Chrysanthemum morifolium-derived exosome-like nano-vesicle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to Hangzhou white chrysanthemum-derived exosome-like nano-vesicles as well as a preparation method and application thereof. Chrysanthemum morifolium ramat nano-vesicles are prepared from compositae plants through a high-speed centrifugation method, the average particle size of the nano-vesicles is 68 nm, and the Zeta potential is-19.50 + / -1.10 mV. The nano vesicles can effectively protect nerve cells and improve anxiety behaviors and cognitive impairment of SAE mice. The compound has a good application prospect in preparation of medicines for treating neuroinflammation and SAE.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an exosome-like nanovesicle derived from Hangzhou white chrysanthemum, its preparation method, and its application. Background Technology

[0002] Sepsis is an organ dysfunction caused by infection with pathogens such as bacteria, fungi, viruses, and parasites. Sepsis-associated encephalopathy (SAE) is a common central nervous system complication in sepsis patients, characterized by brain dysfunction occurring without direct central nervous system infection or structural brain damage. Approximately 70% of sepsis patients are diagnosed with SAE, and its course can be divided into acute and chronic phases based on clinical presentation. In the acute phase, common symptoms of SAE include delirium, seizures, and coma, which are closely associated with increased mortality, complication rates, and cognitive impairment. 25%–50% of sepsis survivors develop cognitive impairment in the chronic phase, manifesting as difficulties with memory, attention, decision-making, and interpersonal communication; severe cases may even develop dementia, mild Alzheimer's disease (AD), and other conditions. Brain damage caused by sepsis also affects mental health, manifesting as post-traumatic stress disorder, anxiety, and depression, which seriously affects long-term quality of life and causes a significant socioeconomic burden.

[0003] Microglia are resident macrophages of the central nervous system (CNS), playing a crucial role in immune surveillance, neuroinflammation, synaptic pruning, and neuronal survival. Under SAE conditions, bacterial material, cytokines, neighboring cells, and imbalanced neurotransmitters can activate microglia, leading to the release of various signaling molecules, including pro-inflammatory cytokines, chemokines, and reactive oxygen species (ROS). The immunomodulatory function and polarization state of macrophages are closely related to the occurrence and development of SAE. Macrophage metabolic reprogramming plays a decisive role in polarization. Lactation, as a novel metabolically relevant post-translational modification, plays a key role in macrophage polarization. In sepsis, enhanced glycolysis leads to lactate accumulation, which in turn regulates histone and non-histone functions through lactation, promoting the activation of pro-inflammatory M1 macrophages and a sustained inflammatory response. Therefore, therapeutic strategies involving microglia reprogramming have shown significant advantages in SAE treatment.

[0004] Many plant-derived products have been shown to have antibacterial, anti-inflammatory, and antioxidant effects. Plant-derived exosome-like nanovesicles (PELNVs), as a natural nanocarrier, contain lipids, proteins, DNA, and miRNAs, and can enter mammalian cells and regulate cellular activity. They exhibit excellent biocompatibility and the ability to cross the blood-brain barrier.

[0005] Hangzhou white chrysanthemum (Chrysanthemum morifolium Ramat.cv.Hangbaiju) is an important cultivated variety of chrysanthemum used for both medicinal and edible purposes. Current research on Hangzhou white chrysanthemum mainly focuses on the chemical composition and bioactivity of its extracts. However, the poor water solubility, rapid metabolism, or insufficient BBB penetration of individual components of Hangzhou white chrysanthemum limit their application in CNS diseases.

[0006] Therefore, it is necessary to develop exosome-like nanovesicles (CRELNVs) derived from Chrysanthemum indicum and explore their potential applications in the treatment of septic encephalopathy. Summary of the Invention

[0007] To address the aforementioned issues, this invention aims to provide a novel and highly effective multi-target therapeutic strategy for SAE, namely, that exosome-like nanovesicles derived from Chrysanthemum morifolium improve SAE by modulating macrophage metabolic reprogramming.

[0008] This invention marks the first successful preparation and isolation of CRELNVs from the medicinal plant *Chrysanthemum indicum*. Verification showed that the CRELNVs obtained by this invention are non-toxic to central nervous system cells and macrophages, and can be efficiently taken up by microglia. They can promote the polarization of the M1 to M2 phenotype, significantly inhibit the generation of ROS and the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α in a neuroinflammatory model, and promote the release of the anti-inflammatory factor IL-10, protecting neurons. Simultaneously, they can intervene in the metabolic reprogramming of microglia, establishing a therapeutic paradigm of immune-metabolic dual-dimensional regulation. In an LPS-induced mouse SAE model experiment, the CRELNVs obtained by this invention significantly reduced inflammatory damage in the hippocampus and effectively alleviated anxiety behavior and cognitive impairment by protecting hippocampal neurons.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] In a first aspect, the present invention provides the application of exosome-like nanovesicles derived from Hangzhou white chrysanthemum in the preparation of drugs for treating septic encephalopathy.

[0011] Furthermore, the exosome-like nanovesicles derived from Hangzhou white chrysanthemum are shaped like tea trays, with a particle size of 30–100 nm and a zeta potential of -19.50 ± 1.10 mV.

[0012] Furthermore, the preparation method of the exosome-like nanovesicles derived from Hangzhou white chrysanthemum includes the following steps:

[0013] (1) Crush the flower heads of Hangzhou white chrysanthemum, homogenize them, filter them, and obtain the filtrate;

[0014] (2) The filtrate was centrifuged at differential speed to obtain crude PELNVs;

[0015] (3) The crude PELNVs were subjected to sucrose gradient ultracentrifugation to obtain purified PELNVs.

[0016] Further, in step (2), the differential centrifugation includes:

[0017] The filtrate was centrifuged at 1000×g and 5000×g for 30 min at 4℃, and the supernatant was collected. The supernatant was centrifuged at 15000×g for 60 min at 4℃, and the supernatant was collected. The supernatant was centrifuged in an ultra-high speed refrigerated centrifuge at 120000×g for 90 min at 4℃ to obtain crude PELNVs.

[0018] Further purification of the obtained crude PELNVs by sucrose gradient ultracentrifugation includes:

[0019] Sucrose solutions with concentrations of 15%, 30%, 45%, and 60% were prepared using Tris-HCl (pH = 7.4). These solutions were added to ultracentrifuge tubes in descending order of concentration, with the crude PELNVs resuspension added last. The tubes were centrifuged at 4°C and 150,000 × g for 2 hours. The 15%–30% sucrose concentration layer was collected, placed in a 100 kDa ultrafiltration tube, and centrifuged at 5,000 × g for 30 minutes to remove the sucrose solution, yielding the purified PELNVs precipitate.

[0020] Furthermore, the application is the use of exosome-like nanovesicles derived from Chrysanthemum morifolium in the preparation of drugs to alleviate septic encephalopathy.

[0021] Furthermore, the relief of septic encephalopathy involves regulating microglial cell metabolic reprogramming to improve SAE by downregulating the secretion of pro-inflammatory factors / upregulating the secretion of anti-inflammatory factors.

[0022] Furthermore, the pro-inflammatory cytokines include IL-1β, IL-6, and TNF-α; the anti-inflammatory cytokines include IL-10.

[0023] Furthermore, the drug for treating septic encephalopathy has at least one of the following effects:

[0024] (1) Promotes microglial phenotype polarization from M1 to M2;

[0025] (2) Reduce the content of lactate, a characteristic metabolite of M1 microglia;

[0026] (3) Reduces inflammation and damage in the hippocampus and protects hippocampal neurons;

[0027] (4) Improve anxiety behavior and cognitive impairment.

[0028] Furthermore, the septic encephalopathy is caused by any one of the systemic inflammatory responses induced by bacteria, fungi, viruses, or LPS.

[0029] The present invention also provides a drug comprising the exosome-like nanovesicles derived from Chrysanthemum morifolium prepared above.

[0030] This invention provides an exosome-like nanovesicle (CRELNVs) derived from Hangzhou white chrysanthemum, wherein the CRELNVs are rich in flavonoids, lignans, coumarins and phenolic acids.

[0031] On the other hand, the CRELNVs were prepared using the "gold standard" of exosome extraction, through differential centrifugation, ultracentrifugation, and sucrose density gradient purification steps. CRELNVs possess exosome morphology, zeta potential, and particle size characteristics that meet the requirements of MISEV2018, and belong to PELNVs.

[0032] The preparation method of this invention can yield exosomes with high purity and high particle number.

[0033] The CRELNVs prepared in this invention were characterized by an average particle size of 68 nm, a zeta potential of -19.50 ± 1.10 mV, and a particle concentration of 9.97 × 10⁻⁶ mV through multi-dimensional characterization. 11 The particles / mL concentration was high, with a vesicle ratio of 97.10%, exhibiting a typical saucer-like structure characteristic of exosomes, and possessing high purity, meeting the basic requirements for PELNVs. Bioactive component identification results showed that CRELNVs were rich in flavonoids, lignans, coumarins, and phenolic acids. Cell experiments confirmed that CRELNVs were non-toxic to central nervous system cells and macrophages, and could be efficiently taken up by BV2 cells.

[0034] The CRELNVs prepared using this invention can effectively inhibit LPS-induced inflammatory activity in microglia. In a neuroinflammation model, they exhibit significant neuroprotective, anti-inflammatory, anti-oxidative stress, and microglia polarization phenotype-regulating effects, reducing the content of lactate, a characteristic metabolite of M1 microglia, and downregulating protein lactation levels. Administered via tail vein, CRELNVs can cross the blood-brain barrier and reach the brain, improving the efficiency of delivery of the active ingredients to the brain. In an LPS-induced mouse SAE model, the CRELNVs obtained using this invention effectively improved anxiety-like behavior and cognitive impairment in SAE mice.

[0035] The beneficial effects of this invention include at least the following:

[0036] (1) The exosome-like nanovesicles derived from Chrysanthemum morifolium prepared in this invention can significantly reduce inflammatory damage in the hippocampus and effectively reduce anxiety behavior and cognitive impairment by protecting neurons in the hippocampus.

[0037] (2) The exosome-like nanovesicles derived from Chrysanthemum morifolium prepared in this invention can exhibit significant neuroprotective, anti-inflammatory, antioxidant stress and microglia polarization phenotype regulation effects in a neuroinflammatory model. They can reduce the content of lactic acid, a characteristic metabolite of M1 microglia, and downregulate the level of protein lactation.

[0038] (3) The exosome-like nanovesicles derived from Chrysanthemum morifolium prepared by this invention can cross the blood-brain barrier, providing a novel delivery system. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the preparation process of CRELNVs in Embodiment 1 of the present invention.

[0040] Figure 2 Characterization of CRELNVs obtained in Example 1 of the present invention. (A) Images of each density layer after purification by sucrose density gradient; (B) Electron micrographs of CRELNVs obtained in Example 1 of the present invention; (C) Zeta potential of CRELNVs obtained in Example 1 of the present invention; (D) Particle size and concentration of CRELNVs obtained in Example 1 of the present invention; (E) Particle size and concentration of CRELNVs after Triton X-100 treatment.

[0041] Figure 3 Identification of the bioactive components contained in CRELNVs prepared in Example 1 of this invention.

[0042] Figure 4 This is a schematic diagram illustrating the construction and treatment plan for a mouse SAE model.

[0043] Figure 5The effects of CRELNVs prepared in Example 1 of this invention on activity levels and anxiety behavior in SAE mice. (A) Mouse activity trajectory diagram; (B) Total distance traveled by the mouse; (C) Activity time of the mouse in the central region.

[0044] Figure 6 The effect of CRELNVs prepared in Example 1 of this invention on cognitive impairment in SAE mice. (A) Heatmap of mouse activity; (B) Novel object recognition index of mice; (C) Spontaneous alternation rate of mice.

[0045] Figure 7 The effects of CRELNVs prepared in Example 1 of this invention on hippocampal neuronal damage and microglial activation in SAE mice. (A) H&E, Nissl, and TUNEL staining to observe the effects of CRELNVs on hippocampal neurons in SAE mice; the effects of CRELNVs on IL-1β (B), IL-6 (C), TNF-α (D), and IL-10 (E) in the hippocampus of SAE mice; (F) Immunofluorescence image of Iba-1 in the hippocampus of mice; (G) 2.4 mm 2 (H) Number of microglia in the field of view; (I) Number of branch nodes of microglia; (H) Total length of branches of microglia.

[0046] Figure 8 The effects of CRELNVs prepared in Example 1 of this invention on the secretion of IL-1β (A), IL-6 (B), TNF-α (C) and IL-10 (D) in BV2 cells.

[0047] Figure 9 The effect of CRELNVs prepared in Example 1 of this invention on the protein expression of BV2 cell polarization markers CD86 and CD206. (A) Flow cytometry results; (B, C) Quantitative analysis.

[0048] Figure 10 The effect of CRELNVs prepared in Example 1 of this invention on lactate content in BV2 cells. Detailed Implementation

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0051] The following specific embodiments illustrate the solution proposed in this invention:

[0052] Example 1: Preparation, Separation and Purification of CRELNVs

[0053] The specific steps are as follows:

[0054] 1) Weigh 50g of the capitulum of a chrysanthemum (Hangzhou white chrysanthemum), soak it in 100mL PBS, homogenize it in a tissue homogenizer for 1min, add 50mL of pre-cooled PBS at 4℃, let it stand for 1min, and repeat 3 times. Filter the homogenate through a 300-mesh sieve and collect the filtrate.

[0055] 2) Centrifuge the filtrate collected in 1) at 4°C with centrifugal forces of 1000×g and 5000×g for 30 minutes respectively.

[0056] Remove large particles such as cells and protoplasts using a sterilizing agent, and collect the supernatant. Incubate the supernatant at 4°C for 15,000 minutes.

[0057] Centrifuge at ×g for 60 min to remove plant fibers, cell debris, and large vesicles, and collect the supernatant. Centrifuge the supernatant in an ultra-high-speed refrigerated centrifuge at 4°C, 120,000 ×g for 90 min to obtain crude PELNVs.

[0058] 3) Prepare sucrose solutions with concentrations of 15%, 30%, 45%, and 60% (m / m) using Tris-HCl (pH = 7.4). Add these solutions to ultracentrifuge tubes in descending order of concentration, and finally add 2 mL of the crude PELNVs resuspension obtained in step 2). Centrifuge at 4°C and 150,000 × g for 2 h using an ultracentrifuge. Collect the 15%–30% sucrose concentration layer and transfer it to 15 mL of centrifuge tube.

[0059] In a 100 kDa ultrafiltration tube, Tris-HCl was added to a final volume of 15 mL. The tube was centrifuged at 5000 × g for 30 min to remove the sucrose solution, yielding purified PELNVs precipitate. The precipitate was resuspended in 2 mL of PBS, filtered through a 0.22 μm filter membrane for sterilization, and stored at -80 °C. Aliquots were then used to avoid repeated freeze-thaw cycles.

[0060] The preparation process diagram is as follows: Figure 1 As shown.

[0061] Characterization analysis of CRELNVs in Example 2

[0062] After purification, CRELNVs were mainly distributed in the 15%–30% sucrose concentration layer, as shown in the results. Figure 2 As shown in Figure A, the CRELNVs prepared in Example 1 were observed by transmission electron microscopy, and the results are as follows: Figure 2 As shown in Figure B, the surface charge distribution of CRELNVs was measured at 25°C using a nanoparticle size zeta potential analyzer, and the results are as follows. Figure 2 As shown in Figure C. The particle size and concentration of CRELNVs were determined using a Coulter nanoparticle size analyzer. After treatment with 10% Triton X-100, the particle size and concentration were measured again to calculate the vesicle ratio. The results are shown in Figure C. Figure 2 As shown in D and 2E.

[0063] The results showed that the CRELNVs prepared by this invention were uniform in size, with an average particle size of 68 nm, a Zeta potential of -19.50 ± 1.10 mV, and a particle concentration of 9.97 × 10⁻⁶. 11 The particles / mL and vesicle ratio were 97.10%, exhibiting the typical saucer-like structure of exosomes, and possessing high purity, yield, and vesicle ratio.

[0064] Example 3: Determination of Bioactive Components in CRELNVs

[0065] The bioactive components in CRELNVs were identified using ultra-high performance liquid chromatography-mass spectrometry, and the results are as follows: Figure 3 As shown, metabolomics detected 1660 small molecule compounds, mainly including flavonoids, phenolic acids, alkaloids, lignans, coumarins, and terpenoids, which possess a variety of pharmacological activities. The synergistic effect of these multi-component, multi-target bioactive components provides a scientific basis for the material foundation and mechanism of action of CRELNVs in the treatment of SAE.

[0066] Example 4: The effect of CRELNVs on improving anxiety behavior in SAE mice

[0067] 1. Laboratory animals

[0068] Male C57BL / 6 mice, weighing 20–22 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All animal experimental procedures were approved and implemented by the Ethics Committee of the Animal Facility of Zhejiang Provincial People's Hospital. Mice were housed in an SPF-grade environment at the Animal Facility of Zhejiang Provincial People's Hospital, with an indoor temperature of 20–26℃, a daily temperature difference of ≤4℃, and a day / night cycle of 12 / 12h.

[0069] 2. Construction of a mouse SAE model and treatment regimen

[0070] According to the literature, an SAE model was established by intraperitoneal injection of 10 mg / kg LPS. Mice were randomly divided into four groups: Control group, LPS group, LPS+CRELNVs-L (4 mg / kg) group, and LPS+CRELNVs-H (8 mg / kg) group. Figure 4 As shown, 2 hours after LPS injection, the LPS group and the CRELNVs administration group received equal volumes of PBS and CRELNVs via tail vein injection, respectively. PBS or CRELNVs were then injected every two days. After behavioral experiments on days 6 and 7, the animals were sacrificed, and samples were collected. A schematic diagram of the SAE model construction and treatment protocol is shown below. Figure 4 As shown.

[0071] 3. Behavioral research

[0072] 3.1 Open Field Experiment

[0073] The experimental setting was an open box measuring 50cm × 50cm × 40cm, with a camera mounted directly above the box to record the mice's movement. The experiment was conducted in a quiet environment. Mice were gently placed in the corner of the open box, facing the lower right corner, and allowed to move freely. Their movement was recorded over 8 minutes. After the experiment, the mice were isolated to avoid disturbing other mice, and the open box was cleaned, disinfected, and odor removed. Tracking Master behavioral analysis software was used to process the videos, specifically analyzing the total distance traveled and the time spent in the central area for each group of mice. Results are as follows: Figure 5 As shown in Figure 5A, the behavioral trajectory images of each group of mice were displayed. The total distance traveled by each group of mice in the open field was statistically analyzed (5B). It was found that the total distance traveled by mice in each group was similar except for the Control group. Furthermore, the total distance traveled by mice in the Control group was not significantly different from that of other groups, indicating that the spontaneous motor ability of the mice had basically recovered after modeling during the behavioral experiment, eliminating errors caused by the mice's motor ability. To evaluate anxiety behavior, the activity time of mice in the central region was statistically analyzed (5C). It was found that the activity time in the central region of mice in the LPS group was significantly lower than that in the Control group (P<0.01), indicating that the mice in this group exhibited anxious emotional behavior. The activity time in the central region of mice in the LPS+CRELNVs-L group was slightly increased compared to the LPS group, but due to large individual differences, the difference was not statistically significant. However, the activity time in the central region of mice in the LPS+CRELNVs-H group was significantly increased (P<0.01), recovering to a level similar to that of the Control group, indicating that high-dose CRELNVs can alleviate anxiety behavior in mice.

[0074] 3.2 New Object Recognition Experiment

[0075] The experimental site was the same as an open field. The day before the formal experiment, mice were placed in an open box and allowed to move freely for 5 minutes to acclimatize to the environment and avoid stress. On the day of the formal experiment, mice were first allowed to move freely in an empty open box for 10 minutes. Then, two identical objects were placed approximately 10 cm from each side wall of the open box, and the mice were allowed to freely explore the two objects for 10 minutes to observe any preference for the same object or its location. After a 1-hour interval, the objects on one side were replaced with different objects of similar shape, and the mice were allowed to freely explore the old and new objects for 5 minutes. When the distance between the mouse's nose and the object was less than 2 cm, it was considered to be in exploratory behavior. Analysis software was used to count the exploration time of the mice for the old and new objects and calculate the recognition index. The experimental results are as follows: Figure 6 As shown in Figure 6A, the motion trajectory images of mice in each group during the novel object recognition experiment are displayed. Further statistical analysis of the recognition ability of various mice for novel objects was conducted (6B). The results showed that compared with the Control group, the LPS group mice exhibited a significant decrease in their ability to recognize novel objects (P<0.001). Compared with the LPS group, the recognition index of the LPS+CRELNVs-L group increased, but the difference was not statistically significant, while the recognition index of the CRELNVs-H group increased significantly (P<0.01), indicating that the cognitive impairment of the LPS+CRELNVs-H group mice was improved.

[0076] 3.3Y Maze Experiment

[0077] The Y-maze consists of three arms of equal length, each 40cm x 10cm, with an angle of 120° between them. The three arms are labeled A, B, and C. A mouse is placed at the end of one arm, facing the central area. The mouse is allowed to freely explore each arm for 8 minutes. The order and number of times the mouse enters each arm are recorded, and the spontaneous alternation rate is calculated. The experimental results are as follows: Figure 6 As shown in Figure C, in the Y maze experiment, the spontaneous alternation rate decreased in the LPS group (P<0.001), while the spontaneous alternation rate increased significantly after treatment with CRELNVs-L and CRELNVs-H compared to the LPS group (P<0.05, P<0.001), indicating that CRELNVs can improve the spatial memory ability of mice.

[0078] Example 5: Effects of CRELNVs on SAE Mouse Hippocampal Nerve Injury and Microglial Cell Activation

[0079] 1. Testing Method

[0080] 1) Brain tissue sample collection

[0081] To avoid interference from autofluorescence in mouse blood during the experiment, the blood vessels were flushed clean via cardiac perfusion before sampling. After blood collection, the mice were secured to a foam board, and the chest cavity was carefully cut open to expose the heart. A blunted IV needle was inserted into the left ventricle from the apex to the upper left, and then the needle was secured with surgical forceps. 20–40 mL of physiological saline was infused into the heart until the liver turned off-white, followed by 20 mL of 4% paraformaldehyde to rapidly fix the brain tissue. Successful perfusion was indicated by limb twitching and tail erection observed after the infusion of 4% paraformaldehyde. After perfusion, the brain tissue was removed; at this stage, the brain tissue should be off-white and without visible blood vessels. The brain tissue was then immersed in 4% paraformaldehyde and fixed at room temperature for 24 hours for later use.

[0082] 2) Preparation of tissue sections

[0083] The fixed tissue was removed, trimmed with a scalpel, and placed in a dehydration box. The dehydration box was placed in a basket and dehydrated in a dehydrator using an ethanol concentration gradient of 70%-80%-90%-100%. Then, the tissue was cleared with a xylene:anhydrous ethanol (1:1) solution and xylene, and finally immersed in paraffin wax. After paraffin immersion, the tissue was placed in an embedding frame and solidified on a -20°C freezing stage. The paraffin block was then removed and trimmed. The paraffin block was cut into 4μm sections using a microtome, the tissue was flattened, and baked in a 60°C oven until the water evaporated and the wax was cured. The sections were then stored at room temperature for later use.

[0084] 3) H&E staining

[0085] Dewax the paraffin sections in xylene, then hydrate them using an ethanol concentration gradient of 100%-90%-80%-70%, followed by rinsing with distilled water. After drying, stain with hematoxylin for 5 minutes. Differentiate with 1% hydrochloric acid and ethanol for 3 seconds, wash with water, and then re-blue with 0.6% ammonia solution, followed by rinsing with distilled water. Add eosin stain and stain for 5 minutes. Next, dehydrate using an ethanol concentration gradient of 80%-90%-95%-100%, clear with xylene, air dry at room temperature, mount with neutral resin, and observe under a microscope.

[0086] 4) Nissl staining

[0087] After dewaxing to water, immerse the sections in Nissl stain for 5 minutes, rinse with distilled water, dehydrate with a gradient of ethanol, clear with xylene, and finally mount for observation.

[0088] 5) TUNEL staining

[0089] Dewax to water, add 20 μg / ml proteinase K. Incubate at 37℃ for 15 min, wash 3 times with PBS, incubate at room temperature with 3% H2O2 for 20 min to inactivate peroxidase, wash 3 times with PBS. Prepare biotin labeling solution, add 50 μL of biotin labeling solution to each sample, incubate at 37℃ in the dark for 60 min, wash once with PBS, add 0.2 mL of stop solution, incubate at room temperature for 10 min, wash with PBS. Prepare Streptavidin-HRP working solution and DAB chromogenic solution, add 50 μL of Streptavidin-HRP working solution to each sample, incubate at room temperature for 30 min, wash with PBS, add 0.3 mL of DAB chromogenic solution, incubate at room temperature for 20 min, wash with PBS. Stain with hematoxylin solution for 5 min, wash with PBS, dehydrate with ethanol gradient, clear with xylene, and finally mount for observation.

[0090] 6) Microglial cell immunofluorescence and 3D remodeling

[0091] Brain tissue was fixed and dehydrated, and frozen sections were prepared by OCT embedding. Sections were washed three times with PBS to remove embedding medium, permeabilized with 0.1% Triton X-100 for 30 min, washed three times, and blocked with immunofluorescence rapid blocking solution for 30 min. Iba-1 primary antibody was diluted 1:200 with immunofluorescence primary antibody dilution buffer and incubated at 4°C for 24 h. The primary antibody was recovered, and the sections were washed three times with PBS. 488 fluorescent secondary antibody was diluted with immunofluorescence secondary antibody dilution buffer and incubated at room temperature in the dark for 1 h, followed by three washes. Brain slices were developed on glass slides using the surface tension of PBS. Excess PBS was aspirated, 10 μL of DAPI-containing anti-quenching mounting medium was added, and covers with a coverslip and fixed with clear nail polish. After drying, the slices were observed under a 63× oil immersion microscope using a super-resolution confocal microscope, with images stacked in 1 μm steps along the z-axis. The fluorescence images were reconstructed in 3D using the "Fliaments" module in Imaris 10.1.0 software, and the number of branch nodes and total branch length of microglia were quantified.

[0092] 2. Test Results

[0093] 2.1 Effects of CRELNVs on hippocampal neurons in SAE mice

[0094] The hippocampus plays a crucial role in learning, memory, and emotion regulation. Highly sensitive to inflammation and hypoxia, it is the most vulnerable site in sepsis, hence cognitive impairment is frequently observed in SAE patients. To investigate the effects of CRELNVs on hippocampal neurons in SAE mice, H&E staining was used to observe pathological changes in the hippocampus, and Nissl and TUNEL staining was used to observe neuronal activity. Figure 7A) H&E staining results showed that the CA1 region in the LPS group had numerous edematous vacuoles caused by inflammatory infiltration and disordered cell arrangement, while CRELNV treatment reduced vacuoles and resulted in more orderly cell arrangement, indicating that CRELNVs can alleviate inflammatory damage. Nissl staining revealed disordered and sparse hippocampal neurons in the CA3 region of the LPS group, with blurred or missing Nissl bodies, while CRELNV treatment increased the number of neurons and Nissl bodies. TUNEL staining observed neuronal apoptosis in the DG region; the LPS group showed an increase in apoptotic positive cells, while CRELNV treatment reduced the number of apoptotic positive cells. Therefore, CRELNVs can improve neuronal damage in the hippocampus.

[0095] 2.2 Effects of CRELNVs on cytokine levels in the hippocampus of SAE mice

[0096] The expression of four cytokines, IL-1β, IL-6, TNF-α, and IL-10, in the mouse hippocampus was detected by ELISA. The results are as follows: Figure 7 As shown in the BE study, the levels of IL-1β, IL-6, TNF-α, and IL-10 proteins in the hippocampus of LPS-treated mice were all elevated (P<0.001), with the elevation of the three pro-inflammatory cytokines being more significant than that of IL-10. However, after CRELNVs treatment, the levels of the three pro-inflammatory cytokines were significantly reduced (P<0.05, P<0.01, P<0.001), while IL-10 expression increased (P<0.05). This indicates that CRELNVs can also inhibit the expression of pro-inflammatory factors and promote the expression of anti-inflammatory factors in vivo.

[0097] 2.3 Effects of CRELNVs on the number and morphology of microglia in the hippocampus of SAE mice

[0098] Iba-1 is a microglia / macrophage-specific calcium-binding protein that participates in cell migration and phagocytosis by binding to F-actin to label its cytoskeleton. Inflammatory stimuli can induce upregulation of Iba-1 expression and promote the transformation of microglia into an amoeboid morphology: increased cell body size, shortened processes, and reduced branching (from 3-4 order long branches in the resting state to a short, thick morphology), while simultaneously secreting inflammatory factors. Resting-state (M0 phenotype) microglia perform immune surveillance and synaptic pruning functions through a highly branched network of processes.

[0099] Iba-1 was used to label microglia to investigate the effects of CRELNVs on the number and morphology of microglia in the hippocampus of SAE mice (7F-G). An increase in microglia was observed in the LPS group (P<0.001), indicating microglia migration to the hippocampus. However, the number of microglia in the hippocampus decreased after CRELNVs treatment compared to the LPS group (P<0.001). A 3D model of microglia was constructed using Imaris to quantify the branch length (7I) and branch node number (7H) of microglia. The Control group showed highly branched microglia, in an M0 state. In contrast, the LPS group showed significantly shortened branch length (P<0.001) and significantly reduced branch node number (P<0.001), exhibiting an amoeboid appearance, indicating activation of microglia in this group. After CRELNVs treatment, the branch length and number of branch nodes of microglia in this group were significantly increased compared with those in the LPS group (P<0.001, P<0.01), indicating that CRELNVs can also play a regulatory role on microglia in vivo.

[0100] Example 6: Experiment on CRELNVs regulating microglia polarization and metabolic reprogramming

[0101] 1. Experimental Grouping

[0102] Control group: PBS buffer.

[0103] LPS+IFN-γ group: LPS solution was prepared with PBS buffer (dosage concentration: 100 ng / mL), and IFN-γ solution was used to dissolve the protein with 5% trehalose solution according to the instructions (dosage concentration: 20 ng / mL). CRELNVs group: The protein concentration of CRELNVs obtained in Example 1 was determined by BCA. Based on the protein concentration, the groups were divided into a low-dose CRELNVs group (100 μg / mL), a medium-dose CRELNVs group (200 μg / mL), and a high-dose CRELNVs group (400 μg / mL).

[0104] Positive drug group: Dexamethasone (DEX)

[0105] 2. Experimental Methods

[0106] 2.1 ELISA detection of cytokine levels secreted by BV2 cells

[0107] BV2 cells were loaded at 5 × 10 5Cells were seeded at a density of 10 cells / well in 6-well plates, with 2 mL of complete culture medium added to each well. After 24 h of incubation, the complete culture medium was discarded. The Control group was replaced with 2 mL of culture medium containing 20 μL PBS, and the inflammation model group was replaced with 2 mL of culture medium containing 100 ng / mL LPS and 20 ng / mL IFN-γ, and incubated for 24 h. The CRELNVs group was pretreated with different concentrations of CRELNVs for 2 h, then replaced with culture medium containing the same concentrations of CRELNVs, LPS, and IFN-γ, and incubated for 24 h. The DEX group was replaced with 2 mL of culture medium containing 10 μM DEX. The cell culture supernatant was collected, and the levels of IL-1β, IL-6, TNF-α, and IL-10 in the supernatant of BV2 cells in each group were detected according to the ELISA kit instructions.

[0108] Experimental results are as follows Figure 8 As shown, the levels of IL-1β (8A), IL-6 (8B), and TNF-α (8C) in the supernatant of BV2 cells in the LPS+IFN-γ group were significantly increased (P<0.001), while the levels of these three cytokines decreased with increasing CRELNVs dosage after administration (P<0.05, P<0.001). 400 μg / mL CRELNVs showed better inhibitory effects on IL-1β and IL-6 than dexamethasone (DEX), while 200 μg / mL CRELNVs showed comparable inhibitory effects to dexamethasone. Furthermore, 200 and 400 μg / mL CRELNVs significantly promoted the secretion of IL-10 (8D) (P<0.01, P<0.001). These results indicate that CRELNVs exert a protective effect on neuronal cells by inhibiting the secretion of pro-inflammatory cytokines and promoting the secretion of anti-inflammatory cytokines.

[0109] 2.2 Flow cytometry detection of CD86 and CD206 protein levels in BV2 cells

[0110] Cells were treated as described in section 2.1, with LPS and IFN-γ treatment groups and IL-4 treatment groups serving as positive controls for CD86 and CD206, respectively. Cells were digested and collected, washed with PBS, and resuspended in 120 μL of PBS in each tube. 20 μL of cell suspension was taken from each sample tube and mixed to serve as a blank tube. The LPS and IFN-γ treatment groups were used as CD86 single-staining tubes, and the IL-4 treatment group as CD206 single-staining tubes, with 100 μL in each tube. 0.1 μL of the cell viability dye FVS510 was added to each sample tube, and the tubes were incubated at room temperature in the dark for 15 min, followed by washing with stain buffer. After resuspending in 100 μL of stain buffer, 2 μL of FC block was added, and the tubes were blocked at room temperature in the dark for 10 min, washed with stain buffer, and resuspended. 1.5 μL of CD86 and 1.5 μL of LCD11b fluorescent antibodies were added to each sample tube. Add 1.5 μL of CD86 fluorescent antibody to the CD86 single-stain tube, mix well, and incubate at 4°C in the dark for 1 h, then wash with stain buffer. Add 250 μL of fixation / permeabilization buffer to the blank tube, single-stain tube, and sample tube, and incubate at 4°C in the dark for 30 min, then centrifuge at 400×g for 6 min and collect the precipitate. Wash twice with 1 mL of washing buffer and resuspend in stain buffer. Add 1.5 μL of CD206 fluorescent antibody to the sample tube and CD206 single-stain tube, mix well, and incubate at 4°C in the dark for 30 min. Wash and resuspend the cells with stain buffer, pass through a 300-mesh filter, and then analyze. The experimental results are shown in Figure 9. In the LPS and IFN-γ groups, the CD86 positivity rate increased to 66.3±5.3% (P<0.001), while the CD206 positivity rate did not change significantly compared with the control group, indicating that most cells in this group were polarized to the pro-inflammatory M1 phenotype. After administration of CRELNVs, the CD86 positivity rate decreased significantly (P<0.001), while the CD206 positivity rate increased significantly (P<0.001), indicating that CRELNVs promoted the polarization of BV2 cells towards the M2 phenotype. Unlike CRELNVs, after DEX treatment, the expression of both CD86 and CD206 decreased, and the number of M0 microglia increased significantly, suggesting that DEX may exert its anti-inflammatory effect by inhibiting microglia activation.

[0111] 2.3 Detection of lactate content in BV2 cells

[0112] Metabolic reprogramming of macrophages is a core regulatory mechanism for their functional phenotype transition. M1 macrophages convert their metabolic phenotype to glycolysis via iNOS-mediated arginine breakdown, while M2 macrophages convert their phenotype to oxidative phosphorylation via Arg-1-mediated arginine hydrolysis. In M1 microglia, glucose is broken down into pyruvate via glycolysis, and pyruvate is further reduced to lactate by lactate dehydrogenase. As a key end product of glycolysis, lactate not only serves as an indicator of energy metabolism but also plays a crucial role in regulating the immune microenvironment. Lactate levels in cells were detected using the NBT method. Results are as follows: Figure 10 As shown, the lactate level in the LPS+IFN-γ group (2) was significantly higher than that in the Control group (1) (P<0.001), indicating that the metabolic phenotype of BV2 cells may be transformed into glycolysis and M1 polarization. However, the lactate level decreased after CRELNVs treatment (P<0.001), inhibiting M1 polarization.

[0113] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0114] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0115] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of exosome-like nanovesicles derived from Hangzhou white chrysanthemum in the preparation of drugs for treating septic encephalopathy.

2. The application according to claim 1, characterized in that, The exosome-like nanovesicles derived from Hangzhou white chrysanthemum are shaped like tea trays, with a particle size of 30–100 nm and a zeta potential of -19.50 ± 1.10 mV.

3. The application according to claim 1, characterized in that, The preparation method of the exosome-like nanovesicles derived from Hangzhou white chrysanthemum includes the following steps: (1) Crush the flower heads of Hangzhou white chrysanthemum, homogenize them, filter them, and obtain the filtrate; (2) The filtrate was centrifuged at differential speed to obtain crude PELNVs; (3) The crude PELNVs were subjected to sucrose gradient ultracentrifugation to obtain purified PELNVs.

4. The application according to claim 3, characterized in that, In step (2), the differential centrifugation includes: The filtrate was centrifuged at 1000×g and 5000×g for 30 min at 4℃, and the supernatant was collected. The supernatant was centrifuged at 15000×g for 60 min at 4℃, and the supernatant was collected. The supernatant was centrifuged in an ultra-high speed refrigerated centrifuge at 120000×g for 90 min at 4℃ to obtain crude PELNVs.

5. The application according to claim 3, characterized in that, In step (3), the process of subjecting the obtained crude PELNVs to sucrose gradient ultracentrifugation includes: Sucrose solutions with concentrations of 15%, 30%, 45%, and 60% were prepared using Tris-HCl at pH 7.

4. These solutions were added to ultracentrifuge tubes in descending order of concentration, with the crude PELNVs resuspension added last. The tubes were centrifuged at 150,000 × g for 2 hours at 4°C. The 15%–30% sucrose concentration layer was collected, placed in a 100 kDa ultrafiltration tube, and centrifuged at 5,000 × g for 30 minutes to remove the sucrose solution, yielding the purified PELNVs precipitate.

6. The application according to claim 1, characterized in that, The application is the use of exosome-like nanovesicles derived from Chrysanthemum morifolium in the preparation of drugs to alleviate septic encephalopathy. The relief of septic encephalopathy is achieved by regulating microglia metabolic reprogramming to improve SAE, downregulating the secretion of pro-inflammatory factors / upregulating the secretion of anti-inflammatory factors.

7. The application according to claim 6, characterized in that, The pro-inflammatory cytokines include IL-1β, IL-6, and TNF-α; the anti-inflammatory cytokines include IL-10.

8. The application according to claim 1, characterized in that, The drug for treating septic encephalopathy has at least one of the following effects: (1) Promotes microglial phenotype polarization from M1 to M2; (2) Reduce the content of lactate, a characteristic metabolite of M1 microglia; (3) Reduces inflammation and damage in the hippocampus and protects hippocampal neurons; (4) Improve anxiety behavior and cognitive impairment.

9. The application according to claim 1, characterized in that, The septic encephalopathy is caused by any one of the systemic inflammatory responses induced by bacteria, fungi, viruses, or LPS.

10. A drug, characterized in that, Including the exosome-like nanovesicles derived from Chrysanthemum morifolium prepared in any one of claims 3 to 5.

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