Application of di-Pal-MTO as a neutrophil extracellular trap inhibitor in the preparation of drugs for the treatment of sepsis

Abstract

The present invention relates to the application of di-Pal-MTO as a neutrophil extracellular capture network inhibitor in the preparation of a drug for treating sepsis, and belongs to the field of biomedicine technology. The present invention provides the application of di-Pal-MTO in the preparation of NETs inhibitors. The NETs inhibitor di-Pal-MTO inhibits the function of neutrophil extracellular capture network NETs, ​​blocks the action of NETs and downstream cells, and effectively inhibits the macrophage inflammatory factor storm caused by NETs in sepsis, thereby significantly reducing the level of inflammation, reducing organ damage and improving survival time, thereby achieving the effect of preventing and treating sepsis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of di-Pal-MTO as a neutrophil extracellular trap inhibitor in the preparation of a drug for treating sepsis. Background Art

[0002] Every year, there are 31.5 million new cases of sepsis and 19.4 million cases of severe sepsis worldwide, with 5.3 million deaths and a hospital mortality rate of 30-50%. The onset of sepsis is accompanied by the recognition and activation of danger signals by innate immunity, which triggers dysregulated systemic inflammation and is the main cause of multi-organ damage and death. Therefore, inhibiting the generation of inflammatory storms is the key to treating sepsis and improving patient survival.

[0003] Neutrophils play an important role in the pathophysiological process of sepsis. They produce a special extracellular structure, namely neutrophil extracellular traps (NETs), which have outstanding characteristics and functions in the progression of sepsis. NETs are DNA mesh structures that are released into the extracellular space by chromatin depolymerization of neutrophils under the stimulation of pathogens, endotoxins, platelets and other factors. Various proteins and enzymes are anchored in them. On the one hand, NETs capture, inhibit and eliminate pathogens, playing an important defensive role in innate immunity. On the other hand, NETs act on immune cells such as macrophages and dendritic cells, promoting the production of cytokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), thereby further amplifying the inflammatory response. In addition, NETs also lead to coagulation disorders, endothelial and tissue damage in sepsis. It can be seen that targeting NETs to treat sepsis will achieve the effect of reducing inflammatory damage.

[0004] Treatment of sepsis mainly relies on comprehensive treatment methods such as infection control, correction of hemodynamic disorders and symptomatic supportive therapy, which can only stabilize the patient's vital signs and alleviate the progression of this acute course to a certain extent. How to further control the deterioration of the disease and effectively improve the prognosis and survival rate is a difficult problem to solve at this stage. The reason is that most conventional treatment plans are aimed at abnormal signs that have appeared and are difficult to correct. They are still symptomatic supportive treatments, lack methods to target the pathogenesis, and cannot curb the progression of the disease early. Therefore, there is an urgent need for drugs that specifically target NETs to effectively inhibit the occurrence and development of diseases such as sepsis. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a drug di-Pal-MTO that specifically targets NETs, ​​and to provide the use of di-Pal-MTO as a neutrophil extracellular trap inhibitor in the preparation of a drug for treating sepsis.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides the use of di-Pal-MTO in the preparation of a neutrophil extracellular trap inhibitor. The CAS number of di-Pal-MTO is 1349197-90-5, and it is a palm oil-based lipid generated by combining mitoxantrone (MTO) with palmitoleic acid.

[0008] The present invention discovers that di-Pal-MTO inhibits the function of neutrophil extracellular capture nets NETs, ​​blocks the action of NETs and downstream cells, and thus treats related diseases with NETs as therapeutic targets.

[0009] In a second aspect, the present invention provides the use of di-Pal-MTO in the preparation of a drug for preventing and / or treating diseases caused by neutrophil extracellular traps.

[0010] Furthermore, the drug is a drug for preventing and / or treating sepsis.

[0011] Furthermore, the drug is a drug for preventing and / or treating inflammatory cytokine storm.

[0012] Furthermore, the drug is a drug that inhibits immune cells from producing inflammatory factors.

[0013] In a specific embodiment of the present invention, the immune cells are macrophages.

[0014] In a specific embodiment of the present invention, the inflammatory factor is at least one of IL-6, IL-1β and TNF-α.

[0015] Furthermore, the drug is a drug for preventing and / or treating organ damage.

[0016] In a specific embodiment of the present invention, the organ is at least one of the lung, liver and kidney.

[0017] Furthermore, the drug is a neutrophil extracellular trap inhibitor.

[0018] Furthermore, the medicine also contains pharmaceutically acceptable additives and / or excipients and / or carriers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a neutrophil extracellular trapping network NETs inhibitor di-Pal-MTO, which inhibits the function of neutrophil extracellular trapping network NETs, ​​blocks the action of NETs and downstream cells, and effectively inhibits the macrophage inflammatory factor storm caused by NETs in sepsis, thereby significantly reducing the level of inflammation, reducing organ damage and improving survival time, thereby achieving the effect of preventing and treating sepsis. The present invention provides a new solution for preventing and treating sepsis, and also lays a foundation for the clinical application of di-Pal-MTO. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of inflammatory factors in macrophages induced by LPS and NETs was inhibited by di-Pal-MTO. A is IL-6; B is IL-1β; C is TNF-α. The number of repetitions n=3 biological independent experiments, the data are expressed as mean standard deviation, and the variance analysis was performed by One-way ANOVA, LSD-t test, *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 2 The formation of NETs in the lung, liver and kidney tissues of mice in the control group and the sepsis group. MPO is represented in red, H3Cit is represented in green, and nuclear DAPI is represented in blue. Scale bar: 20 μm, and the number of mice in each group is n=5.

[0023] Figure 3 The effect of di-Pal-MTO on the survival time of mice with sepsis. The number of mice in the control group was n=7, and the number of mice in the di-Pal-MTO group was n=7. Kaplan-meier survival curve analysis and Log-rank test were used, *P<0.05.

[0024] Figure 4 The effect of di-Pal-MTO on inflammatory indicators in septic mice. A is IL-6; B is IL-1β; C is TNF-α. The number of mice in each group is n=5. Data are expressed as mean standard deviation, and independent sample two-sided t test was used, *P<0.05, **P<0.01.

[0025] Figure 5 Representative HE-stained sections showing the effect of di-Pal-MTO on histological damage in septic mice. Scale bar: 40 μm.

[0026] Figure 6HE staining scores of the effects of di-Pal-MTO on histological damage in septic mice. A is the lung; B is the liver; C is the kidney. The number of mice in each group is n=5, and 3 to 5 fields of view were randomly selected for observation and scoring for each slice, and the average value was taken. The data are expressed as mean standard deviation, and the independent sample two-sided t test was used, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0027] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0028] Example 1 Inducing macrophages from human peripheral blood and extracting neutrophil extracellular traps (NETs)

[0029] 1. Inducing macrophages from human peripheral blood

[0030] (1) Pour 15 mL of human peripheral blood lymphocyte separation solution Ficoll (Cat. No.: LTS1077, Brand: TBD) into a 50 mL centrifuge tube, slightly dilute healthy human peripheral blood with saline (2 to 3 times) to obtain diluted human peripheral blood, and slowly add the diluted human peripheral blood dropwise into Ficoll to form layers.

[0031] (2) The human peripheral blood treated in step (1) was subjected to density gradient centrifugation at 800 g for 20 min, with the ascending speed adjusted to 5 and the descending speed adjusted to 0; after centrifugation, the liquid in the tube was separated into a plasma layer, a buffy coat layer, a Ficoll layer, and a mixed layer of red blood cells and granulocytes.

[0032] (3) Carefully aspirate the buffy coat layer (mainly containing mononuclear cells of peripheral blood) in step (2) using a Pasteur tube, add physiological saline and wash three times to obtain a washed buffy coat layer; centrifuge the washed buffy coat layer at 400 g for 5 min, adjust the speed of increase to 10, and adjust the speed of decrease to 10, and retain the cell pellet.

[0033] (4) The cell pellet from step (3) was resuspended in serum-free Dulbecco's Modified Eagle Medium (DMEM), inoculated into two 24-well plates, and cultured in a 37° C., 5% CO 2 incubator for 20 min to promote cell adhesion.

[0034] (5) After the incubation in step (4) was completed, the 24-well plate was taken out, the supernatant was aspirated, the plate was washed twice with saline, and DMEM complete medium containing 10% (v / v) fetal bovine serum (FBS), 2% (v / v) penicillin / streptomycin and 10 ng / mL macrophage colony stimulating factor (M-CSF) (Cat. No.: 300-25, Brand: Peprotech) was added and cultured in a 37°C, 5% CO2 incubator.

[0035] (6) Step (5): The culture medium was changed on the third and fifth days of culture, and macrophages derived from human peripheral blood were induced after one week.

[0036] 2. Extraction of human NETs

[0037] (1) The same as steps (1) and (2) of inducing macrophages derived from human peripheral blood in this example.

[0038] (2) Aspirate the buffy coat layer from step (1), remove all the supernatant above the red blood cell layer, add 3 times the volume of red blood cell lysis buffer (Cat. No.: P004003, Brand: Eskin Biotech), and lyse on ice for 25 min, shaking thoroughly during the process.

[0039] (3) The red blood cell layer treated in step (2) was centrifuged at 400 g for 5 min, with the speed increased to 10 and the speed decreased to 10, and the supernatant was removed to obtain the granulocyte precipitate.

[0040] (4) Add 5 mL of red blood cell lysis solution to the granulocyte precipitate in step (3), and further lyse the granulocyte precipitate at room temperature (25°C) for 5 min to obtain a lysis product; centrifuge the lysis product at 400g for 5 min, adjust the speed of increase to 10, adjust the speed of decrease to 10, remove the supernatant, and obtain a neutrophil precipitate.

[0041] (5) The neutrophils obtained in step (4) were resuspended in 7 mL of complete culture medium, inoculated into a 10 cm culture dish, and phorbol ester (name: Phorbol-12-myristate-13-acetate, PMA, catalog number: S7791, brand: Selleck) with a final concentration of 500 nM was added, and cultured in an incubator at 37° C., 5% CO 2 for 6 to 8 h.

[0042] (6) After the cell culture supernatant in step (5) is removed, the viscous NETs component can be seen at the bottom of the dish. 1 mL of pre-cooled phosphate buffered saline (PBS) is added to the NETs and the mixture is mixed by pipetting repeatedly. The mixture is transferred to a centrifuge tube and centrifuged at 4°C and 1000 g for 10 min. The speed is adjusted to 10 and the speed is adjusted to 10. The supernatant is collected and the DNA concentration of the NETs is measured on an ultra-micro spectrophotometer (NanoDrop One, Thermo Fisher Scientific) and stored in a -80°C refrigerator for later use.

[0043] Example 2 Detection of the effect of di-Pal-MTO on the production of inflammatory factors in macrophages by quantitative real-time PCR (qPCR)

[0044] 1. Experimental Methods

[0045] 1. Resuspend the macrophages isolated from human peripheral blood in Example 1 with DMEM complete medium to a cell density of 1×10 6 / mL, macrophage fluid from human peripheral blood was obtained and inoculated into 24-well plates.

[0046] 2. Add di-Pal-MTO (final concentration 0.1 nM, volume ratio of di-Pal-MTO to macrophage fluid from human peripheral blood is 1:1000) to the macrophages derived from human peripheral blood in step 1, incubate for more than 1 hour, add the same volume of dimethyl sulfoxide (DMSO) to the control (Vehicle) group, and add the same volume of physiological saline to the Con group; then add lipopolysaccharide (LPS) (final concentration 100 ng / mL, volume ratio of LPS to macrophage fluid from human peripheral blood is 1:1000) and NETs extracted in Example 1 (final concentration 10 μg / mL, volume ratio of NETs to macrophage fluid from human peripheral blood is 1:50) to each group, and culture in an incubator at 37°C, 5% CO2 for 6 to 8 hours.

[0047] 3. Extract the total RNA of the cells cultured in step 2, perform reverse transcription and real-time fluorescence quantitative PCR analysis, and detect the expression of various inflammatory factors.

[0048] (1) Using an RNA rapid extraction kit (Cat. No.: RN001, Brand: YiShan Biotech), according to the instructions of the kit, lysis buffer was added to the cells cultured in step 2, and then anhydrous ethanol was added. Total cellular RNA was extracted by column centrifugation, and the RNA concentration was measured on an ultra-micro spectrophotometer (NanoDrop One, Thermo Fisher).

[0049] (2) Prepare 10 μL RNA reverse transcription system (Cat. No.: RR036A, Brand: TaKaRa) according to the instructions. For each sample, mix 500 ng of RNA from step (2) with 2 μL reverse transcription system. Perform reverse transcription at 37°C for 15 min. Perform reverse transcriptase inactivation at 85°C for 5 s to obtain cell reverse transcribed cDNA, which was then stored at 4°C.

[0050] (3) The total system per well is 10 μL. Add 5 μL of TB Green Premix Ex Taq II (Cat. No.: RR820A, Brand: TaKaRa), 1 μL of the cell reverse transcribed cDNA from step (2), 3.2 μL of water, 0.4 μL of the internal reference gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) and the upstream and downstream primers of each target gene, with the final concentration of each primer being 0.4 μM, to a 384-well qPCR plate. Make three replicate wells for each gene in each sample to obtain the total reaction system.

[0051] (4) Using a real-time fluorescence quantitative PCR instrument (model LC480, brand: Roche) to detect the total reaction system of step (3), after pre-denaturation, deformation, annealing and extension, the fluorescence intensity of the amplified DNA is detected to obtain the Ct value.

[0052] The PCR reaction conditions are shown in Table 1.

[0053] Table 1

[0054]

[0055] (5) The Ct values ​​of each group in step (4) were normalized, and the target gene value minus the GAPDH value was compared with the control group. -ΔΔCt The relative multiple of each group was calculated (the control group was 1).

[0056] The primer sequences used are:

[0057] Human IL6_F: 5'-ACTCACCTCTTCAGAACGAATTG-3'

[0058] Human IL6_R: 5'-CCATCTTTGGAAGGTTCAGGTTG-3'

[0059] Human IL1B_F: 5'-ATGATGGCTTATTACAGTGGCAA-3'

[0060] Human IL1B_R: 5'-GTCGGAGATTCGTAGCTGGA-3'

[0061] Human TNF_F: 5'-GAGGCCAAGCCCTGGTATG-3'

[0062] Human TNF_R: 5'-CGGGCCGATTGATCTCAGC-3'

[0063] Human GAPDH_F: 5'-ACAACTTTGGTATCGTGGAAGG-3'

[0064] Human GAPDH_R: 5'-GCCATCACGCCACAGTTTC-3'

[0065] 2. Experimental Results

[0066] like Figure 1 A. Figure 1 B and Figure 1 As shown in C, after treatment with di-Pal-MTO, the levels of IL-6, IL-1β, and TNF-α produced by macrophages stimulated by LPS and NETs were reduced.

[0067] Example 3 The therapeutic effect of di-Pal-MTO on sepsis in mice

[0068] 1. Experimental Methods

[0069] 1. C57BL / 6 mice aged 6 to 8 weeks and weighing 18 to 20 g were selected and intraperitoneally injected with di-Pal-MTO (5 mg / kg) one day in advance. The control group (Vehicle) was injected with the same volume of drug solvent (DMSO).

[0070] 2. Perform cecal ligation and puncture (CLP) on the mice treated in step 1 to establish a sepsis mouse model, and the control group undergoes sham surgery.

[0071] The method of cecal ligation and puncture is as follows:

[0072] (1) Mice were anesthetized by inhalation of 2% (v / v) isoflurane to obtain anesthetized mice; the anesthetized mice were fixed in a supine position, the lower abdomen was shaved, disinfected, and a midline incision was made in the lower abdomen.

[0073] (2) For mice treated in step (1), the cecum was located, separated and externalized, the intestinal contents were squeezed as far as possible toward the blind end, and the cecum was ligated below the ileocecal valve (the range from the ligation site to the blind end of the cecum accounted for 75% of the entire cecum). In the middle of the ligated intestinal section, a 21-gauge needle was used to penetrate the intestinal wall and squeeze out a small amount of intestinal contents to ensure that the perforation was unobstructed. During the entire process, care was taken not to damage the mesenteric blood vessels.

[0074] (3) After step (2) is completed, the cecum is returned, the wound is cleaned with a saline cotton ball, and the mouse peritoneum, muscularis, and skin are sutured layer by layer with 4-0 absorbable sutures.

[0075] (4) After the operation in step (3), 1 mL of warm saline was subcutaneously injected into the mouse for fluid resuscitation.

[0076] 3. Step 2: Administer the drug once 6 hours after surgery according to the method in step 1, and then administer it by intraperitoneal injection once every other day.

[0077] 4. After the completion of step 3, the state of the mice was closely observed for 7 days, during which the survival time of each mouse was recorded and analyzed; 24 hours after surgery, blood was collected from the orbits to collect serum for detection of inflammatory factors by enzyme-linked immunosorbent assay (ELISA); lung, liver and kidney tissues were collected for hematoxylin-eosin staining (HE) to evaluate the damage, and immunofluorescence staining was performed to observe the formation of NETs in various organs of the sepsis mouse model. When the mice showed obvious weight loss, decreased activity, respiratory depression, decreased body temperature and weakened heartbeat, and no obvious response to external stimuli, it was considered to have reached the humane endpoint and euthanasia was performed.

[0078] (1) Collecting serum

[0079] ① Collect the mouse orbital blood in an EP tube and place it in a 4°C refrigerator overnight (12-16 hours) to allow the blood to coagulate and precipitate the serum.

[0080] ② Centrifuge the precipitated serum in step ① at 4°C, 2000g for 20 min, adjust the ascending speed to 10, and adjust the descending speed to 10, collect the light yellow supernatant as serum, and store it in a -80°C refrigerator for later use.

[0081] (2) ELISA to detect the levels of IL-6, IL-1β and TNF-α inflammatory factors in serum

[0082] Use commercial IL-6 (Cat. No.: E-MSEL-M0001, Brand: Elabscience), IL-1β (Cat. No.: E-MSEL-M0003, Brand: Elabscience) and TNF-α (Cat. No.: E-MSEL-M0002, Brand: Elabscience) ELISA detection kits according to the manufacturer's instructions.

[0083] (3) HE staining to observe and evaluate the damage to various organs in sepsis

[0084] ① Take the liver, lung, kidney and other organs of mice, fix them in 4% (v / v) paraformaldehyde to obtain fixed organ tissues.

[0085] ② Embed the organ tissue fixed in step ① in paraffin and slice it to obtain paraffin sections.

[0086] ③ Take the paraffin sections from step ② and bake them at 65℃ for 1h to obtain baked sections. Dewax the baked sections in xylene I and II for 2 times, 10min each time; then dewax them in anhydrous ethanol for 2 times, 8min each time; hydrate them with 95% (v / v), 85% (v / v), 75% (v / v) and 55% (v / v) graded alcohol and rehydrate them with distilled water for 2 times to obtain rehydrated sections; stain the rehydrated sections with hematoxylin stain for 3-5min, rinse with tap water for 10s to obtain hematoxylin stain stained sections.

[0087] ④ Use 1% (v / v) hydrochloric acid ethanol differentiation solution to differentiate the sections stained with hematoxylin solution in step ③ for 1-5 seconds, rinse with tap water for 10-20 seconds, wash away the differentiation solution, and obtain sections treated with the differentiation solution.

[0088] ⑤ Use bluing solution to bluing the slices treated with differentiation solution in step ④ for 10 to 30 seconds, rinse with tap water for 10 to 20 seconds, wash away the bluing solution, and obtain slices treated with bluing solution.

[0089] ⑥ Place the slices treated with the bluing solution in step ⑤ in eosin staining solution for 30 seconds to 2 minutes, rinse with tap water for 2 to 3 seconds, and obtain eosin staining solution slices.

[0090] ⑦ Dehydrate the sections stained with eosin solution in step ⑥ against an alcohol gradient with alcohol concentrations of 55% (v / v), 75% (v / v), 85% (v / v) and 95% (v / v) to obtain transparent dehydrated sections, seal the dehydrated sections with neutral gum, obtain HE-stained sections, and observe under a microscope.

[0091] ⑧For each HE-stained section of mouse tissue, 3 to 5 fields were randomly selected for scoring to quantify the degree of organ damage. Two researchers were blinded to evaluate each section without knowing the grouping and treatment.

[0092] Lung injury assessment was scored according to the following criteria:

[0093] 1 point, normal tissue;

[0094] 2 points, focal (<50% of the visual field) interstitial congestion and inflammatory cell infiltration;

[0095] 3 points, diffuse (>50% of the visual field) interstitial congestion and inflammatory cell infiltration;

[0096] 4 points, focal (<50% of the visual field) consolidation and inflammatory cell infiltration;

[0097] 5 points, diffuse (>50% of the visual field) consolidation and inflammatory cell infiltration.

[0098] Liver injury assessment was scored according to the following criteria:

[0099] Fatty degeneration: <25% = 1 point; <50% = 2 points; <75% = 3 points; >75% = 4 points;

[0100] Inflammation and necrosis: 1 lesion = 1 point; 2 or more lesions = 2 points.

[0101] Renal injury assessment was scored according to the following criteria:

[0102] The renal tubules were observed for tubular dilatation, protein cast formation, and loss of the brush border;

[0103] 0 points, normal;

[0104] 1 point, injury involving <25% of the renal tubules;

[0105] 2 points, damage involving 25-50% of the renal tubules;

[0106] 3 points, damage involving 50-75% of the renal tubules;

[0107] 4 points: damage involves the renal tubules 75% to 100% of the alcohol.

[0108] (4) Immunofluorescence staining to observe the formation of NETs in various organs of sepsis mouse model

[0109] ① Take the liver, lung and kidney organs of the mouse and obtain the rehydrated sections according to the methods ① to ③ in step 4 (3) of HE staining.

[0110] ② Place the rehydrated slices in step ① in ethylenediaminetetraacetic acid (EDTA) antigen retrieval solution (manufacturer: Zhongshan Jinqiao) at pH 8.0 and repair them in an electric rice cooker under high pressure for 4 to 5 minutes, and wait for cooling to room temperature (25°C) to obtain repaired slices; block the repaired slices with 5% (v / v) bovine serum albumin (BSA) at room temperature for 20 minutes to obtain blocked slices; dilute rabbit-H3Cit (rabbit citrullinated histone) (the volume ratio of rabbit-H3Cit to 1% (v / v) BSA is 1:100, catalog number: ab5103, brand: Abcam) and goat-MPO (goat myeloperoxidase) (the volume ratio of goat-MPO to 1% (v / v) BSA is 1:75, catalog number: AF3667, brand: R&D) with 1% (v / v) BSA as primary antibodies, incubate the blocked slices overnight (12 to 16 hours) at 4°C to obtain primary antibody incubated slices.

[0111] ③Take out the sections incubated with the primary antibody in step ②, wash them three times with 1× phosphate-saline-Tween buffer (PBST), 5 min each time, to obtain the washed sections incubated with the primary antibody; dilute Alexa Fluor TM 488-labeled donkey anti-rabbit IgG antibody conjugate (Alexa Fluor TM 488 labeled donkey anti-rabbit IgG antibody conjugate and 1% (v / v) BSA volume ratio of 1:200, catalog number: A21206, brand: Thermo Fisher) and 1% (v / v) BSA diluted Alexa Fluor TM 555-labeled donkey anti-goat IgG antibody conjugate (Alexa Fluor TM 555-labeled donkey anti-goat IgG antibody conjugate and 1% (v / v) BSA (volume ratio of 1:200, catalog number: A21432, brand: Thermo Fisher) were used as secondary antibody. After incubation and washing at room temperature (25°C), the sections were incubated with the primary antibody for 1 to 2 hours to obtain the sections incubated with the secondary antibody. The sections incubated with the secondary antibody were washed 3 times with 1×PBST, each time for 5 minutes to obtain the sections incubated with the secondary antibody after washing.

[0112] ④ After washing in step ③, drop 10 μg / mL of 4',6-diamidino-2-phenylindole (DAPI) working solution (brand: Eskin Bio) on the sections incubated with secondary antibodies to stain the cell nuclei, incubate at room temperature (25°C) for 15 minutes to obtain DAPI-stained sections.

[0113] ⑤ Wash with 1×PBST. ④ DAPI stain the sections 3 times, 5 min each time, to obtain DAPI stained sections after washing; seal the DAPI stained sections after washing with anti-fluorescence quenching sealing agent, and observe the production of NETs under a laser confocal microscope (Cat. No.: LSM800, Brand: Carl Zeiss) at 63 times.

[0114] 2. Experimental Results

[0115] like Figure 2 As shown, the expression levels of MPO and H3Cit, representative indicators of NETs in septic mice, were significantly increased in the lungs, liver, and kidneys of mice.

[0116] like Figure 3 As shown, di-Pal-MTO prolonged the survival time of septic mice.

[0117] like Figure 4 A. Figure 4 B and Figure 4 As shown in C, di-Pal-MTO reduced inflammatory markers (IL-6, IL-1β, and TNF-α) in septic mice.

[0118] like Figure 5 , Figure 6 A. Figure 6 B and Figure 6 As shown in C, di-Pal-MTO alleviated histological damage in the lung, liver, and kidney of septic mice.

[0119] It can be seen that di-Pal-MTO inhibits NETs and downstream cell effects, and plays a good therapeutic role in sepsis caused by NETs.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of di-Pal-MTO in the preparation of a drug for preventing and / or treating sepsis caused by neutrophil extracellular traps, wherein the CAS number of the di-Pal-MTO is 1349197-90-5.

2. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and / or treating inflammatory cytokine storm.

3. The use according to claim 2, characterized in that: The drug is a drug that inhibits immune cells from producing inflammatory factors.

4. The use according to claim 3, characterized in that: The immune cells are macrophages.

5. The use according to claim 3, characterized in that: The inflammatory factor is at least one of IL-6, IL-1β and TNF-α.

6. The use according to claim 2, characterized in that: The medicine is a medicine for preventing and / or treating organ damage.

7. The use according to claim 6, characterized in that: The organ is at least one of a lung, a liver and a kidney.

8. The use according to any one of claims 1 to 7, characterized in that: The drug is a neutrophil extracellular trap inhibitor.

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