Use of kaval leaf exosome-like nanovesicles in the treatment of sjogren's syndrome

Abstract

The application discloses application of a Gypsophila oldhamiana exosome-like nanovesicle in treatment of Sjogren's syndrome. The Gypsophila oldhamiana exosome-like nanovesicle is prepared by differential centrifugation and ultracentrifugation, and toxicology evaluation shows that the Gypsophila oldhamiana exosome-like nanovesicle has good safety and can be used for in-vivo and in-vitro treatment. In-vivo experiments prove that the saliva secretion of Sjogren's syndrome model mice treated by the Gypsophila oldhamiana exosome-like nanovesicle is significantly increased, and the xerostomia symptom is significantly improved; in-vitro experiments further prove that the nanovesicle can also significantly improve the viability and water secretion function of salivary gland epithelial cells in Sjogren's syndrome. The Gypsophila oldhamiana exosome-like nanovesicle is successfully extracted from Gypsophila oldhamiana, and is creatively used for treating Sjogren's syndrome, thereby providing a new idea and material source for drug research and development of Sjogren's syndrome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of *Gnaphalium affine* leaf exosome-like nanovesicles in the treatment of Sjögren's syndrome. Background Technology

[0002] Sjögren's syndrome (SS) is a chronic, progressive systemic autoimmune disease characterized by dysfunction of exocrine glands (especially salivary and lacrimal glands) and lymphocytic infiltration. Clinical manifestations primarily include intractable dry mouth, dry eyes, dysphagia, rampant dental caries, and recurrent parotid gland swelling, severely impacting patients' daily lives, nutritional status, and mental health. Furthermore, approximately one-third of patients may experience multi-system involvement outside the glands, such as joint pain, fatigue, interstitial lung disease, and peripheral neuropathy, even increasing the risk of developing lymphoma.

[0003] The exact etiology and pathogenesis of Sjögren's syndrome are not yet fully understood, but it is generally believed to be related to the interaction of multiple factors, including genetic susceptibility, environmental factors, infection triggers, and abnormal immune regulation. Current treatments primarily aim to relieve symptoms and control systemic lesions, lacking a radical cure. For dryness symptoms, artificial saliva and artificial tears are often used as replacement therapies, or cholinergic drugs (such as pilocarpine and cevimeline) are used to stimulate glandular secretion, but the effects are unsatisfactory and may cause side effects such as sweating and gastrointestinal discomfort. For systemic manifestations, immunosuppressants or biologics such as hydroxychloroquine, methotrexate, and rituximab are required; however, these drugs are expensive, carry potential risks such as infection and bone marrow suppression, and are not effective for all patients. Therefore, developing novel, highly effective, and low-toxicity therapeutic drugs, especially those that can fundamentally improve salivary gland secretion function and repair acinar epithelial cells, has become an urgent need and an important direction in the current research field of Sjögren's syndrome.

[0004] In recent years, plant-derived exosome-like nanovesicles (PELNVs) have attracted widespread attention due to their unique advantages. Naturally occurring in plants, they serve as important mediators for intercellular communication, carrying various bioactive components such as proteins, lipids, RNA, and metabolites. Compared to animal-derived exosomes or artificially synthesized nanocarriers, plant exosome-like nanovesicles offer significant advantages, including wide availability, low cost, lack of ethical controversy, low immunogenicity, good biocompatibility, ease of storage, and large-scale production. Studies have shown that nanovesicles derived from some medicinal plants (such as ginger, grapes, and broccoli) exhibit significant activities in anti-inflammatory, antioxidant, immunomodulatory, and tissue repair-promoting effects, demonstrating great potential in drug delivery systems and disease treatment.

[0005] Melia azedarach leaves, also known as bitter neem leaves, are a traditional Chinese medicine with the effects of clearing heat and drying dampness, killing parasites and relieving itching, and promoting qi circulation and relieving pain. They are commonly used to treat eczema, scabies, snake and insect bites, trichomoniasis, and hernia pain. Modern pharmacological studies suggest that they have anti-inflammatory, antibacterial, antiviral, and immunomodulatory effects. However, to date, there are no reports on the extraction of exosome-like nanovesicles from Melia azedarach leaves and their application in the treatment of Sjögren's syndrome. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of *Gnaphalium affine* exosome-like nanovesicles in the preparation of drugs for treating or improving Sjögren's syndrome.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention first extracts exosome-like nanovesicles from *Gnaphalium affine* leaves using physical methods such as leaf washing, low-temperature homogenization, differential centrifugation, and ultracentrifugation. The nanovesicles exhibit intact morphology, uniform particle size distribution, primarily between 100-200 nm, with a peak particle size of 152 nm and an average particle size of approximately 150 nm. The particle size distribution is uniform (polydispersity index PDI < 0.2). Characterization by transmission electron microscopy and nanoparticle tracking analysis confirms that the nanovesicles possess typical cup-shaped or spherical morphology and intact membrane structures. BCA assay shows a protein concentration of 1.5 mg / mL and a particle concentration of 3.2 × 10^10 particles / mL. The extraction method for *Gnaphalium affine* leaf exosome-like nanovesicles provided by this invention is simple, mild, and reproducible, without involving organic solvents or complex chemical modifications. It is suitable for large-scale preparation and clinical translation, and is low-cost while also exhibiting good safety and stability.

[0009] This invention pioneered the use of *Gnaphalium affine* leaf exosome-like nanovesicles for the treatment of Sjögren's syndrome, providing a novel approach and material source for drug development and clinical treatment of this syndrome. Toxicological evaluation showed that *Gnaphalium affine* leaf exosome-like nanovesicles have good safety profiles and can be used for in vitro and in vivo treatment. In vivo experiments confirmed that *Gnaphalium affine* leaf exosome-like nanovesicles significantly increased salivary secretion in Sjögren's syndrome model mice, and significantly improved symptoms such as dry mouth. HE staining, AQP5 histochemical staining, and blood SSA and SSB levels were also observed. AQP5 The mRNA levels of the samples all showed improvement in Sjögren's syndrome, and no obvious toxic reactions or side effects were observed. In vitro experiments further demonstrated that these nanovesicles significantly improved the viability and water secretion function of salivary gland epithelial cells in Sjögren's syndrome in a concentration-dependent manner. The exosome-like nanovesicles from *Gnaphalium affine* leaves also promoted… AQP5The restoration of mRNA and protein levels indicates that *Gnaphalium affine* exosome-like nanovesicles can effectively improve water secretion dysfunction in a Sjögren's syndrome cell model. In summary, rigorous evaluation using cell and animal models confirms that *Gnaphalium affine* exosome-like nanovesicles can significantly improve the survival rate and function (water secretion capacity) of salivary gland epithelial cells in Sjögren's syndrome, significantly increase salivary flow in Sjögren's syndrome model mice, fundamentally alleviate core symptoms such as dry mouth, and improve the pathological condition of salivary gland tissue, demonstrating a clear and significant therapeutic effect. This invention provides a novel natural drug option for the clinical treatment of Sjögren's syndrome, possessing significant application value and development prospects.

[0010] Therefore, the present invention provides the use of *Gnaphalium affine* leaf exosome-like nanovesicles in the preparation of medicaments for the treatment or improvement of Sjögren's syndrome.

[0011] Furthermore, the extraction method of the exosome-like nanovesicles from the bitter clover leaves is to homogenize the bitter clover leaves by differential centrifugation and ultracentrifugation, followed by washing and resuspending.

[0012] Furthermore, the homogenate is prepared by mixing tartary leaves with PBS at a weight-to-volume ratio of 1:3 (g / mL) and then homogenizing.

[0013] Furthermore, the differential centrifugation includes coarse centrifugation and medium-speed centrifugation; the coarse centrifugation is 500-1500×g for 5-15 minutes; 2500-3500×g for 15-25 minutes; and the medium-speed centrifugation is 9000-11000×g for 35-45 minutes.

[0014] Furthermore, the ultracentrifugation is 100,000 to 200,000 × g for 170 to 190 minutes.

[0015] Specifically, the leaves of *Gnaphalium affine* were mixed with PBS at a weight-to-volume ratio of 1:3 (g / mL) and homogenized. The resulting slurry was centrifuged at 1000×g for 10 min at 4°C, and the precipitate was discarded. Then, it was centrifuged at 3000×g for 20 min, and the supernatant was collected. At 4°C, it was centrifuged at 10000×g for 40 min, and the supernatant was collected. At 4°C, it was ultracentrifuged at 150000×g for 180 min, and the supernatant was discarded. The precipitate was washed, resuspended, and centrifuged again at 4°C and 100000×g for 70 min. After resuspending, it was filtered through a 0.22 μm microporous membrane for sterilization.

[0016] Furthermore, the particle size of the *Gnaphalium affine* leaf exosome-like nanovesicles is 140–160 nm.

[0017] Specifically, the peak particle size of the *Gnaphalium affine* leaf exosome-like nanovesicles is 152 nm, and the average particle size is approximately 150 nm.

[0018] Furthermore, the drug achieves its therapeutic effect by protecting and / or repairing the function of salivary gland epithelial cells, increasing saliva secretion, and thereby improving dry mouth symptoms.

[0019] Furthermore, the effective dose range of the *Gnaphalium affine* leaf exosome-like nanovesicles is 0.1–100 mg / kg body weight / day. The determination of the dosage needs to comprehensively consider factors such as the severity of the patient's condition, weight, and route of administration.

[0020] Furthermore, the effective dose range of the *Gnaphalium affine* leaf exosome-like nanovesicles is 1–20 mg / kg body weight / day, and can be administered once or in divided doses.

[0021] Furthermore, the drug may also include other pharmaceutically acceptable carriers, diluents, or excipients. Plant exosomes themselves are a natural library of active ingredients, potentially exerting comprehensive therapeutic effects through multiple targets and pathways. In addition, their excellent carrier properties give them the potential to load other drugs (such as small-molecule anti-inflammatory drugs, nucleic acid drugs, etc.), achieving multifunctional and synergistic therapeutic goals.

[0022] Furthermore, the dosage form of the drug is an injection, oral liquid, capsule, oral spray, or gel.

[0023] Drugs containing exosome-like nanovesicles from *Gnaphalium affine* leaves can be formulated into various dosage forms for local or systemic administration to safely and effectively treat Sjögren's syndrome. For example:

[0024] Injectable formulation: Dissolve the exosome-like nanovesicles of the bitter clover leaf in physiological saline or phosphate buffer, adjust to the required concentration, and use for intravenous injection, intraperitoneal injection, or local intraglandular injection.

[0025] Oral liquid or capsule: Mix the exosome-like nanovesicles of bitter ginseng leaf with flavoring agents and stabilizers (such as trehalose) to make an oral liquid, or fill it into enteric-coated capsules to avoid destruction by gastric acid.

[0026] Oral sprays or gels: Mixing exosome-like nanovesicles from the leaves of the bitter gourd with an adhesive (such as sodium carboxymethyl cellulose) and a humectant (such as glycerin) to form oral sprays or gels for direct application to the oral mucosa and local action on the salivary glands.

[0027] Furthermore, the oral spray is composed of the above-mentioned *Gnaphalium affine* leaf exosome-like nanovesicles, thickener, humectant, and preservative in a mass ratio of 1.3–1.7:9–11:98–102:1.

[0028] Specifically, the oral spray is prepared by mixing 10 mL of *Gnaphalium affine* leaf exosome-like nanovesicles (protein concentration 1.5 mg / mL) with 0.1 g sodium carboxymethyl cellulose (thickener), 1.0 g glycerin (humectant), and 0.01 g benzalkonium chloride (preservative), stirring thoroughly to dissolve, and then adjusting the volume to 100 mL with purified water.

[0029] Therefore, the present invention also provides a pharmaceutical composition comprising the above-mentioned *Gnaphalium affine* leaf exosome-like nanovesicles.

[0030] The leaves of *Salvia splendens* are known to be safe medicinal plants, and the nanovesicles derived from them are natural products with good biocompatibility and high safety. Detailed in vitro and in vivo toxicological evaluations (including cytotoxicity tests, acute toxicity tests, and long-term toxicity tests) have shown that they do not exhibit significant toxic side effects at effective doses, demonstrating a safety profile superior to many chemically synthesized drugs and immunosuppressants. Furthermore, the exosome-like nanovesicles from *Salvia splendens* leaves maintain their physical stability and biological activity over extended periods at 4°C or -80°C, which is beneficial for drug storage, transportation, and clinical application. Therefore, the application of the aforementioned exosome-like nanovesicles from *Salvia splendens* leaves or the aforementioned pharmaceutical compositions in the preparation of drugs for treating or improving Sjögren's syndrome is of significant clinical translational value.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention provides the application of *Gnaphalium affine* leaf exosome-like nanovesicles in the treatment of Sjögren's syndrome. This invention prepares *Gnaphalium affine* leaf exosome-like nanovesicles using differential centrifugation and ultracentrifugation. Toxicological evaluation shows that the *Gnaphalium affine* leaf exosome-like nanovesicles have good safety and can be used for in vitro and in vivo treatment. In vivo experiments verified that the salivary secretion of Sjögren's syndrome model mice treated with these nanovesicles significantly increased, and dry mouth symptoms significantly improved. In vitro experiments further showed that these nanovesicles can also significantly improve the vitality and water secretion function of salivary gland epithelial cells in Sjögren's syndrome. This invention is the first to successfully extract exosome-like nanovesicles from *Salvia splendens* leaves and pioneering its use in the treatment of Sjögren's syndrome. It demonstrates that *Salvia splendens* leaf exosome-like nanovesicles can significantly improve the survival rate and function (water secretion capacity) of salivary gland epithelial cells in Sjögren's syndrome, significantly increase saliva flow in Sjögren's syndrome model mice, fundamentally alleviate core symptoms such as dry mouth, and improve the pathological condition of salivary gland tissue. The therapeutic effect is clear and significant, providing a novel approach and material source for drug development in Sjögren's syndrome. Attached Figure Description

[0033] Figure 1 Characterization of exosome-like nanovesicles from *Gnaphalium affine* leaves. Among them, Figure 1In the diagram, A represents the preparation process; B is a TEM image (scale bar = 200 nm); and C is the NTA particle size distribution.

[0034] Figure 2 These are the results of in vitro and in vivo toxicity tests on exosome-like nanovesicles from *Gnaphalium affine* leaves. Figure 2 In the figures, A represents HE staining images of major mouse organs (heart, liver, brain, lung, and kidney) (scale bar = 50 μm); B represents mouse blood parameters; and C represents cytotoxicity assays of A253 cells. ns indicates no significant difference.

[0035] Figure 3 This study evaluates the effectiveness of *Gnaphalium affine* leaf exosome-like nanovesicles in treating NOD mice. Among them, Figure 3 In the diagram, A represents the effect of salivary flow; B represents HE staining of the salivary glands; C represents AQP5 histochemical staining of the salivary glands; D represents HE statistical plot; E represents AQP5 histochemical statistical plot; F represents changes in blood SSA levels; G represents changes in blood SSB levels; and H represents salivary glands. AQP5 qPCR. *p<0.001, **p<0.01, ***p<0.001 vs SS Model group.

[0036] Figure 4 The effect of *Gnaphalium affine* leaf exosome-like nanovesicles on water secretion function in an SS cell model. Figure 4 In the diagram, A represents the phagocytosis of exosome-like nanovesicles by salivary gland epithelial cells; B represents the cell viability assay under different concentrations of exovesicles; and C represents... AQP5 D represents qPCR detection; E represents Western blotting (WB) detection of AQP5; F represents immunofluorescence staining of AQP5; G represents immunofluorescence staining statistics of AQP5. *p<0.05, **p<0.01, ***p<0.001.

[0037] Figure 5 The results show the effect of oral spray on saliva flow in NOD / Ltj mice. ns indicates no significant difference, ***p<0.001. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Data statistical methods:

[0041] All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 8.0 software. Student's t-test was used for comparisons between two groups, and one-way ANOVA, followed by Tukey's post hoc test, was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0042] Example 1: Extraction and characterization of exosome-like nanovesicles from *Gnaphalium affine* leaves

[0043] I. Experimental Methods

[0044] 1. Extraction of exosome-like nanovesicles from *Gnaphalium affine* leaves

[0045] (1) Raw material pretreatment: Pick fresh, disease-free leaves of the bitter jujube, rinse them with running pure water to remove surface dust, and then use sterile filter paper to absorb the surface moisture.

[0046] (2) Homogenization: The drained leaves of the bitter elm were mixed with pre-cooled phosphate buffer (PBS, 0.01 M, pH 7.4) at a weight-to-volume ratio of 1:3 (g / mL), placed in an ice-water bath, and mechanically homogenized using a tissue homogenizer until a homogeneous slurry was formed.

[0047] (3) Coarse centrifugation: Centrifuge the obtained slurry at 1000×g for 10 min at 4℃ and discard the precipitate; then centrifuge at 3000×g for 20 min, carefully aspirate the supernatant and discard the precipitate (mainly tissue residue and unbroken cells).

[0048] (4) Medium speed centrifugation: Centrifuge the supernatant obtained in the previous step at 4°C at 10000×g for 40 minutes, and aspirate the supernatant again to discard the precipitate (mainly cell debris and larger organelles).

[0049] (5) Ultracentrifugation: Transfer the supernatant to an ultracentrifuge tube and centrifuge at 150,000 × g for 180 minutes at 4°C. Carefully discard the supernatant. The milky white precipitate obtained at the bottom of the tube is the exosome-like nanovesicle of the bitter clover leaf.

[0050] (6) Washing and resuspension: Gently resuspend the above precipitate with pre-cooled PBS, and centrifuge again at 4°C and 100,000 × g for 70 minutes to remove any soluble proteins that may be contaminating it. Finally, resuspend the precipitate with an appropriate amount of sterile PBS and filter it through a 0.22 μm microporous membrane for sterilization.

[0051] (7) Preservation: The final obtained *Gnaphalium affine* leaf exosome-like nanovesicle suspension was aliquoted into sterile EP tubes and could be stored at 4°C for short-term use (within one week) or at -80°C for long-term storage. The protein concentration was determined using a BCA protein quantification kit.

[0052] 2. Characterization and quality control of exosome-like nanovesicles from *Gnaphalium affine* leaves

[0053] (1) Morphological observation: 10 μL of nanovesicle suspension was dropped onto a copper grid, negatively stained (e.g., 2% phosphotungstic acid solution), and observed and photographed using a transmission electron microscope (TEM).

[0054] (2) Particle size distribution and concentration determination: The nanovesicle suspension after appropriate dilution with PBS was detected using a nanoparticle tracking analyzer (NTA) to analyze its particle size distribution range and particle concentration.

[0055] (3) Microbial limits and endotoxin testing: Ensure that the final product meets the relevant standards for injectables or pharmaceutical excipients.

[0056] II. Experimental Results

[0057] Nanovesicles were extracted from 100g of fresh bitter melon leaves. Figure 1 The protein (A) was finally resuspended in 1 mL of PBS to obtain a milky white suspension, and its protein concentration was determined to be 1.5 mg / mL by the BCA method.

[0058] TEM observation of 10 μL of the suspension showed that the extracted *Gnaphalium affine* leaf exosome-like nanovesicles were relatively uniform in size, spherical or cup-shaped, and had a complete membrane, i.e., a complete lipid bilayer membrane structure. Figure 1 (B in the middle).

[0059] NTA analysis showed that the extracted *Gnaphalium affine* leaf exosome-like nanovesicles were mainly distributed in the range of 100-200 nm, with a peak particle size of 152 nm and an average particle size of approximately 150 nm. The particle size distribution was uniform (polydispersity index PDI < 0.2), and the particle concentration was 3.2 × 10^10 particles / mL. Figure 1 (C in the middle).

[0060] Example 2: In vitro and in vivo safety evaluation of exosome-like nanovesicles from *Gnaphalium affine* leaves.

[0061] I. Experimental Methods

[0062] 1. In vivo safety evaluation: Ten-week-old female NOD / Ltj mice (classic SS model) were used as experimental animals. NOD mice were randomly divided into two groups (n=8): Model group (SS model, PBS group): daily intraperitoneal injection of an equal volume of PBS. Treatment group (SS+PELNVs, ELNs(1200) group): daily intraperitoneal injection of *Gnaphalium affine* exosome-like nanovesicles (1200 μg / kg body weight, dissolved in PBS). After 4 weeks of continuous administration, the experimental results were observed. After the last administration, organs were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed blinded by pathologists; and hematological indicators were tested.

[0063] 2. In vitro safety evaluation: Cytotoxicity assays were performed using the human salivary gland epithelial cell line A253. Cells were seeded in 96-well plates and cultured overnight. The medium was then replaced with fresh medium containing different concentrations of *Gnaphalium affine* leaf exosome-like nanovesicles (0, 5, 20, 80 μg / mL), with six replicates for each concentration. After 24, 48, and 72 hours of further culture, CCK-8 reagent was added to each well, and after 1 hour of incubation, the absorbance (OD) at 450 nm was measured using a microplate reader. 450 ), calculate the relative cell viability.

[0064] II. Experimental Results

[0065] In vivo safety evaluation, such as Figure 2 As shown in Figures A and B, during the administration period, all mice maintained normal mental state, activity, and appetite, with no deaths. HE staining results of organs (heart, liver, brain, lungs, and kidneys) after the last administration, observed blinded by a pathologist, revealed no significant pathological changes related to the drug administration. Figure 2 (A) Hematological indicators showed that after administration, the relative alanine aminotransferase (ALT), relative aspartate aminotransferase (AST), relative ALT / AST ratio, and relative serum creatinine were not significantly different from the control group. Figure 2 (B in the middle).

[0066] In vitro safety evaluation, such as Figure 2 As shown in Figure C, there was no statistically significant difference in cell survival rates among the drug-treated groups compared to the blank control group (p>0.05). These results indicate that within this concentration range, *Gnaphalium affine* leaf exosome-like nanovesicles have no significant toxicity to A253 cells.

[0067] The exosome-like nanovesicles from the bitter clover leaf have good safety profiles and can be used for in vitro and in vivo treatment.

[0068] Example 3: Therapeutic effect of *Gnaphalium affine* leaf exosome-like nanovesicles on Sjögren's syndrome model mice.

[0069] I. Experimental Methods

[0070] Ten-week-old female NOD / Ltj mice (classical Sjögren's syndrome (SS) model) were used as experimental animals, and ICR mice were used as healthy controls. NOD mice were randomly divided into 5 groups (n=8):

[0071] NOD mouse group: No treatment was given.

[0072] Blank control group (SS Model) (NOD+PBS group): Daily intraperitoneal injection of an equal volume of PBS.

[0073] Positive control group (SS+HCQ) (NOD+HCQ group): Daily intraperitoneal injection of hydroxychloroquine (60 mg / kg body weight, dissolved in PBS).

[0074] Low-dose treatment group (SS+PELNVs) (NOD+ELNs (800) group): daily intraperitoneal injection of *Gnaphalium affine* exosome-like nanovesicles (800 μg / kg body weight, dissolved in PBS).

[0075] High-dose treatment group (SS+PELNVs) (NOD+ELNs (1200) group): daily intraperitoneal injection of *Gnaphalium affine* exosome-like nanovesicles (1200 μg / kg body weight, dissolved in PBS).

[0076] A separate ICR healthy control group was set up.

[0077] AQP5 is a marker protein for changes in SS disease; AQP5 levels decrease after the onset of the disease. The following tests were performed after 4 weeks of continuous drug administration:

[0078] (1) Saliva flow rate measurement: All mice were injected intraperitoneally with pilocarpine (1 mg / kg) to stimulate saliva secretion. A pre-weighed sterile cotton ball was placed in the mouth of the mouse to collect saliva for 8 minutes. The saliva secretion volume (mg / 8min) was calculated by weighing the cotton ball again.

[0079] (2) HE staining: The tissue was fixed in 4% paraformaldehyde for 48 h, dehydrated with graded ethanol solutions, and embedded in paraffin. Sample sections with a thickness of 5 μm were prepared, stained with hematoxylin and eosin staining kits, and observed under an optical microscope.

[0080] (3) Immunohistochemical detection of AQP5: After dewaxing, tissue sections were citrate-retended for 16 hours, washed with PBS, and incubated with 3% hydrogen peroxide solution at room temperature for 10 minutes. After washing with PBS, the sections were blocked with sheep serum at room temperature for half an hour, and then incubated with AQP5 primary antibody at 4°C overnight. The primary antibody was recovered, washed with PBS, and then incubated with secondary antibody at room temperature for 1 hour. After washing with PBS, the sections were developed with DAB, counterstained with hematoxylin, and mounted with neutral resin.

[0081] (4) WB detection of AQP5: cell lysis buffer, tissue lysis buffer, PAGE electrophoresis, after membrane transfer and blocking, incubate with primary antibody at 4℃ overnight, wash with TBST, incubate with secondary antibody at 37℃ for 1 hour, wash with PBS, and perform ECL chemiluminescence imaging.

[0082] (5) Blood SSA and SSB detection: Mouse serum was collected and the expression levels of anti-SSA / Ro and anti-SSB / La antibodies in mice were quantitatively detected by ELISA.

[0083] (6) AQP5 qPCR detection: Total RNA was extracted and cDNA was prepared using the PrimeScript™ RT kit. Real-time quantitative PCR was performed using the SuperReal PreMix Plus (SYBR Green) kit. Each reaction contained 8.5 μL of 2×SYBR Green. ® The premixed Ex Taq™ (using SYBR Green I), 300 nM oligonucleotide primers, and a final volume of 10 μL were used. Reaction conditions included denaturation at 95°C for 30 s; 95°C for 5 s; 40 cycles; and 60°C for 30 s.

[0084] II. Experimental Results

[0085] Saliva flow measurement results are as follows Figure 3 As shown in Figure A, the salivary flow rate of mice in the treatment group was significantly higher than that in the model group (p<0.001). Although it did not fully recover to the level of healthy controls, it was greatly improved, and the effect was significantly better than that of hydroxychloroquine.

[0086] HE staining results are as follows Figure 3 As shown in B and D, the degree of lymphocyte inflammatory infiltration was significantly reduced in the treatment group.

[0087] AQP5 immunohistochemical staining results are as follows Figure 3 As shown in C and E, the treatment group AQP5 Expression levels were significantly increased; qPCR results Figure 3 As shown by H in the figure, the treatment group AQP5 Gene expression levels were significantly increased.

[0088] Blood SSA and SSB test results as follows Figure 3 As shown in F and G, the antibody levels of SSA and SSB in the treatment group were significantly reduced.

[0089] In summary, exosome-like nanovesicles from the leaves of the bitter clover can be used to treat Sjögren's syndrome.

[0090] Example 4: Effects of *Gnaphalium affine* leaf exosome-like nanovesicles on water secretion function in a Sjögren's syndrome cell model

[0091] I. Experimental Methods

[0092] An in vitro SS model was established by stimulating A253 cells with IFN-γ (50 ng / mL) for 72 hours. Cells were divided into three groups: (1) normal control group (Control); (2) SS model group (IFN-γ); and (3) SS model + *Gnaphalium affine* exosome-like nanovesicle treatment group (IFN-γ + PELNVs (20 μg / mL)). After treatment, the water secretion function of the cells was measured using a water permeability assay kit based on the YFP-H148Q / V163S fluorescence quenching principle.

[0093] II. Experimental Results

[0094] The results are as follows Figure 4 As shown in Figures A and B, salivary gland epithelial cells can phagocytose *P. sarcodactylis* leaf exosome-like nanovesicles, and cell viability increases with increasing *P. sarcodactylis* leaf exosome-like nanovesicle concentration. However, there was no difference in cell viability recovery between PELNVs concentrations of 20 μg / mL and 80 μg / mL; therefore, a PELNVs concentration of 20 μg / mL was chosen for subsequent experiments. Figure 4 According to CG, *Gnaphalium affine* leaf exosome-like nanovesicles can promote... AQP5 The mRNA and protein levels of the cells were restored. Compared with the normal control group, the water permeability of the SS model group was significantly reduced (p<0.01); while compared with the SS model group, the water permeability of the nanovesicle treatment group was significantly restored (p<0.01), indicating that it can effectively improve the water secretion dysfunction of the SS cell model.

[0095] In summary, both in vivo and in vitro experiments have demonstrated that *Gnaphalium affine* leaf exosome-like nanovesicles can be used to treat Sjögren's syndrome.

[0096] Example 5: Preparation of a bitter clover leaf exosome-like nanovesicle oral spray

[0097] I. Experimental Methods

[0098] Take 10 mL of *Gnaphalium affine* leaf exosome-like nanovesicles (protein concentration 1.5 mg / mL), mix with 0.1 g sodium carboxymethyl cellulose (thickener), 1.0 g glycerin (humectant), and 0.01 g benzalkonium chloride (preservative), stir thoroughly to dissolve, and then dilute to 100 mL with purified water. Stir well and dispense into sterile spray bottles to obtain *Gnaphalium affine* leaf exosome-like nanovesicle oral spray. This preparation can be used for daily oral care of SS patients, sprayed directly onto the oral mucosa several times a day to relieve dry mouth discomfort.

[0099] Ten-week-old female NOD / Ltj mice were selected as experimental animals, and the NOD mice were randomly divided into two groups (n=8).

[0100] Control group: The control oral spray (containing all components except ectosome-like vesicles) was sprayed several times daily for 4 weeks.

[0101] Treatment group: Spray the oral spray containing exosome-like nanovesicles of bitter clover leaves several times a day for 4 weeks.

[0102] Saliva flow rate measurement: All mice were injected intraperitoneally with pilocarpine (1 mg / kg) to stimulate saliva secretion. A pre-weighed sterile cotton ball was placed in the mouth of the mouse to collect saliva for 8 minutes. The saliva secretion volume (mg / 8min) was calculated by weighing the cotton ball again.

[0103] II. Experimental Results

[0104] Saliva flow measurement results are as follows Figure 5 As shown, the saliva flow rate in the treatment group mice was significantly higher than that in the control group (p<0.001). This indicates that the oral spray prepared using exosome-like nanovesicles from *Gnaphalium affine* leaves can also be used to treat Sjögren's syndrome.

Claims

1. The application of *Gnaphalium affine* leaf exosome-like nanovesicles in the preparation of drugs for treating or improving Sjögren's syndrome, characterized in that... The extraction method of the exosome-like nanovesicles from the leaves of *Gnaphalium affine* involves homogenizing the leaves, subjecting the mixture to differential centrifugation and ultracentrifugation, followed by washing and resuspending. The differential centrifugation includes coarse centrifugation and medium centrifugation. The coarse centrifugation is performed at 500–1500 × g for 5–15 minutes, and at 2500–3500 × g for 15–25 minutes. The medium centrifugation is performed at 9000–11000 × g for 35–45 minutes. The ultracentrifugation is performed at 100000–200000 × g for 170–190 minutes.

2. The application according to claim 1, characterized in that, The particle size of the exosome-like nanovesicles from the bitter clover leaves is 140–160 nm.

3. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by protecting and / or repairing the function of salivary gland epithelial cells, increasing saliva secretion, and thus improving dry mouth symptoms.

4. The application according to claim 1, characterized in that, The effective dose range of the *Gnaphalium affine* leaf exosome-like nanovesicles is 0.1–100 mg / kg body weight / day.

5. The application according to claim 1, characterized in that, The drug also includes other pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The dosage form of the drug is injection, oral liquid, capsule, oral spray or gel.

7. The application according to claim 6, characterized in that, The oral spray is composed of *Gnaphalium affine* leaf exosome-like nanovesicles, thickener, humectant, and preservative in a mass ratio of 1.3–1.7:9–11:98–102:1.

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