Preparation method and application of exosome-like nano vesicle

Abstract

The invention relates to a preparation method of exosome-like nano vesicles. The preparation method comprises the following steps: extracting beet juice; performing low-speed centrifugal treatment on the beet juice, and taking supernate; carrying out medium-speed centrifugation treatment on the supernatant obtained by low-speed centrifugation, and taking the supernatant; carrying out high-speed centrifugation treatment on the supernatant obtained by medium-speed centrifugation to obtain a precipitate containing exosome-like nano vesicles; and carrying out sucrose density gradient ultracentrifugation on the obtained precipitate containing the exosome-like nano-vesicles to obtain the purified exosome-like nano-vesicles. The BELNV prepared by the invention can significantly improve the survival rate of sepsis mice, increase blood perfusion of tissues and organs of the sepsis mice, reduce pathological changes, macrophage infiltration and fibrin deposition in lung and liver tissues of the sepsis mice, and reduce blood coagulation indexes and inflammation indexes. The invention provides a novel treatment target and strategy for a treatment means of sepsis-related blood coagulation dysfunction.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for preparing exosome-like nanovesicles and applications thereof, and specifically to a method for preparing exosome-like nanovesicles derived from beets and applications thereof. Background Art

[0002] Exosomes (EVs), double-layer membrane vesicles with a diameter of approximately 40-160 nm, are important vehicles for intercellular communication. They participate in physiological and pathological regulation by carrying bioactive molecules such as proteins, nucleic acids, and lipids. Plant-derived extracellular vesicles (such as plant exosome-like nanoparticles (ELNVs)) share a high degree of similarity with mammalian exosomes, including a double-layer membrane structure, negative surface charge, and a complex active composition (spherical, ellipsoidal, or cup-shaped morphology and negative surface charge, along with a complex array of naturally occurring bioactive compounds). This provides a theoretical basis for the development of plant-derived exosome therapeutics. Beet (Beta vulgaris), a typical medicinal plant, has extracts that have been shown to improve cardiovascular and liver function through antioxidant and anti-inflammatory mechanisms. However, the potential value of delivery vehicles for its active ingredients, such as beet exosomes, in disease treatment has not been fully explored.

[0003] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, and coagulation disturbances are a key feature of sepsis. Current treatments for sepsis-related coagulation disturbances (such as anticoagulants and plasma exchange) have limited efficacy and carry the risk of bleeding. There is an urgent need to develop novel therapeutic strategies that combine anti-inflammatory and coagulation-regulating properties.

[0004] Exosomes, due to their natural targeted delivery properties and multi-component synergistic effects, exhibit unique advantages in regulating immune responses and endothelial function. CN105861430A discloses exosomes, a method for preparing exosomes, and their use in preparing drugs or preparations for treating sepsis. The exosomes are derived from human umbilical cord mesenchymal stem cells and are optimized for IL-1β. However, animal-derived exosomes may carry host-specific antigens, posing a potential risk of immune rejection.

[0005] Plant exosome-like nanoparticles have attracted attention due to their low immunogenicity and diverse natural active ingredients. While existing research on ginger-derived exosomes has been limited to anti-inflammatory applications, the application of plant exosomes, particularly sugar beet ELNV, in the treatment of coagulation disorders associated with sepsis remains unexplored. Given the natural anti-inflammatory and antioxidant properties of sugar beet exosomes and their potential association with coagulation-related regulatory functions, exploring their mechanisms of action in intervening in sepsis-related coagulation disorders is of great clinical significance. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the first aspect of the present invention provides a method for preparing exosome-like nanovesicles, which comprises the following steps:

[0007] extracting beet juice;

[0008] The beet juice was centrifuged at low speed and the supernatant was collected;

[0009] The supernatant obtained by low-speed centrifugation is subjected to medium-speed centrifugation and the supernatant is taken;

[0010] The supernatant obtained by medium-speed centrifugation was subjected to high-speed centrifugation to obtain a precipitate containing exosome-like nanovesicles;

[0011] The obtained precipitate containing exosome-like nanovesicles was subjected to sucrose density gradient ultracentrifugation to obtain purified exosome-like nanovesicles.

[0012] According to a preferred embodiment, the low-speed centrifugation step is a differential centrifugation at 4°C and 3,000×g for 30 min.

[0013] According to a preferred embodiment, the intermediate speed centrifugation step is a differential centrifugation at 4°C and 10,000×g for 60 min.

[0014] According to a preferred embodiment, the high-speed centrifugation step is a differential centrifugation at 4°C and 120,000×g for 90 min.

[0015] According to a preferred embodiment, in the sucrose density gradient ultracentrifugation step, the sucrose gradient density system used is a sucrose solution of 60%, 45%, 30%, and 15% sucrose solution layered from bottom to top to obtain a sucrose density gradient layered interface stable configuration system.

[0016] According to a preferred embodiment, in the step of obtaining purified exosome-like nanovesicles, the precipitate is first resuspended in an 8% sucrose solution to obtain a resuspension, and then the resuspension is added to the top layer of the sucrose density gradient layered interface stable configuration system.

[0017] According to a preferred embodiment, the sucrose density gradient ultracentrifugation step is performed at 4° C. and 120,000×g for 90 min.

[0018] According to a preferred embodiment, the prepared exosome-like nanovesicles are cup-shaped.

[0019] The second aspect of the present invention provides exosome-like nanovesicles prepared by the preparation method provided by the first aspect of the present invention.

[0020] The third aspect of the present invention provides the use of the preparation method provided by the first aspect of the present invention and the exosome-like nanovesicles provided by the second aspect of the present invention in the preparation of a drug for treating sepsis.

[0021] The third aspect of the present invention provides the use of the preparation method provided in the first aspect of the present invention and the exosome-like nanovesicles provided in the second aspect of the present invention in the preparation of a medicament for improving sepsis-induced coagulation disorder.

[0022] The fourth aspect of the present invention provides the use of the preparation method provided in the first aspect of the present invention and the exosome-like nanovesicles provided in the second aspect of the present invention in the preparation of a medicament for improving organ damage.

[0023] The fifth aspect of the present invention provides the use of the preparation method provided in the first aspect of the present invention and the exosome-like nanovesicles provided in the second aspect of the present invention in the preparation of a drug for improving blood perfusion.

[0024] In a sixth aspect, the present invention provides the use of the preparation method provided in the first aspect of the present invention and the exosome-like nanovesicles provided in the second aspect of the present invention in the preparation of a medicament for improving macrophage infiltration and fibrin deposition in the lungs and liver caused by sepsis.

[0025] The seventh aspect of the present invention provides the use of the preparation method provided in the first aspect of the present invention and the exosome-like nanovesicles provided in the second aspect of the present invention in the preparation of a drug for increasing platelet and Fbg levels.

[0026] In an eighth aspect, the present invention provides the use of the preparation method provided in the first aspect of the present invention and the exosome-like nanovesicles provided in the second aspect of the present invention in the preparation of a drug for reducing plasma TAT and DD levels.

[0027] Technical effects of the present invention:

[0028] The present invention extracts BELNV from sugar beets by differential centrifugation and sucrose density gradient centrifugation. BELNV is cup-shaped with an average size of 125.2 nm. At the cellular level, BELNV significantly reduces coagulation and inflammatory indicators in RAW264.7 cells after LPS stimulation. Coagulation indicators refer to platelet count, TAT, Fbg or DD. At the animal level, BELNV significantly improves the survival rate of septic mice, increases blood perfusion of tissues and organs of septic mice, reduces pathological changes, macrophage infiltration and fibrin deposition in the lung and liver tissues of septic mice, and reduces coagulation and inflammatory indicators.

[0029] The present invention found that IER3 was significantly upregulated in sepsis and mouse mononuclear macrophage RAW264.7 inflammatory models, and is a key gene for sepsis, affecting the prognosis of sepsis. The GSEA analysis results of IER3 in clinical data sets showed that IER3 is closely related to human immune regulation. In addition, the protein interaction network results in the STRING database showed that there are extensive molecular interactions between IER3, inflammatory factors, coagulation indicators, and ferroptosis markers. The results of protein molecular docking analysis showed that IER3 may exert its biological function through protein interactions with Hmox1, SLC7A11 and GPX4. Therefore, the present invention provides a theoretical basis for IER3 as a biomarker and therapeutic target for coagulation dysfunction in sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the extraction route of sugar beet exosomes;

[0031] Figure 2 This is the identification result of BELNV;

[0032] Figure 3 CCK8 assay results;

[0033] FIG4 shows that BELNV reduces LPS-induced morphological changes in RAW264.7 cells;

[0034] Figure 5 The results show the effect of BELNV on the mRNA expression of inflammatory factors induced by LPS in RAW264.7 cells;

[0035] Figure 6 The results show the effect of BELNV on the expression of procoagulant factors induced by LPS in RAW264.7 cells;

[0036] Figure 7 The results showed that BELNV improved the survival rate of septic mice;

[0037] Figure 8 The laser speckle imaging system detects the blood perfusion results of mouse tissues and organs;

[0038] Figure 9 HE staining results of BELNV-induced improvement of pathological damage in the lungs, liver, heart, kidneys, and spleen of septic mice;

[0039] Figure 10 The immunohistochemical results showed that BELNV improved macrophage infiltration and fibrin deposition in the lungs and livers of septic mice;

[0040] Figure 11 Improve the outcome of sepsis-induced coagulopathy for BELNV;

[0041] Figure 12 The results showed that BELNV reduced the levels of inflammatory factors in the lung and liver tissues of septic mice;

[0042] Figure 13 To detect the relative mRNA expression of TF and PAI-1 in lung and liver tissues of each group by RT-qPCR;

[0043] FIG14 shows the protein levels of TF, PAI-1 and Thrombin in lung and liver tissues of each group detected by WB;

[0044] Figure 15 The expression of IER3, inflammatory factors, coagulation markers, and ferroptosis markers in tissue cells;

[0045] Figure 16 This is the result that LPS is cytotoxic in RAW264.7 cells;

[0046] Figure 17 The concentration gradient changes of IER3, TF, GPX4, IL-1β, IL-6 and TNF-α mRNA expression in RAW264.7 cells after LPS stimulation;

[0047] Figure 18 The temporal gradient changes of IER3 and TF mRNA expression in RAW264.7 cells after LPS stimulation;

[0048] Figure 19 The results show the changes in IER3 and TF protein expression in RAW264.7 cells after LPS stimulation;

[0049] Figure 20 Fluorescence staining was used to detect the level of ROS in cells;

[0050] Figure 21 The temporal gradient changes of IER3 and TF protein expression in RAW264.7 cells after LPS stimulation;

[0051] Figure 22 is the expression level of IER3 in the plasma of patients with sepsis;

[0052] FIG23 shows the evaluation results of iron death markers in whole blood of patients with sepsis;

[0053] FIG24 shows the time-dependent changes in the expression of ferroptosis marker mRNA in RAW264.7 cells after LPS stimulation;

[0054] Figure 25 The results show the effect of LPS stimulation time on the expression of NRF2, Hmoxl, xCT, GPX4, and Fth1 proteins;

[0055] Figure 26 Evaluation of knockdown effects of 4 si-IER sequences;

[0056] Figure 27 The results show the effect of si-IER3 transfection on the expression of IER3 mRNA and protein in RAW264.7 cells;

[0057] Figure 28 IER3 knockdown increased ROS levels in RAW264.7 cells and RAW264.7 cells after LPS stimulation;

[0058] Figure 29 The results show the effect of IER3 knockdown on the mRNA expression of TF, PAI-1, IL-1β, IL-6, and TNF-α in RAW264.7 cells after LPS stimulation;

[0059] Figure 30 The results show the effect of IER3 knockdown on TF protein expression in RAW264.7 cells after LPS stimulation;

[0060] Figure 31 The results show the effect of IER3 knockdown on the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins in RAW264.7 cells after LPS stimulation;

[0061] Figure 32 This is the validation result of IER3-overexpressing RAW264.7 cell line;

[0062] Figure 33 To evaluate the ROS level in each group of cells, the MDA content in each group of cells was detected by malondialdehyde assay kit;

[0063] Figure 34 The results show the effect of IER3 overexpression on the mRNA expression of TF, PAI-1, IL-1β, IL-6 and TNF-α in RAW264.7 cells after LPS stimulation;

[0064] Figure 35 The results show the effect of IER3 overexpression on TF protein expression in RAW264.7 cells after LPS stimulation;

[0065] Figure 36 The results show the effect of IER3 overexpression on the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins in RAW264.7 cells after LPS stimulation;

[0066] Figure 37 The results of IER3 mRNA and protein expression changes after IER3 overexpression plasmid was transfected into RAW264.7 cells;

[0067] Figure 38The expression results of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins in RAW264.7 cells after transfection with IER3 overexpression plasmid;

[0068] Figure 39 The results show that high-dose RSL3 and Erastin exhibit significant cytotoxicity in RAW264.7 cells;

[0069] Figure 40 The concentration gradient changes of TF and PAI-1 mRNA expression in RAW264.7 cells after stimulation with RSL3 and Erastin;

[0070] Figure 41 The temporal gradient changes of TF and PAI-1 mRNA expression in RAW264.7 cells after stimulation with RSL3 and Erastin;

[0071] Figure 42 The expression level of TF protein in RAW264.7 cells after stimulation with RSL3 and Erastin;

[0072] Figure 43 RSL3 and Erastin stimulation increased the ROS level in RAW264.7 cells;

[0073] Figure 44 Effects of ferroptosis inhibitors Fer-1 and MitoQ treatment on the mRNA and protein expression of IER3 in RAW264.7 cells;

[0074] Figure 45 The results show the effects of Fer-1 and MitoQ treatment on the mRNA expression of IL-1β, IL-6, and TNF-α in RAW264.7 cells;

[0075] Figure 46 The results show the effects of ferroptosis inhibitors and ferroptosis inducers on the mRNA and protein expressions of TF in LPS-stimulated RAW264.7 cells;

[0076] Figure 47 The results show that BELNV reduces LPS-induced iron accumulation in RAW264.7 cells;

[0077] Figure 48 The results showed that BELNV reduced LPS-induced lipid peroxidation in RAW264.7 cells;

[0078] Figure 49 The results show the effect of BELNV on IER3 expression in LPS-stimulated RAW264.7 cells;

[0079] Figure 50The results showed that BELNV reduced LPS-induced NRF2, Hmox1, xCT, GPX4, and Fth1 protein expression in RAW264.7 cells;

[0080] Figure 51 The results showed that BELNV improved the ferroptosis-related indicators in septic mice;

[0081] Figure 52 The results show the effect of BELNV on IER3 expression in the lung and liver tissues of septic mice;

[0082] FIG53 shows the results of WB detection of the protein levels of NRF2, xCT, Hmox1, GPX4, and Fth1 in the lung and liver tissues of each group. DETAILED DESCRIPTION

[0083] If the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, for which the manufacturer is not specified, are all conventional products that can be purchased commercially. Plant exosomes refer to extracellular vesicles (plant extracellular vesicles, plant EVs) of a specified size range secreted by plant cells. In the present invention, the meaning of beet exosome-like nanovesicles (Betavulgaris-derived exosome-like nanovesicles, BELNV) is equivalent to exosomes and nanovesicles. Plant exosome-like nanovesicles (exosome-like nanovesicles, ELNV) in the present invention also refer to beet exosome-like nanovesicles. In the present invention, the control group (Control group) was treated by adding the same dose of PBS as the other treatment groups. Statistical data are expressed as mean ± standard deviation (SD) and analyzed using GraphPad 8.0 (GraphPad Software, USA). Comparisons among the three groups were assessed by one-way ANOVA followed by Tukey's test for multiple comparisons by comparing the mean of each column with the mean of every other column. Statistical significance for all comparisons was set at p < 0.05. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. Cecal ligation and puncture (CLP) was performed on experimental animals.

[0084] Example 1

[0085] This embodiment provides a method for preparing exosome-like nanovesicles, the preparation steps are as follows: Figure 1 As shown, Figure A shows the separation of BELNV from beet juice by differential centrifugation, and Figure B shows the purification of BELNV by sucrose density gradient centrifugation, which specifically includes the following steps:

[0086] 1. Sample pretreatment (differential centrifugation to separate BELNV)

[0087] Beet processing: select fresh beets, wash them 3 times with distilled water to remove soil and dust; peel and cut into pieces, crush them in a blender to extract the juice, and filter with a colander.

[0088] Centrifuge at low speed and perform differential centrifugation at 4°C and 3,000×g for 30 min to remove cells and debris (cell debris, starch granules, and large particle contaminants), and take the supernatant.

[0089] The supernatant obtained by low-speed centrifugation was subjected to differential centrifugation at 4°C and 10,000×g for 60 min to remove cells and debris (subcellular organelles), and the supernatant was collected.

[0090] The supernatant of the medium-speed centrifugation was subjected to differential centrifugation at 4°C and 120,000×g for 90 min, the supernatant was discarded, and the precipitate containing exosome-like nanovesicles was obtained.

[0091] 2. Configure the sucrose gradient density system

[0092] Prepare 8%, 15%, 30%, 45%, and 60% sucrose solutions in ultrapure water. Layer the 60%, 45%, 30%, and 15% sucrose solutions in this order from bottom to top in an ultracentrifuge tube suitable for an ultracentrifuge bucket rotor to create a sucrose density gradient. Add gently to maintain a stable interface.

[0093] 3. Sucrose Density Gradient Ultracentrifugation Purification

[0094] After sample loading and centrifugation, the pellet containing BELNV was resuspended in 8% sucrose solution (pre-cooled to 4°C) and added to the top layer of the sucrose density gradient prepared above (i.e., the resuspension was added to the top layer of the 15% sucrose concentration). The sucrose density gradient was then ultracentrifuged at 120,000 × g for 90 minutes at 4°C for further purification.

[0095] BELNV collection and purification: BELNV is usually distributed in the 30%-45% sucrose layer, and the 30% / 45% layer of the sucrose gradient is collected.

[0096] The sucrose was removed and the 30% / 45% layer of sucrose was resuspended in sterile PBS (pre-cooled to 4°C) to obtain the BELNV sample. The sample was stored at -80°C until use.

[0097] This example further characterizes the prepared BELNV, and the steps are as follows:

[0098] 10 μL of the BELNV sample prepared in the above steps was taken and dropped into the cup-shaped coating grid. The sample was absorbed for 5 minutes and the suspension was absorbed with filter paper.

[0099] The grid was stained with a drop of phosphotungstic acid for 1 minute, and the floating liquid was removed with filter paper. The grid was then dried at room temperature for 20 minutes.

[0100] After drying, the samples were observed in a Tecnai G2 Spirit BioTwin electron microscope (TEM) at 100 kV.

[0101] The results are as follows Figure 2 As shown, Figure A is the shape result of BELNV contained in the 30% / 45% sucrose layer identified by TEM, scale bar = 100 nm. The result shows that BELNV is cup-shaped, proving that BELNV was successfully extracted in this example.

[0102] This example also uses nanoparticle tracking analysis (NTA) technology (ZetaVIWES / N17-310 nanoparticle tracking analyzer) to analyze the concentration and size distribution of the prepared exosomes, and uses ZetaView 8.04.02 software to analyze the diluted nanovesicles. The specific steps are as follows:

[0103] The frozen samples of BELNV were taken and thawed in a 25°C water bath. The thawed exosome samples were diluted with 1×PBS and then placed on a ZetaVIWES / N17-310 nanoparticle tracking analyzer for detection. After detection, the diluted nanovesicles were analyzed using ZetaView8.04.02 software. The results are shown in Figure 2. Figure 2 As shown, Figure B shows the results of using NTA to identify the size and concentration of BELNV contained in the 30% / 45% sucrose layer, the abscissa represents the particle size (diameter) of the prepared BELNV, and the ordinate represents the concentration of BELNV (number of particles / mL).

[0104] Example 2

[0105] In this example, the safety of BELNV was tested. The specific steps included: taking an appropriate amount of cells according to the cell density and culturing them in a 96-well plate for 24 hours; incubating BELNV with RAW264.7 cells for 24 to 48 hours, and performing a CCK8 assay; in a clean bench, discarding the original culture medium, adding 100 μL of CCK8 working solution (CCK8 reagent: complete culture medium = 1:10) to each well, and incubating in an incubator for 1 to 4 hours; measuring the absorbance of each well at a wavelength of 450 nm using a microplate reader; and calculating the cell viability. The results are shown in FIG. Figure 3 As shown (the briefing also mentioned the half-maximal inhibitory concentration, but there was no corresponding result).

[0106] Figure 3 The horizontal axis is the concentration of different BELNV treatments (number of particles / mL), and the vertical axis is the cell viability. 8 particles / mL, 1×10 9 particles / mL, 1×10 10 There was no significant statistical difference in the BELNV treatment with 1×10 particles / mL compared with the control group (denoted as “0” in the figure); compared with the control group, 11 After treatment with BELNV at a concentration of 100 particles / mL, cell viability increased significantly, indicating that BELNV has no significant cytotoxicity.

[0107] Example 3

[0108] This example provides an exploration process of the effect of BELNV prepared in Example 1 on improving coagulation dysfunction in sepsis in a RAW264.7 cell inflammation model. This example conducted a cell morphology fluorescence staining test, specifically comprising the following steps: in a clean bench, taking a 12-well plate and placing a cell slide, adding an appropriate amount of cells and culturing for 24 hours, and then treating the cells according to the following groupings. The cell test grouping and treatment steps are as follows:

[0109] 1. Grouping of BELNV concentration gradient experiments.

[0110] Mouse mononuclear macrophage RAW264.7 cells were divided into 5 groups: control group (control group); BELNV 1×10 8 particles / mL group ( Figure 3 Indicated as 10 8 ); BELNV 1×10 9 particles / mL group ( Figure 3 Indicated as 10 9 ); BELNV 1×10 10 particles / mL group ( Figure 3 Indicated as 1O1 0); BELNV 1×10 1111 particles / mL group ( Figure 3 Indicated as 10 1111 ), and each group was added with the same dose of PBS.

[0111] 2. LPS induces morphological changes in RAW264.7 cells.

[0112] After macrophages are stimulated by LPS, they undergo significant morphological changes, which are important markers of macrophage activation and immune response. To evaluate the effect of LPS on the morphology of RAW264.7 cells, this example stimulated RAW264.7 cells with LPS, where the concentration gradient of LPS included 0μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, and 10μg / mL. RAW264.7 cells were incubated with LPS for 24 hours and then fluorescently stained and observed. The results are shown in Figure 2. Figure 4.1 As shown, after LPS stimulation, RAW264.7 transformed into an irregular shape and developed pseudopodia-like protrusions.

[0113] 3. BELNV treatment experimental grouping.

[0114] Mouse mononuclear macrophage RAW264.7 cells were divided into 5 groups: control group; BELNV control group (BELNV 1×10 11 particles / mL group); LPS stimulation group (10 μg / mL); low-dose BELNV treatment group (BELNV 1×10 10 particles / mL+LPS10μg / mL group); high-dose BELNV treatment group (BELNV 1×10 11 particles / ml+LPS10μg / mL group).

[0115] In a clean hood, discard the original culture medium and rinse the cells three times with PBS buffer for 3 minutes each. Fix the cells with tissue cell fixative for 1 minute and rinse the cells three times with PBS for 3 minutes each. Permeabilize the membrane with 0.3% Triton X-100 / PBS (prepared by adding 3 μL Triton X-100 to 1 mL PBS) for 5 minutes at room temperature. Rinse the cells three times with PBS for 3 minutes each. After aspirating the PBS, add 1% blocking goat serum (0.1 mL goat serum + 10 mL PBS) dropwise and block at room temperature for 40 minutes. Add 2 μL of FITC-phalloidin stock solution to 100 μL PBS to prepare a working solution, which is then used to stain the cells. Stain the cells in a humidified chamber at room temperature for 30-60 minutes. Rinse the cells again with PBS for 3 minutes each, aspirate the solution, and evenly apply mounting solution containing DAPI to the center groove of the dish. Confocal images were acquired 5 minutes later, as shown in Figure 4.

[0116] This example also uses a red fluorescent cell attachment kit and uses the fluorescent dye PKH26 to label the purified BELNV. RAW264.7 cells and BELNV were co-incubated, and red fluorescent BELNV was observed in the green RAW264.7 cells. Figure 4.2 (Scale bar = 50 μm) As shown in the RAW264.7 cell control group that received complete medium, no BELNV was observed in the cells. The results indicate that PKH26-labeled BELNV was significantly taken up by RAW264.7 cells.

[0117] Figure 4.1 (Scale bar = 50 μm) and Figure 4.3 (Scale bar = 50 μm) The results show that macrophages undergo significant morphological and functional changes after LPS stimulation. Specifically, in the LPS stimulation group (represented as "LPS" in the figure), RAW264.7 cells transformed into irregular shapes and developed pseudopodia-like protrusions; while pseudopodia-like protrusions decreased after BELNV treatment. Pseudopodia are the structural basis for macrophage migration and phagocytic activity. Excessive pseudopodia formation may promote the spread of inflammation (such as sepsis). LPS stimulation leads to pseudopodia formation in RAW264.7 cells, and BELNV can improve LPS-induced macrophage activation by blocking pro-inflammatory signals and promoting phenotypic conversion.

[0118] This example also explored the effect of BELNV on the mRNA expression of inflammatory factors induced by LPS in RAW264.7 cells, including the following steps: 6 h after group administration (same as above, no further details are given here), the relative mRNA expression of inflammatory factors (IL-1β, IL-6, TNF-α) in each group of cells was detected by RT-qPCR. The results are shown in Figure 2. Figure 5As shown in the figure, the horizontal axis represents different treatment groups, the vertical axis represents the relative mRNA expression level, "-" represents that the treatment is not added, and "+" represents that the treatment is added, among which Figure A is the relative mRNA expression level of IL-1β; Figure B is the relative mRNA expression level of IL-6; Figure C is the relative mRNA expression level of TNF-α.

[0119] Figure 5 The results showed that when the relative mRNA expression levels of IL-1β, IL-6, and TNF-α produced by LPS stimulation increased significantly, the low-dose BELNV treatment group and the high-dose BELNV treatment group were able to significantly reduce the mRNA expression levels of IL-1β, IL-6, and TNF-α in the cells, that is, the expression levels of IL-1β, IL-6, and TNF-α were significantly reduced after BELNV treatment, indicating that BELNV improved LPS-induced macrophage inflammation.

[0120] This example also explored the effect of BELNV on LPS-induced procoagulant factor expression in RAW264.7 cells, including the following steps: 6 h after administration of the drugs in groups (same as above, no further details will be given here), the relative mRNA expression levels of procoagulant factors (TF, PAI-1) in each group of cells were detected by RT-qPCR. The results are shown in Table 1. Figure 6 As shown in the figure, "-" indicates that the treatment was not added, and "+" indicates that the treatment was added. Figure A is the relative mRNA expression level of TF, and Figure B is the relative mRNA expression level of PAI-1. The horizontal axes of Figures A and B represent different treatment groups, and the vertical axes represent the relative mRNA expression levels; Figure C is the WB band of TF; Figure D is the relative protein expression level of TF, the horizontal axes represent different treatment groups, and the vertical axes represent the relative protein expression levels.

[0121] Figure 6 The results showed that in the LPS-stimulated group, TF and PAI-1 mRNA levels were significantly increased compared with the control group. After treatment with (low-dose and high-dose) BELNV, the mRNA expression of TF and PAI-1 was significantly decreased (Figures A and B). Western blot analysis of TF protein levels in each group of cells showed that in the LPS-stimulated group, TF protein expression was significantly increased compared with the control group, while TF expression was significantly decreased after treatment with (low-dose and high-dose) BELNV. TF is the initiator of the coagulation cascade (coagulation factor III). Decreased TF expression inhibits the initiation of the coagulation cascade, while reduced PAI-1 enhances fibrinolytic activity, jointly alleviating the tendency of LPS-induced disseminated intravascular coagulation (DIC). High TF / PAI-1 expression is a key factor in sepsis-related mortality. After BELNV treatment, the expression of TF and PAI-1 was significantly decreased, demonstrating that BELNV can improve coagulation disorders and organ damage.

[0122] Example 4

[0123] This example provides an investigation into the effect of BELNV on improving coagulation disorders in CLP-induced sepsis mice. First, a CLP-induced sepsis mouse model was established using adult C57BL / 6N male mice (8 weeks old, 18-22 g).

[0124] The modeling method includes the following steps: after the mouse is anesthetized with isoflurane inhalation, the abdominal skin is prepared and disinfected; the skin and peritoneum are incised to expose the cecum; the cecum is ligated 1 cm distal to the ligature with sutures; a 21G needle is used to puncture the distal end of the ligature and gently squeeze the cecum to allow a small amount of intestinal contents to overflow; after the cecum is repositioned, the peritoneum and skin are sutured; 1 mL of normal saline is injected subcutaneously immediately after the operation to prevent dehydration; the animal is placed on a heating pad to maintain body temperature until it wakes up; after the mouse recovers, it is returned to the cage and continued to be raised and given a standard diet.

[0125] In this example, a BELNV survival analysis experiment was conducted, and the groups were as follows.

[0126] The mice were randomly divided into 5 groups: control group; sepsis model group (CLP group); low-dose BELNV treatment group (BELNV 2×10 11 particles / kg body weight+CLP group); medium-dose BELNV treatment group (BELNV 5×10 11 particles / kg body weight+CLP group) and high-dose BELNV treatment group (BELNV 2×10 12 particles / kg body weight + CLP group).

[0127] After the sepsis model was established, the status of all mice (8 per group) was closely observed, and all mice were recorded once a day. Euthanasia was performed in the dying stage and the observation was carried out for 28 days. The results of the BELNV survival analysis experiment are shown in Figure 7 , where the horizontal axis is the number of days and the vertical axis is the survival probability. The survival status of mice was observed for 28 days after CLP surgery. Figure 7 It can be seen that the survival rate of mice in the sepsis model group was significantly lower than that of mice in the control group, and all died within the second day of modeling. However, the survival rate of mice in the three BELNV treatment groups was higher than that in the CLP group. Therefore, BELNV can improve the survival rate of septic mice. Subsequent experiments selected a high BELNV dose (2×10 12 particles / kg) for treatment.

[0128] In this example, a BELNV treatment experiment was conducted. Adult C57BL / 6N male mice (8 weeks old, 18-22 g) were randomly divided into three groups: a control group (control group); a sepsis model group (CLP group); and a high-dose BELNV treatment group (BELNV 2×10 12 particles / kg body weight + CLP group).

[0129] Treatment method of the control group: mice were only opened and sutured without CLP treatment, and 1 mL of normal saline was injected subcutaneously after the operation.

[0130] Treatment of the CLP group: Mice were given CLP model but did not receive BELNV treatment.

[0131] High-dose BELNV treatment group: mice were given 2×10 BELNV once after CLP model establishment. 12 particles / kg body weight treatment.

[0132] Each group of mice was treated accordingly, and blood perfusion was tested 12 hours later. The head, abdomen, and lower limbs were exposed, the hair was shaved, depilated, and disinfected. The skin of the mice was cut open to expose the brain, liver, mesentery, lower limbs and other tissues and organs. The blood perfusion of the mouse tissues and organs was tested using a laser speckle imaging system. Figure 8 Figure 1 shows the perfusion results for the brain (A), liver (B), intestinal (C), and lower limbs (D). These results indicate that blood perfusion in the tissues and organs of mice in the CLP group was significantly lower than that in the control group. However, after BELNV treatment, blood perfusion in each tissue and organ was significantly higher than in the CLP group, indicating that BELNV treatment can improve blood perfusion.

[0133] After the laser speckle experiment, mice were euthanized and specimens were collected. The collection method included: mice in each group were anesthetized with isoflurane inhalation after treatment; eyeballs were removed and blood was collected using anticoagulant tubes; blood samples were analyzed for platelets and then centrifuged at 3,000 rpm at 4°C for 15 minutes. The supernatant was collected as the plasma sample. Mice were euthanized by overdose of anesthesia; the heart, lungs, liver, spleen, and kidneys were removed after apical cardiac puncture and lavage, and tissues were collected for pathological examination. Pathological examination steps included: tissues were fixed in histological fixative for 60 ± 6 hours and dehydrated in a Leica automatic dehydrator; then immersed in 50%, 60%, 70%, 80%, and 90% alcohol for 30 minutes in sequence; immersed in xylene I for 10 minutes; immersed in xylene II for 10 minutes; immersed in paraffin at 68°C for 60 minutes; embedded in a Leica tissue embedding table; and sectioned (5 μm thickness) using a Leica manual rotary microtome.

[0134] To investigate the protective effect of BELNV on tissues and organs, tissue and organ specimens from each group of mice were collected for HE staining. To evaluate the safety of BELNV administration in vivo, a BELNV control group was also established. Mice in this group underwent only abdominal laparotomy and suture without CLP. After the operation, 1 mL of normal saline was injected subcutaneously, and 2×10 BELNV was administered. 12 / kg body weight.

[0135] HE staining: bake the sections at 68°C for 1 hour; soak in xylene I for 10 minutes; soak in xylene II for 10 minutes; soak in 100%, 95%, 90%, 80%, and 70% alcohol for 5 minutes in sequence for hydration; soak in hematoxylin solution for 5 minutes to stain the nuclei; soak in hydrochloric acid and alcohol for differentiation; rinse with water to return to blue; soak in eosin solution for 3 minutes for staining; soak in 80%, 90%, 95%, and 100% alcohol for 5 minutes in sequence for dehydration; soak in xylene I for 10 minutes; soak in xylene II for 10 minutes; and seal the sections with neutral gum.

[0136] HE staining results Figure 9 Figure A shows the staining results of the lungs, scale bar = 100 μm; Figure B shows the staining results of the liver, scale bar = 100 μm; Figure C shows the staining results of the heart, scale bar = 100 μm; Figure D shows the staining results of the kidneys, scale bar = 100 μm; Figure E shows the staining results of the spleen, scale bar = 50 μm, n ≥ 3 for each group. The results showed that the lungs, livers, hearts, and kidneys of mice in the CLP group had severe inflammation and damage, and hemosiderin deposition was found in the spleen; BELNV (BELNV 2×10 12 particles / kg body weight+CLP group) treatment group could improve the above tissue and organ damage. 12 particles / mL group) showed no obvious organ damage, indicating that BELNV has good safety in vivo administration.

[0137] Immunohistochemistry: Repeat drying, dewaxing, and hydration steps are the same as "HE staining"; add an appropriate amount of trypsin containing EDTA to the tissue on the glass slide, incubate at 37℃ for 30 minutes, and wash with PBS buffer for antigen retrieval; incubate in 3% hydrogen peroxide solution at room temperature for 10 minutes. Wash with PBS buffer; incubate with BSA at room temperature for 30 minutes; add diluted primary antibody, incubate in a 4℃ refrigerator overnight, and wash with PBS buffer; add enzyme-labeled goat anti-rabbit IgG polymer, incubate at 37℃ for 1 hour, and wash with PBS buffer; add DAB color development solution, observe the tissue staining under a microscope, and wash with PBS buffer after color development; soak in hematoxylin stain for 5 minutes to stain the nucleus; soak in hydrochloric acid alcohol for differentiation; rinse with water to return to blue; dehydration, transparency, and sealing steps are the same as "HE staining", observe under a microscope and take pictures, the results are as follows Figure 10 Figure 4 (scale bar = 50 μm). Panel A shows lung macrophage infiltration; Panel B shows liver macrophage infiltration; Panel C shows lung fibrin deposition; Panel D shows liver fibrin deposition; and Panel E shows the statistical results of fibrin deposition area. n = 4 per group. F4 / 80 is a commonly used macrophage marker in mice. To assess macrophage infiltration in each group of mice, this example used anti-F4 / 80 antibody immunohistochemical staining to label macrophages. Fibrinogen is a key substrate in thrombosis. To assess fibrinogen deposition in each group of mice, this example used anti-fibrin antibody immunohistochemical staining to assess tissue fibrin deposition.

[0138] Immunohistochemistry results showed that the number of macrophages in the lung and liver tissues of mice in the CLP group increased, while BELNV treatment (BELNV 2×10 12 BELNV treatment significantly reduced macrophage infiltration in the lungs and livers of septic mice (Figures A and B). Furthermore, CLP-treated mice showed extensive fibrin deposition in the lungs and livers, whereas BELNV treatment significantly reduced fibrin deposition (Figures C-E). These results suggest that BELNV treatment can reduce macrophage and fibrin deposition in the lungs and livers of septic mice. Therefore, BELNV can improve macrophage infiltration and fibrin deposition in the lungs and livers of septic mice.

[0139] Platelets, thrombin-antithrombin complex (TAT), plasma fibrinogen (Fbg), and the fibrin breakdown product D-dimer (DD) are important indicators for assessing coagulation disorders. Changes in these indicators can reflect the status of the coagulation and fibrinolytic systems during sepsis. In this example, whole blood platelet count was used to determine platelet count, and ELISA was used to measure the concentrations of TAT, Fbg, and DD in the plasma of each group of mice to assess the extent of coagulation disorders in septic mice. The ELISA test includes: according to the instructions, dilute the plasma sample with sample diluent; add the sample to be tested and the standard to the ELISA plate and incubate, wash the ELISA plate three times with wash buffer; add the detection antibody working solution to the ELISA plate and incubate, wash the ELISA plate three times with wash buffer; add HRP working solution to the ELISA plate and incubate, wash the ELISA plate three times with wash buffer; add the color development solution to the ELISA plate and incubate; add the stop solution to the ELISA plate; detect the absorbance of each well in the ELISA plate with an ELISA reader; calculate the concentration of the detection index in the sample to be tested, and the result is as follows Figure 11 As shown, the horizontal axes represent different treatments, and the vertical axes represent the concentration levels of coagulation-related indicators, among which Figure A represents the platelet level; Figure B represents the TAT level; Figure C represents the Fbg level; and Figure D represents the DD level.

[0140] according to Figure 11The results showed that compared with normal mice, the platelet and Fbg levels of CLP group mice were significantly decreased (as shown in Figures A and C), and the TAT and DD levels of CLP group mice were significantly increased (as shown in Figures B and D), that is, the coagulation indicators of CLP group mice were significantly abnormal; after BELNV treatment (BELNV 2×10 12 After BELNV administration (particles / kg body weight + CLP group), the above coagulation parameters were improved. Therefore, BELNV improved sepsis-induced coagulation disorder.

[0141] This example also explored the effects of BELNV on inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the lung and liver tissues of septic mice. Specifically, this example measured the relative mRNA expression of IL-1β, IL-6, and TNF-α in the lung and liver tissues of each group using RT-qPCR.

[0142] First, the total RNA extraction operation of the tissue was performed, and the specific operation was as follows: about 50 mg of tissue was placed in 1 mL of lysis solution RZ, homogenized, and transferred to an enzyme-free EP tube; after standing for 5 minutes, 200 μL of 1,3-bromochloropropane was added to each enzyme-free EP tube and vortexed thoroughly; after standing for 3 minutes, centrifuged at 4°C and 12,000 rpm for 10 minutes; 200 μL of the upper aqueous phase liquid was aspirated into a new enzyme-free EP tube, 100 μL of anhydrous ethanol was added, mixed, and added to the inner cannula; centrifuged at 4°C and 12,000 rpm for 30 seconds, and the waste liquid was discarded; 500 μL of RD solution was added to the inner cannula, centrifuged at 4°C and 12,000 rpm for 30 seconds, and the waste liquid was discarded; 500 μL of the inner cannula Prepare the RW solution, let it sit for 2 minutes, centrifuge it at 4°C and 12,000 rpm for 30 seconds, discard the waste liquid, and repeat this step once; transfer the inner tube to a new enzyme-free EP tube, add an appropriate amount of DEPC water, let it sit for 2 minutes, and centrifuge it at 4°C and 12,000 rpm for 2 minutes. The liquid in the EP tube is the total cellular RNA solution. Prepare the reverse transcription reaction system according to Table 1, mix thoroughly, and place it in the reverse transcription instrument for reverse transcription. Primers were designed and synthesized by China Sangon Biotechnology (Shanghai) Co., Ltd. and Jierui Biotechnology Co., Ltd.; the primer sequences are shown in Table 2. Prepare the RT-qPCR reaction system according to Table 3. Place the PCR plate containing the RT-qPCR reaction system into the qPCR machine, set the reaction conditions according to Table 4, and start the run.

[0143] Table 1

[0144]

[0145] Table 2

[0146]

[0147] Table 3

[0148]

[0149] Table 4

[0150]

[0151] Melting curve reaction conditions: 9°C for 15 s; 60°C for 15 s; 95°C for 15 s.

[0152] Figure 12 Figure 3: BELNV reduces the levels of inflammatory factors in the lung and liver tissues of septic mice. The horizontal axes represent different treatment groups, and the vertical axes represent relative mRNA expression levels. Figure A shows the mRNA expression levels of IL-1β, IL-6, and TNF-α in lung tissue; Figure B shows the mRNA expression levels of IL-1β, IL-6, and TNF-α in liver tissue.

[0153] Figure 12 The results showed that the RNA levels of IL-1β, IL-6, and TNF-α in the lungs and livers of CLP mice were significantly increased compared with those of normal mice; after BELNV treatment (BELNV 2×10 12 After administration of BELNV (particles / kg body weight + CLP group), the mRNA expression levels of IL-1β, IL-6, and TNF-α were significantly decreased compared with the CLP group. Therefore, BELNV can improve sepsis-induced inflammation.

[0154] In this example, the relative mRNA expression levels of TF and PAI-1 in lung and liver tissues of each group were detected by RT-qPCR. Figure 13 As shown, the horizontal axes are all different treatment groups, and the vertical axes are all relative mRNA expression levels. Figure A shows the mRNA expression levels of TF and PAI-1 in lung tissue; Figure B shows the mRNA expression levels of TF and PAI-1 in liver tissue. The results show that the mRNA levels of TF and PAI-1 in the lungs and livers of mice in the CLP group were significantly increased compared with those in the control group, and the mRNA expression levels of TF and PAI-1 were significantly reduced after BELNV treatment. Furthermore, in this example, WB was used to detect the protein levels of TF, PAI-1 and Thrombin in the lung and liver tissues of each group. The results are shown in Figure 14. Figure 14.1 In the figure, Panel A shows the WB bands of TF, PAI-1, and Thrombin in lung tissue, and Panel B shows the WB bands of TF, PAI-1, and Thrombin in liver tissue; Figure 14.2Figure C shows the protein expression of TF, PAI-1, and Thrombin in the lungs, with the horizontal axis representing the different treatment groups and the vertical axis representing protein expression. Figure D shows the protein expression of TF, PAI-1, and Thrombin in the liver, with the horizontal axis representing the different treatment groups and the vertical axis representing protein expression. Figure 14 shows that the protein expression of TF, PAI-1, and Thrombin in the lungs and livers of mice in the CLP group was significantly increased compared to the control group, while the protein expression of TF, PAI-1, and Thrombin was significantly decreased after BELNV treatment. Therefore, BELNV can improve sepsis-induced coagulation abnormalities.

[0155] Example 5

[0156] Sepsis-induced coagulation dysfunction plays a crucial role in the development and progression of sepsis. IER3 is significantly upregulated in sepsis and macrophage inflammation models, while TF and PAI-1 are important indicators of sepsis-induced coagulation dysfunction. Furthermore, current research has confirmed that ferroptosis plays a crucial role in sepsis. Ferritin heavy chain 1 (Fth1), ferritin light chain (Ftl), nuclear receptor coactivator 4 (Ncoa4), Hmox1, acetyl-CoA synthetase long-chain family member 4 (Acsl4), lysophosphatidylcholine acyltransferase 3 (Lpcat3), arachidonate lipoxygenase 15 (Alox15), solute carrier family 7 member 11 (SLC7A11, also known as xCT), solute carrier family 3 member 2 (Slc3a2), prostaglandin-endoperoxide synthase 2 synthase2, Ptgs2), glutathione peroxidase 4 (Glutathione peroxidase 4, GPX4), apoptosis-inducing factor mitochondrion-associated 2 (Apoptosis-inducing factor mitochondrion-associated 2, AIFM2) and upstream oxidative stress key regulatory protein NRF2 (Nuclear factor erythroid2-related factor 2, nuclear factor E2-related factor 2) are commonly used biomarkers in ferroptosis research. Therefore, this example further analyzes the expression of IER3, inflammatory factors (IL-1β, IL-6 and TNF-a), coagulation indexes (TF, PAI-1) and ferroptosis markers (Fth1, Ftl, Ncoa4, Hmox1, Acsl4, Lpcat3, Alox15, xCT, Slc3a2, Ptgs2, GPX4 (glutathione peroxidase 4), AIFM2 and NRF2) in tissue cells in the single-cell transcriptomics database. The expression of IER3, inflammatory factors, coagulation markers, and ferroptosis markers in tissue cells is as follows Figure 15As shown, NFE2L2 is NRF2 and SERPINE1 is PAI-1.

[0157] Example 6

[0158] The mouse mononuclear macrophage cell line RAW264.7 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. IER3 overexpression and control RAW264.7 cells were purchased from APExBIO, USA. All siRNAs (shown in Table 5) were designed and synthesized by Jima Gene Co., Ltd., China. The final concentration of the stock solution was 20 μM. All overexpression plasmids were designed and synthesized by Jima Gene Co., Ltd., China. The final concentration of the stock solution was 500 ng / μL.

[0159] Table 5

[0160]

[0161] The cell test grouping and processing of this embodiment are as follows:

[0162] (1) LPS concentration gradient experiment grouping. Mouse mononuclear macrophage RAW264.7 cells were divided into 7 groups: control group, LPS 1μg / mL stimulation group, LPS 2μg / mL stimulation group, LPS 5μg / mL stimulation group, LPS 10μg / mL stimulation group, LPS 20μg / mL stimulation group, and LPS 50μg / mL stimulation group.

[0163] (2) Grouping for LPS time gradient experiment. Mouse mononuclear macrophage RAW264.7 cells were divided into 8 groups: control group, 10 μg / mL LPS stimulation for 2 h group, LPS stimulation for 4 h group, LPS stimulation for 6 h group, LPS stimulation for 12 h group, LPS stimulation for 24 h group, LPS stimulation for 36 h group, and LPS stimulation for 48 h group.

[0164] (3) Ferropoptosis inhibitor ferrostatin-1 (Fer-1) concentration gradient experiment grouping. Mouse mononuclear macrophage RAW264.7 cells were divided into 6 groups: control group, Fer-1 0.5 μM stimulation group, Fer-1 1 μM stimulation group, Fer-1 2 μM stimulation group, Fer-1 5 μM stimulation group, and Fer-1 10 μM stimulation group. DMSO was added to each group to make the final DMSO concentration the same.

[0165] (4) Grouping for the ferroptosis inhibitor mitochondrial-targeted coenzyme Q10 (Mitoquinone, MitoQ) concentration gradient experiment. Mouse mononuclear macrophage RAW264.7 cells were divided into 6 groups: control group, MitoQ 0.005μM stimulation group, MitoQ 0.01μM stimulation group, MitoQ 0.02μM stimulation group, MitoQ 0.05μM stimulation group, and MitoQ 0.1μM stimulation group. DMSO was added to each group to ensure the same final DMSO concentration.

[0166] (5) Grouping for the concentration gradient experiment of the ferroptosis inducer GSH peroxidase 4 inhibitor ((1S, 3R)-RSL3, RSL3). Mouse mononuclear macrophage RAW264.7 cells were divided into 7 groups: control group, RSL3 1μM stimulation group, RSL3 2μM stimulation group, RSL3 5μM stimulation group, RSL3 10μM stimulation group, RSL3 20μM stimulation group, and RSL3 50μM stimulation group. DMSO was added to each group to make the final DMSO concentration the same.

[0167] (6) Grouping for the ferroptosis inducer Erastin concentration gradient experiment. Mouse mononuclear macrophage RAW264.7 cells were divided into 7 groups: control group, erastin 1μM stimulation group, erastin 2μM stimulation group, erastin 5μM stimulation group, erastin 10μM stimulation group, erastin 20μM stimulation group, and erastin 50μM stimulation group. DMSO was added to each group to ensure the same final DMSO concentration.

[0168] (7) Grouping for RSL3 time gradient experiment. Mouse mononuclear macrophage RAW264.7 cells were divided into 7 groups: control group, 10 μM RSL3 stimulation group for 2 h, RSL3 stimulation group for 6 h, RSL3 stimulation group for 12 h, RSL3 stimulation group for 24 h, RSL3 stimulation group for 36 h, and RSL3 stimulation group for 48 h. DMSO was added to each group to make the final DMSO concentration the same.

[0169] (8) Erastin time gradient experiment grouping. Erastin time gradient experiment grouping: Mouse mononuclear macrophage RAW264.7 cells were divided into 7 groups: control group, 30 μM Erastin stimulation for 2 h group, Erastin stimulation for 6 h group, Erastin stimulation for 12 h group, Erastin stimulation for 24 h group, Erastin stimulation for 36 h group, and Erastin stimulation for 48 h group. DMSO was added to each group to make the final DMSO concentration the same in each group.

[0170] (9) Experimental grouping of the effects of ferroptosis inhibitors and ferroptosis inducers in sepsis cell models. Mouse mononuclear macrophage RAW264.7 cells were divided into 8 groups: control group, Fer-1 control group, MitoQ control group, LPS stimulation group, Fer-1 treatment group, MitoQ treatment group, LPS+RSL3 stimulation group, and LPS+Erastin stimulation group. The Fer-1 control group was given Fer-1 (10 μM), the MitoQ control group was given MitoQ (0.1 μM), the LPS stimulation group was given LPS (10 μg / mL), the Fer-1 treatment group was given LPS (10 μg / mL) + Fer-1 (10 μM), the MitoQ treatment group was given LPS (10 μg / mL) + MitoQ (0.1 μM), the LPS + RSL3 stimulation group was given LPS (10 μg / mL) + RSL3 (10 μM), and the LPS + Erastin stimulation group was given LPS (10 μg / mL) + 30 μM Erastin. Each group was administered for 6 hours, and DMSO was added to each group to achieve the same final DMSO concentration.

[0171] (10) Grouping of siRNA transfection experiments. Mouse mononuclear macrophage RAW264.7 cells were divided into four groups: si-NC group, si-IER3 group, si-NC+LPS group, and si-IER3+LPS group. The si-NC+LPS group and si-IER3+LPS group were stimulated with LPS (10 μg / mL) for 6 h.

[0172] (11) Experimental groups of IER3 overexpressing cell lines: control group (vector), IER3 overexpressing cell line group (OE-IER3), vector+LPS group, and OE-IER3+LPS group. Among them, the vector+LPS group and OE-IER3+LPS group were stimulated with LPS (10 μg / mL) for 6 hours.

[0173] (12) IER3 plasmid overexpression experimental groups: control group (vector) and IER3 plasmid overexpression group (OE-IER3).

[0174] This example provides the safety effects of LPS on RAW264.7 cells.

[0175] To evaluate LPS toxicity and calculate IC50, RAW264.7 cells were stimulated with LPS at a concentration gradient of 0 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL. CCK8 assay was performed after incubation for 48 h. At 48 h, after LPS stimulation, RAW264.7 cells proliferated significantly at a concentration of 1 μg / mL, but as the LPS concentration increased, the number of RAW264.7 cells decreased significantly starting from 5 μg / mL. The LPS IC50 calculated based on the CCK8 assay was 9.56 μg / mL. Figure 16 Therefore, in subsequent studies, 10 μg / mL was used to induce an in vitro inflammatory model. This result indicates that high-dose LPS is cytotoxic in RAW264.7 cells.

[0176] GPX4 is one of the most common markers in current ferroptosis research. Therefore, this example selects GPX4 as a ferroptosis indicator. In order to evaluate the effect of LPS stimulation concentration on the expression of IER3, TF, GPX4, IL-1β, IL-6 and TNF-α mRNA in RAW264.7 cells, RAW264.7 cells were stimulated with LPS. The concentration gradient was set to 0μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, and 10μg / mL, and RT-qPCR detection was performed after incubation for 6 hours. The results of RT-qPCR detection showed that with the increase of LPS stimulation concentration, IER3, coagulation index TF and inflammatory indicators IL-1β, IL-6 and TNF-α in RAW264.7 cells were significantly upregulated, and the expression of ferroptosis indicator GPX4 decreased, as shown in Figure 2. Figure 17 As shown, Figure A shows the changes in IER3; Figure B shows the changes in TF; Figure C shows the changes in GPX4; Figure D shows the changes in IL-1β; Figure E shows the changes in IL-6; and Figure F shows the changes in TNF-α.

[0177] To evaluate the effect of LPS stimulation time on the expression of IER3 and TF mRNA in RAW264.7 cells, this example used LPS to stimulate RAW264.7 cells at a time gradient of 0h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, and 48h, followed by RT-qPCR detection. The RT-qPCR detection results showed that after LPS stimulation, IER3 and TF mRNA in RAW264.7 cells were significantly upregulated, reaching a peak at 6h, and then decreased. Figure 18 As shown, Figure A shows the time gradient change of IER3 expression; Figure B shows the time gradient change of TF expression.

[0178] According to the results of LPS CCK8 and the changes in IER3 and TF mRNA expression in the concentration gradient and time gradient, the protein levels of IER3 and TF in each group of cells were detected by WB after LPS 10μg / mL treatment for 6h. The WB test results showed that IER3 and TF proteins in RAW264.7 cells were significantly upregulated after LPS stimulation. Figure 19 Figure 1 shows the Western blotting of IER3 and TF, Figure A shows the Western blotting of IER3 and TF, Figure B shows the statistical graph of the relative protein levels of IER3, and Figure C shows the statistical graph of the relative protein levels of TF. These results indicate that the expression of IER3 and TF proteins is upregulated in RAW264.7 cells after LPS stimulation.

[0179] The C11 BODIPY581 / 591 probe is an important tool for assessing ROS in cells. Under normal physiological conditions, the C11 BODIPY581 / 591 probe exists in its non-oxidized form and emits red fluorescence. When cells undergo ferroptosis, lipid peroxidation levels increase, and the C11 chain in the probe is oxidized, shifting its red fluorescence to green. This change in fluorescence color and intensity can be used to assess the extent of intracellular lipid peroxidation and determine the occurrence of ferroptosis.

[0180] In this example, C11BODIPY581 / 591 probes were used for fluorescence staining to detect ROS levels in cells. After 6 hours of LPS 10 μg / mL treatment, the non-oxidized state (590 nm) fluorescence (red) was significantly weakened, while the oxidized state (510 nm) fluorescence (green) was significantly enhanced, indicating that the ROS level in the cells was significantly increased. Figure 20 shown.

[0181] In order to evaluate the effect of LPS stimulation time on the expression of IER3 and TF proteins, RAW264.7 cells were stimulated with LPS, and the time gradient was set to 0h, 6h, 12h, 24h, and 48h, followed by WB detection. The test results showed that after LPS stimulation, the expression of IER3 protein in RAW264.7 cells was first significantly upregulated, reached a peak at 6h, and then decreased. After LPS stimulation, the expression of TF protein in RAW264.7 cells was first significantly upregulated, reached a peak at 12h, and then decreased. The results are as follows Figure 21 As shown, Figure A is the WB bands of IER3 and TF; Figure B is the statistical graph of the relative protein amount of IER3; Figure C is the statistical graph of the relative protein amount of TF.

[0182] This example used samples collected from previous research projects with informed consent for a secondary study, divided into a healthy control group and a sepsis group. ELISA was used to detect IER3 expression in the plasma of each group, including 26 people in the healthy control group and 181 people in the sepsis group. The ELISA test results showed that IER3 expression was significantly upregulated in the sepsis group. Figure 22 shown.

[0183] This example also conducted a secondary study using specimens collected with informed consent from previous research projects. The subjects were divided into a healthy control group and a sepsis patient group. RT-qPCR was used to detect the iron death markers Fth1, Ft1, Hmox1, Ptgs2, Ncoa4, Acsl4, Lpcat3, Alox15, S1c3a2, xCT, GPX4, and AIFM2 in the whole blood of the healthy control group and the sepsis patient group. Figure 23.1 Figure A shows the relative mRNA expression of Fthl, Figure B shows the relative mRNA expression of Ftl, Figure C shows the relative mRNA expression of Hmox1, Figure D shows the relative mRNA expression of Ptgs2, Figure E shows the relative mRNA expression of Ncoa4, and Figure F shows the relative mRNA expression of Acsl4; Figure 23.2 Figure G is the relative expression level of Lpcat3 mRNA, Figure H is the relative expression level of Alox15 mRNA, Figure I is the relative expression level of Slc3a2 mRNA, Figure J is the relative expression level of xCT mRNA, and Figure K is the relative expression level of GPX4 mRNA. Figure L RT-qPCR results showed that the mRNA expression of Fth1, Ft1, Hmox1, Ncoa4, Acsl4, Lpcat3, xCT, and GPX4 was significantly downregulated, while the mRNA expression of AIFM2 was significantly upregulated, as shown in Figure 23.

[0184] To evaluate the effect of LPS stimulation time on the mRNA expression of ferroptosis markers, RAW264.7 cells were stimulated with LPS at a time gradient of 0 h, 6 h, 12 h, 24 h, and 48 h, followed by RT-qPCR detection. Figure 24.1 Figure A shows the relative mRNA expression of Fth1, Figure B shows the relative mRNA expression of Ftl, Figure C shows the relative mRNA expression of Hmox1, Figure D shows the relative mRNA expression of Ptgs2, Figure E shows the relative mRNA expression of Ncoa4, and Figure F shows the relative mRNA expression of Acsl4; Figure 24.2 Figure G is the relative expression level of Lpcat3 mRNA, Figure H is the relative expression level of Alox15 mRNA, Figure I is the relative expression level of Slc3a2 mRNA, Figure J is the relative expression level of xCT mRNA, and Figure K is the relative expression level of GPX4 mRNA. Figure L is the relative expression level of AIFM2 mRNA.

[0185] RT-qPCR results showed that the mRNA expression levels of Fth1, Ft1, Ncoa4, Lpcat3, Alox15, and GPX4 were significantly downregulated, while the mRNA expression levels of Ptgs2, Acsl4, and AIFM2 were significantly upregulated. The mRNA expression levels of Hmox1, SLC3A2, and xCT were first significantly upregulated and then decreased, as shown in Figure 24.

[0186] In order to evaluate the effect of LPS stimulation time on the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins, RAW264.7 cells were stimulated with LPS at 0h, 6h, 12h, 24h, and 48h, followed by WB detection. Unexpectedly, the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins was significantly upregulated after LPS stimulation, reaching a peak at 12h, and then significantly decreased at 48h. Figure 25 shown.

[0187] Example 7

[0188] In this example, four si-IER3 sequences were designed and synthesized, namely si-IER3-mus-94, si-IER3-mus-234, si-IER3-mus-368, and si-IER3-mus-435. These four si-IER3s were used to transfect RAW264.7 cells to knock down IER3 expression, and the IER3 knockdown level was detected by RT-qPCR. The RT-qPCR test results are shown in Figure 2. Figure 26 The results showed that si-IER3-mus-94 had the highest knockdown effect, so si-IER3-mus-94 was selected for subsequent experiments.

[0189] RT-qPCR was used to detect IER3 mRNA in RAW264.7 cells after si-IER3 transfection, and WB was used to detect IER3 protein expression in RAW264.7 cells after si-IER3 transfection. The results showed that IER3 knockdown significantly reduced the mRNA and protein expression of IER3 in RAW264.7 cells. Figure 27 shown.

[0190] In this example, si-IER3 was used to transfect RAW264.7 cells to knock down IER3 expression. The MDA content of each group of cells was detected by a malondialdehyde assay kit in the si-NC group, si-IER3 group, si-NC+LPS group, and si-IER3+LPS group to evaluate the ROS level of each group of cells. The test results showed that the MDA content in the si-IER3 group was significantly higher than that in the si-NC group, and the MDA content in the si-IER3+LPS group was significantly higher than that in the si-NC+LPS group. These results indicate that IER3 knockdown increases the ROS level of RAW264.7 cells and RAW264.7 cells after LPS stimulation. The results are shown in Figure 2. Figure 28 shown.

[0191] The mRNA expressions of IER3, TF, PAI-1, IL-1β, IL-6, and TNF-α in the si-NC group, si-IER3 group, si-NC+LPS group, and si-IER3+LPS group were detected by RT-qPCR. The results showed that IER3 knockdown significantly reduced the mRNA expression of IER3 in RAW264.7 cells after LPS stimulation, and significantly upregulated the mRNA expression of TF, PAI-1, IL-1β, IL-6, and TNF-α. Figure 29 The results showed that IER3 knockdown aggravated LPS-induced macrophage coagulation abnormalities and inflammation.

[0192] In this example, WB was used to detect the expression of IER3 and TF proteins in the si-NC group, si-IER3 group, si-NC+LPS group, and si-IER3+LPS group. The results showed that IER3 knockdown significantly reduced the expression of IER3 protein in RAW264.7 cells after LPS stimulation and significantly upregulated the expression of TF protein. Figure 30 The results showed that IER3 knockdown aggravated LPS-induced coagulation abnormalities in macrophages.

[0193] In this example, the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins in the si-NC group, si-IER3 group, si-NC+LPS group, and si-IER3+LPS group was detected by WB. The results showed that IER3 knockdown significantly reduced the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins in RAW264.7 cells after LPS stimulation. Figure 31 The results showed that IER3 knockdown aggravated LPS-induced ferroptosis.

[0194] Example 8

[0195] IER3 overexpression and control RAW264.7 cell lines were purchased from APExBIO. RT-qPCR was used to detect IER3 mRNA levels in the vector group and OE-IER3 group, and WB was used to detect IER3 protein expression in the vector group and OE-IER3 group. The results showed that IER3 mRNA and protein expression in OE-IER3 cells were significantly upregulated, as shown in Figure 2. Figure 32 shown.

[0196] In the vector group, OE-IER3 group, vector+LPS group and OE-IER3+LPS group, the MDA content of each group of cells was detected by malondialdehyde assay kit to evaluate the ROS level of each group of cells. The test results showed that the MDA content in the OE-IER3 group was significantly lower than that in the vector group, and the MDA content in the OE-IER3+LPS group was significantly lower than that in the vector+LPS group. These results indicate that IER3 overexpression reduces the ROS level of RAW264.7 cells and RAW264.7 cells after LPS stimulation. Figure 33 shown.

[0197] RT-qPCR was used to detect the mRNA expressions of IER3, TF, PAI-1, IL-1β, IL-6, and TNF-α in the vector group, OE-IER3 group, vector+LPS group, and OE-IER3+LPS group. The results showed that IER3 overexpression significantly increased the mRNA expression of IER3 in RAW264.7 cells after LPS stimulation, and significantly downregulated the mRNA expressions of TF, PAI-1, IL-1β, IL-6, and TNF-α. Figure 34 The results showed that IER3 overexpression improved LPS-induced coagulation abnormalities and inflammation in macrophages.

[0198] In this example, WB was used to detect the expression of IER3 and TF proteins in the vector group, OE-IER3 group, vector+LPS group, and OE-IER3+LPS group. The test results showed that IER3 overexpression significantly upregulated the expression of IER3 protein in RAW264.7 cells after LPS stimulation and significantly downregulated the expression of TF protein. Figure 35 The results showed that IER3 overexpression improved LPS-induced coagulation abnormalities in macrophages.

[0199] The protein expressions of INRF2, Hmox1, xCT, GPX4, and Fth1 in the vector group, OE-IER3 group, vector+LPS group, and OE-IER3+LPS group were detected by WB. The test results showed that IER3 overexpression significantly upregulated the protein expression of NRF2, Hmox1, xCT, GPX4, and Fth1 in RAW264.7 cells after LPS stimulation. These results indicate that IER3 overexpression improves LPS-induced ferroptosis. The expression levels of NRF2, Hmoxl, xCT, GPX4, and Fth1 in the IER3 overexpression RAW264.7 cell line were also significantly higher than those in the vector group. Figure 36 Therefore, IER3 improves LPS-induced ferroptosis through the NRF2-Hmox1 signaling pathway.

[0200] This example also uses IER3 overexpression plasmid transfection experiments to verify the effects of IER3 overexpression on the NRF2-Hmox1 pathway and downstream ferroptosis markers xCT, GPX4, and Fth1. This example first evaluates the transfection efficiency of the IER3 overexpression plasmid. After the IER3 overexpression plasmid was transfected into RAW264.7 cells, the IER3 mRNA expression in the vector group and the OE-IER3 group was detected by RT-qPCR, and the IER3 protein expression in the vector group and the OE-IER3 group was detected by WB. The results showed that the mRNA and protein expression of ER3 were significantly upregulated after the IER3 overexpression plasmid was transfected into RAW264.7 cells, and the results are as follows: Figure 37 shown.

[0201] The protein expressions of INRF2, Hmox1, xCT, GPX4, and Fth1 in the vector group and OE-IER3 group were detected by WB. Figure 38 It was shown that IER3 overexpression plasmid transfection significantly upregulated the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 proteins in RAW264.7 cells. Figure 41 It was verified that IER3 improved LPS-induced ferroptosis through the NRF2-Hmox1 signaling pathway. Figure 39 It was demonstrated that Fer-1 and MitoQ had no obvious cytotoxicity in RAW264.7 cells, while high doses of RSL3 and Erastin showed significant cytotoxicity in RAW264.7 cells. Figure 40 The concentration gradient changes of TF and PAI-1 mRNA expression in RAW264.7 cells after stimulation with RSL3 and Erastin. Figure 41 The time gradient changes of TF and PAI-1 mRNA expression in RAW264.7 cells after stimulation with RSL3 and Erastin. Figure 42The expression levels of TF proteins in RAW264.7 cells were changed after stimulation with RSL3 and Erastin. Figure 43 RSL3 and Erastin stimulated the increase of ROS levels in RAW264.7 cells. n = 4 in each group. Figure 44 Effects of ferroptosis inhibitors Fer-1 and MitoQ treatment on the mRNA and protein expression levels of IER3 in RAW264.7 cells.

[0202] The relative expression levels of IL-1β, IL-6, and TNF-α mRNA in each group of cells were detected by RT-qPCR. The mRNA levels of IL-1β, IL-6, and TNF-α in the LPS stimulation group were significantly increased compared with the control group, while the mRNA expression levels of IL-1β, IL-6, and TNF-α were significantly decreased after Fer-1 and MitoQ treatment. Figure 45 The results showed that Fer-1 and MitoQ improved LPS-induced macrophage inflammation.

[0203] RT-qPCR was used to detect the relative expression of TF mRNA in each group of cells, and WB was used to detect the relative expression of TF protein. The mRNA and protein expression of TF in the ferroptosis inhibitor Fer-1 and MitoQ treatment groups were significantly higher than those in the LPS stimulation group. The mRNA and protein expression of TF in the LPS+RSL3 stimulation group and the LPS+Erastin stimulation group were significantly higher than those in the LPS stimulation group. Figure 46 The results showed that inhibiting ferroptosis improved LPS-induced coagulation abnormalities in macrophages, while inducing ferroptosis exacerbated coagulation abnormalities, indicating that ferroptosis plays an important role in sepsis-induced coagulation abnormalities. IER3 inhibited ferroptosis and improved sepsis-induced coagulation disorders by upregulating the NRF2-Hmoxl signaling pathway.

[0204] Example 9

[0205] This example explores whether the effect of BELNV in improving coagulation disorders in sepsis is related to IER3 and ferroptosis.

[0206] Mouse mononuclear macrophage RAW264.7 cells were divided into 5 groups: control group (control group); BELNV 1×10 8 particles / mL group (shown in the figure as 10 8 ); BELNV 1×10 9 particles / mL group (shown in the figure as 10 9 ); BELNV 1×10 10 particles / mL group (shown in the figure as 10 10 ); BELNV 1×10 11particles / mL group (shown in the figure as 10 11 ), and each group was added with the same dose of PBS.

[0207] Adult C57BL / 6N male mice (8 weeks old, 18-22 g) were randomly divided into three groups: control group (Control group), sepsis model group (CLP group) and high-dose BELNV treatment group (BELNV 2×10 12 particles / kg body weight + CLP group).

[0208] In this example, the total iron content of each group of cells was detected by tissue iron determination kit. The tissue iron level in the LPS stimulation group was significantly higher than that in the control group, while the tissue iron level was significantly reduced after BELNV treatment. Figure 47 shown.

[0209] In this example, the ROS fluorescence intensity of each group of cells was detected by fluorescence staining and flow cytometry. The ROS fluorescence intensity of the LPS-stimulated group was significantly higher than that of the control group, while the ROS fluorescence intensity was significantly reduced after BELNV treatment. Figure 48 As shown. The results show that BELNV improves LPS-induced macrophage lipid peroxidation and improves ferroptosis. This example also uses a malondialdehyde assay kit to detect the MDA content of each group of cells. The MDA content in the LPS stimulation group was significantly higher than that in the control group, while after BELNV treatment, the MDA content was significantly reduced, as shown in Figure 2. Figure 48 As shown in Figure D in . Figure 48 The results showed that BELNV improved LPS-induced lipid peroxidation and ferroptosis in macrophages.

[0210] Six hours after administration, the mRNA and protein expressions of IER3 in each group of cells were detected by RT-qPCR and WB. The mRNA and protein expressions of IER3 in the BELNV control group were significantly higher than those in the control group, and the IER3 in the LPS stimulation group was significantly higher than that in the control group. The IER3 expression in the BELNV treatment group was significantly higher than that in the LPS stimulation group. Figure 49 The results showed that BELNV improved LPS-induced macrophage injury, which may be related to the upregulation of IER3.

[0211] After 48 hours of drug administration, the protein levels of NRF2, xCT, Hmox1, GPX4, and Fth1 in each group of cells were detected by WB. The expression of NRF2, Hmox1, xCT, GPX4, and Fth1 in the LPS stimulation group was significantly lower than that in the control group, while the expression of NRF2, Hmox1, xCT, GPX4, and Fth1 was significantly increased after BELNV treatment. Figure 50The results showed that BELNV upregulated the NRF2 and Hmox1 pathways and the downstream key ferroptosis proteins xCT, GPX4, and Fth1 in an LPS-induced in vitro inflammation model, thereby inhibiting ferroptosis.

[0212] Figure 51 The results show that BELNV improves ferroptosis-related indicators in septic mice. The total iron content in the lungs and livers of each group of mice was measured using a tissue iron assay kit. The total iron content in the lungs and livers of the sepsis group was significantly increased compared with the control group, while the total iron content in the lungs and livers of mice treated with BELNV was significantly decreased, as shown in Figure A. The MDA content in the lungs and livers of each group of mice was measured using a malondialdehyde assay kit. The MDA content in the lungs and livers of the sepsis group was significantly increased compared with the control group, while the MDA content in the lungs and livers of mice treated with BELNV was significantly decreased, as shown in Figure B. These results indicate that BELNV improves sepsis-induced ferroptosis.

[0213] RT-qPCR and WB were used to detect the mRNA and protein expression of IER3 in the lung and liver tissues of each group. The IER3 expression in the lung and liver of the sepsis group was significantly increased compared with that in the control group, and the IER3 expression in the BELNV treatment group was significantly higher than that in the sepsis group. Figure 52 The results showed that BELNV improved sepsis-induced tissue damage, which may be related to the upregulation of IER3.

[0214] Western blot was used to detect the protein levels of NRF2, xCT, Hmox1, GPX4, and Fth1 in the lung and liver tissues of each group. NRF2 in the lung and liver tissues of the sepsis group was significantly upregulated compared with the control group, while the expression of NRF2 was significantly decreased after BELNV treatment. Figure 53.1 As shown in Figure 3, the expression of Hmox1, xCT, GPX4, and Fth1 in the lung and liver tissues of the sepsis group was significantly lower than that in the control group, while the expression of Hmox1, xCT, GPX4, and Fth1 was significantly increased after BELNV treatment. Figure 53.2 The results in Figure 53 suggest that BELNV can inhibit ferroptosis in a sepsis model by upregulating the IER3-NRF2-Hmox1 axis, thereby improving sepsis-induced coagulopathy.

Claims

1. A method for preparing exosome-like nanovesicles, characterized in that: The preparation method comprises the following steps: extracting beet juice; The beet juice was centrifuged at low speed and the supernatant was collected; The supernatant obtained by low-speed centrifugation is subjected to medium-speed centrifugation and the supernatant is taken; The supernatant obtained by medium-speed centrifugation was subjected to high-speed centrifugation to obtain a precipitate containing exosome-like nanovesicles; The obtained precipitate containing exosome-like nanovesicles was subjected to sucrose density gradient ultracentrifugation to obtain purified exosome-like nanovesicles.

2. The preparation method according to claim 1, characterized in that The low-speed centrifugation step was performed by differential centrifugation at 3,000×g for 30 min at 4°C.

3. The preparation method according to claim 1, characterized in that The intermediate speed centrifugation step was performed by differential centrifugation at 10,000×g for 60 min at 4°C.

4. The preparation method according to claim 1, characterized in that The high-speed centrifugation step was performed by differential centrifugation at 120,000×g for 90 min at 4°C.

5. The exosome-like nanovesicles prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the preparation method according to any one of claims 1 to 4 or the exosome-like nanovesicles according to claim 5 in the preparation of a drug for treating sepsis.

7. Use of the preparation method according to any one of claims 1 to 4 and the exosome-like nanovesicles according to claim 5 in the preparation of a drug for improving sepsis-induced coagulation disorder.

8. Use of the preparation method according to any one of claims 1 to 4 and the exosome-like nanovesicles according to claim 5 in the preparation of a drug for improving macrophage infiltration and fibrin deposition in the lungs and liver caused by sepsis.

9. Use of the preparation method according to any one of claims 1 to 4 and the exosome-like nanovesicles according to claim 5 in the preparation of a drug for increasing platelet and Fbg levels.

10. Use of the preparation method according to any one of claims 1 to 4 and the exosome-like nanovesicles according to claim 5 in the preparation of a drug for reducing plasma TAT and DD levels.

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