 group. *: p <0.05,**: p <0.01.

[0026] Figure 5 rEfeB ​​and rHemH inhibited sepsis-induced renal and hepatic inflammation levels. DETAILED DESCRIPTION

[0027] Example 1 Synthesis of ferroptosis inhibitors EfeB and HemH Preparation method step 1: efeB and hemH Gene amplification: 1. Uropathogenic Escherichia coli Escherichia coli , UPEC) CFT073 strain culture The UPEC CFT073 strain (kindly donated by Academician Xu Jianguo, Institute of Infectious Diseases, Chinese Center for Disease Control and Prevention) was cultured in LB liquid medium at 37°C and shaken at 220 rpm overnight.

[0028] 2. Extraction of UPEC CFT073 genomic DNA The procedure was followed according to the bacterial genomic DNA extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-MN-MS-GDNA-50).

[0029] (1) Collect the logarithmic phase UPEC CFT073 culture in a 2 ml centrifuge tube, centrifuge at 12,000 × g for 30 s, and discard the supernatant; resuspend the precipitate in 150 μl of Buffer S containing RNase A; add 20 μl of lysozyme storage solution, mix well, and let stand at room temperature for 5 min; (2) Add 30 μl of 0.25 M EDTA (pH 8.0), mix well, and incubate on ice for 5 min; (3) Add 450 μl of Buffer GA, vortex for 15 s, and incubate in a 65°C water bath for 10 min; add 400 μl of Buffer GB and 1 ml of 4°C pre-cooled Buffer DV, mix well, and centrifuge at 12,000 × g for 2 min; (4) Discard the upper phase, retain the time precipitate and the lower phase; add 1 ml of 4°C pre-cooled Buffer DV, mix vigorously, and centrifuge at 12,000 × g for 2 min; discard the upper phase, transfer the lower phase to a filter, and centrifuge at 12,000 × g for 1 min; discard the filter, add 400 μl of Buffer BV to the filtrate, and mix evenly; (5) Place the preparation tube in a 2 ml centrifuge tube, transfer the liquid from step (4) into the preparation tube, and centrifuge at 12,000 × g for 1 min; discard the filtrate, place the preparation tube back into the 2 ml centrifuge tube, add 500 μl of Buffer W1, and centrifuge at 12,000 × g for 1 min; (6) Discard the filtrate, place the preparation tube back into a 2 ml centrifuge tube, add 700 μl of Buffer W2, and centrifuge at 12,000 × g for 1 min; wash once more with 700 μl of Buffer W2 using the same method; (7) Discard the filtrate, place the preparation tube back into a 2 ml centrifuge tube, and centrifuge at 12,000 × g for 1 min. (8) Place the preparation tube in a 1.5 ml centrifuge tube, add 150 μl of elution buffer to the center of the silica gel membrane, and let it stand at room temperature for 1 minute; centrifuge at 12,000 × g for 1 minute to elute the DNA.

[0030] UPEC CFT073 strain efeB and hemH Gene amplification and purification of amplification products (1) Primer design Obtained from GenBank efeB gene (sequence number NZ_CP051263.1: 1124808-1126079 bp) and hemH The gene sequence (sequence number is NZ_CP051263.1: 575517-576479 bp) was analyzed by Primer 6.0 software, and primers were designed by ourselves. The endonuclease sites were selected as follows: Nde I (CATATG) and Xho I (CTCGAG), the primer sequences are shown in Table 1, and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0031] Table 1 UPEC CFT073 strain efeB and hemH Gene amplification primers

[0032] F: forward primer, R: reverse primer.

[0033] (2) Amplification of target gene UPEC CFT073 genomic DNA was used as a template to amplify efeB and hemH Gene, prepare PCR reaction system according to Table 2.

[0034] Table 2 efeB and hemH Gene PCR reaction system

[0035] PCR parameters: 94°C for 5 min; 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 180 s; and 72°C for 10 min. Amplified products were detected by electrophoresis on 1.5% agarose gels prestained with ethidium bromide.

[0036] (3) Purification of PCR amplification products The PCR amplified target fragment was purified using the PCR Clean-up Kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-PCR-50).

[0037] 1) Add 3 volumes of Buffer PCR-A to the PCR reaction mixture; mix thoroughly and transfer to the preparation tube; 2) Place the prepared tube in a 2 ml centrifuge tube, centrifuge at 12,000 × g for 1 min, and discard the filtrate; 3) Place the prepared tube back into a 2 ml centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. 4) Return the prepared tube to a 2 ml centrifuge tube, add 400 μl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. 5) Place the preparation tube in a 1.5 ml centrifuge tube, add 30 μl of elution buffer to the center of the preparation tube membrane, and let it stand at room temperature for 1 minute. Centrifuge at 12,000 × g for 1 minute to elute the DNA.

[0038] The experimental results showed that the expected size of the gene was successfully amplified from the genomic DNA of UPEC CFT073 strain. efeB (1272 bp) and hemH (963 bp) gene fragment ( Figure 1 A in ).

[0039] Preparation method step 2: efeB and hemH TA cloning of genes (1) Preparation of competent cells of Escherichia coli strains DH5α (Beijing Beina Chuanglian Biotechnology Research Institute, BNCC353719) and BL21 (DE3) (Beijing Beina Chuanglian Biotechnology Research Institute, BNCC353806) 1) Streak DH5α or BL21 (DE3) onto LB plates and incubate at 37°C for 16 h. 2) Pick a single DH5α or BL21 (DE3) colony and place it in 10 ml of LB liquid medium. Incubate with shaking at 37°C for 16 hours. 3) Inoculate 1% of the culture into 30 ml of LB medium and culture at 37°C with shaking for 2.5 h until the OD 600 =0.4; 4) Transfer the culture medium into a 50 ml sterile centrifuge tube and place on ice for 10 minutes; 5) Centrifuge at 3000 × g for 10 min at 4°C; discard the supernatant to remove any remaining culture medium. 6) Add 20 ml of 4°C pre-cooled 0.1 M CaCl2 solution to resuspend the precipitate and place on ice for 30 min. Centrifuge at 3000 × g for 10 min at 4°C. Discard the supernatant to remove any remaining culture medium. 7) Add 2 ml of 4°C pre-cooled 0.1 M CaCl2 solution containing 15% glycerol to resuspend the precipitate and aliquot into 100 μl tubes for later use.

[0040] (2) Connection The TA cloning kit (Baori Biotechnology (Beijing) Co., Ltd., 3271) was used to prepare the ligation system according to Table 3 in a centrifuge tube. After mixing, the ligation was carried out at 16°C for 12 h.

[0041] Table 3 UPEC CFT073 strain efeB and hemH Gene TA cloning and ligation system

[0042] (3) Conversion 1) Use a pre-chilled pipette to draw up 100 µl of DH5α competent cells into an ice-cold 1.5 ml microcentrifuge tube. Add 10 µl of the ligation reaction product, swirl gently to mix, and incubate on ice for 30 min. 2) Transfer the ice-cold tubes to a rack in a 42°C circulating water bath for exactly 90 seconds. 3) Quickly transfer the tube to ice and cool for 2 minutes; 4) Add 890 µl of 37°C preheated SOC liquid medium and shake at 160 rpm / min at 37°C for 1 h. 5) Evenly spread 200 μl of the bacterial solution onto an LB plate containing 20 mg / ml X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside), 0.1 M IPTG (isopropyl-β-D-thiogalactoside), and 100 μg / ml ampicillin. After the plate is almost dry, incubate the plate upside down at 37°C for 16 h until colonies appear. Recombinant cells will appear as white colonies, while non-recombinant cells will appear as blue colonies.

[0043] (4) Extraction of target recombinant plasmid Plasmid DNA was extracted according to the procedure of the plasmid extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-MN-P-50).

[0044] 1) Take 1 ml of bacterial culture, centrifuge at 12,000 × g for 1 min, and discard the supernatant; 2) Add 250 µl of Buffer S1 to evenly suspend the bacterial pellet; 3) Add 250 µl of Buffer S2 and mix thoroughly by gently inverting the tube six times to fully lyse the cells until a clear solution is formed. 4) Add 350 µl of Buffer S3, mix thoroughly by gently inverting the tube eight times, and centrifuge at 12,000 × g for 10 min. 5) Pipette the supernatant from step 4) into a preparation tube, centrifuge at 12,000 × g for 1 min, and discard the filtrate; 6) Return the preparation tube to the centrifuge tube, add 500 µl of Buffer W1, centrifuge at 12,000 × g for 1 min, and discard the filtrate.

[0045] 7) Return the preparation tube to the centrifuge tube, add 700 µl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. Wash once more with 700 µl of Buffer W2 using the same method, and discard the filtrate. 8) Place the prepared tube back into a 2 ml centrifuge tube and centrifuge at 12,000 × g for 1 min. 9) Transfer the preparation tube to a new 1.5 ml centrifuge tube, add 60 µl of elution buffer to the center of the preparation tube membrane, let it stand at room temperature for 1 minute, and centrifuge at 12,000 × g for 1 minute to elute the plasmid DNA.

[0046] (5) Plasmid pMD19-T efeB and pMD19-T hemH Double enzyme digestion identification 1) Prepare the enzyme digestion reaction system according to Table 4 for the extracted target gene recombinant clone plasmid: Table 4 Plasmid pMD19-T efeBand pMD19-T hemH Double enzyme digestion reaction system

[0047] 2) Digest the enzyme in a 37°C water bath for 4 h. Add the digested reaction solution to 10× Loading Buffer and run electrophoresis on a 1.5% agarose gel to confirm the location of each target fragment.

[0048] (6) DNA sequence determination The bacterial solution with positive enzyme digestion results was commissioned to Sangon Biotech (Shanghai) Co., Ltd. to determine the nucleotide sequence of the inserted fragment in the positive bacterial solution.

[0049] The experimental results showed that pMD19-T efeB and pMD19-T hemH The expected target gene band was cut out by double enzyme in the recombinant cloning plasmid ( Figure 1 B in ).

[0050] Preparation method step 3: efeB and hemH Gene subcloning and prokaryotic expression system construction (1) Recovery of target gene fragments DNA was extracted according to the procedure of the gel extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-GX-50).

[0051] 1) Cut the agarose gel containing the target DNA under UV light, absorb the liquid on the surface of the gel with a paper towel, and mince it. Calculate the weight of the gel and use this weight as the gel volume. 2) Add Buffer DE-A (3 times the volume of the gel), mix well, and heat at 75°C with intermittent mixing until the gel is completely melted. 3) Add 0.5 times the volume of Buffer DE-A to Buffer DE-B and mix well; 4) Pipette the mixture from step 3 and transfer it to a DNA preparation tube. Centrifuge at 12,000 × g for 1 min and discard the filtrate. 5) Return the preparation tube to the centrifuge tube, add 500 µl of Buffer W1, centrifuge at 12,000 × g for 30 s, and discard the filtrate. 6) Return the preparation tube to the centrifuge tube, add 700 µl of Buffer W2, centrifuge at 12,000 × g for 30 s, and discard the filtrate. Wash once with 700 µl of Buffer W2 and centrifuge at 12,000 × g for 1 min. 7) Place the prepared tube in a 2 ml centrifuge tube and centrifuge at 12,000 × g for 1 min. 8) Place the preparation tube in a clean 1.5 ml centrifuge tube. Add 25 µl of elution buffer to the center of the DNA preparation membrane. Let it sit at room temperature for 1 minute. Centrifuge at 12,000 × g for 1 minute to elute the DNA.

[0052] (2) Connection Prepare the ligation system according to Table 5 in a centrifuge tube, mix well, and ligate at 16°C overnight.

[0053] Table 5 UPEC CFT073 efeB and hemH Gene subcloning ligation system

[0054] (3) Conversion 1) Use a pre-chilled pipette to draw up 100 µl of BL21 (DE3) competent cells into an ice-cold 1.5 ml microcentrifuge tube. Add 10 µl of the ligation reaction product, swirl gently to mix, and incubate on ice for 30 min. 2) Transfer the ice-cold tubes to a rack in a 42°C circulating water bath for exactly 90 seconds. 3) Quickly transfer the tube to ice and cool for 2 minutes; 4) Add 890 µl of 37°C preheated SOC liquid medium and shake at 160 rpm / min at 37°C for 1 h. 5) Spread 200 μl of the bacterial solution evenly on an LB plate containing 100 μg / ml kanamycin. After the liquid is almost completely absorbed at room temperature, incubate the plate upside down at 37°C for 16 h.

[0055] (4) Extraction of recombinant expression plasmid Follow the procedure of the kit to extract plasmid DNA.

[0056] 1) Take 1 ml of the bacterial suspension grown overnight in LB medium and centrifuge at 12,000 × g for 1 min. 2) Add 250 µl of Buffer S1 to evenly suspend the bacterial pellet; 3) Add 250 µl of Buffer S2 and mix thoroughly by gently inverting the tube six times to fully lyse the cells until a clear solution is formed. 4) Add 350 µl of Buffer S3, mix thoroughly by gently inverting the tube eight times, and centrifuge at 12,000 × g for 10 min. 5) Pipette the supernatant from step 4) and transfer it to a preparation tube. Centrifuge at 12,000 × g for 1 min and discard the filtrate. 6) Return the preparation tube to the centrifuge tube, add 500 µl of Buffer W1, centrifuge at 12,000 × g for 1 min, and discard the filtrate. 7) Return the preparation tube to the centrifuge tube, add 700 µl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. Wash once more with 700 µl of Buffer W2 using the same method, and discard the filtrate. 8) Place the prepared tube back into a 2 ml centrifuge tube and centrifuge at 12,000 × g for 1 min. 9) Transfer the preparation tube to a new 1.5 ml centrifuge tube, add 60 µl of elution buffer to the center of the preparation tube membrane, let it stand at room temperature for 1 minute, and centrifuge at 12,000 × g for 1 minute to elute the plasmid DNA.

[0057] (5) Plasmid pET-42a efeB , pET-42a hemH Double enzyme digestion identification 1) Prepare the enzyme digestion reaction system according to Table 6 for the extracted target gene recombinant clone plasmid: Table 6 Plasmid pET-42a efeB , pET-42a hemH Double enzyme digestion reaction system

[0058] 2) Digest the enzyme in a 37°C water bath for 4 h. Add the digested reaction mixture to 10× loading buffer and run electrophoresis on a 1.5% agarose gel to confirm the location of each target fragment.

[0059] (6) DNA sequence determination The bacterial solution with positive enzyme digestion results was commissioned to Sangon Biotech (Shanghai) Co., Ltd. to determine the nucleotide sequence of the inserted fragment in the positive bacterial solution.

[0060] The results showed that the electrophoresis results after double enzyme digestion showed that: in pET-42a efeB and pET-42a hemH The target gene band of the expected size was cut out by double enzymes in the plasmid, and the prokaryotic expression system was successfully constructed ( Figure 1 C in ).

[0061] Preparation method step 4: efeB and hemH Gene induction and purification (1) efeB and hemH Inducible gene expression 1) Transfect Escherichia coli BL21 (DE3) containing the correct plasmid pET42a-efeB BL21 (DE3) pET42a-hemH Inoculate into 10 ml of LB liquid medium containing 50 μg / ml kanamycin and culture at 37°C with shaking at 220 rpm overnight; 2) After streaking, pick a single, well-isolated, translucent white colony with a diameter of 2 mm from the plate and incubate in 10 ml of LB liquid medium containing 50 μg / ml kanamycin at 37°C with shaking at 220 rpm overnight. 3) Pipette 100 μl of the overnight culture into 10 ml of LB liquid medium containing 50 μg / ml kanamycin and shake-culture at 37°C at 220 rpm until the OD value reaches 0.8. 4) Add IPTG to a final concentration of 0.5 mM and induce the culture at 37°C with shaking at 220 rpm for 6 h. 5) Pipette 1 ml of the induced bacterial solution, centrifuge at 12,000 × g for 1 min, and discard the supernatant. 6) Resuspend the pellet in 1 ml of 0.01 mM PBS, pH 7.4, and wash twice by centrifugation at 12,000 × g for 1 min. Discard the supernatant. 7) Repeat step 6) once and resuspend the pellet in 200 μl of 0.01 mM PBS, pH 7.4; 8) Ultrasonicate the cells at 300 W, 5 s on / off, 10 s interval, 5 times. Keep on ice. 9) Centrifuge the disrupted cells at 12,000 × g for 5 min at 4°C. Collect the supernatant and precipitate separately. Resuspend each precipitate in 80 μl of 0.01 mM PBS, pH 7.4.

[0062] (2) SDS-polyacrylamide gel electrophoresis 1) Set up the SDS-PAGE electrophoresis apparatus: Use two clean electrophoresis glass plates and seals to create a vertical plate trough and place it vertically. 2) Prepare 5 ml of 12% separating gel: 1.6 ml of deionized water, 2.0 ml of 30% acrylamide-bisacrylamide, 1.3 ml of Tris-HCl (pH 8.8), 50 μl of 10% (w / v) SDS, 50 μl of 10% (w / v) ammonium persulfate, and 4 μl of TEMED; pour the separating gel into the well and block with deionized water. 3) After the separation gel has solidified at room temperature for 120 minutes, discard the deionized water used for blocking and remove any remaining liquid with the edge of a filter paper. 4) Prepare 2 ml of 5% stacking gel: 1.4 ml of deionized water, 0.33 ml of 30% acrylamide-bisacrylamide, 0.25 ml of Tris-HCl (pH 6.8), 20 μl of 10% (w / v) SDS, 20 μl of 10% (w / v) ammonium persulfate, and 2 μl of TEMED. Pour the stacking gel onto the polymerized separating gel and immediately insert a clean 10-well comb into the stacking gel solution. Let it solidify at room temperature for approximately 90 minutes. 5) After the stacking gel is fully polymerized, place the electrophoresis plate in the electrophoresis tank. Add an appropriate amount of 1× Tris-glycine running buffer to the inside and outside of the plate, and carefully remove the comb. 6) Turn on the power supply and run electrophoresis at a constant voltage of 80 V. When the leading edge of the bromophenol blue indicator enters the separation gel, adjust the voltage to 120 V and continue running at a constant voltage until the bromophenol blue indicator reaches the bottom of the gel. Turn off the power supply. 7) Staining: Carefully peel the SDS-PAGE gel from the electrophoresis plate and stain with Coomassie Brilliant Blue R250 staining solution for 30 minutes, then destain with destaining solution for 2-3 hours.

[0063] The results showed that the SDS-PAGE gel electrophoresis lanes contained high concentrations of the expected recombinant proteins rEfeB ​​(46.5 kDa) and rHemH (35.2 kDa) ( Figure 1 D in the figure).

[0064] Preparation method step 5: Isolation and purification of recombinant proteins rEfeB ​​and rHemH 1) Pick out a single well-separated BL21 (DE3) with a diameter of about 3 mm from the plate. pET42a-hemH BL21 (DE3) pET42a-efeB Translucent white colonies were cultured in 10 ml of LB liquid medium containing 50 μg / ml kanamycin at 37°C with shaking at 220 rpm overnight; 2) Pipette 5 ml of the overnight culture into 500 ml of fresh LB liquid medium containing 50 μg / ml kanamycin and shake-culture at 37°C at 220 rpm until the OD value reaches 0.8. 3) Add IPTG to a final concentration of 0.5 mM and induce culture at 37°C with shaking at 220 rpm for 6 h; 4) Centrifuge at 12,000 × g for 30 min at 4°C to collect the cells. 5) After washing three times with PBS, add 1 / 20 the bacterial growth volume of NTA-0 buffer and 125 μl of 200 mM PMSF to suspend the cells and disrupt the cells by ultrasonication on ice. 6) Centrifuge at 12,000 × g for 30 min at 4°C, remove the supernatant, and place on ice. 7) Load the NTA resin into a glass chromatography column and wash with NTA-0 Buffer (10 times the volume of NTA) at a flow rate of approximately 30 ml / h. Monitor and record the protein outflow using a nucleic acid protein detector and a nucleic acid protein detection recorder. 8) Add the sample to the NTA column at a flow rate of approximately 15 ml / h and collect the breakthrough peak; 9) Elute with 5 times the volume of NTA-0 Buffer at a flow rate of about 30 ml / h; 10) Elute with 5 times the volume of NTA using NTA-20, NTA-40, NTA-60, NTA-100, or NTA-500 at a flow rate of approximately 15 ml / h. Collect the eluate, collecting one NTA volume per tube. 11) Concentrate the purified target protein using an ultrafiltration tube and determine its concentration using the BCA assay; 12) Aliquot the target protein into sterile centrifuge tubes and store at -80°C; 13) Detect and confirm the purification effect of target protein by SDS-PAGE.

[0065] The experimental results showed that the purified recombinant proteins rEfeB ​​and rHemH were successfully obtained ( Figure 1 EG in).

[0066] Preparation method step 6: Removal of bacterial LPS from recombinant proteins rEfeB ​​and rHemH (1) Use Detoxi-Gel chromatography to remove LPS that may be present in rEfeB ​​and rHemH: 1) Add 5 column volumes of 1% sodium deoxycholate to the chromatography column, and then remove the excess sodium deoxycholate with 5 column volumes of ultrapure water; 2) Equilibrate the column with 5 column volumes of ultrapure water; 3) Add rEfeB ​​and rHemH to the chromatography column, seal the outlet, and collect the sample after standing for 1 hour; 4) The LPS content in rEfeB ​​and rHemH was measured using an endotoxin quantification kit (GenScript Biotech Co., Ltd., L00350).

[0067] (2) Preparation of endotoxin standard curve: 1) Prepare 200 μl of 1.0 EU / ml endotoxin solution. 2) Add 100 μl of LPS-free water to three test tubes and perform three replicates. 3) Add 100 μl of endotoxin solution to three test tubes in sequence using a two-fold dilution method, so that the endotoxin concentrations in the four test tubes are 1.0, 0.5, 0.25, and 0.1 EU / ml, respectively; (3) Quantitative determination of endotoxins: 1) Incubate the test reagent at 37°C for 10 min; 2) Add substrate solution and mix with the sample to be tested, then incubate at 37°C for 6 min; 3) Add stop reagent; 4) Measure the absorbance of the sample at 405 nm. LPS-free water and 0.5 ng of E. coli LPS were used as controls. The experimental results showed that after the Detoxi-Gel chromatography column method was used to remove LPS, the quantitative detection results showed that LPS in 10 μg and 50 μg rEfeB ​​and rHemH were negative ( Figure 1 H in ).

[0068] Example 2: EfeB and HemH promote cell survival (1) Human macrophage cell line (ATCC, TIB-202) (THP-1) (1×10 7 / ml) were plated in 96-well plates, and phorbol ester PMA (100 ng / ml) was used to induce mononuclear THP-1 cells into macrophages; (2) The cells were divided into control group, RSL3 (10 μM), rEfeB ​​(2 μg / ml), rHemH (2 μg / ml), RSL3 (10 μM) + rEfeB ​​(2 μg / ml), and RSL3 (10 μM) + rHemH (2 μg / ml). After adding the reagents, the cells were cultured at 37°C for 24 h. The control group was not treated. (3) Cell viability was detected using CCK8 (Shanghai Biotech Co., Ltd., C0037) and MTT kit (Shanghai Biotech Co., Ltd., C0009S); (4) 2 h after adding CCK8 reagent, measure the absorbance of the cell suspension at 450 nm using a microplate reader; 4 h after adding MTT reagent, measure the absorbance of the cell suspension at 570 nm using a microplate reader; (5) Analyze and compare the changes in relative cell viability.

[0069] The experimental results showed that rEfeB ​​and rHemH had no significant effect on THP-1 cell viability, suggesting that both rEfeB ​​and rHemH had low cytotoxicity; both rEfeB ​​and rHemH could promote the survival of THP-1 cells induced by RSL3 ( Figure 2 AB in).

[0070] Experimental Example 3: EfeB and HemH promote GPX4 expression in macrophages (1) Sample preparation 1) Cell preparation 1. Transfect human macrophage cell line (THP-1) (1×10 7 / ml) were plated in 96-well plates, and phorbol ester PMA (100ng / ml) was used to induce mononuclear THP-1 cells into macrophages; 2. Cells were divided into control group, RSL3 (10 μM), rEfeB ​​(2 μg / ml), rHemH (2 μg / ml), RSL3 (10 μM) + rEfeB ​​(2 μg / ml), and RSL3 (10 μM) + rHemH (2 μg / ml). After adding the reagents, culture at 37°C for 24 hours. The control group did not receive treatment.

[0071] 2) Cell lysis 1. Remove the supernatant from the cultured cells and wash them three times with PBS; 2. Add 1× SDS-PAGE protein loading buffer, fully lyse, and boil the sample for 15 minutes; (2) SDS-PAGE gel electrophoresis 1) PAGE gel preparation 1. Preparation of Separation Gel 1.1 Stack the two glass plates neatly and clamp them vertically on the base. Clamp them tightly and prepare to glue. 1.2 Prepare 10 ml of 12% separating gel: 3.3 ml of deionized water, 4 ml of 30% acrylamide-bisacrylamide, 2.5 ml of Tris-HCl (pH 8.8), 0.1 ml of 10% (w / v) SDS, 0.1 ml of 10% (w / v) ammonium persulfate, and 4 μl of TEMED. Pour the separating gel into the gel and seal with deionized water. 1.3 Let it stand for 120 minutes, pour off the water on the top of the glue and absorb the remaining liquid with the edge of the filter paper.

[0072] 2. Preparation of Stacking Gel Prepare 3 ml of 5% stacking gel: 2.1 ml of deionized water, 0.5 ml of 30% acrylamide-bisacrylamide, 0.38 ml of Tris-HCl (pH 6.8), 30 μl of 10% (w / v) SDS, 30 μl of 10% (w / v) ammonium persulfate, and 3 μl of TEMED. Pour the stacking gel onto the polymerized separating gel and immediately insert a comb into the stacking gel solution. Allow the gel to solidify at room temperature for approximately 90 minutes. After the stacking gel has fully polymerized, remove the seal and wipe off any excess liquid from the bottom and edges of the electrophoresis plate with filter paper.

[0073] 2) Sample loading for electrophoresis 1. Place the electrophoresis plate in the electrophoresis tank; add appropriate amounts of 1× Tris-glycine running buffer to the inside and outside of the plate, and carefully remove the comb; 2. Boil the prepared protein sample in water for 5 minutes and load 25 μl into each well. 3. Turn on the power supply and perform electrophoresis at a constant voltage of 80 V. When the leading edge of the bromophenol blue indicator enters the separation gel, adjust the voltage to 120 V and continue electrophoresis at a constant voltage until the bromophenol blue indicator reaches the bottom of the gel. Turn off the power supply.

[0074] (3) Protein transfer 1. Prepare 1× transfer buffer in advance and soak the filter paper and sponge in the transfer buffer. 2. Soak the PVDF membrane in methanol for 3 minutes, then soak the PVDF membrane in transfer buffer for 5 minutes; 3. Remove the gel after electrophoresis, gently pry open the glass plate, and soak the gel in transfer buffer; 4. Prepare a "sandwich" by placing the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in that order, ensuring there are no bubbles between the layers. Place the prepared "sandwich" into the transfer tank and add an appropriate amount of 1× transfer buffer. 5. Turn on the power supply and transfer the membrane at 330 mA for 25 minutes.

[0075] (4) Membrane blocking and antibody incubation 1. After transfer, place the PVDF membrane in a small box, add 5% skim milk powder blocking solution, and block on a shaker at room temperature for 1 hour; 2. Discard the blocking solution and wash with TBST three times, 5 min each time; add GPX4-IgG antibody dilution solution and incubate on a shaker at 4°C overnight; 3. Recover the GPX4-IgG antibody dilution solution and wash with TBST three times, 5 min each time; 4. Add fluorescently labeled anti-rabbit Ig-G and incubate on a shaker at room temperature for 1 h; 5. Recover Ig-G and wash with TBST 4 times, 5 min each time; 6. Discard TBST for the final time and scan the membrane using an imager.

[0076] The experimental results showed that rEfeB ​​and rHemH promoted the expression level of GPX4 in THP-1 cells ( Figure 2 CD in the ).

[0077] Experimental Example 4: EfeB and HemH inhibit ROS levels in macrophages (1) Cell preparation 1) Human macrophage cell line (THP-1) (1×10 7 / ml) were plated in 96-well plates, and phorbol ester PMA (100ng / ml) was used to induce mononuclear THP-1 cells into macrophages; 2) The cells were divided into control group, RSL3 (10 μM), rEfeB ​​(2 μg / ml), rHemH (2 μg / ml), RSL3 (10 μM) + rEfeB ​​(2 μg / ml), and RSL3 (10 μM) + rHemH (2 μg / ml). After adding the reagents, they were cultured at 37°C for 24 h. The control group was not treated. 3) Collect the cells by centrifugation at 800 × g for 10 min and wash three times with serum-free culture medium.

[0078] (2) Detection of ROS levels in cells by flow cytometry 1) Resuspend cells in 10 μM DCFH-DA fluorescent probe and incubate at 37°C for 20 min, shaking every 5 min to mix thoroughly. 2) Collect the cells by centrifugation at 800 × g for 10 min and wash three times with serum-free culture medium; 3) Detection of cellular ROS levels by flow cytometry.

[0079] (3) Detection of cellular ROS levels using fluorescence microscopy 1) Collect the treated cells and wash them three times with serum-free cell culture medium; 2) In situ loading of DCFH-DA (10 μM) fluorescent probe and incubation at 37°C for 20 min; 3) After washing the cells three times with serum-free cell culture medium, the cellular ROS levels were observed under a fluorescence microscope.

[0080] The experimental results showed that EfeB and HemH inhibited the level of ROS in THP-1 cells ( Figure 3 ).

[0081] Experimental Example 5: EfeB and HemH reduce Fe in macrophages 2+ level (1) Cell preparation 1) Human macrophage cell line (THP-1) (1×10 7 / ml) were plated in 96-well plates, and phorbol ester PMA (100ng / ml) was used to induce mononuclear THP-1 cells into macrophages; 2) The cells were divided into a control group, Fe(NH4)2(SO4)2 (100 μM), rEfeB ​​(2 μg / ml), and rHemH (2 μg / ml). After adding the reagents, they were cultured at 37°C for 24 h. The control group was not treated.

[0082] (2) Detection of Fe in cells by fluorescence microscopy 2+ level 1) Collect the treated cells and wash them three times with HBSS; 2) In situ loading of FerroOrange (1 μM) fluorescent probe (ferrous ion fluorescent probe) and incubation at 37°C for 30 min; 3) Wash the cells three times with HBSS and observe them under a fluorescence microscope.

[0083] The experimental results showed that EfeB and HemH inhibited the expression of Fe in THP-1 cells. 2+ level( Figure 4 AB in).

[0084] Experimental Example 6: EfeB and HemH inhibit intracellular lipid peroxide levels (1) Cell preparation 1) Human macrophage cell line (THP-1) (1×10 7 / ml) were plated in 96-well plates, and phorbol ester PMA (100ng / ml) was used to induce mononuclear THP-1 cells into macrophages; 2) The cells were divided into control group, RSL3 (10 μM), rEfeB ​​(2 μg / ml), rHemH (2 μg / ml), RSL3 (10 μM) + rEfeB ​​(2 μg / ml), and RSL3 (10 μM) + rHemH (2 μg / ml). After adding the reagents, they were cultured at 37°C for 24 h. The control group did not receive treatment.

[0085] (2) Detection of lipid peroxide levels in cells using fluorescence microscopy 1) Collect the treated cells and wash them three times with serum-free medium; 2) In situ loading of BDP 581 / 591 C11 (10 μM) (a fluorescent probe for lipid peroxidation) and incubation at 37°C for 30 min; 3) Wash the cells three times with serum-free medium and observe under a fluorescence microscope.

[0086] The experimental results showed that rEfeB ​​and rHemH inhibited the levels of lipid peroxides in THP-1 cells ( Figure 4 C in ).

[0087] Experimental Example 7: EfeB and HemH reduce renal and hepatic inflammation in LPS-induced sepsis (1) Establishment of a mouse sepsis model and the anti-sepsis effects of rEfeB ​​and rHemH 1) Thirty C57BL / 6 mice weighing 18-22 g were divided into a control group (PBS buffer injection), LPS, rEfeB, rHemH, LPS + rEfeB, and LPS + rHemH injection groups; 2) rEfeB, rHemH (4 mg / ml), or PBS were injected into the tail vein for 7 consecutive days, and LPS (10 mg / kg) was injected intraperitoneally after the last injection; 3) Mice were sacrificed by cervical dislocation, and the kidneys and livers were removed after dissection. Histological changes were observed using HE staining. 3.1) Fix kidney tissue with 4% paraformaldehyde for 24 h; 3.2) Dehydrate the fixed tissue using a gradient of ethanol (70%, 80%, 95%, 100%) for 1–2 h. 3.3) Transparent the tissue in xylene and then immerse in wax for 2-3 hours. 3.4) Embedding and sectioning: After embedding with wax in an embedding tank, the tissue is trimmed into wax blocks; 3.5) Sectioning: Use a microtome to trim the wax block to a thickness of 5-7 μm. 3.6) Adhesive Slide: Place the slide in a slide box, flatten it, and attach it to 2 / 3 of the glass slide. Allow it to air dry. 3.7) Dewaxing and hydration: Treat sections with xylene, graded alcohol (100%, 95%, 90%, 80%, 70%), and distilled water in sequence. 3.8) Staining: Use hematoxylin and eosin to stain the nuclei and cytoplasm of tissue cells, respectively; 3.9) Dehydration and mounting: Treat sections with a gradient of alcohol (70%, 80%, 95%, 100%), xylene, and resin, and cover with a coverslip. 3.10) Observe tissue pathological changes under a microscope.

[0088] The experimental results showed that rEfeB ​​and rHemH significantly reduced the levels of renal and liver inflammation in LPS-induced sepsis ( Figure 5 ).

Claims

1. A ferroptosis inhibitor, characterized in that The ferroptosis inhibitor is a protein consisting of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.

2.

2. An isolated polynucleotide, characterized in that The nucleotide sequence encodes the protein according to claim 1.

3. The polynucleotide according to claim 2, wherein The sequence of the polynucleotide is shown as SEQ ID No. 3 or SEQ ID No.

4.

4. Use of the ferroptosis inhibitor according to claim 1 or the polynucleotide according to claim 2 or 3 in the preparation of a drug for inhibiting cell ferroptosis.

5. The use according to claim 4, characterized in that The cells are macrophages.

6. Use of the ferroptosis inhibitor according to claim 1 or the polynucleotide according to claim 2 or 3 in the preparation or screening of drugs for preventing and / or treating sepsis.

7. The use according to claim 6, characterized in that When used, the drug can inhibit sepsis-induced ferroptosis, reduce inflammatory response and / or alleviate organ damage.

8. The use according to claim 7, characterized in that The organ is a kidney or a liver.

Images