Novel ferroptosis inhibitors and their application in the preparation of drugs for the prevention and / or treatment of sepsis

Abstract

This invention discloses a novel ferroptosis inhibitor and its application in the preparation of drugs for the prevention and / or treatment of sepsis, belonging to the fields of pharmaceutical formulation and biomedicine. The invention discloses a method for preparing the novel ferroptosis inhibitor, which can be used to develop highly efficient and low-toxicity ferroptosis inhibitors. This agent can be used to prepare drugs for the prevention of sepsis-related organ damage and is a potential drug for the treatment of sepsis. It can inhibit ferroptosis in sepsis-induced kidney, liver, lung, and cardiomyopathy, and reduce inflammatory responses and organ damage. Furthermore, this invention has the outstanding advantages of high specificity, high bioavailability, significant efficacy, and a stable preparation method, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical formulation and biomedical technology, and to novel ferroptosis inhibitors and their application in the preparation of drugs for the prevention and / or treatment of sepsis. Background Technology

[0002] Ferroplasmosis is an iron-dependent form of cell death, biochemically characterized by the excessive accumulation of reactive oxygen species and lipid peroxides, and decreased glutathione peroxidase 4 activity. This leads to intracellular redox imbalance and cell membrane damage, ultimately resulting in cell death. Morphologically, ferroptosis is characterized by cell membrane rupture, reduced mitochondrial volume, increased membrane density, decreased or absent cristae, and outer membrane rupture. Iron is an essential trace element in organisms, participating in key metabolic processes in many pathogenic microorganisms. The virulence of some bacteria depends on iron uptake; iron can enhance bacterial virulence and promote their proliferation. Current research shows that ferroptosis plays an increasingly important role in organ dysfunction and has a significant impact on disease treatment and prognosis.

[0003] Sepsis is a severe, systemic disease caused by an excessive immune-inflammatory response to infection, surgery, or trauma, leading to multiple organ dysfunction. Sepsis can originate in community settings or be acquired in hospitals or other healthcare facilities; approximately 80% of hospital-treated sepsis cases occur in the community. Upon pathogen invasion, macrophages recognize pathogen-associated receptors on the pathogen's surface, activating other immune cells and releasing inflammatory cytokines, triggering an intracellular cytokine storm and an excessive inflammatory response, ultimately leading to immunosuppression. Sepsis can disrupt intracellular iron metabolism, including increased intracellular iron transport and uptake, and decreased iron output, resulting in intracellular iron overload. Ferric death can release damage-associated pattern molecules or lipid oxides, thereby activating inflammation-related signaling pathways, further promoting the cytokine storm and exacerbating organ damage.

[0004] In recent years, the incidence of sepsis has been increasing at a rate of 8% to 11%, with an overall mortality rate approaching 30%, making it a major global threat to life. Therefore, exploring therapeutic targets for sepsis is of great significance in reducing its mortality rate. Current advances in sepsis treatment are limited to symptomatic care, including organ support and fluid resuscitation, primarily focusing on eliminating pathogens, clearing inflammatory mediators, and restoring cellular function. There are still gaps in the development of effective therapeutic drugs for sepsis, making the exploration of new treatment directions and potential therapeutic targets crucial. Intervening in the progression of sepsis from a trace element perspective holds promise as a method for precision medicine.

[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, inhibitors of acyl-CoA synthase family member 4, and lipoxygenase inhibitors, but they suffer from drawbacks such as 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 application in the treatment of sepsis.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] First, through bioinformatics analysis, it was predicted that EfeB can bind free iron ions and has the function of trans-ion iron transport, while HemH can bind free iron ions and insert them into porphyrins in cells, thereby more efficiently reducing the level of intracellular iron ions and inhibiting ferroptosis.

[0010] On the other hand, through bioinformatics analysis, it is predicted that EfeB and HemH can be exocrine.

[0011] On the other hand, successful construction using molecular cloning methods efeB and hemH A prokaryotic gene expression system was developed, and recombinant proteins rEfeB ​​and rHemH were expressed and purified.

[0012] That is, a ferroptosis inhibitor, which is the protein described in a) or b) below:

[0013] a) A protein consisting of the amino acid sequences shown in SEQ ID No. 1 and / or SEQ ID No. 2; or

[0014] b) Proteins derived from a) that have undergone substitution, deletion and / or addition of one or more amino acids in the amino acid sequence defined in a) and have the same ferroptosis inhibitory activity as the amino acid sequence defined in a).

[0015] An isolated polynucleotide, said polynucleotide encoding the aforementioned protein.

[0016] Specifically, the polynucleotide preferably has the following nucleotide sequence:

[0017] 1) A nucleotide sequence represented by SEQ ID No. 3 or SEQ ID No. 4;

[0018] 2) A nucleotide sequence that hybridizes to the nucleotide sequence defined in 1) under stringent conditions; or

[0019] 3) A nucleotide sequence that has at least 90% homology with 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.

[0020] This invention also provides the application of the aforementioned ferroptosis inhibitor and the aforementioned polynucleotide in the preparation of drugs that inhibit ferroptosis.

[0021] Preferably, the cells are macrophages. Specifically, this manifests as: reducing the concentration of iron ions in the cells, decreasing the production of ferroptosis-related molecules ROS and lipid peroxides, and promoting the expression of the key negative regulatory protein GPX4 of ferroptosis, thereby inhibiting the occurrence of ferroptosis.

[0022] The present invention also provides the use of the aforementioned ferroptosis inhibitor and the aforementioned polynucleotide in the preparation or screening of medicaments for the prevention and / or treatment of sepsis.

[0023] Preferably, when applied, the drug can inhibit sepsis-induced ferroptosis, reduce inflammatory response, and / or reduce organ damage.

[0024] In some embodiments of the present invention, the organ is the kidney or the liver.

[0025] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0026] 1. The ferroptosis inhibitor provided by this invention has high purity, strong specificity, and low cytotoxicity.

[0027] 2. The ferroptosis inhibitor provided by this invention has functional sites for binding to iron ions in its molecular structural domains and possesses iron ion transport capabilities, enabling it to bind to and transport free iron in cells. Compared to other ferroptosis inhibitors, this invention exhibits higher inhibitory efficiency and can effectively reduce the concentration of iron ions in cells. Compared to other ferroptosis inhibitors, it can reduce the production of ferroptosis-related molecules such as reactive oxygen species and lipid peroxides, and promote the expression of glutathione peroxidase 4, thereby inhibiting the occurrence of ferroptosis.

[0028] 3. The ferroptosis inhibitor provided by this invention can inhibit sepsis-induced ferroptosis and reduce inflammation and organ damage. Attached Figure Description

[0029] Figure 1 Expression and purification of EfeB and HemH. (A) Amplification of the expected size of EfeB and HemH from DNA of strain CFT073. efeB (1272 bp) and hemH(963 bp) gene fragment. Note: M: DNA marker; 1: hemH Gene amplification band; 2: efeB Gene amplification bands. (BC) Construction of TA clones and subclones of the target gene fragment: Double digestion and 1.5% agarose gel electrophoresis yielded DNA fragments of the expected size and location. Note: In B, M: DNA marker; 1: pMD19-T hemH Depend on Nde I and Xho PMD19-T after I enzyme digestion and hemH ;2: pMD19-T efeB Depend on Nde I and Xho PMD19-T after I enzyme digestion and efeB In C, M stands for DNA marker; and 1 stands for pET-42a. hemH Depend on Nde I and Xho pET-42a digested with enzyme I and hemH ;2: pET-42a efeB Depend on Nde I and Xho pET-42a digested with enzyme I and efeB (D) High-efficiency expression of recombinant proteins rEfeB ​​and rHemH in a prokaryotic expression system induced by IPTG: SDS-PAGE gel electrophoresis showed the expected sizes 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) Successfully purified recombinant protein: Automated protein purification system showed a single peak, and SDS-PAGE showed a single bright band. Note: M: protein marker; 1: purified rHemH protein; 2: purified rEfeB ​​protein. (H) Detoxi-Gel chromatography was used to remove bacterial endotoxins (LPS) from rEfeB ​​and rHemH; the discrete dots represent parallel samples.

[0030] Figure 2EfeB and HemH were used to promote cell survival and GPX4 expression levels. (A) CCK8 assay of THP-1 cell viability. (B) MTT assay of 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 low cytotoxicity; compared with the RSL3-induced ferroptosis-positive group, rEfeB ​​and rHemH promoted the survival of THP-1 cells. (C) Western blot analysis of the effect of rEfeB ​​and rHemH on GPX4 expression. (D) ImageJ software analysis of GPX4 band grayscale in three experiments. Compared with the RSL3 group, rEfeB ​​and rHemH promoted the expression level of GPX4 in THP-1 cells. Discrete circles represent parallel samples; *: p <0.05, **: p <0.01, ns: p >0.05.

[0031] Figure 3 EfeB and HemH were used to inhibit ROS levels in macrophages. (A) Intracellular ROS levels were detected by flow cytometry. (B) Statistical analysis of the mean fluorescence intensity of ROS in experiment (A) was performed using ImageJ software, with discrete circles representing parallel samples. (C) ROS levels were observed using fluorescence microscopy. 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.

[0032] Figure 4 EfeB and HemH inhibit intracellular Fe in macrophages 2+ And lipid peroxide levels. (A) FerroOrange fluorescent probe detects intracellular Fe 2+ Horizontal fluorescence, blue fluorescence is the color of DAPI, and orange fluorescence is the color of FerroOrange after binding to free intracellular ferrous ions. (B) ImageJ software on Fe in experiment (A). 2+ Statistical analysis of mean fluorescence intensity was performed, with discrete circles representing parallel samples. Results showed that, compared to the control group, rEfeB ​​and rHemH could inhibit Fe in THP-1 cells. 2+The levels of lipid peroxides in THP-1 cells were measured using the BDP 581 / 591 C11 probe. (C) The red fluorescence represents the color of the BDP 581 / 591 C11 probe, and the green fluorescence represents the color of the probe after binding with lipid peroxides. Results showed that, compared to the RSL3 group, rEfeB ​​and rHemH inhibited lipid peroxide levels in THP-1 cells. * p <0.05, **: p <0.01.

[0033] Figure 5 rEfeB ​​and rHemH inhibit sepsis-induced levels of kidney and liver inflammation. Detailed Implementation

[0034] Example 1: Synthesis of ferroptosis inhibitors EfeB and HemH

[0035] Preparation method step 1: efeB and hemH Gene amplification:

[0036] I. Uropathogenic Escherichia coli Escherichia coli Culture of UPEC strain CFT073

[0037] The UPEC CFT073 strain (gifted by Academician Xu Jianguo of the Institute of Infectious Diseases, Chinese Center for Disease Control and Prevention) was cultured overnight in LB liquid culture at 37°C and 220 r / min.

[0038] II. Extraction of UPEC CFT073 genomic DNA

[0039] Follow the procedure outlined in the bacterial genomic DNA extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-MN-MS-GDNA-50).

[0040] (1) Collect the logarithmic phase UPEC CFT073 culture in a 2 ml centrifuge tube, centrifuge at 12000 × g for 30 s, and discard the supernatant; suspend the precipitate in 150 μl of Buffer S containing RNase A; add 20 μl of lysozyme stock solution, mix well, and let stand at room temperature for 5 min;

[0041] (2) Add 30 μl of 0.25 M EDTA (pH 8.0), mix well, and incubate on ice for 5 min;

[0042] (3) Add 450 μl Buffer GA, vortex for 15 s, and incubate in a 65℃ water bath for 10 min; add 400 μl Buffer GB and 1 ml Buffer DV pre-cooled at 4℃, mix evenly, and centrifuge at 12000 ×g for 2 min.

[0043] (4) Discard the upper phase, retain the time precipitate and the lower phase; add 1 ml of 4℃ pre-cooled Buffer DV, mix vigorously, and centrifuge at 12000×g for 2 min; discard the upper phase, transfer the lower phase to the filter, and centrifuge at 12000×g for 1 min; discard the filter, add 400 μl of Buffer BV to the filtrate, and mix evenly;

[0044] (5) Place the preparation tube into a 2 ml centrifuge tube, transfer the liquid from step (4) into the preparation tube, centrifuge at 12000 ×g for 1 min; discard the filtrate, place the preparation tube back into the 2 ml centrifuge tube, add 500 μl Buffer W1, centrifuge at 12000 ×g for 1 min;

[0045] (6) Discard the filtrate, place the preparation tube back into a 2 ml centrifuge tube, add 700 μl Buffer W2, and centrifuge at 12000 ×g for 1 min; wash again with 700 μl Buffer W2 in the same way;

[0046] (7) Discard the filtrate, place the preparation tube back into a 2 ml centrifuge tube, and centrifuge at 12000 × g for 1 min;

[0047] (8) Place the preparation tube in a 1.5 ml centrifuge tube, add 150 μl of elution buffer to the center of the silica membrane, and let stand at room temperature for 1 min; centrifuge at 12000 ×g for 1 min to elute the DNA.

[0048] III. UPEC CFT073 strain efeB and hemH Gene amplification and purification of amplification products

[0049] (1) Primer design

[0050] Obtained from GenBank efeB Gene (sequence NZ_CP051263.1: 1124808-1126079bp) and hemH The gene sequence (NZ_CP051263.1: 575517-576479bp) was analyzed using Primer 6.0 software to determine its restriction endonuclease map. Primers were then designed, and the selected endonuclease sites were... Nde I (CATATG) and XhoI (CTCGAG), primer sequences are shown in Table 1, synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0051] Table 1 UPEC CFT073 strain efeB and hemH Gene amplification primers

[0052]

[0053] F: forward primer, R: reverse primer.

[0054] (2) Amplification of the target gene

[0055] Using UPEC CFT073 genomic DNA as a template, amplification efeB and hemH Genes, prepare PCR reaction system according to Table 2.

[0056] Table 2 efeB and hemH PCR reaction system for genes

[0057]

[0058] PCR parameters: 94℃ for 5 min; 94℃ for 30 s, 50℃ for 30 s, 72℃ for 180 s, 30 cycles; 72℃ for 10 min. Amplified products were detected by 1.5% agarose gel electrophoresis pre-stained with ethidium bromide.

[0059] (3) Purification of PCR amplification products

[0060] Purify the target fragment for PCR amplification using the PCR cleaning kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-PCR-50).

[0061] 1) Add 3 volumes of Buffer PCR-A to the PCR reaction solution; mix thoroughly and then transfer to a preparation tube;

[0062] 2) Place the preparation tube into a 2 ml centrifuge tube, centrifuge at 12000 × g for 1 min, and discard the filtrate;

[0063] 3) Place the preparation tube back into the 2 ml centrifuge tube, add 700 μl Buffer W2, centrifuge at 12000 ×g for 1 min, and discard the filtrate;

[0064] 4) Place the preparation tube back into a 2 ml centrifuge tube, add 400 μl Buffer W2, centrifuge at 12000 ×g for 1 min, and discard the filtrate;

[0065] 5) Place the preparation tube into a 1.5 ml centrifuge tube, add 30 μl of elution buffer to the center of the membrane in the preparation tube, and let it stand at room temperature for 1 min; then centrifuge at 12000 ×g for 1 min to elute the DNA.

[0066] Experimental results showed that the expected size of [a specific type of DNA] was successfully amplified from the genomic DNA of UPEC strain CFT073. efeB (1272 bp) and hemH (963 bp) gene fragment ( Figure 1 (A in the middle).

[0067] Preparation method step 2: efeB and hemH TA cloning of genes

[0068] (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)

[0069] 1) Streak DH5α or BL21 (DE3) onto LB plates and incubate at 37°C for 16 h;

[0070] 2) Pick a single colony of DH5α or BL21(DE3) and place it in 10 ml of LB liquid medium. Incubate at 37°C with shaking for 16 h.

[0071] 3) Inoculate the culture into 30 ml of LB medium at a 1% inoculum rate, and incubate at 37°C with shaking for 2.5 h, until OD reaches 1. 600 =0.4;

[0072] 4) Transfer the culture medium into a 50 ml sterile centrifuge tube and incubate on ice for 10 min;

[0073] 5) Centrifuge at 3000 ×g for 10 min at 4℃; discard the supernatant to allow any remaining trace amounts of culture medium to drain completely;

[0074] 6) Add 20 ml of 0.1 M CaCl2 solution pre-cooled at 4℃ to suspend the precipitate, and let it stand on ice for 30 min; centrifuge at 3000×g at 4℃ for 10 min; discard the supernatant to allow any remaining trace amounts of culture medium to drain completely;

[0075] 7) Add 2 ml of 0.1 M CaCl2 solution containing 15% glycerol pre-cooled at 4℃, suspend the precipitate, and dispense into 100 μl tubes for later use.

[0076] (2) Connection

[0077] The ligation system was prepared in centrifuge tubes according to Table 3 using the TA cloning kit (Baori Biotechnology (Beijing) Co., Ltd., 3271), and after mixing, the ligation was carried out at 16℃ for 12 h.

[0078] Table 3 UPEC CFT073 strain efeB and hemH Gene TA cloning ligation system

[0079]

[0080] (3) Transformation

[0081] 1) Using a pre-cooled pipette tip, aspirate 100 µl of DH5α competent cells and transfer them into an ice-cold 1.5 ml centrifuge tube. Add 10 µl of the ligation reaction product, gently swirl to mix, and incubate on ice for 30 min.

[0082] 2) Transfer the cold tube to a rack in a 42°C circulating water bath and position it precisely for 90 seconds;

[0083] 3) Quickly transfer the tube to ice and allow it to cool for 2 minutes;

[0084] 4) Add 890 µl of preheated SOC liquid medium at 37℃, and incubate at 37℃ with shaking at 160 rpm / min for 1 h;

[0085] 5) Take 200 μl of bacterial suspension and spread it evenly on an LB agar 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 spread liquid is basically dry, incubate it upside down at 37°C for 16 h. Colonies will appear. Recombinants will show white colonies, and non-recombinants will show blue colonies.

[0086] (4) Extraction of the target recombinant plasmid

[0087] Follow the procedure for extracting plasmid DNA using the plasmid extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-MN-P-50).

[0088] 1) Take 1 ml of bacterial culture, centrifuge at 12000 ×g for 1 min, and discard the supernatant;

[0089] 2) Add 250 µl of Buffer S1 to resuspend the bacterial precipitate evenly;

[0090] 3) Add 250 µl of Buffer S2, gently and thoroughly mix by turning the container up and down 6 times to ensure complete lysis of the bacteria until a clear solution is formed;

[0091] 4) Add 350 µl of Buffer S3, gently and thoroughly mix by inverting the container 8 times, and centrifuge at 12000 ×g for 10 min;

[0092] 5) Transfer the supernatant from step 4) into the preparation tube, centrifuge at 12000 ×g for 1 min, and discard the filtrate;

[0093] 6) Place the preparation tube back into the centrifuge tube, add 500 µl of Buffer W1, centrifuge at 12000 ×g for 1 min, and discard the filtrate.

[0094] 7) Place the preparation tube back into the centrifuge tube, add 700 µl of Buffer W2, centrifuge at 12000 ×g for 1 min, and discard the filtrate; wash again with 700 µl of Buffer W2 in the same way, and discard the filtrate.

[0095] 8) Place the preparation tube back into a 2 ml centrifuge tube and centrifuge at 12000 × g for 1 min;

[0096] 9) Transfer the preparation tube into a new 1.5 ml centrifuge tube, add 60 µl of elution buffer to the center of the membrane in the preparation tube, and let stand at room temperature for 1 min; centrifuge at 12000 ×g for 1 min to elute the plasmid DNA.

[0097] (5) plasmid pMD19-T efeB and pMD19-T hemH Double enzyme digestion identification

[0098] 1) Prepare the enzyme digestion reaction system according to Table 4 using the extracted recombinant cloning plasmid of the target gene:

[0099] Table 4. Plasmid pMD19-T efeB and pMD19-T hemH Double enzyme digestion reaction system

[0100]

[0101] 2) Incubate the enzyme solution in a 37℃ water bath for 4 h; add the digested solution to 10× Loading Buffer and electrophore it on a 1.5% agarose gel to confirm the location of each target fragment.

[0102] (6) DNA sequencing

[0103] The bacterial solutions that showed positive enzyme digestion results were sent to Sangon Biotech (Shanghai) Co., Ltd. to determine the nucleotide sequence of the inserted fragment in the positive bacterial solutions.

[0104] Experimental results show that pMD19-T efeB and pMD19-T hemHThe expected target gene band was obtained by double enzyme digestion in the recombinant cloning plasmid. Figure 1 (B in the middle).

[0105] Preparation method step 3: efeB and hemH Subcloning of genes and construction of prokaryotic expression systems

[0106] (1) Recovery of the target gene fragment

[0107] Follow the DNA extraction procedure using the gel extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., AP-GX-50).

[0108] 1) Cut the agarose gel containing the target DNA under UV light, blot the liquid off the surface of the gel with a paper towel and cut it into pieces; calculate the weight of the gel, which is taken as a gel volume;

[0109] 2) Add 3 times the volume of Buffer DE-A, mix well, and heat at 75°C, mixing intermittently, until the gel block is completely melted;

[0110] 3) Add 0.5 times the volume of Buffer DE-A to Buffer DE-B and mix thoroughly;

[0111] 4) Take the mixture from step 3, transfer it to a DNA preparation tube, centrifuge at 12000 ×g for 1 min, and discard the filtrate;

[0112] 5) Place the preparation tube back into the centrifuge tube, add 500 µl of Buffer W1, centrifuge at 12000 ×g for 30 s, and discard the filtrate;

[0113] 6) Place the preparation tube back into the centrifuge tube, add 700 µl of Buffer W2, centrifuge at 12000 ×g for 30 s, and discard the filtrate; wash once with 700 µl of Buffer W2 and centrifuge at 12000 ×g for 1 min in the same manner.

[0114] 7) Place the preparation tube into a 2 ml centrifuge tube and centrifuge at 12000 × g for 1 min;

[0115] 8) Place the preparation tube into a clean 1.5 ml centrifuge tube, add 25 µl of elution buffer to the center of the DNA preparation membrane, and let stand at room temperature for 1 min. Centrifuge at 12000 ×g for 1 min to elute the DNA.

[0116] (2) Connection

[0117] Prepare the connection system in centrifuge tubes according to Table 5, mix well, and connect overnight at 16°C.

[0118] Table 5 UPEC CFT073 efeB and hemH Gene subcloning ligation system

[0119]

[0120] (3) Transformation

[0121] 1) Using a pre-cooled pipette tip, aspirate 100 µl of BL21(DE3) competent cells and transfer them into an ice-cold 1.5 ml centrifuge tube. Add 10 µl of the ligation reaction product, gently swirl to mix, and incubate on ice for 30 min.

[0122] 2) Transfer the cold tube to a rack in a 42°C circulating water bath and position it precisely for 90 seconds;

[0123] 3) Quickly transfer the tube to ice and allow it to cool for 2 minutes;

[0124] 4) Add 890 µl of preheated SOC liquid medium at 37℃, and incubate at 37℃ with shaking at 160 rpm / min for 1 h;

[0125] 5) Take 200 μl of bacterial suspension and spread it evenly on an LB agar plate containing 100 μg / ml kanamycin. After the liquid on the plate has been basically dried at room temperature, incubate it upside down at 37°C for 16 h.

[0126] (4) Extraction of recombinant expression plasmids

[0127] Follow the instructions in the kit to extract plasmid DNA.

[0128] 1) Take 1 ml of bacterial culture that has been cultured overnight in LB medium and centrifuge at 12000 ×g for 1 min;

[0129] 2) Add 250 µl of Buffer S1 to resuspend the bacterial precipitate evenly;

[0130] 3) Add 250 µl of Buffer S2, gently and thoroughly mix by turning the container up and down 6 times to ensure complete lysis of the bacteria until a clear solution is formed;

[0131] 4) Add 350 µl of Buffer S3, gently and thoroughly mix by inverting the container 8 times, and centrifuge at 12000 ×g for 10 min;

[0132] 5) Take the supernatant from step 4) and transfer it to the preparation tube. Centrifuge at 12000 ×g for 1 min and discard the filtrate.

[0133] 6) Place the preparation tube back into the centrifuge tube, add 500 µl of Buffer W1, centrifuge at 12000 ×g for 1 min, and discard the filtrate;

[0134] 7) Place the preparation tube back into the centrifuge tube, add 700 µl of Buffer W2, centrifuge at 12000 ×g for 1 min, and discard the filtrate; wash again with 700 µl of Buffer W2 in the same way, and discard the filtrate.

[0135] 8) Place the preparation tube back into a 2 ml centrifuge tube and centrifuge at 12000 × g for 1 min;

[0136] 9) Transfer the preparation tube into a new 1.5 ml centrifuge tube, add 60 µl of elution buffer to the center of the membrane in the preparation tube, and let stand at room temperature for 1 min; centrifuge at 12000 ×g for 1 min to elute the plasmid DNA.

[0137] (5) plasmid pET-42a efeB pET-42a hemH Double enzyme digestion identification

[0138] 1) Prepare the enzyme digestion reaction system according to Table 6 using the extracted recombinant cloning plasmid of the target gene:

[0139] Table 6. Plasmid pET-42a efeB pET-42a hemH Double enzyme digestion reaction system

[0140]

[0141] 2) Incubate the enzyme solution in a 37℃ water bath for 4 h. Add the digested solution to 10× loading buffer and electrophoresis on a 1.5% agarose gel to confirm the location of each target fragment.

[0142] (6) DNA sequencing

[0143] The bacterial solutions that showed positive enzyme digestion results were sent to Sangon Biotech (Shanghai) Co., Ltd. to determine the nucleotide sequence of the inserted fragment in the positive bacterial solutions.

[0144] The results showed that, after double enzyme digestion, electrophoresis revealed that pET-42a efeB and pET-42a hemH The target gene band of the expected size was successfully extracted from the plasmid by double enzyme digestion, and a prokaryotic expression system was successfully constructed. Figure 1 (C in the middle).

[0145] Preparation method step 4: efeB and hemH Induced expression and purification of genes

[0146] (1) efeB and hemH Induced expression of genes

[0147] 1) Introduce 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 incubate overnight at 37°C with shaking at 220 rpm;

[0148] 2) After streak isolation, pick a single well-separated, translucent white colony with a diameter of 2 mm from the plate and place it in 10 ml of LB liquid medium containing 50 μg / ml kanamycin. Incubate overnight at 37°C with shaking at 220 rpm.

[0149] 3) Take 100 μl of the overnight culture and add it to 10 ml of LB liquid medium containing 50 μg / ml kanamycin. Incubate at 37℃ with shaking at 220 rpm until the OD value is 0.8.

[0150] 4) Add IPTG to a final concentration of 0.5 mM, and induce incubation at 37℃ with shaking at 220 rpm for 6 h;

[0151] 5) Take 1 ml of the induced bacterial culture, centrifuge at 12000 ×g for 1 min, and discard the supernatant;

[0152] 6) Resuspend the precipitate in 1 ml of 0.01 mM pH7.4 PBS, centrifuge at 12000 ×g for 1 min, wash twice, and discard the supernatant;

[0153] 7) Repeat step 6) once, resuspending the precipitate in 200 μl of 0.01 mM pH 7.4 PBS;

[0154] 8) Use an ultrasonic cell disruptor to disrupt bacterial cells. Conditions: 300 W, operating time 5 s, interval 10 s, 5 ultrasonic cycles. Operate on ice.

[0155] 9) The lysed bacterial cells were centrifuged at 12000 ×g for 5 min at 4℃. The supernatant and precipitate were collected separately, and each precipitate was resuspended in 80 μl of 0.01 mM pH7.4 PBS.

[0156] (2) SDS-polyacrylamide gel electrophoresis

[0157] 1) Install the SDS-PAGE electrophoresis apparatus: Use two clean electrophoresis glass plates and sealing strips to make a vertical plate groove, and place it vertically;

[0158] 2) Prepare 5 ml of 12% separating gel: 1.6 ml deionized water, 2.0 ml 30% acrylamide-bisacrylamide, 1.3 ml pH 8.8 Tris-HCl, 50 μl 10% (w / v) SDS, 50 μl 10% (w / v) ammonium persulfate, and 4 μl TEMED; pour the separating gel and seal with deionized water;

[0159] 3) After the separating gel solidifies at room temperature for 120 min, pour off the deionized water used for sealing, and then use the edge of filter paper to absorb the remaining liquid.

[0160] 4) Prepare 2 ml of 5% stacking gel: 1.4 ml deionized water, 0.33 ml 30% acrylamide-bisacrylamide, 0.25 ml pH 6.8 Tris-HCl, 20 μl 10% (w / v) SDS, 20 μl 10% (w / v) ammonium persulfate, and 2 μl TEMED; pour the stacking gel onto the polymerized separating gel, and immediately insert a clean 10-hole comb into the stacking gel solution. Let it stand at room temperature for about 90 minutes to solidify.

[0161] 5) After the stacking gel has fully polymerized, place the electrophoresis plate into the electrophoresis tank, add an appropriate amount of 1×Tris-glycine electrophoresis buffer to both the inside and outside of the electrophoresis plate, and carefully remove the comb.

[0162] 6) Turn on the power and perform 80 V constant voltage electrophoresis. When the leading edge of the bromophenol blue indicator enters the separating gel, adjust the voltage to 120 V and continue constant voltage electrophoresis until the bromophenol blue indicator reaches the bottom of the gel. Then turn off the power.

[0163] 7) Staining: Carefully peel off the SDS-PAGE gel from the electrophoresis plate and stain with Coomassie Brilliant Blue R250 staining solution for 30 min, then destain with destaining solution for 2-3 h.

[0164] The results showed high concentrations of the expected-size recombinant proteins rEfeB ​​(46.5 kDa) and rHemH (35.2 kDa) in the SDS-PAGE gel electrophoresis lanes. Figure 1 (D in the middle).

[0165] Preparation method step 5: Isolation and purification of recombinant proteins rEfeB ​​and rHemH

[0166] 1) Pick out individual BL21 (DE3) samples that are well separated and have a diameter of about 3 mm from the plate. pET42a-hemH BL21 (DE3) pET42a-efeB Translucent white colonies were cultured overnight at 37°C with shaking at 220 rpm in 10 ml of LB liquid medium containing 50 μg / ml kanamycin.

[0167] 2) Take 5 ml of the overnight culture and add it to 500 ml of fresh LB liquid medium containing 50 μg / ml kanamycin. Incubate at 37°C with shaking at 220 rpm until the OD value is 0.8.

[0168] 3) Add IPTG to a final concentration of 0.5 mM, and induce incubation at 37℃ with shaking at 220 rpm for 6 h;

[0169] 4) Collect bacterial cells by centrifugation at 12000 ×g for 30 min at 4℃;

[0170] 5) After washing three times with PBS, add 1 / 20 of the bacterial growth volume of NTA-0 Buffer and 125 μl of 200 mM PMSF to suspend the cells, and sonicate the cells on ice.

[0171] 6) Centrifuge at 12000 ×g for 30 min at 4℃, collect the supernatant and place it on ice;

[0172] 7) Pack NTA resin into a glass chromatography column, wash with NTA-0 buffer at 10 times the volume of NTA, control the flow rate at about 30 ml / h, and monitor and record the amount of protein elution using a nucleic acid protein detector and a nucleic acid protein detection recorder.

[0173] 8) Add the sample to the NTA chromatography column at a flow rate of about 15 ml / h and collect the breakthrough peak;

[0174] 9) Elute with 5 times the volume of NTA-0 Buffer at a flow rate of approximately 30 ml / h;

[0175] 10) Elute with 5 NTA volumes of NTA-20, NTA-40, NTA-60, NTA-100, and NTA-500 at a flow rate of approximately 15 ml / h. Collect the eluent, one NTA volume per tube.

[0176] 11) The purified target protein was concentrated using an ultrafiltration tube, and the concentration was determined using the BCA method;

[0177] 12) Aliquot the target protein into sterile centrifuge tubes and store at -80°C;

[0178] 13) SDS-PAGE detection and determination of the purification effect of the target protein.

[0179] Experimental results showed that purified recombinant proteins rEfeB ​​and rHemH were successfully obtained. Figure 1 (EG in the middle).

[0180] Preparation method step 6: Removal of bacterial LPS from recombinant proteins rEfeB ​​and rHemH

[0181] (1) Remove LPS that may be present in rEfeB ​​and rHemH using Detoxi-Gel chromatography:

[0182] 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.

[0183] 2) Use an ultrapure water equilibrium chromatography column with a column volume of 5 times;

[0184] 3) Add rEfeB ​​and rHemH to the chromatography column, seal the outlet, let stand for 1 h, and then collect the sample;

[0185] 4) The LPS content in rEfeB ​​and rHemH was determined using an endotoxin quantitative kit (GenScript Biotech Inc., L00350).

[0186] (2) Preparation of the endotoxin standard curve:

[0187] 1) Prepare 200 μl of 1.0 EU / ml endotoxin solution;

[0188] 2) Add 100 μl of LPS-free water to 3 test tubes, with 3 replicates;

[0189] 3) Add 100 μl of endotoxin solution to three test tubes sequentially using the 2-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;

[0190] (3) Quantitative determination of endotoxins:

[0191] 1) Incubate the test reagent at 37℃ for 10 min;

[0192] 2) After adding the substrate solution and mixing it with the sample to be tested, incubate at 37°C for 6 min;

[0193] 3) Add the termination reagent;

[0194] 4) Measure the absorbance of the sample at 405 nm. Water without LPS and 0.5 ng of Escherichia coli LPS were used as controls in the experiment.

[0195] Experimental results showed that after LPS removal by Detoxi-Gel chromatography, quantitative detection results showed that LPS was negative in both 10 μg and 50 μg EfeB and rHemH. Figure 1 (H in the text).

[0196] Example 2: EfeB and HemH promote cell survival

[0197] (1) The human macrophage cell line (ATCC, TIB-202) (THP-1) (1×10 7The cells were placed in 96-well plates with phorbol ester PMA (100 ng / ml) to induce THP-1 mononuclear cells to become macrophages;

[0198] (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℃ for 24 h. The control group was not treated.

[0199] (3) Cell viability was detected using CCK8 (Shanghai Beyotime Biotechnology Co., Ltd., C0037) and MTT kit (Shanghai Beyotime Biotechnology Co., Ltd., C0009S), respectively.

[0200] (4) After adding CCK8 reagent for 2 h, the absorbance of the cell suspension at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader; after adding MTT reagent for 4 h, the absorbance of the cell suspension at 570 nm was measured using an ELISA reader.

[0201] (5) Analyze and compare the changes in relative cell viability.

[0202] The experimental results showed that rEfeB ​​and rHemH had no significant effect on the viability of THP-1 cells, 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 the middle).

[0203] Experiment 3: EfeB and HemH promote GPX4 expression in macrophages

[0204] (1) Sample preparation

[0205] 1) Cell preparation

[0206] 1. The human macrophage cell line (THP-1) (1×10⁻⁶) was used. 7 The cells were placed in 96-well plates with phorbol ester PMA (100 ng / ml) to induce THP-1 mononuclear cells to become macrophages;

[0207] 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, the cells were cultured at 37℃ for 24 h. The control group was not treated.

[0208] 2) Cell lysis

[0209] 1. After removing the supernatant from the cultured cells, wash them three times with PBS;

[0210] 2. Add 1×SDS-PAGE protein loading buffer, lyse thoroughly, and then boil the sample for 15 min.

[0211] (2) SDS-PAGE gel electrophoresis

[0212] 1) Preparation of PAGE adhesive

[0213] 1. Preparation of separating gel

[0214] 1.1 After stacking the two glass plates neatly, clamp them vertically onto the base and tighten the clamps in preparation for applying adhesive;

[0215] 1.2 Prepare 10 ml of 12% separating gel: 3.3 ml deionized water, 4 ml 30% acrylamide-bisacrylamide, 2.5 ml pH 8.8 Tris-HCl, 0.1 ml 10% (w / v) SDS, 0.1 ml 10% (w / v) ammonium persulfate, and 4 μl TEMED. Pour the separating gel and seal with deionized water.

[0216] 1.3 Let stand for 120 min, pour off the water on the top of the gel, and use the edge of filter paper to absorb the remaining liquid.

[0217] 2. Preparation of Concentrated Gel

[0218] Prepare 3 ml of 5% stacking gel: 2.1 ml deionized water, 0.5 ml 30% acrylamide-bisacrylamide, 0.38 ml pH 6.8 Tris-HCl, 30 μl 10% (w / v) SDS, 30 μl 10% (w / v) ammonium persulfate, and 3 μl TEMED. Pour the stacking gel onto the polymerized separating gel and immediately insert a comb into the stacking gel solution. Let it stand at room temperature for approximately 90 min to solidify. After the stacking gel has completely polymerized, remove the seal and wipe away excess liquid from the bottom and edges of the electrophoresis plate with filter paper.

[0219] 2) Sample loading and electrophoresis

[0220] 1. Place the electrophoresis plate into the electrophoresis tank; add an appropriate amount of 1×Tris-glycine electrophoresis buffer to both the inside and outside of the electrophoresis plate, and carefully remove the comb;

[0221] 2. Boil the prepared protein sample in a water bath for 5 min, and load 25 μl of the sample into each well;

[0222] 3. Turn on the power and perform 80 V constant voltage electrophoresis. When the leading edge of the bromophenol blue indicator enters the separating gel, adjust the voltage to 120 V and continue constant voltage electrophoresis until the bromophenol blue indicator reaches the bottom of the gel. Then turn off the power.

[0223] (3) Protein transfer

[0224] 1. Prepare 1× transfer buffer in advance, and soak the filter paper and sponge in the transfer buffer;

[0225] 2. Soak the PVDF membrane in methanol for 3 min, then soak the PVDF membrane in transfer buffer for 5 min;

[0226] 3. Remove the gel after electrophoresis, gently pry open the glass plate, and immerse the gel in transfer buffer;

[0227] 4. Prepare the "sandwich" in the following order: sponge, filter paper, PVDF membrane, gel, filter paper, sponge, ensuring there are no air bubbles between each layer; place the prepared "sandwich" into the transfer tank and add an appropriate amount of 1× transfer buffer.

[0228] 5. Turn on the power and transfer the membrane at 330 mA for 25 minutes.

[0229] (4) Membrane blocking and antibody incubation

[0230] 1. After the membrane transfer is complete, place the PVDF membrane in a small box, add a 5% skim milk powder blocking solution, and seal on a shaker at room temperature for 1 hour;

[0231] 2. Discard the blocking solution, wash three times with TBST for 5 min each time; add GPX4-IgG antibody dilution buffer and incubate overnight at 4°C on a shaker;

[0232] 3. Recover the GPX4-IgG antibody dilution buffer, wash three times with TBST for 5 min each time;

[0233] 4. Add fluorescently labeled anti-rabbit Ig-G and incubate on a shaker at room temperature for 1 h;

[0234] 5. Recover IgG, wash with TBST 4 times, 5 min each time;

[0235] 6. Finally, discard the TBST and use an imager to scan the membrane for imaging.

[0236] Experimental results showed that rEfeB ​​and rHemH promoted the expression level of GPX4 in THP-1 cells. Figure 2 (CD in the middle).

[0237] Experiment Example 4: EfeB and HemH inhibit ROS levels in macrophages

[0238] (1) Cell preparation

[0239] 1) The human macrophage cell line (THP-1) (1×10 7 The cells were placed in 96-well plates with phorbol ester PMA (100 ng / ml) to induce THP-1 mononuclear cells to become macrophages;

[0240] 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, the cells were cultured at 37℃ for 24 h. The control group was not treated.

[0241] 3) Collect the above cells by centrifugation at 800 ×g for 10 min, and wash them 3 times with serum-free culture medium.

[0242] (2) Flow cytometry detection of ROS levels in cells

[0243] 1) Resuspend cells in 10 μM DCFH-DA fluorescent probe, incubate at 37℃ for 20 min, and shake to mix once every 5 min;

[0244] 2) Collect the above cells by centrifugation at 800 ×g for 10 min, and wash them 3 times with serum-free cell culture medium;

[0245] 3) Flow cytometry was used to detect the ROS level in cells.

[0246] (3) Detection of cell ROS levels by fluorescence microscopy

[0247] 1) Collect the treated cells and wash them three times with serum-free cell culture medium;

[0248] 2) Load DCFH-DA (10 μM) fluorescent probe in situ and incubate at 37℃ for 20 min;

[0249] 3) After washing the cells three times with serum-free cell culture medium, observe the ROS level of the cells under a fluorescence microscope.

[0250] Experimental results showed that EfeB and HemH inhibited ROS levels in THP-1 cells ( Figure 3 ).

[0251] Experimental Example 5: EfeB and HemH reduce intracellular Fe in macrophages 2+ level

[0252] (1) Cell preparation

[0253] 1) The human macrophage cell line (THP-1) (1×10 7The cells were placed in 96-well plates with phorbol ester PMA (100 ng / ml) to induce THP-1 mononuclear cells to become macrophages;

[0254] 2) The cells were divided into control group, Fe(NH4)2(SO4)2 (100 μM), rEfeB ​​(2 μg / ml), and rHemH (2 μg / ml). After adding the reagents, the cells were cultured at 37℃ for 24 h. The control group was not treated.

[0255] (2) Detection of Fe in cells using fluorescence microscopy 2+ level

[0256] 1) Collect the treated cells and wash them three times with HBSS;

[0257] 2) Load FerroOrange (1 μM) fluorescent probe (ferrous ion fluorescent probe) in situ and incubate at 37°C for 30 min;

[0258] 3) After washing the cells three times with HBSS, observe them under a fluorescence microscope.

[0259] Experimental results showed that EfeB and HemH inhibited Fe in THP-1 cells. 2+ level( Figure 4 (AB in the middle).

[0260] Experiment Example 6: EfeB and HemH inhibit intracellular lipid peroxidation levels

[0261] (1) Cell preparation

[0262] 1) The human macrophage cell line (THP-1) (1×10 7 The cells were placed in 96-well plates with phorbol ester PMA (100 ng / ml) to induce THP-1 mononuclear cells to become macrophages;

[0263] 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, the cells were cultured at 37℃ for 24 h. The control group was not treated.

[0264] (2) Detection of lipid peroxide levels in cells using fluorescence microscopy

[0265] 1) Collect the processed cells and wash them three times with serum-free culture medium;

[0266] 2) Load BDP 581 / 591 C11 (10 μM) (lipid peroxidation fluorescent probe) in situ and incubate at 37°C for 30 min;

[0267] 3) After washing the cells three times with serum-free culture medium, observe them under a fluorescence microscope.

[0268] Experimental results showed that rEfeB ​​and rHemH inhibited lipid peroxidation levels in THP-1 cells ( Figure 4 (C in the middle).

[0269] Experiment Example 7: EfeB and HemH reduce renal and hepatic inflammation levels in LPS-induced sepsis

[0270] (1) Establishment of a mouse sepsis model and the antiseptic effects of rEfeB ​​and rHemH

[0271] 1) Thirty C57BL / 6 mice weighing 18-22 g were divided into control group (PBS buffer injection), LPS, rEfeB, rHemH, LPS + rEfeB, and LPS + rHemH injection groups.

[0272] 2) rEfeB, rHemH (4 mg / ml) or PBS were administered via tail vein injection for 7 consecutive days, followed by intraperitoneal injection of LPS (10 mg / kg) after the last injection.

[0273] 3) Mice were euthanized by cervical dislocation, and the kidneys and livers were removed and stained with hematoxylin and eosin (HE) to observe tissue lesions.

[0274] 3.1) Kidney tissue was fixed with 4% paraformaldehyde for 24 h;

[0275] 3.2) Dehydrate the fixed tissue sequentially with gradient alcohols (70%, 80%, 95%, 100%) for 1-2 hours.

[0276] 3.3) Clear the tissue in xylene; then treat with paraffin for 2-3 hours;

[0277] 3.4) Embedding and sectioning: After embedding the tissue in the embedding tank with paraffin solution, the tissue is trimmed into a paraffin block;

[0278] 3.5) Sectioning: The microtome trims the paraffin block to a thickness of 5~7 μm;

[0279] 3.6) Attaching slides: Place the slides in the slide box, flatten them, and attach the slides to 2 / 3 of the slide. Let them air dry slightly in the air.

[0280] 3.7) Dewaxing and hydration: The sections were treated sequentially with xylene, a gradient of alcohols (100%, 95%, 90%, 80%, 70%), and distilled water;

[0281] 3.8) Staining: The nuclei and cytoplasm of the tissue cells were stained with hematoxylin and eosin, respectively;

[0282] 3.9) Dehydration and mounting: After treating the sections with a gradient of alcohols (70%, 80%, 95%, 100%), xylene, and resin, cover with a coverslip;

[0283] 3.10) Observe the histopathological changes under a microscope.

[0284] Experimental results showed that rEfeB ​​and rHemH significantly reduced the levels of renal and hepatic inflammation in LPS-induced sepsis. Figure 5 ).

Claims

1. The use of ferroptosis inhibitors or polynucleotides in the preparation of medicaments for the treatment of sepsis, wherein the ferroptosis inhibitor is a protein consisting of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2; and the polynucleotide encodes the protein, the sequence of which is shown in SEQ ID No. 3 or SEQ ID No.

4.

2. The application as described in claim 1, characterized in that, When applied, the drug can inhibit sepsis-induced ferroptosis, reduce inflammatory response, and / or reduce organ damage.

3. The application as described in claim 2, characterized in that, The organ in question is either the kidney or the liver.

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