Specific dermatitis treatment uses of bidens pilosa exosomes

Abstract

The application relates to the technical field of the application of bidens pilosa exosome, and specifically discloses a specific dermatitis treatment purpose of bidens pilosa exosome. The bidens pilosa exosome is secreted by bidens pilosa tissue, and the bidens pilosa tissue is selected from any one or more of bidens pilosa leaves, stems and roots. The bidens pilosa exosome has a significantly excellent treatment effect on specific dermatitis.

Description

Technical Field

[0001] This application relates to the technical field of Bidens pilosa exosomes, and more specifically, it relates to the specific therapeutic use of Bidens pilosa exosomes for dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a prevalent, genetically predisposed skin disease in children, characterized by intense itching and a high recurrence rate. The causes of AD are complex, closely related to environmental factors, autoimmune disorders, and genetics. The main clinical manifestations include acute and chronic eczematous lesions, dry skin, and significant itching, severely impacting patients' daily lives. Currently, topical medications are commonly used for treatment, with corticosteroids being the most widely applied. However, these medications are prone to relapse upon discontinuation. Other topical corticosteroids and immunosuppressants can alleviate symptoms, but long-term use can lead to side effects such as rashes, stinging, and atrophy. Therefore, developing treatments for AD that are free of side effects and prone to relapse after discontinuation is essential.

[0003] Regarding the side effects, plant extracts, due to their biocompatibility, hold promise for treating atopic dermatitis with no or low side effects. Among them, Bidens pilosa (Bidens pilosa)... Bidens pilosa L Bidens pilosa is routinely used in food and medicine with no obvious side effects. Many parts of the plant, including its flowers and stems, can be used to treat various ailments and have multiple medicinal functions, such as acting as an antipyretic and analgesic, and treating gastrointestinal bleeding and eczema. Pharmacological studies have shown that Bidens pilosa extract possesses a wide range of biological activities, including antioxidant, anticancer, antidiabetic, anti-inflammatory, antimicrobial, and immunomodulatory activities.

[0004] Therefore, it is necessary to develop the therapeutic applications of Bidens pilosa exosomes for treating atopic dermatitis. Summary of the Invention

[0005] To address the above issues, this application provides a specific therapeutic use of Bidens pilosa exosomes for dermatitis.

[0006] The application of Bidens pilosa exosomes for the treatment of atopic dermatitis provided in this application adopts the following technical solution:

[0007] The use of Bidens pilosa exosomes for the treatment of atopic dermatitis, wherein the Bidens pilosa exosomes are secreted by Bidens pilosa tissue, which is selected from any one or more of Bidens pilosa leaves, stems, and roots.

[0008] This application demonstrates through cell experiments that Bidens pilosa exosomes can reduce the expression of chemokines (TNFα, CCL22, IL-6, and RANTES) in cells, proving the alleviating effect of Bidens pilosa exosomes on atopic dermatitis. Furthermore, animal experiments, using skin lesion scoring and measuring ear swelling thickness, showed that after administration of Bidens pilosa exosomes to mice with atopic dermatitis, the area of ​​dorsal skin lesions decreased, wound healing was better, and the skin lesion score was significantly lower than that of untreated mice, with a significant reduction in ear swelling thickness; these results demonstrate that Bidens pilosa exosomes can significantly alleviate the symptoms of atopic dermatitis. In addition, H&E staining results revealed that the dorsal skin lesions of mice with atopic dermatitis showed parakeratosis or hyperkeratosis, significant epidermal thickening, and extensive inflammatory cell infiltration, with significant edema in the lesion tissue; these symptoms were significantly improved in mice treated with Bidens pilosa exosomes, proving the ameliorative effect of Bidens pilosa exosomes on atopic dermatitis in mice.

[0009] The above experimental results fully demonstrate the therapeutic potential of Bidens pilosa exosomes for atopic dermatitis.

[0010] Optionally, the method for preparing the Bidens pilosa exosomes includes the following steps:

[0011] Bidens pilosa tissue was taken, added to buffer solution and crushed. After solid-liquid separation, the supernatant was collected and microfiltered to obtain a Bidens pilosa pretreated sample.

[0012] Bidens pilosa pretreated samples were subjected to ultracentrifugation to obtain Bidens pilosa exosomes.

[0013] Optionally, the ultracentrifugation specifically includes the following steps:

[0014] The pretreated Bidens pilosa sample was centrifuged at 1500-2500g for 25-35 min; the supernatant was then centrifuged at 8000-12000g for 40-50 min and microfiltered; the filtrate was centrifuged at 80000-120000g for 65-75 min; the precipitate was resuspended in buffer and then centrifuged at 80000-120000g for 65-75 min to obtain Bidens pilosa exosomes.

[0015] Optionally, the average particle size of the Bidens pilosa exosomes was 79.0 nm, and the concentration was (1.2-1.4)×10⁻⁶. 9 per ml.

[0016] Optionally, the solid-liquid separation of the Bidens pilosa tissue after pulverization specifically includes the following steps:

[0017] After the Bidens pilosa tissue was crushed and filtered, the supernatant was centrifuged at 8000-12000 rpm for 15-25 min.

[0018] Optionally, the Bidens pilosa exosomes are used to prepare a composition for treating atopic dermatitis, wherein the composition further comprises any one or more excipients, dispersants, pharmaceutical additives, and probiotics.

[0019] Optionally, the Bidens pilosa exosomes are used to prepare drugs for treating atopic dermatitis or as adjunctive therapy.

[0020] Optionally, the dosage form of the drug and adjuvant therapy drug is selected from any one or more of microneedle preparations, topical liquid preparations, topical solid preparations, topical semi-solid preparations, plasters, patches, and topical gaseous preparations.

[0021] In summary, this application has the following beneficial effects:

[0022] This application provides a novel use of Bidens pilosa exosomes in the treatment of atopic dermatitis, which can significantly improve skin symptoms in atopic dermatitis model mice and has excellent therapeutic effects on atopic dermatitis. Attached Figure Description

[0023] Figure 1 This is a flowchart of a cell experiment.

[0024] Figure 2 This is a flowchart illustrating the process of animal experiments;

[0025] Figure 3 This is an electron micrograph of the Bidens pilosa exosomes from Example 2;

[0026] Figure 4 This is a particle size and concentration analysis diagram of Bidens pilosa exosomes from Example 2;

[0027] Figure 5 This is a graph showing the cytokine expression results of different treatment groups;

[0028] Figure 6 These are photographs of the backs and ears of mice in different treatment groups;

[0029] Figure 7 The skin lesion scores and ear swelling thickness of mice in different treatment groups;

[0030] Figure 8 These are images showing the H&E staining results of the ears and backs of mice in different treatment groups. Detailed Implementation

[0031] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that: unless otherwise specified in the following embodiments, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources.

[0032] 1. Transmission electron microscopy observation of Bidens pilosa exosomes

[0033] The specific experimental steps are as follows: Take 10 μL of the obtained Bidens pilosa exosomes and drop them onto a copper grid to precipitate for 1 min, then use filter paper to absorb the floating liquid; drop 10 μL of uranium acetate onto a copper grid to precipitate for 1 min, then use filter paper to absorb the floating liquid; then air dry at room temperature for 5 min, and then place the processed sample on a transmission electron microscope for electron microscopy detection and imaging at 100 kV.

[0034] 2. Particle size analysis of Bidens pilosa exosomes

[0035] The specific method is as follows: Take 5 μL of the obtained Bidens pilosa exosomes and dilute them to 30 μL; first, use a standard to test the instrument performance. After passing the test, load the Bidens pilosa exosome sample for testing. The instrument will detect the particle size and concentration information of the exosomes.

[0036] 3. Determination of protein concentration in Bidens pilosa exosomes

[0037] The specific method is as follows: Bidens pilosa exosomes were rapidly lysed at 37℃, and 5× RIPA lysis buffer was quickly added. After mixing, the mixture was incubated on ice for 30 min and then mixed again to obtain the sample to be tested. Standard samples for protein concentration determination using the BCA method were prepared, and 5 μL of the sample was added to the BCA mixture and mixed well. Subsequently, each sample was incubated at 37℃ for 30 min, and the results were analyzed using a microplate reader and OD... 562nm The absorbance values ​​of the test standard and the sample to be tested are recorded to obtain a standard curve. Based on the absorbance value of the sample to be tested and the standard curve, the protein concentration of the sample to be tested is calculated.

[0038] 4. Cell experiments

[0039] BPNP refers to Bidens pilosa exosomes, TNFα refers to α tumor necrosis factor, IFNγ refers to interferon-γ, CCL22 is also known as macrophage-derived chemokine, and RANTES is also known as chemokine CCL5.

[0040] The specific experimental procedure is as follows: Figure 1 As shown, HACAT cells (purchased from Cybio Biotechnology Co., Ltd.) were used at a concentration of 1×10⁻⁶. 5Cells were seeded at a density of 10 cells / mL in 12-well plates and divided into three groups: control group, M group, and BPNP group, with three wells in each group. The culture medium (high glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, purchased from Shanghai Peiyuan Biotechnology Co., Ltd., and fetal bovine serum purchased from Yikesai Biotechnology Co., Ltd.) was 1 mL in volume. After overnight culture, the BPNP group was given 10 μg of BPNP, while the other two groups were given 100 μL of PBS buffer. After 1 h of culture, the M group and BPNP group were given 1 μL each of TNFα and IFNγ at a concentration of 10 μg / mL. After 23 h of continued culture, RNA was extracted, reverse transcribed into cDNA, and the expression changes of chemokines (TNFα, CCL22, IL-6, and RANTES) in each group were measured by RT-qPCR.

[0041] 5. Animal experiments

[0042] DNFB refers to dinitrofluorobenzene, DXM refers to dexamethasone, and BPNP refers to Bidens pilosa exosomes.

[0043] Reference Figure 2Eight-week-old BALB / c mice (purchased from the Animal Center of Xiamen University) were divided into four groups: control group, DNFB group, DNFB+DXM group, and DNFB+BPNP group, with five mice in each group. Except for the control group, the other three groups were sensitized on day 1 by uniformly applying 100 μL of 1.0% DNFB (w / v, solvent is acetone / olive oil in a volume ratio of 3:1) to the hairless area on the back of the mice. Sensitization was intensified on day 3. From week 2 to week 4, on days 7, 10, 14, 17, 21, 24, and 28, 0.5% DNFB solution (w / v, solvent is acetone / olive oil in a volume ratio of 3:1) was uniformly applied to the back and both sides of the right ear of the mice. Among them, 50 μL was applied to the back and 20 μL to the right ear to induce dermatitis. The control group received an equal volume of acetone-olive oil matrix (acetone:olive oil = 3:1, v / v) applied to the hair removal area on their backs, with the same treatment time and dosage as the other three groups. Starting on day 7, the control and DNFB groups received a blank solvent (ethanol:saline = 1:99, v / v) via gavage at a dose of 0.1 mL / 10 g body weight, three times a week; the DNFB+DXM group received 0.1 mg / kg dexamethasone via injection (the solvent was a blank solvent, specifically ethanol:saline = 1:99, v / v), three times a week; and the DNFB+BPNP group received 200 μL of BPNP via gavage, with a concentration of 0.5 μg / μL of Bidens pilosa exosomes in the BPNP (the solvent was a blank solvent, specifically ethanol:saline = 1:99, v / v), three times a week.

[0044] Semi-quantitative scoring of skin lesions and ear swelling thickness are important indicators of atopic dermatitis in mice. Therefore, the following analysis was performed on mice.

[0045] 5.1 Skin lesion analysis

[0046] Semi-quantitative scoring was performed on the skin lesions on the back of mice on days 1, 3, 7, 10, 14, 17, 21, 24, and 29 after modeling.

[0047] The evaluation is based on the clinical manifestation criteria, as follows: 0 points: no damage; 1 point: mild damage; 2 points: moderate damage; 3 points and above: severe damage; damage evaluation is based on the following four clinical symptoms: (1) erythema; (2) edema / papules; (3) epidermal peeling / scratching; (4) dryness / scabs.

[0048] 5.2 Measurement of Ear Swelling Thickness

[0049] On days 3, 10, 17, 24 and 29 of the modeling experiment, the thickness of the right and left ears of mice was precisely measured at the same position using an electronic micrometer, and the thickness difference (μm) was used to reflect ear swelling.

[0050] 5.3 H&E staining

[0051] The specific experimental procedure is as follows:

[0052] On day 29 of the experiment, mice were euthanized and skin and ear tissue specimens were obtained.

[0053] 1. Dehydration and wax impregnation

[0054] Before embedding, the tissue was rinsed overnight with running water to remove residual fixative, and then dehydrated, cleared, and impregnated with paraffin according to the procedure shown in Table 1.

[0055] Table 1 Dehydration and waxing procedures

[0056] 50% ethanol 1 h 75% ethanol 1 hour (or long-term storage at 4°C) 85% ethanol 30 min 95% Ethanol I 30 min 95% Ethanol II 90 min 100% Ethanol I 10 min 100% Ethanol II 10 min Xylene I 5 min Xylene II 5 min Xylene III 5 min Paraffin I 20 min Paraffin II 10 min Paraffin III 10 min

[0057] 2. Staining

[0058] (1) Paraffin section preparation: Set the section thickness to 5μm and perform sectioning. After sectioning, bleach the sections in 30% ethanol solution, then spread them in 48℃ warm water, and after scooping them out, bake them in a 60℃ constant temperature oven for 2 hours.

[0059] (2) Dewaxing and rehydrating the sections according to Table 2.

[0060] Table 2 Dewaxing and Rehydration Procedure

[0061] Xylene I 10min Xylene II 10min Xylene 1:100% alcohol I 5min 100% alcohol I 5min 100% Alcohol II 5min 95% alcohol 5min 80% alcohol 5min 70% alcohol 5min tap water 5min

[0062] (3) Stain with hematoxylin and eosin solution for 5 minutes, then rinse quickly with tap water;

[0063] (4) Separate the colors with 0.5wt% hydrochloric acid and ethanol for 1~2s, then wash quickly with distilled water;

[0064] (5) 1% ammonia solution turns blue: Immerse the slice in 1% ammonia solution for a few seconds or until the tissue turns blue, wash with tap water for 45 seconds, and examine the degree of color separation of cell nuclei under a microscope;

[0065] (6) 1% eosin for 15 seconds, then rinse quickly with distilled water;

[0066] (7) Wash rapidly with 80%, 90%, and 95% ethanol solutions, 15 seconds per wash. Monitor the color contrast between the cell nucleus and cytoplasm under a microscope;

[0067] (8) Wash twice with 100% ethanol, 2 min each time;

[0068] (9) Wash twice with xylene for 2 minutes each time, and then seal with neutral resin;

[0069] (10) Take photos for record-keeping, observe the staining results, and calculate the skin epidermal thickness.

[0070] 7. Statistical Analysis

[0071] SPSS software was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (x ± s). The t-test was used, and one-way ANOVA was used for comparisons between groups. P < 0.05 was considered to be statistically significant. Example

[0072] Experimental verification in this application confirms that Bidens pilosa exosomes have a therapeutic effect on atopic dermatitis. As a plant, Bidens pilosa generally secretes exosomes (also known as extracellular vesicles) from its various tissues; the tissues capable of secreting exosomes in Bidens pilosa can be its leaves, stems, rhizomes, and roots. Therefore, in this application, Bidens pilosa exosomes can be extracted from any one or more of the leaves, stems, and roots of Bidens pilosa. For example, Bidens pilosa exosomes can be obtained from the rhizomes; or from the stems and leaves.

[0073] The preparation method of Bidens pilosa exosomes includes the following steps:

[0074] Bidens pilosa tissue was taken, added to buffer solution and crushed. After solid-liquid separation, the liquid was collected to obtain a Bidens pilosa pretreated sample.

[0075] Bidens pilosa pretreated samples were subjected to ultracentrifugation to obtain Bidens pilosa exosomes.

[0076] In the above method, the buffer solution can be PBS buffer. Adding the buffer solution facilitates the distribution of exosomes into the buffer solution after pulverization, and the exosomes and tissue residues are separated through subsequent solid-liquid separation. The solid-liquid separation can be achieved through centrifugation, filtration, vacuum filtration, microfiltration, or any combination of methods capable of separating Bidens pilosa exosomes and Bidens pilosa tissue residues. For example, a combination of filtration + centrifugation + microfiltration can achieve this solid-liquid separation. Further, gauze filtration is used to remove large tissue particles; then further centrifugation is performed, specifically at 8000-12000 rpm for 15-25 min; finally, microfiltration is used to remove small solid impurities.

[0077] Based on solid-liquid separation, exosomes of *Gnaphalium affine* were further separated from the liquid by ultracentrifugation.

[0078] Ultracentrifugation specifically includes the following steps:

[0079] The pretreated Bidens pilosa sample was centrifuged at 1500-2500g for 25-35 min; the supernatant was then centrifuged at 8000-12000g for 40-50 min and microfiltered; the filtrate was centrifuged at 80000-120000g for 65-75 min; the precipitate was resuspended in buffer and then centrifuged at 80000-120000g for 65-75 min to obtain Bidens pilosa exosomes.

[0080] Based on its therapeutic use for atopic dermatitis, Bidens pilosa exosomes can be used to prepare compositions for treating atopic dermatitis, with Bidens pilosa exosomes serving as the main pharmaceutical component to achieve the therapeutic effect.

[0081] The composition also includes any one or more of excipients, dispersants, pharmaceutical additives, and probiotics.

[0082] Excipients, also known as excipients, can be the base component in semi-solid preparations (such as ointments and creams); or preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations. General requirements for excipients include: stability, no incompatibility with the active pharmaceutical ingredient (API), no side effects, no impact on efficacy, resistance to deformation, cracking, mold, and insect infestation at room temperature, harmlessness to humans, no physiological effects, no chemical or physical reactions with the API, and no interference with the API's content determination.

[0083] Based on its therapeutic use for atopic dermatitis, Bidens pilosa exosomes can be used to prepare drugs for treating atopic dermatitis or as adjunctive therapy.

[0084] The dosage forms of the drugs and adjuvant therapeutic drugs are selected from any one or more of the following: microneedles, topical liquids, topical solids, topical semi-solids, plasters, patches, and topical gaseous preparations.

[0085] Example 1

[0086] The specific method for preparing Bidens pilosa exosomes is as follows:

[0087] Take tender stems and leaves of Bidens pilosa, chop them (1mm×1mm), soak 10g in PBS buffer, then filter with gauze, centrifuge the liquid at 8000rpm for 25min, and microfilter the liquid (0.45μm) to obtain the Bidens pilosa pretreated sample.

[0088] Bidens pilosa pretreated samples were subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The specific steps of ultracentrifugation are as follows:

[0089] Centrifuge the sample at 1500×g, 4℃ for 35 min. Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 8000×g, 4℃ for 50 min to remove larger vesicles. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate. Transfer the filtrate to a new centrifuge tube and centrifuge at 4℃, 80000×g for 75 min using an ultracentrifuge rotor. Remove the supernatant, resuspend the sample in 10 mL of pre-chilled 1×PBS, and centrifuge again at 4℃, 80000×g for 75 min using an ultracentrifuge rotor. Remove the supernatant again and resuspend the sample in 150 μL of pre-chilled 1×PBS.

[0090] Example 2

[0091] The specific method for preparing Bidens pilosa exosomes is as follows:

[0092] Take tender stems and roots of Bidens pilosa, wash them clean, chop them (1mm×1mm), soak 10g in PBS buffer, filter with gauze, centrifuge the liquid at 10000rpm for 20min, and microfilter the liquid (0.45μm) to obtain the Bidens pilosa pretreated sample.

[0093] Bidens pilosa pretreated samples were subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The specific steps of ultracentrifugation are as follows:

[0094] Centrifuge the sample at 2000×g, 4℃ for 30 min. Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 10000×g, 4℃ for 45 min to remove larger vesicles. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate. Transfer the filtrate to a new centrifuge tube and centrifuge at 4℃, 100000×g for 70 min using an ultracentrifuge rotor. Remove the supernatant, resuspend the sample in 10 mL of pre-chilled 1×PBS, and centrifuge again at 4℃, 100000×g for 70 min using an ultracentrifuge rotor. Remove the supernatant again and resuspend the sample in 150 μL of pre-chilled 1×PBS.

[0095] Example 3

[0096] The specific method for preparing Bidens pilosa exosomes is as follows:

[0097] Take tender stems and roots of Bidens pilosa, clean them, chop them (1mm×1mm), soak 10g in PBS buffer, filter with gauze, centrifuge the liquid at 12000rpm for 15min, and microfilter the liquid (0.45μm) to obtain the Bidens pilosa pretreated sample.

[0098] Bidens pilosa pretreated samples were subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The specific steps of ultracentrifugation are as follows:

[0099] Centrifuge the sample at 2500×g, 4℃ for 25 min. Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 12000×g, 4℃ for 40 min to remove larger vesicles. Filter the supernatant through a 0.45 μm filter and collect the filtrate. Transfer the filtrate to a new centrifuge tube and centrifuge at 4℃, 120000×g for 65 min using an ultracentrifuge rotor. Remove the supernatant, resuspend the sample in 10 mL of pre-chilled 1×PBS, and centrifuge again at 4℃, 120000×g for 65 min using an ultracentrifuge rotor. Remove the supernatant again and resuspend the sample in 150 μL of pre-chilled 1×PBS.

[0100] Test results

[0101] 1. Transmission electron microscopy observation of Bidens pilosa exosomes: The Bidens pilosa exosomes from Example 2 were used as samples for the experiment, and the results are shown in [the table below]. Figure 3 . Figure 3 This indicates that Bidens pilosa exosomes were obtained using this method. The electron microscopy morphology of these exosomes is cup-shaped, and their particle size is approximately 79.0 nm.

[0102] 2. Particle size analysis of Bidens pilosa exosomes: The Bidens pilosa exosomes from Example 2 were used as samples for the experiment, and the results are shown in [the table below]. Figure 4 . Figure 4 This indicates that the average particle size of the exosomes is 79.0 nm, and the concentration of the exosomes is 1.31 × 10⁻⁶. 9 The particle size and concentration of the exosomes are within the range of exosomes, indicating that the obtained exosomes are Bidens pilosa exosomes.

[0103] 3. The protein concentration of the Bidens pilosa exosomes from Example 2 was determined to be 16.57 μg / μL. Subsequent cell experiments were conducted based on this concentration result.

[0104] 4. Cell experiments:

[0105] The BPNP group used Bidens pilosa exosomes from Example 2 as samples for experiments, measuring the expression levels of various cytokines including TNFα, CCL22, IL-6, and RANTES. Specific results are shown below. Figure 5 The expression of TNFα, CCL22, IL-6 and RANTES was upregulated in group M, while the expression in group BPNP was improved compared to group M.

[0106] Atopic dermatitis is associated with the expression of chemokines, and the expression levels of chemokines in the atopic dermatitis cell model were higher than those in normal cells. Therefore, the above results indicate that Bidens pilosa exosomes have an effect in alleviating atopic dermatitis.

[0107] 5. Animal experiments

[0108] In the following experiments, the BPNP group used Bidens pilosa exosomes from Example 2 as samples.

[0109] 5.1 Photos of the back and ears

[0110] Photos of the mouse's back and ears taken on day 29, such as... Figure 6 As shown, the DNFB group had severe back and ear injuries, while the DNFB+DXM and DNFB+BPNP groups showed improvement in back and ear injuries compared to the DNFB group. These results indicate that Bidens pilosa exosomes have a significant effect in improving the symptoms of atopic dermatitis.

[0111] 5.2 Skin lesion score and ear swelling thickness

[0112] like Figure 7 As shown, on day 29, the skin lesion score and ear swelling thickness in the DNFB group were significantly higher than those in the Control group, with significant differences; the DNFB+DXM group and the DNFB+BPNP group showed significant differences from the DNFB group, indicating that Bidens pilosa exosomes can alleviate the symptoms of atopic dermatitis in mice.

[0113] 5.3 H&E staining results

[0114] like Figure 8 As shown, the H&E staining results of skin and ear tissue sections from different groups of mice revealed that: in the Control group, the skin layers on the back of mice were clearly defined, with normal epidermal and dermal structures and no obvious inflammatory cell infiltration between or within cells; in the DNFB group, the skin lesions on the back of mice showed parakeratosis or hyperkeratosis, significant epidermal thickening, and a large number of inflammatory cell infiltrations, with significant edema in the lesions; the DNFB+DXM group and the DNFB+BPNP group showed improvement compared to the DNFB group, demonstrating the ameliorative effect of BPNP on atopic dermatitis in mice.

[0115] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. The use of Bidens pilosa exosomes in the preparation of compositions for treating atopic dermatitis, characterized in that, The Bidens pilosa exosomes are secreted by Bidens pilosa tissue, which is selected from any one or more of Bidens pilosa leaves, stems, and roots. The method for preparing the Bidens pilosa exosomes includes the following steps: Bidens pilosa tissue was taken, added to buffer solution and crushed. After solid-liquid separation, the supernatant was collected and microfiltered to obtain a Bidens pilosa pretreated sample. The Bidens pilosa pretreated sample was subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The ultracentrifugation specifically includes the following steps: The pretreated Bidens pilosa sample was centrifuged at 1500-2500g for 25-35 min; the supernatant was then centrifuged at 8000-12000g for 40-50 min and then microfiltered through a 0.45μm filter membrane; the filtrate was centrifuged at 80000-120000g for 65-75 min; the precipitate was resuspended in buffer and then centrifuged at 80000-120000g for 65-75 min to obtain Bidens pilosa exosomes; The average particle size of Bidens pilosa exosomes was 79.0 nm, and the concentration was (1.2-1.4)×10⁻⁶. 9 pcs / ml; The solid-liquid separation of Bidens pilosa tissue after pulverization specifically includes the following steps: After the Bidens pilosa tissue was crushed and filtered, the supernatant was centrifuged at 8000-12000 rpm for 15-25 min.

2. The use of the Bidens pilosa exosomes according to claim 1 for preparing a composition for treating atopic dermatitis, characterized in that, The Bidens pilosa exosomes are used to prepare a composition for treating atopic dermatitis, and the composition further includes excipients.

3. The use of the Bidens pilosa exosomes according to claim 1 for preparing a composition for treating atopic dermatitis, characterized in that, The Bidens pilosa exosomes are used to prepare drugs for treating atopic dermatitis or as adjunctive therapy.

4. The use of the Bidens pilosa exosomes according to claim 3 for preparing a composition for treating atopic dermatitis, characterized in that, The dosage forms of the drugs and adjuvant therapeutic drugs are selected from any one or more of the following: topical liquids, topical solids, topical semi-solids, and topical gaseous preparations.

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