Zanthoxylum nitidum-sourced external vesicle, preparation method thereof and application of external vesicle in preparation of acute lung injury prevention and treatment medicine

Abstract

The invention belongs to the technical field of biological medicine, and discloses a zanthoxylum nitidum-sourced external vesicle, a preparation method and an application in preparation of a medicine for preventing and treating acute lung injury, and the external vesicle with the particle size of 60-100 nm and a lipid bilayer structure is extracted and purified from the root of zanthoxylum nitidum for the first time. The external vesicle can be administered in modes of aerosol inhalation and the like, and shows an excellent curative effect in an acute lung injury model: lung tissue edema and inflammatory cell infiltration can be remarkably relieved, levels of key proinflammatory factors such as IL-6, TNF-alpha, IL-1beta and the like in serum are reduced, macrophages are promoted to be differentiated to an anti-inflammatory M2 type, and the curative effect is remarkable. The treatment effect is better than that of traditional dexamethasone, and the side effect is lower. The invention provides a brand-new, efficient and safe candidate drug and a natural nano treatment strategy for preventing and treating acute lung injury and ARDS.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an external vesicle derived from Zanthoxylum nitidum, its preparation method, and its application in the preparation of drugs for the prevention and treatment of acute lung injury. Background Technology

[0002] Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are among the leading causes of death in intensive care patients. Its core pathological features include damage to alveolar epithelial cells and capillary endothelial cells, massive release of inflammatory factors, and inflammatory cell infiltration, leading to a rapid decline in lung function. Despite ongoing research into the pathogenesis of ALI / ARDS, highly effective and precise treatment methods remain lacking in clinical practice. Existing treatment strategies, such as mechanical ventilation, may cause nonspecific lung tissue damage, while hormone therapy, due to immunosuppression, is prone to complications such as infection, making it difficult to achieve a balanced regulation of "inflammation resolution-tissue repair."

[0003] Zanthoxylum nitidum (Roxb.) DC., a plant belonging to the genus Zanthoxylum in the Rutaceae family, is used medicinally for its roots. It is included in the Chinese Pharmacopoeia and is one of the "Ten Delicacies of Guangxi," a traditional Chinese medicine with the functions of promoting blood circulation, removing blood stasis, detoxifying, and reducing swelling. Modern pharmacological studies have shown that the pharmacological activity of Zanthoxylum nitidum mainly comes from alkaloid components (such as nitidine chloride and chelidonine), which have anti-inflammatory, antiviral, and immunomodulatory effects. However, as a traditional Chinese medicine, Zanthoxylum nitidum faces many limitations in its clinical application: traditional dosage forms (decoctions, pills, etc.) have complex components, low bioavailability, significant first-pass effect, and difficulty in fully releasing and absorbing the active ingredients; it lacks a targeting mechanism, resulting in uneven drug distribution in the body and difficulty in accurately targeting diseased lung tissue; the preparation process is complex, with large quality differences between batches, making standardized production and quality control difficult.

[0004] Plant-derived exosome-like nanoparticles (PELNs) are nanoscale vesicles (30-150 nm) encapsulated in a lipid bilayer, carrying active ingredients such as nucleic acids and proteins. They possess advantages such as wide availability, low immunogenicity, high biocompatibility, and natural targeting, and can mediate intercellular communication and precisely regulate target tissue function, showing broad prospects in the treatment of diseases such as inflammation and tumors, as well as drug delivery. Currently, there are no reports on the extraction of exosomes from *Zanthoxylum nitidum* and their application in the prevention and treatment of acute lung injury. Therefore, developing a method for efficiently extracting high-purity exosomes from *Zanthoxylum nitidum* and clarifying their application value in the treatment of acute lung injury has significant clinical significance and application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses an external vesicle derived from Zanthoxylum nitidum, its preparation method, and its application in the preparation of drugs for the prevention and treatment of acute lung injury.

[0006] This invention includes the following specific technical solutions:

[0007] An external vesicle derived from Zanthoxylum nitidum, wherein the external vesicle is a nanoparticle with a lipid bilayer structure extracted from the root of Zanthoxylum nitidum.

[0008] This invention creatively extracts and purifies Zanthoxylum nitidum external vesicles (ZNEXO) from the root of Zanthoxylum nitidum. These external vesicles are typically round or elliptical, have a complete lipid bilayer structure, uniform particle size ranging from 60 to 100 nm, stable composition, and good biocompatibility.

[0009] In some embodiments, it has been demonstrated that the extravesicles of *Zanthoxylum nitidum* can be effectively taken up by target cells such as macrophages, and exert a role in preventing and treating acute lung injury through the following mechanism:

[0010] 1. Significantly reduces inflammatory cell infiltration in the alveolar spaces, and improves lung tissue edema and structural damage;

[0011] 2. Effectively reduces serum levels of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β;

[0012] 3. Promotes the differentiation of macrophages into anti-inflammatory (M2) types and regulates the balance of the inflammatory microenvironment;

[0013] 4. Improves the rate of weight recovery in mice with acute lung injury, reduces the lung coefficient, and improves pathological damage to lung tissue.

[0014] Furthermore, the present invention also discloses that the above-mentioned external vesicles derived from Zanthoxylum nitidum are prepared by a method comprising the following steps:

[0015] (a) Enzymatic hydrolysis: After the root tissue of Zanthoxylum nitidum is chopped, it is enzymatically hydrolyzed in a solution containing cellulase and pectinase at 45-55℃.

[0016] (b) Preliminary purification: Centrifuge the enzyme hydrolysate and collect the supernatant;

[0017] (c) Exovesicle separation: The supernatant from step (b) was subjected to low-speed centrifugation, high-speed centrifugation and filtration in sequence to remove cell debris and large particulate impurities;

[0018] (d) Ultracentrifugation enrichment: The filtrate from step (c) is subjected to ultracentrifugation, and the precipitate is collected to obtain the external vesicles derived from the two-sided needle.

[0019] Furthermore, in the above application, in step (a), the enzymatic hydrolysis solution contains 2-4% (w / v) cellulase, 1-3% (w / v) pectinase and 0.5-0.7 mol / L mannitol, with a pH of 5.5-6.0.

[0020] Furthermore, in the above application, in step (c), the conditions for low-speed centrifugation are 1500-2500 ×g, and the conditions for high-speed centrifugation are 8000-12000 ×g; in step (d), the conditions for ultracentrifugation are 80,000-120,000 ×g.

[0021] Furthermore, the two-sided needle-like vesicles of the present invention can be obtained by the following preferred extraction method, which is simple to operate, has good repeatability, and can effectively improve the yield and purity of the vesicles:

[0022] (1) Sample pretreatment: Wash the roots of Zanthoxylum nitidum with distilled water, dry the surface moisture with filter paper, cut into small pieces of 1mm×1mm, soak in 75% alcohol for 1 minute for sterilization, rinse with distilled water to remove alcohol residue, and wipe dry with sterile paper; add 50mL enzyme solution (containing 4% cellulase, 2% pectinase, 0.6mol / L mannitol, pH5.8), and enzymatically hydrolyze at 50℃ for 6h; then centrifuge at 16000×g for 1h to allow the tissue residue from enzymatic hydrolysis to settle, collect the supernatant, and freeze at -80℃.

[0023] (2) Gradient centrifugation purification: Thaw the frozen supernatant at 37℃ at medium speed, transfer it to a centrifuge tube, centrifuge at 2000×g and 4℃ for 30 min, and collect the supernatant; transfer the supernatant to a new centrifuge tube, centrifuge at 10000×g and 4℃ for 45 min to remove larger vesicles and impurities, and collect the supernatant; filter the supernatant through a 0.45μm microporous membrane and collect the filtrate; transfer the filtrate to an ultracentrifuge tube, ultracentrifuge at 100000×g and 4℃ for 70 min, discard the supernatant, and collect the precipitate.

[0024] (3) Resuspension and purification: Add 10 mL of pre-cooled 1×PBS buffer to the precipitate for resuspension, and centrifuge again at 100000×g and 4℃ for 70 min. Discard the supernatant. Resuspend the precipitate with 150 μL of pre-cooled 1×PBS buffer and filter through a 0.22-0.45 μm microporous membrane to obtain the Zanthoxylum nitidum exovesicles. Aliquot the obtained exovesicles and store them at -80℃ for later use.

[0025] The present invention also discloses the use of the above-mentioned external vesicles derived from Zanthoxylum nitidum in the preparation of medicaments for the prevention and / or treatment of acute lung injury (ALI).

[0026] Furthermore, in the above applications, the acute lung injury includes acute diffuse lung injury conditions such as acute respiratory distress syndrome (ARDS).

[0027] Furthermore, in the above applications, the dosage form of the drug is a pulmonary administration formulation; preferably, the pulmonary administration formulation is a nebulized inhaler, a dry powder inhaler, or an aerosol. Furthermore, the drug for preventing and treating acute lung injury according to the present invention can be prepared into various dosage forms suitable for clinical use, preferably nebulizers, and also includes solutions, injections, aerosols, or sprays. The drug may also contain pharmaceutically acceptable excipients, such as diluents (e.g., physiological saline, glucose solution), stabilizers (e.g., albumin), preservatives (e.g., benzalkonium chloride), or solubilizers (e.g., polyethylene glycol), etc. The selection and dosage of excipients can be conventionally determined according to dosage form requirements.

[0028] The present invention also discloses a pharmaceutical composition comprising a therapeutically effective amount of Zanthoxylum nitidum-derived exovesicles and a pharmaceutically acceptable carrier, wherein the particle size of the Zanthoxylum nitidum-derived exovesicles ranges from 60 to 100 nm; preferably, the pharmaceutical composition is used for the prevention and / or treatment of acute lung injury.

[0029] This invention also discloses the use of Zanthoxylum nitidum-derived vesicles in the preparation of formulations for promoting the differentiation of anti-inflammatory macrophages or inhibiting the polarization of pro-inflammatory macrophages. In some embodiments, Zanthoxylum nitidum vesicles can promote the differentiation of macrophages (such as RAW264.7 cells) into anti-inflammatory (M2 type) types, regulate the inflammatory microenvironment, and provide new targets and formulation options for the treatment of inflammatory diseases.

[0030] The present invention also discloses a method for non-therapeutic purposes, the method comprising: contacting cells or tissues requiring modulation of inflammatory response with an effective amount of external vesicles derived from Zanthoxylum nitidum, thereby reducing the level of one or more pro-inflammatory factors in the cells or tissues, and / or promoting the differentiation of anti-inflammatory macrophages;

[0031] The pro-inflammatory factors are selected from IL-6, TNF-α, and IL-1β.

[0032] Compared with the prior art, the present invention has the following outstanding advantages:

[0033] 1. Provides new active substances and clear characterization: This invention is the first to successfully isolate and purify the outer vesicles from the root of Zanthoxylum nitidum, clarifying its basic physical characteristics such as lipid bilayer structure and particle size mainly distributed in 60-100 nanometers. Through experiments, its morphology, particle size distribution and composition were systematically characterized, laying the foundation for in-depth research on this substance.

[0034] 2. A stable and feasible extraction and purification process was established: This invention provides an extraction and purification method that includes enzymatic hydrolysis, gradient centrifugation, and ultracentrifugation enrichment. This method has a clear process route and well-defined operating parameters, enabling relatively efficient extraction of high-purity, uniformly sized exovesicles from plant tissues, providing a reference scheme for potential large-scale preparation and quality control.

[0035] 3. Demonstrating therapeutic potential in disease models: Experimental results show that the Zanthoxylum nitidum vesicles obtained by the method of this invention can reduce pulmonary edema, decrease inflammatory cell infiltration, and downregulate serum levels of pro-inflammatory factors such as IL-6 and TNF-α in a mouse model of lipopolysaccharide-induced acute lung injury. Cellular experiments further suggest that it may exert its anti-inflammatory effect by influencing the polarization state of macrophages.

[0036] 4. This study expands new avenues for the utilization of traditional Chinese medicine resources: It combines the traditional Chinese medicine *Zanthoxylum nitidum* with modern nanovesicle research, providing new ideas and material carriers for exploring its potential medicinal value. The obtained exovesicles exhibit good biocompatibility and are suitable for preparation into pulmonary drug delivery formulations such as nebulized inhalation, exploring a possible direction for its application in the field of respiratory diseases. Attached Figure Description

[0037] Figure 1 Characterization results of Znexocysts (ZNEXO): A. Morphology of the vesicles under transmission electron microscopy (typical round / elliptical lipid bilayer structure); B. Particle size distribution of the vesicles detected by a nanoparticle tracer (particle size range 60-100 nm); C. Statistical analysis of the average concentration of vesicles extracted three times; D. Results of protein expression detection in vesicles; E. Results of miRNA expression detection in vesicles.

[0038] Figure 2 HE staining images of mouse lung tissue sections: in order, control group (Ctrl), LPS model group (LPS), dexamethasone positive control group (LPS+DEX), and Zanthoxylum bungeanum vesicle administration group (LPS+ZNEXO). The images show that the ZNEXO group has reduced alveolar structure destruction and reduced inflammatory cell infiltration.

[0039] Figure 3 The statistical results of lung coefficients in each group of mice are shown in the figure: the lung coefficient of the LPS+ZNEXO group is significantly lower than that of the model group, indicating that the degree of lung edema and inflammation is reduced;

[0040] Figure 4 The image shows the pathological scoring results of lung tissue stained with HE: the pathological score of the LPS+ZNEXO group was significantly lower than that of the model group, and the degree of lung injury was significantly improved;

[0041] Figure 5The daily weight gain trend graphs for each group of mice show that the LPS+ZNEXO group recovered weight faster than the LPS+DEX group and the model group.

[0042] Figure 6 Figure 1 shows the results of serum inflammatory factor levels in mice of each group: A. IL-6 level; B. TNF-α level; C. IL-1β level; The results show that the levels of each inflammatory factor in the LPS+ZNEXO group were significantly lower than those in the model group and better than those in the LPS+DEX group.

[0043] Figure 7 Western Blot results for promoting the differentiation of anti-inflammatory (M2 type) macrophages by external vesicles of Zanthoxylum nitidum: The LPS+ZNEXO group showed upregulation of Arg1, a marker of M2 type macrophages, and downregulation of iNOS, a marker of M1 type macrophages.

[0044] Figure 8 Biosafety evaluation showed that, at effective doses, ZNEXO had no significant effect on zebrafish embryonic development. Figure 8 A), no significant pathological changes were observed in the major organs (heart, liver, spleen, lungs, kidneys, and brain) of mice. Figure 8 B). Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0046] Example 1

[0047] Extraction and characterization of Zanthoxylum nitidum exovesicles (ZNEXO)

[0048] This embodiment aims to illustrate a specific method for efficiently extracting high-purity exovesicles from the root of Zanthoxylum nitidum and to characterize their basic physicochemical properties.

[0049] 1.1 Raw Materials and Reagents

[0050] Fresh roots of Zanthoxylum nitidum were purchased from a local medicinal herb cultivation base in Guangxi and identified as roots of Zanthoxylum nitidum (Roxb.) DC., a plant belonging to the Rutaceae family and the Zanthoxylum genus. Cellulase and pectinase were purchased from Yuanye Biotechnology, and D-mannitol was purchased from Shanghai Sangon Biotech. Phosphate-buffered saline (PBS), RIPA lysis buffer, and BCA protein concentration assay kit were all commercially available biochemical reagents.

[0051] 1.2 Methods for extracting external vesicles The specific steps are as follows: (1) Sample pretreatment: Take 500g of fresh Zanthoxylum nitidum roots, wash with distilled water, and blot dry with filter paper. In a sterile operating table, chop the roots into small pieces of about 1 mm³. Immerse the tissue pieces in 75% ethanol for 1 minute for surface sterilization, and then rinse 3 times with sterile distilled water to remove ethanol residue. Transfer the treated tissue to a container, add 50 mL of enzymatic hydrolysis solution (containing 4% w / v cellulase, 2% w / v pectinase, 0.6 mol / L mannitol, and adjust the pH to 5.8 with hydrochloric acid). Incubate in a 50℃ constant temperature water bath for 6 hours for enzymatic hydrolysis.

[0052] (2) Preliminary clarification: After the enzymatic hydrolysis is completed, the mixture is centrifuged at 4℃ and 16,000 ×g for 1 hour. The supernatant is carefully collected and frozen in a -80℃ freezer for later use.

[0053] (3) Gradient centrifugation purification: Thaw the frozen supernatant in a 37°C water bath. First, centrifuge at 4°C and 2,000 ×g for 30 minutes and collect the supernatant; centrifuge the supernatant again at 4°C and 10,000 ×g for 45 minutes to further remove large particles and organelles; collect the supernatant and filter it through a microporous membrane with a pore size of 0.45 μm.

[0054] (4) Ultracentrifugation enrichment: Transfer the filtrate into an ultracentrifuge tube and ultracentrifuge at 4℃ and 100,000 ×g for 70 minutes. Discard the supernatant and the precipitate at the bottom of the tube is the crude extracted vesicle.

[0055] (5) Washing and purification: Gently resuspend the above precipitate in 10 mL of pre-chilled 1×PBS buffer. Wash again by ultracentrifugation at 4°C and 100,000 ×g for 70 minutes, discarding the supernatant to remove soluble protein impurities. Finally, resuspend the precipitate in 150 μL of pre-chilled 1×PBS buffer and filter through a 0.22 μm sterile filter membrane. The resulting filtrate is the purified ZnexO (zanthoxylum nitidum) suspension. Aliquot and store at -80°C.

[0056] 1.3 Characterization results of external vesicles

[0057] (1) Morphology and size: 10 μL of ZNEXO suspension was negatively stained with 2% phosphotungstic acid and observed using a transmission electron microscope (TEM, Hitachi HT-7700). Figure 1 As shown in Figure A, ZNEXO exhibits a typical round or near-round vesicle structure with a clearly defined lipid bilayer membrane. Particle size analysis was performed using a nanoflow cytometer (NanoFCM N30E), and the results showed ( Figure 1B) The particle size of ZNEXO is mainly distributed in the range of 60-100 nm, with an average particle size of about 72.8 nm and a uniform distribution. (2) Concentration and protein content: The average particle concentration of ZNEXO extracted three times independently was approximately 6.74 × 10⁻⁶ using nanoflow cytometry. 9 particles / mL ( Figure 1 C). Protein concentration was determined using the BCA method, and the protein concentration of the extracted ZNEXO stock solution was approximately 8-9 μg / μL (corresponding to experimental report data). (3) Preliminary component analysis: Extravesicle-related protein markers can be detected by Western blotting ( Figure 1 D). Total RNA was extracted from vesicles, and small RNA (miRNA) bands were detected by electrophoresis and Qsep100 analysis. Figure 1 E), indicating that it carries nucleic acid components. (4) Sterility: ZNEXO was added to LB medium and cultured for 24 hours. No bacterial growth was observed, which proved that the extraction process and the final product were sterile.

[0058] Example 2

[0059] The therapeutic effect of Zanthoxylum nitidum external vesicles on a mouse model of acute lung injury

[0060] This embodiment uses a lipopolysaccharide (LPS)-induced acute lung injury mouse model to verify the in vivo therapeutic effect of ZNEXO.

[0061] 2.1 Animal model construction and drug administration

[0062] SPF-grade male C57BL / 6 mice (6-8 weeks old, weighing 20-25 g) were selected and randomly divided into 4 groups (n=8) after one week of acclimatization:

[0063] Control group (Ctrl): Intraperitoneal injection of an equal volume of normal saline.

[0064] Model group (LPS): LPS (5 mg / kg, dissolved in sterile PBS) was injected intraperitoneally to induce acute lung injury.

[0065] Positive drug group (LPS+DEX): Dexamethasone (5 mg / kg) was injected intraperitoneally 1 hour before LPS injection.

[0066] Treatment group (LPS+ZNEXO): One hour before LPS injection, ZNEXO nebulized solution (at a dose equivalent to 10 mg / kg) was administered via a nebulizer. Nebulized administration allows the drug to be deposited directly in the lungs.

[0067] All mice were observed for 7 days after modeling, and their weight changes were recorded daily.

[0068] 2.2 Efficacy evaluation indicators and results

[0069] (1) Lung coefficient: Mice were sacrificed 24 hours after the last administration, and intact lung tissue was removed. After blotting away surface blood with filter paper, the wet weight was measured. The lung coefficient (lung wet weight / body weight × 100%) was calculated. Figure 3 As shown, the lung coefficient in the LPS model group was significantly higher than that in the control group (P<0.01), indicating significant pulmonary edema. The lung coefficient in the LPS+ZNEXO treatment group was significantly lower than that in the model group (P<0.05) and also lower than that in the LPS+DEX positive drug group, suggesting that ZNEXO can effectively reduce pulmonary edema and inflammatory exudation.

[0070] (2) Pathological analysis of lung tissue: Left lung lobe was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Figure 2 It was observed that the alveolar structure in the control group remained intact; the alveolar structure in the model group was severely damaged, with thickened septa and extensive inflammatory cell infiltration. In contrast, the alveolar structural integrity was well maintained in the LPS+ZNEXO treatment group, and inflammatory cell infiltration was significantly reduced. Statistical analysis was performed using a semi-quantitative pathological score (0-3 points). Figure 4 The results showed that the pathological score of the LPS+ZNEXO group was significantly lower than that of the model group (P<0.01), and the efficacy was better than that of the dexamethasone group.

[0071] (3) Weight recovery status: such as Figure 5 As shown, mice experienced weight loss after LPS modeling. The LPS+ZNEXO treatment group recovered significantly faster than the model group and LPS+DEX group from day 3, with the highest weight recovery rate on day 7, indicating that ZNEXO can promote the overall recovery of the model animals.

[0072] (4) Serum inflammatory factor levels: Mouse serum was collected, and the levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β were detected using an ELISA kit. Results are as follows: Figure 6 As shown, the levels of various inflammatory factors in the LPS model group increased sharply. LPS+ZNEXO treatment significantly reduced the levels of IL-6, TNF-α, and IL-1β in serum (all P<0.01 vs. model group), and the reduction was greater than that in the dexamethasone group, demonstrating its strong systemic anti-inflammatory effect.

[0073] Example 3

[0074] Regulatory role of Zanthoxylum bungeanum outer vesicles on macrophage polarization

[0075] This embodiment elucidates part of the mechanism by which ZNEXO exerts its anti-inflammatory effect at the cellular level, namely, regulating the transformation of macrophages from a pro-inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2).

[0076] 3.1 Cellular Experimental Methods

[0077] The mouse mononuclear macrophage cell line RAW264.7 was used. The experiment was divided into 4 groups:

[0078] Control group: Routine culture.

[0079] Model group (LPS): M1 polarization was induced by stimulation with 1 μg / mL LPS for 24 hours.

[0080] Positive control group (LPS+DEX): 100 nmol / L dexamethasone was added during LPS stimulation.

[0081] Experimental group (LPS+ZNEs): ZNEXO (final concentration 1×10⁻⁶) was added concurrently with LPS stimulation. 9 (particles / mL) were co-cultured for 24 hours.

[0082] 3.2 Test Results

[0083] (1) Cell phenotype conversion: After culture, total cell protein was extracted, and the expression of inducible nitric oxide synthase (iNOS), a marker of M1 macrophages, and arginase 1 (Arg1), a marker of M2 macrophages, was detected by Western blotting. The results are as follows: Figure 7 As shown, iNOS expression was significantly upregulated and Arg1 expression was downregulated in the LPS group. Compared with the LPS group, iNOS expression was significantly inhibited and Arg1 expression was significantly upregulated in the LPS+ZNEs group (P<0.05). This effect was superior to the LPS+DEX group, indicating that ZNEXO can actively reprogram macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type.

[0084] (2) Secretion of inflammatory factors: Cell culture supernatant was collected for detection. Consistent with in vivo experimental results, ZNEXO treatment significantly inhibited the secretion of TNF-α, IL-6 and IL-1β by LPS-induced RAW264.7 cells, while promoting the secretion of anti-inflammatory factor IL-10, thus confirming its ability to remodel the anti-inflammatory microenvironment at the cellular level.

[0085] Example 4

[0086] Other bioactivities and pharmaceutical formulations of Zanthoxylum nitidum outer vesicles

[0087] 4.1 Other protection mechanisms

[0088] Further research indicates that ZNEXO also possesses the following multi-target protective effects:

[0089] Protecting the endothelial barrier: ZNEXO can inhibit LPS-induced increase in pulmonary microvascular endothelial cell barrier permeability.

[0090] Anti-fibrotic potential: In the inflammatory microenvironment, ZNEXO can inhibit the transformation of fibroblasts into myofibroblasts and reduce the deposition of extracellular matrix collagen, suggesting that it may delay the progression of acute lung injury to pulmonary fibrosis.

[0091] Protection of alveolar organoids: In a three-dimensional alveolar organoid model, ZNEXO can alleviate LPS-induced inflammatory responses and tissue damage.

[0092] 4.2 Pharmaceutical Preparations

[0093] Based on the above-mentioned activities, the ZNEXO prepared in Example 1 can be combined with pharmaceutically acceptable excipients to formulate a dosage form suitable for clinical administration. A preferred dosage form is a direct pulmonary administration formulation to maximize local drug concentration and reduce systemic exposure.

[0094] Nebulized inhalation preparation: Mix ZNEXO suspension with physiological saline, or add a trace amount of stabilizer (such as human serum albumin), and load it into a nebulizer. This is suitable for the administration method described in Example 2.

[0095] Dry powder inhaler: ZNEXO is prepared into powder using freeze-drying technology, mixed with carriers such as inhaled lactose, and then filled into capsules or blister packs for administration using a dry powder inhaler.

[0096] Aerosol: ZNEXO is dispersed in a propellant and stabilizer and packaged in a pressurized metered-dose inhalation canister.

[0097] In addition, it can also be prepared into injections (such as liposome-encapsulated formulations for intravenous injection) or solutions, as needed. The preparation of all these dosage forms can be carried out with reference to conventional techniques in this field.

[0098] 4.3 Security

[0099] like Figure 8 Preliminary biosafety assessments showed that, at effective doses, ZNEXO had no significant effect on zebrafish embryonic development. Figure 8 A), no significant pathological changes were observed in the major organs (heart, liver, spleen, lungs, kidneys, and brain) of mice. Figure 8 B) indicates that it has a good biosafety basis.

[0100] In summary, this invention is the first to successfully extract extravesicles with specific particle size and structure from Zanthoxylum nitidum, and its remarkable efficacy in preventing and treating acute lung injury has been fully demonstrated through in vitro and in vivo experiments. Its mechanism is related to multiple functions, including regulating macrophage polarization, inhibiting excessive inflammatory responses, and protecting the lung tissue barrier. This invention provides a solid technical solution and experimental basis for developing novel nanomedicines derived from traditional Chinese medicine to treat acute lung injury.

[0101] Summary of Examples: This invention fully verifies the preparation, characterization, and therapeutic effects and mechanisms of Zanthoxylum nitidum external vesicles in treating acute lung injury through specific examples. Example 1 details the process of efficiently extracting Zanthoxylum nitidum external vesicles using enzymatic hydrolysis combined with differential / ultracentrifugation, and confirms that they possess typical nanovesicle morphology and uniform particle size. Example 2 demonstrates in an LPS-induced mouse model of acute lung injury that nebulized inhalation of these external vesicles significantly improves pathological damage to lung tissue, reduces the lung coefficient, promotes weight recovery, and effectively inhibits systemic inflammation. Example 3 reveals at the cellular level that these external vesicles can be taken up by macrophages and drive macrophages to anti-inflammatory M2 polarization by upregulating Arg1 and downregulating iNOS expression, thereby reshaping the inflammatory microenvironment.

[0102] The foregoing embodiments are merely illustrative examples of preferred embodiments of the present invention and do not constitute any limitation on the scope of protection of the present invention. For those skilled in the art, any modifications, equivalent variations, and alterations made to the present invention without departing from the principles and spirit of the invention should be considered to fall within the scope of protection defined by the appended claims.

Claims

1. An external vesicle derived from Zanthoxylum nitidum, characterized in that, The external vesicles are nanoparticles with a lipid bilayer structure extracted from the root of Zanthoxylum nitidum.

2. The method for preparing exovesicles derived from *Zanthoxylum nitidum* as described in claim 1, characterized in that, Includes the following steps: (a) Enzymatic hydrolysis: After the root tissue of Zanthoxylum nitidum is chopped, it is enzymatically hydrolyzed in a solution containing cellulase and pectinase at 45-55℃. (b) Preliminary purification: Centrifuge the enzyme hydrolysate and collect the supernatant; (c) Exovesicle separation: The supernatant from step (b) was subjected to low-speed centrifugation, high-speed centrifugation and filtration in sequence to remove cell debris and large particulate impurities; (d) Ultracentrifugation enrichment: The filtrate from step (c) is subjected to ultracentrifugation, and the precipitate is collected to obtain the external vesicles derived from the two-sided needle.

3. The method according to claim 2, characterized in that, In step (a), the enzymatic hydrolysis solution contains 2-4% (w / v) cellulase, 1-3% (w / v) pectinase and 0.5-0.7 mol / L mannitol, with a pH of 5.5-6.

0.

4. The method according to claim 2, characterized in that, In step (c), the conditions for low-speed centrifugation are 1500-2500 ×g, and the conditions for high-speed centrifugation are 8000-12000 ×g. In step (d), the ultracentrifugation conditions are 80,000-120,000 ×g.

5. The use of the external vesicles derived from Zanthoxylum nitidum as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of acute lung injury (ALI).

6. The application according to claim 5, characterized in that, The acute lung injury includes acute diffuse lung injury conditions such as acute respiratory distress syndrome (ARDS).

7. The application according to claim 5, characterized in that, The dosage form of the drug is a pulmonary administration preparation; preferably, the pulmonary administration preparation is a nebulized inhaler, a dry powder inhaler, or an aerosol.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the vesicles derived from *Zanthoxylum nitidum* as described in claim 1, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is used for the prevention and / or treatment of acute lung injury.

9. The use of the external vesicles derived from Zanthoxylum nitidum as described in claim 1 in the preparation of formulations for promoting the differentiation of anti-inflammatory macrophages.

10. A method for non-therapeutic purposes, characterized in that, The method includes: under in vitro conditions, contacting macrophages with an effective amount of external vesicles derived from *Zanthoxylum nitidum* as described in claim 1, thereby detecting their effect on macrophage polarization or inflammatory factor secretion.

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