Extracellular vesicles of intestinal flora intervened by phellodendri chinensis cortex and atractylodis rhizoma pair and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine biological medicine, and particularly relates to a cortex phellodendri-cangzhu drug pair intervention intestinal flora extracellular vesicle and application thereof. The extracellular vesicle is an extracellular vesicle obtained by separation and purification after the intestinal flora is intervened by the cortex phellodendri-cangzhu drug pair. The present application clearly regulates the control mechanism of the cortex phellodendri-cangzhu drug pair intervention intestinal flora, and directionally induces the intestinal flora to secrete specific extracellular vesicles with high anti-inflammatory and high immune regulation activity. Compared with the disorder intestinal flora of the blank control group, after being intervened by the optimal proportion of cortex phellodendri-cangzhu drug pair extract, the precise intestinal flora structure remodeling effect can be achieved, the imbalance state of flora diversity is effectively repaired, and the systemic immune inflammatory cascade reaction triggered by the intestinal flora disorder is blocked.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine biomedicine technology, and in particular to an extracellular vesicle of intestinal flora intervened by the Phellodendron bark-Atractylodes rhizome and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, symmetrical polyarticular synovitis, which can lead to cartilage destruction, bone erosion, and even joint deformities in severe cases. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying antirheumatic drugs (DMARDs) are commonly used in clinical practice, but these have limitations, significant side effects, and poor patient tolerance. In recent years, traditional Chinese medicine and gut microbiota regulation strategies have provided new insights into the treatment of RA.

[0003] Current technologies for treating rheumatoid arthritis (RA) with traditional Chinese medicine primarily focus on single-herb or compound preparations. Classic combinations, such as the Phellodendron amurense-Atractylodes lancea pair, are often prepared into oral formulations like decoctions, pills, and granules using conventional processes such as decoction, alcohol extraction, and spray drying. These formulations rely on the active ingredients, such as berberine, scutellarin, and atractylodes lancea, to directly exert their anti-inflammatory, analgesic, and immunomodulatory effects. For example, patents such as "A Traditional Chinese Medicine Composition for Treating Rheumatoid Arthritis and Its Application" and "A Traditional Chinese Medicine Composition for Treating Rheumatoid Arthritis and Its Preparation and Application" employ this approach. However, these technologies only focus on the direct absorption and systemic pharmacological effects of the herbal components, neglecting the regulation of the gut microbiota and failing to utilize active carriers derived from the gut microbiota (such as extracellular vesicles). This results in inherent drawbacks such as low bioavailability, poor targeting, and easy degradation of the active ingredients by gastrointestinal metabolism.

[0004] Existing technologies for treating rheumatoid arthritis (RA) through simple gut microbiota intervention focus on microecological regulation. Core approaches include: screening beneficial strains such as Bifidobacteria and Lactobacillus from RA patients or animal models, and preparing live or inactivated bacterial preparations via anaerobic fermentation; or using fecal microbiota transplantation (FMT) to directly adjust the gut microecological balance. For example, the study "Methods for Constructing a Rheumatoid Arthritis Model Through Fecal Microbiota Transplantation" falls into this category. These technologies regulate systemic immune responses by improving intestinal barrier function and inhibiting intestinal inflammation, but they primarily rely on the entire gut microbiota or its metabolites, without combining it with traditional Chinese medicine pairs for targeted regulation, nor is the key active carrier—extracellular vesicles secreted by the microbiota—isolated and enriched. Furthermore, live bacterial preparations suffer from low colonization rates, poor storage stability, and potential pathogenic risks, while FMT faces biosafety concerns and ethical controversies.

[0005] Extracellular vesicles (EVs) derived from microorganisms or host cells have emerged as a novel direction in the treatment of rheumatoid arthritis (RA) in recent years. EVs are nanoscale lipid bilayer structures capable of carrying nucleic acids, proteins, and other bioactive substances to mediate intercellular communication. Current technologies primarily isolate EVs directly from host immune cells, mesenchymal stem cells, or single standard bacterial strains, preparing vesicle formulations using processes such as ultracentrifugation and density gradient centrifugation, and administering them intravenously or intra-articularly to exert immunomodulatory effects. Related patents include "An Engineered Apoptotic Extracellular Vesicle for Regulating the Microenvironment of Rheumatoid Arthritis and Its Preparation Method and Application" and "A Plant Nanovesicle Loaded with Paeoniflorin and Its Application." However, these technologies rely on a single source of EVs and have fixed active ingredients, making it difficult to achieve multi-target synergistic regulation. Furthermore, they lack the integration of traditional Chinese medicine compound interventions for targeted optimization of the EV microbiota, thus limiting their therapeutic efficacy.

[0006] In summary, there is currently no integrated technical solution that organically combines "intervention with the Phellodendron amurense-Atractylodes lancea drug pair," "intestinal flora regulation," and "preparation of extracellular vesicles from the flora." Therefore, developing a novel strategy based on the Phellodendron amurense-Atractylodes lancea drug pair for targeted intervention of the intestinal flora, thereby enriching specific and highly active extracellular vesicles, and applying it to the treatment of rheumatoid arthritis (RA), has significant clinical value and innovative implications. Summary of the Invention

[0007] The purpose of this invention is to provide an extracellular vesicle of the intestinal flora intervened by the Phellodendron amurense-Atractylodes lancea herb pair and its application. This invention addresses the inherent shortcomings of traditional Chinese medicine herb pair preparations, simple intestinal flora regulation techniques, and single-source microbial extracellular vesicle preparations in the current treatment of rheumatoid arthritis. Combining the classic theory of herb pair compatibility in traditional Chinese medicine with modern microbial extracellular vesicle biotechnology, and focusing on the intrinsic regulatory relationship between the Phellodendron amurense-Atractylodes lancea herb pair, intestinal flora, and extracellular vesicles, this invention proposes a novel technical solution. It aims to fundamentally overcome the shortcomings of existing technologies and break through the technical bottleneck of combining targeted Chinese medicine therapy with microecological regulation in rheumatoid arthritis.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides extracellular vesicles of intestinal flora intervened by the Phellodendron bark-Atractylodes rhizome drug pair. The extracellular vesicles are obtained by isolation and purification after intervention of intestinal flora by the Phellodendron bark-Atractylodes rhizome drug pair.

[0009] Preferably, the extract of the Phellodendron bark and Atractylodes lancea drug pair is given to the donor, the donor's feces are collected, and the extracellular vesicles of intestinal flora are isolated and purified from the feces.

[0010] Preferably, the donor is a rheumatoid arthritis model animal.

[0011] Preferably, the preparation method of the Phellodendron bark and Atractylodes lancea extract is as follows: weigh equal amounts of Phellodendron bark and Atractylodes lancea, crush and sieve them, mix them, add 8-12 times the weight of water and soak for 25-35 minutes, decoct for 25-35 minutes, filter, and concentrate to a raw drug content of 0.2-0.4 g / mL.

[0012] Preferably, the intervention dose is 1-5 g / kg body weight per day, and the administration period is 21-28 days.

[0013] Preferably, the separation and purification method is differential centrifugation and ultracentrifugation.

[0014] Preferably, the differential centrifugation method is as follows: centrifugation at 700-1000g for 10-20 min, 3600-4000g for 10-20 min, 7800-8000g for 10-20 min, and 10000-12000g for 10-20 min at 3-5℃; the ultracentrifugation conditions are as follows: centrifugation at 150000-160000g for 1.5-2.5 h at 3-5℃.

[0015] This invention provides the use of the aforementioned extracellular vesicles in the preparation of a medicament for treating rheumatoid arthritis.

[0016] Preferably, the drug is used to achieve one or more of the following functions: (a) Reduce the joint swelling index in individuals with rheumatoid arthritis; (b) Reduce the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum or synovial fluid of individuals with rheumatoid arthritis; (c) Increase the expression level of the anti-inflammatory factor IL-10 in the serum of individuals with rheumatoid arthritis; (d) Inhibits synovial hyperplasia, cartilage erosion and bone destruction in individuals with rheumatoid arthritis; (e) Repair the intestinal barrier function.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a composition of extracellular vesicles of gut microbiota based on the intervention and regulation of the classic Phellodendron amurense-Atractylodes lancea herb pair, and clarifies its novel application in the preparation of drugs for treating rheumatoid arthritis. This approach bridges the gap between traditional Chinese medicine theory and modern biomedical technology, developing novel therapeutic agents with superior efficacy, stronger targeting, higher safety, and a more comprehensive mechanism of action, filling the technological gap in the treatment of autoimmune diseases through intervention of gut microbiota-specific extracellular vesicles using traditional Chinese medicine. Specific beneficial effects are reflected in the following aspects.

[0018] I. Core Overall Technical Effects

[0019] This invention utilizes the Phellodendron bark and Atractylodes lancea herb pair for targeted intervention in the gut microbiota, resulting in the preparation of specific gut microbiota extracellular vesicles. In the treatment of rheumatoid arthritis, this invention achieves a core overall effect of highly effective anti-inflammatory action, precise targeting, addressing both the symptoms and the root cause, and ensuring safety and stability. It completely overcomes the limitations of traditional Chinese medicine pairs, simple microecological preparations, and single extracellular vesicles. It not only rapidly relieves external symptoms of rheumatoid arthritis such as joint redness, swelling, pain, and limited mobility, but also reverses the systemic autoimmune imbalance mediated by gut microbiota dysbiosis at its source, inhibiting synovial inflammation and proliferation, cartilage destruction, and bone erosion. This achieves a fundamental intervention in rheumatoid arthritis, rather than simply providing symptomatic relief. The overall therapeutic effect and safety are far superior to existing similar technologies.

[0020] II. Technological Effects of Subdivided Dimensions

[0021] 1. Breaking through the limitations of traditional Chinese medicine drug pairs, exploring novel active carriers.

[0022] Addressing the shortcomings of traditional Phellodendron amurense-Atractylodes lancea herb pair—low bioavailability after direct administration, exerting only direct pharmacological effects, and neglecting the gut microbiota-mediated pathway—this invention clarifies the regulatory mechanism of the Phellodendron amurense-Atractylodes lancea herb pair in intervening in the gut microbiota, and directionally induces the gut microbiota to secrete specific extracellular vesicles with high anti-inflammatory and immunomodulatory activity. Compared to the disordered gut microbiota in the blank control group, intervention with the optimally proportioned Phellodendron amurense-Atractylodes lancea herb pair extracts can achieve precise gut microbiota remodeling, effectively repairing the imbalance of microbiota diversity and blocking the systemic immune inflammatory cascade response triggered by gut microbiota disorder. This effect differs from simple microbiota culture technology, achieving targeted microbiota optimization mediated by traditional Chinese medicine, laying a core foundation for subsequent highly active vesicle secretion, and transforming the therapeutic efficacy of the traditional Chinese medicine pair into a more stable, easily absorbed, and more targeted extracellular vesicle active carrier, replacing traditional crude Chinese medicine extracts and improving the in vivo utilization efficiency of the drug.

[0023] 2. Optimize the source and activity of extracellular vesicles to address the issue of limited therapeutic efficacy for vesicles from single sources.

[0024] To address the shortcomings of existing single-strain or cell-derived extracellular vesicles with limited active ingredients, capable of regulating only a single inflammatory pathway, and unable to adapt to the complex pathogenesis of rheumatoid arthritis, this invention utilizes the multi-component synergistic regulation of the Phellodendron bark-Atractylodes rhizome pair to enrich diverse and highly active extracellular vesicles of gut microbiota, optimize the composition of active substances such as nucleic acids, proteins, and lipids within the vesicles, and achieve multi-target, multi-pathway simultaneous regulation of rheumatoid arthritis-related immune inflammatory responses. The therapeutic effect of the vesicle formulation of this invention was verified through experiments using a rat model of CIA: after 4 weeks of administration, the joint swelling index of rats was significantly reduced compared with the model group, the pain response was significantly relieved, and the joint mobility was greatly improved; the levels of core pro-inflammatory factors such as TNF-α and IL-6 in rat serum decreased, while the level of anti-inflammatory factor IL-10 was upregulated by 2-3 times; pathological sections showed that the model group had severe synovial hyperplasia, large-area cartilage erosion, and obvious bone destruction, while the vesicle treatment group showed significant inhibition of synovial hyperplasia, intact cartilage structure, and significant reversal of bone erosion. The joint tissue pathological score was reduced by more than 80% compared with the model group. At the same time, it can effectively repair the intestinal mucosal barrier, reduce intestinal permeability, and block the spread of intestinal inflammation to the whole body.

[0025] 3. Avoiding the safety and stability risks of simple gut microbiota intervention.

[0026] To address the shortcomings of live bacteria preparations (low colonization rate), fecal microbiota transplantation (FMT) with significant biosafety risks, and slow onset of action from simple microbial regulation, this invention abandons direct administration of live bacteria and FMT methods, instead using high-purity, live bacteria-free extracellular vesicles as the core active ingredient. Acute and chronic toxicity experiments have verified that during administration, experimental animals showed no mortality, significant weight abnormalities, liver or kidney damage, gastrointestinal irritation, immune rejection, or pathogenic bacterial infection. Long-term continuous administration demonstrated good tolerability, with a safety profile far exceeding that of live bacteria preparations and traditional chemical antirheumatic drugs. Furthermore, the drug-pair extract, after microbial transformation, avoids the irritating side effects of crude Chinese medicine extracts, mitigating risks such as pathogenic bacterial infection, gastrointestinal irritation, and immune rejection. This enhances the storage and in vivo stability of the formulation, making it suitable for large-scale clinical production and long-term use.

[0027] 4. Enhance treatment targeting and strengthen the reversal effect on the core lesions of rheumatoid arthritis.

[0028] Addressing the limitations of existing technologies in precisely targeting joint inflammation, providing only superficial symptom relief, and failing to reverse articular cartilage damage and bone erosion, this invention leverages the natural targeted delivery advantages of extracellular vesicles from gut microbiota, combined with the anti-inflammatory and bone-protective effects of traditional Chinese medicine pairs. Compared to traditional oral preparations using the Phellodendron amurense-Atractylodes lancea pair, this invention uses gut microbiota extracellular vesicles as active carriers, eliminating the need for gastrointestinal metabolism and degradation, thus avoiding the destruction of active substances by gastric acid and bile. Simultaneously, the gut microbiota extracellular vesicles possess natural inflammatory targeting properties, allowing for precise accumulation at the affected joints in rheumatoid arthritis via blood circulation. The local drug concentration in the joint is 5-8 times higher than in other tissues throughout the body, achieving targeted drug delivery. This effectively inhibits the inflammatory storm in the joint, blocks excessive osteoclast activation, and repairs the damaged intestinal barrier, fundamentally improving the systemic autoimmune imbalance mediated by gut microbiota dysbiosis, achieving both symptomatic and radical treatment of rheumatoid arthritis. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 The effect of extracellular vesicles on pathological sections of CIA rats. Detailed Implementation

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] I. Experimental Objective

[0034] To investigate the effect of extracellular vesicles of gut microbiota after intervention with the Phellodendron bark-Atractylodes rhizome on collagen-induced arthritis (CIA) rats.

[0035] II. Experimental Materials

[0036] Phellodendron bark (from Sichuan, batch number 290252) and Atractylodes lancea (from Inner Mongolia, batch number 266142) were both purchased from Zixuantang Medicinal Materials Co., Ltd.

[0037] III. Laboratory Animals

[0038] Male SD rats, weighing 170–200 g, were purchased from Spifort (Beijing) Biotechnology Co., Ltd. and housed in the animal platform of the Experimental Management Center of Shanxi University of Traditional Chinese Medicine. The temperature was maintained at (23±2)℃, the relative humidity at (55±5)%, and the day / night cycle was 12h. They had free access to food and water throughout the experiment.

[0039] IV. Experimental Methods

[0040] 4.1 Preparation of extract of Phellodendron bark and Atractylodes lancea herb pair

[0041] Weigh 50g each of stir-fried Phellodendron bark and stir-fried Atractylodes lancea (soaked in rice water), grind them into powder, sieve, and mix evenly. Weigh 100g of the powder, add 10 times the amount of water and soak for 30 minutes, then decoct for 30 minutes. After cooling, filter. Add 8 times the amount of water to the dregs and continue to decoct for 20 minutes. After cooling, filter and combine the two filtrates. Concentrate the filtrate on a rotary evaporator to a crude drug content of 0.3g / mL. The volume of the drug administered by gavage at this concentration is 1mL / 100g.

[0042] 4.2 Establishment of the CIA Model

[0043] After one week of acclimatization, equal volumes of Freund's complete adjuvant (CFA) and bovine type II collagen (CCII) solution were thoroughly mixed to a thick, creamy consistency to prepare a 1 mg / mL emulsion. This emulsion was injected into five locations on the back, tail base, and heel of each rat, at a rate of 0.1 mL per location. Seven days later, equal volumes of Freund's incomplete adjuvant (IFA) and bovine type II collagen (CCII) were mixed to prepare a 1 mg / mL emulsion. 0.3 mL of this emulsion was injected intraperitoneally into each rat as a booster immunization. Seven days after that, the rats were observed for typical arthritis symptoms in their limbs, and the development of collagen-induced arthritis was assessed according to the arthritis scoring criteria.

[0044] 4.3 Preparation, identification and characterization of extracellular vesicles of intestinal flora induced by the Phellodendron bark-Atractylodes lancea drug pair

[0045] CIA rats were administered 3 g / kg of Ermiao Powder (the extract of Phellodendron bark and Atractylodes lancea prepared in 4.1) by gavage (this dose was determined by conversion of clinical doses recorded in "Danxi Xinfa" and previous experimental optimization by the research group). Feces were collected 21 days after administration. Fresh feces from the Ermiao Powder-treated donors were weighed, and 10 times the volume of PBS was added. The mixture was vortexed for 2 min and then allowed to stand at 4℃ for 2 h. Subsequently, the mixture was centrifuged at 4℃ and 700g for 15 min, and the supernatant was collected. The same process was repeated: centrifuging at 4℃ and 3600g for 15 min, collecting the supernatant; centrifuging at 4℃ and 7800g for 15 min, collecting the supernatant; and centrifuging at 4℃ and 10000g for 15 min, collecting the supernatant. The resulting filtrate was then subjected to ultracentrifugation at 150000g for 2 h, the supernatant was discarded, and the mixture was resuspended in 200 μL of PBS, aliquoted, and stored at -80℃. The results of nanoparticle tracking analysis and transmission electron microscopy showed that the obtained bacterial extracellular vesicles had smooth surfaces, were round / elliptical in shape, and had a double-membrane vesicular structure. The particle size was observed to be 177.6±15.1 nm, and the particle concentration was 1.30e+11 particles / mL.

[0046] 4.4 Effects of extracellular vesicles of gut microbiota on CIA rats

[0047] 4.4.1 Model establishment, grouping, and drug administration

[0048] A rat model of CIA was established according to the method described in "4.2". Rats with successfully established models were randomly divided into a model control group and a high-dose extracellular vesicle group (2×10⁻⁶). 10 Particles / each / dose), low-dose extracellular vesicle group (1×10) 10 The treatment regimens included: Particles (per particle per dose), the Phellodendron amurense-Atractylodes lancea herb pair group (3g / kg / dose), and a positive control group (methotrexate, 0.9mg / kg). A blank control group was also included. The blank control group and the model control group received normal saline via gavage daily. The extracellular vesicle group received the corresponding dose via gavage three times a week. The Phellodendron amurense-Atractylodes lancea herb pair group received the corresponding dose via gavage once a day. The positive control group received the medication via gavage once a week. Treatment lasted for 4 weeks.

[0049] 4.4.2 Index Measurement

[0050] The transverse diameter of the ankle joint was measured using calipers on days 0, 7, 14, 21, and 28 of treatment, and arthritis scores were recorded. After gavage, the rats were fasted for 12 hours, then anesthetized with isoflurane gas, and blood was collected from the abdominal aorta. One ankle joint was harvested, and excess skin, flesh, and tissue were carefully removed using surgical scissors, taking care to preserve the integrity of the joint structure. The joint was then placed in a tissue fixation solution for decalcification and pathological staining.

[0051] V. Experimental Results

[0052] 5.1 Effect of extracellular vesicles on the thickness of paw swelling in CIA rats

[0053] As shown in Table 1, the swelling thickness in the blank control group remained stable (3.41-3.65 mm) throughout the experimental period, with no obvious inflammatory response. The paw thickness in the model control group reached its peak at day 14 (9.43±0.58 mm), an increase of 158.4% compared to the blank control group (3.62±0.20 mm). All treatment groups significantly inhibited paw swelling, and the effect gradually increased over time. The improvement rate at day 42 was ranked as follows: positive control group (30.3%) > high-dose vesicle group (26.6%) > Phellodendron bark-Atractylodes rhizome pair group (17.7%) > low-dose vesicle group (15.8%). Extracellular vesicles showed a clear dose-dependent effect (high-dose was more effective than low-dose).

[0054] Table 1. Effects of extracellular vesicles on joint swelling thickness in CIA rats

[0055] Note: Compared with the blank control group P <0.001; compared with the model control group, ## P <0.01, ### P <0.001

[0056] 5.2 Effects of extracellular vesicles on arthritis scores in CIA rats

[0057] As shown in Table 2, the blank control group had a score of 0 throughout the entire treatment period and showed no signs of arthritis. The model control group developed significant arthritis symptoms starting on day 7, reaching a peak score of 10.8 ± 1.92 points on day 28. The improvement rates in each treatment group over 42 days were excellent: high-dose vesicle group (43.7%) = positive control group (43.7%) > low-dose vesicle group (31.2%) > Phellodendron bark-Atractylodes rhizome pair group (27.1%). The high-dose vesicle group showed accelerated improvement between days 35 and 42, achieving efficacy comparable to the positive control group. All treatment groups effectively delayed arthritis progression and prevented worsening of joint lesions.

[0058] Table 2. Effects of extracellular vesicles on arthritis scores in CIA rats

[0059] Note: Compared with the blank control group P <0.001; compared with the model control group, ## P <0.01, ### P <0.001

[0060] 5.3 Effects of extracellular vesicles on serum inflammatory factors in the joints of CIA rats

[0061] As shown in Table 3, the model control group exhibited a typical state of inflammatory imbalance (elevated pro-inflammatory factors and decreased anti-inflammatory factors). The high-dose vesicle group was able to simultaneously achieve bidirectional regulation of "decreasing pro-inflammatory factors and increasing anti-inflammatory factors," and the effect was significantly better than other treatment groups. Details are as follows: Table 3. Effects of extracellular vesicles on the levels of inflammatory factors in CIA rats.

[0062] Note: Compared with the blank control group P <0.001 P <0.01; compared with the model control group, # P <0.05, ### P <0.001

[0063] According to the experimental data, the model control group showed a typical state of inflammatory imbalance, with pro-inflammatory factors IL-6 and TNF-α increasing by 26.9% and 83.0%, respectively, while anti-inflammatory factor IL-10 decreased by 46.8%. In contrast, the high-dose vesicle group achieved significant bidirectional regulation, with improvement rates of 27.0% and 48.3% for IL-6 and TNF-α, respectively, both of which were the best among the groups. At the same time, the improvement in IL-10 was as high as 83.4%, which was significantly better than other treatment groups.

[0064] 5.4 Effects of extracellular vesicles on pathological sections of CIA rats

[0065] like Figure 1 As shown, inflammatory cell infiltration and cartilage structure were assessed through histopathological examination. H&E staining of the ankle joint revealed no related arthritis pathological symptoms in the blank control group rats; the articular surface was smooth and even, with tightly packed and orderly cell arrangement. In contrast, the model control group rats showed significant changes in ankle joint tissue, including inflammatory cell infiltration, abnormal synovial cell proliferation, pannus formation, and cartilage erosion. Compared with the model control group, oral administration of extracellular vesicles significantly prevented cartilage surface erosion and joint degeneration, inflammatory cell infiltration, and synovial hyperplasia in rats in a dose-dependent manner.

[0066] Simultaneously, observation of safranin O-Fix Green stained sections of rat knee joints revealed the following: Blank control group: Cartilage matrix stained a uniform deep red, subchondral bone fibrous layer a light blue, sections showed uniform staining, and abundant proteoglycans; the junction between cartilage matrix and subchondral bone was distinct, and the tidal line was clearly visible. Model control group: Cartilage matrix staining was lighter, proteoglycan content decreased; cartilage matrix collapse, disordered structural layers, uneven tidal line, and serrated adhesion lines were observed. High-dose extracellular vesicle group: Cartilage structure intact, cartilage matrix staining deepened, comparable to the blank group, abundant proteoglycans, and neatly visible tidal line. Low-dose extracellular vesicle group: Safranin staining was lighter, tidal line was clearly visible, but adhesion lines were disordered and wavy.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An extracellular vesicle of intestinal flora induced by the interaction of Phellodendron bark and Atractylodes lancea, characterized in that, The extracellular vesicles were obtained by separating and purifying extracellular vesicles after intervening in the intestinal flora with the Phellodendron bark-Atractylodes rhizome drug pair.

2. The extracellular vesicle according to claim 1, characterized in that, The extract of Phellodendron bark and Atractylodes lancea was given to the donor, and the donor's feces were collected. Extracellular vesicles of intestinal flora were isolated and purified from the feces.

3. The extracellular vesicle according to claim 2, characterized in that, The donors were animal models of rheumatoid arthritis.

4. The extracellular vesicle according to claim 2, characterized in that, The preparation method of the extract of the Phellodendron bark and Atractylodes lancea is as follows: weigh equal amounts of Phellodendron bark and Atractylodes lancea, crush and sieve them, mix them, add 8 to 12 times the weight of water and soak for 25 to 35 minutes, decoct for 25 to 35 minutes, filter, and concentrate to a raw drug content of 0.2 to 0.4 g / mL.

5. The extracellular vesicle according to claim 4, characterized in that, The intervention was administered at a dose of 1-5 g / kg body weight per day for a period of 21-28 days.

6. The extracellular vesicle according to claim 2, characterized in that, The separation and purification methods are: differential centrifugation and ultracentrifugation.

7. The extracellular vesicle according to claim 6, characterized in that, The differential centrifugation method is as follows: centrifuge at 700-1000g for 10-20 min, 3600-4000g for 10-20 min, 7800-8000g for 10-20 min, and 10000-12000g for 10-20 min at 3-5℃; the ultracentrifugation conditions are as follows: centrifuge at 150000-160000g for 1.5-2.5 h at 3-5℃.

8. Use of the extracellular vesicles according to any one of claims 1 to 7 in the preparation of a medicament for treating rheumatoid arthritis.

9. The application according to claim 8, characterized in that, The drug is used to achieve one or more of the following functions: (a) Reduce the joint swelling index in individuals with rheumatoid arthritis; (b) Reduce the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum or synovial fluid of individuals with rheumatoid arthritis; (c) Increase the expression level of the anti-inflammatory factor IL-10 in the serum of individuals with rheumatoid arthritis; (d) Inhibits synovial hyperplasia, cartilage erosion and bone destruction in individuals with rheumatoid arthritis; (e) Repair the intestinal barrier function.

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