Gastro-resistant tablets of amlodipine

Abstract

This invention discloses an extracellular vesicle derived from Gastrodia elata, its preparation method, and its use in preparing drugs for treating glaucoma, belonging to the field of biomedical technology. This invention claims protection for an extracellular vesicle prepared from dried Gastrodia elata. The extracellular vesicles provided by this invention have no significant cytotoxicity and good biocompatibility. Studies have shown that these extracellular vesicles can significantly inhibit apoptosis of R28 cells and loss of Brn3a⁺ cells in the mouse retina in a glutamate excitotoxicity model. In a glaucoma model, these extracellular vesicles can effectively protect the survival of retinal ganglion cells (RGCs) and retinal function, and can significantly reduce retinal neuroinflammatory responses in the glaucoma model. Therefore, the extracellular vesicles provided by this invention have a clear anti-glaucoma-related retinal damage effect and possess great potential for development into drugs for treating glaucoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an extracellular vesicle derived from Gastrodia elata, its preparation method, and its use in preparing drugs for treating glaucoma. Background Technology

[0002] Glaucoma is the leading cause of irreversible blindness worldwide, affecting nearly 95 million people globally. Its clinical features mainly include optic nerve damage, progressive visual field defects, and vision loss [Reference: Jayaram, H., Kolko, M., Friedman, DS, & Gazzard, G. (2023). Glaucoma: now and beyond. Lancet, 402(10414), 1788–1801. https: / / doi.org / 10.1016 / S0140-6736(23)01289-8]. Due to factors such as slow vision decline, asymmetrical disease progression in both eyes, and compensatory mechanisms of the visual system, patients often seek medical attention only in the late stages of the disease, leading to severe health damage, life distress, and social burden.

[0003] Although intraocular pressure (IOP) is currently the only modifiable risk factor confirmed by large-scale clinical trials, nearly half of glaucoma patients do not have elevated IOP, and IOP-lowering treatments have limitations in clinical application, such as postoperative complications and long-term medication side effects. Therefore, current research has shifted to neuroprotective treatment strategies unrelated to IOP [Reference: Weinreb, RN, Aung, T., & Medeiros, FA (2014). The pathophysiology and treatment of glaucoma: a review. JAMA, 311(18), 1901–1911. https: / / doi.org / 10.1001 / jama.2014.3192].

[0004] The mechanisms underlying retinal ganglion cell (RGC) loss during glaucoma pathogenesis are complex and diverse, and not yet fully elucidated; however, glutamate excitotoxicity is widely considered to be one of the major damaging mechanisms [Reference: Miao, Y., Zhao, GL, Cheng, S., Wang, Z., & Yang, XL (2023). Activation of retinal glial cells contributes to the degeneration of ganglion cells in experimental glaucoma. Progress in retinal and eye research, 93, 101169. https: / / doi.org / 10.1016 / j.preteyeres.2023.101169]. Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system, mainly located intracellularly, and extracellularly regulates neuronal excitability by binding to ionotropic glutamate receptors. Under pathological conditions such as ischemia / reperfusion injury and oxidative stress, the activity of the glutamate uptake / transport system decreases, leading to an increase in extracellular glutamate concentration. This, in turn, produces excitotoxicity by directly acting on retinal glial cells (RGCs) or indirectly causes changes in RGCs by activating glial cells [Reference: Miao, Y., Zhao, GL, Cheng, S., Wang, Z., & Yang, XL (2023). Activation of retinal glial cells contributes to the degeneration of ganglion cells in experimental glaucoma. Progress in retinal and eye research, 93, 101169. https: / / doi.org / 10.1016 / j.preteyeres.2023.101169].

[0005] Microglia are resident immune cells in the central nervous system, performing functions such as immune surveillance, phagocytosis, and neuroprotection under physiological conditions. However, when the central nervous system is damaged, uncontrolled microglia responses may lead to excessive inflammation, threatening neuronal survival [Reference: Ramirez, AI, de Hoz, R., Salobrar-Garcia, E., Salazar, JJ, Rojas, B., Ajoy, D., López-Cuenca, I., Rojas, P., Triviño, A., & Ramírez, JM (2017). The Role of Microglia in Retinal Neurodegeneration: Alzheimer's Disease, Parkinson, and Glaucoma. Frontiers inaging neuroscience, 9, 214. https: / / doi.org / 10.3389 / fnagi.2017.00214]. Neuroinflammation is considered a significant contributing factor to degenerative diseases of the central nervous system, and microglia play a crucial role in neurodegenerative diseases such as glaucoma. Therefore, therapeutic strategies that inhibit the activation of microglia into a pro-inflammatory phenotype show potential in the treatment of glaucoma.

[0006] Exosomes, as the smallest and most widely studied extracellular vesicles (EVs), have been widely applied in disease diagnosis, treatment, and drug delivery due to their excellent biocompatibility, stability, and targeting capabilities. Early exosome research primarily focused on stem cell and tumor cell-derived exosomes. However, with the expansion of research, the existence and function of plant-derived extracellular vesicles (PDEVs) have gradually attracted widespread attention. Studies have shown that PDEVs typically have a diameter of 30–200 nm, possess a lipid bilayer membrane structure, and are similar in size and morphology to animal-derived exosomes. They encapsulate bioactive molecules such as proteins, RNA, lipids, and secondary metabolites, participating in intercellular communication and molecular transport. Furthermore, a large number of microRNAs (miRNAs) have been found in PDEVs. These miRNAs can be absorbed by the human body and exert therapeutic effects, suggesting that PDEVs may be a potential pathway for miRNA transfer from plants to animals.

[0007] There are currently no reports on the use of extracellular vesicles derived from Gastrodia elata in the treatment of glaucoma. Summary of the Invention

[0008] The first objective of this invention is to provide an extracellular vesicle derived from Gastrodia elata, the second objective is to provide a method for preparing the extracellular vesicle, and the third objective is to provide the use of the extracellular vesicle in the preparation of a drug for treating glaucoma.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides an extracellular vesicle prepared from dried Gastrodia elata. The dried Gastrodia elata is preferably a medicinal material conforming to the standards of the Pharmacopoeia of the People's Republic of China, which is prepared by removing impurities, washing, and drying before use. Its effective component content is stable, ensuring that the prepared extracellular vesicles have stable biological activity.

[0010] This invention provides a method for preparing the above-mentioned extracellular vesicles, specifically including the following steps: Step (1): Pulverize the dried Gastrodia elata, preferably to a particle size of 50-100 mesh, to improve extraction efficiency; add buffer salt solution to the pulverized dried Gastrodia elata powder, mix well, and place on a low-temperature shaker overnight. The purpose of this step is to allow the dried Gastrodia elata cells to fully rupture and release the vesicles inside the cells through low-temperature and gentle shaking, while avoiding the damage of high temperature to the vesicle structure and active ingredients.

[0011] Step (2): Filter the mixture obtained in step (1) using a regular filter screen. The main purpose is to obtain a crude extract containing extracellular vesicles.

[0012] Step (3): Place the filtrate collected in step (2) under low temperature conditions for gradient centrifugation. The purpose of gradient centrifugation is to gradually remove impurities from the filtrate. After centrifugation, collect the supernatant, which contains preliminarily purified extracellular vesicles.

[0013] Step (4): Filter the supernatant collected in step (3) to obtain a purer extracellular vesicle solution; centrifuge the filtered solution under low temperature conditions. After centrifugation, discard the supernatant and the precipitate is the crude extracellular vesicle extract; add buffer salt solution to the precipitate for resuspension. The volume of the resuspension is adjusted according to the amount of precipitate to ensure that the vesicles are evenly dispersed.

[0014] Step (5): Centrifuge the resuspension obtained in step (4) under low temperature conditions. The centrifugation parameters are the same as those in step (4). After centrifugation, discard the supernatant, and the precipitate is the target extracellular vesicle. This step can further remove residual impurities and obtain high-purity extracellular vesicles.

[0015] In one specific embodiment, the buffer salt solution in steps (1) and (4) is PBS phosphate buffer solution.

[0016] In one specific embodiment, the low temperature in steps (1), (3), (4), and (5) is 4°C.

[0017] In one specific embodiment, the parameters for gradient centrifugation in step (3) are: 800×g, 10 minutes; 2000×g, 10 minutes; 10000×g, 20 minutes.

[0018] In one specific embodiment, a 0.45 μm filter is used in step (4).

[0019] In one specific embodiment, the centrifugation parameters for low-temperature centrifugation in steps (4) and (5) are 130000×g for 70 min.

[0020] The present invention provides the use of the above-mentioned extracellular vesicles, specifically the use of the above-mentioned extracellular vesicles in the preparation of drugs for treating glaucoma.

[0021] Beneficial effects: 1. This invention is the first to successfully prepare extracellular vesicles using dried Gastrodia elata as raw material. This not only effectively expands the range of raw material sources for extracellular vesicles, but also fully explores and utilizes the medicinal potential of Gastrodia elata, realizing the efficient development and high-value utilization of traditional Chinese medicine resources.

[0022] 2. The extracellular vesicle preparation method provided by this invention is simple to operate and has mild reaction conditions. It does not rely on complex experimental equipment and expensive reagents, which effectively reduces production costs. At the same time, this method can efficiently extract high-purity and highly bioactive extracellular vesicles with good reproducibility and high stability, which meets the technical requirements of large-scale industrial production and has good prospects for industrial application.

[0023] 3. The extracellular vesicles provided by this invention exhibit no significant cytotoxicity and good biocompatibility. Studies have shown that these extracellular vesicles can significantly inhibit apoptosis of R28 cells and loss of Brn3a⁺ cells in the mouse retina in a glutamate excitotoxicity model. Furthermore, these extracellular vesicles effectively protect the survival of retinal ganglion cells (RGCs) and retinal function in a glaucoma model, and significantly reduce retinal neuroinflammatory responses in the glaucoma model. In summary, the extracellular vesicles provided by this invention have a clear anti-glaucoma-related retinal damage effect and possess great potential for development into a glaucoma treatment drug. Attached Figure Description

[0024] Figure 1 Gas-Ex extraction process; Figure 2Characterization of Gas-Ex: A, B: Nanoparticle tracking analysis (NTA) was used to determine the particle size distribution and concentration of Gas-Ex. The results showed that the particle size was mainly distributed in the range of 80 nm to 200 nm, with a peak at approximately 120 nm; the particle concentration was approximately 5.94 × 10¹. 0 -4.58×10¹² particles / mL; C: Nanoscale flow cytometry analysis of Gas-Ex particle size distribution. The results show that the particle size is mainly distributed in the range of 50nm to 100nm, with a peak of about 70nm; the particle concentration is about 1.22×10¹² particles / mL; D: Nanoscale flow cytometry combined with membrane dye staining analysis of Gas-Ex purity. The results show that 89.2% of the particles have membrane dye fluorescence signals, indicating that there are few non-vesicular impurities in the sample and the purity is high; E: Zeta potential measurement results. The average Zeta potential value of Gas-Ex is -4.31mV (25℃); F, G: Transmission electron microscopy observation of the morphology and structure of Gas-Ex.

[0025] Figure 3 Protein composition analysis of Gas-Ex: A: Coomassie Brilliant Blue stained gel image, showing that Gas-Ex contains a certain amount of protein; B: Protein subcellular localization analysis map. Gas-Ex protein sources include: cytoplasm (22.95%), nucleus (18.03%), chloroplasts (16.39%), endoplasmic reticulum (13.11%), mitochondria (9.84%), etc., confirming that it is a plant-derived extracellular vesicle; C: GO analysis map, showing that Gas-Ex protein is mainly enriched in the "Global and Overview Map" and multiple core metabolic pathways; D: KEGG analysis map, showing that Gas-Ex protein is mainly enriched in carbohydrate metabolism, amino acid metabolism, lipid metabolism, energy metabolism, and cofactor and vitamin metabolism pathways; Figure 4 Validation of Gas-Ex uptake: A: Confocal microscopy image. Did-labeled Gas-Ex (red) colocalizes with the cytoskeleton (Actin-tracker Green) and nucleus (DAPI, blue) of R28 cells, indicating that Gas-Ex is internalized and distributed in the cytoplasm; B–D: Immunofluorescence staining images of mouse retinal tissue. DiD-labeled Gas-Ex (red) colocalizes with retinal ganglion cell marker Brn3a (green, B), astrocyte marker GFAP (green, C), and microglia marker IBA-1 (green, D), demonstrating that Gas-Ex can be taken up by various retinal cells in vivo; Figure 5To demonstrate the protective effect of Gas-Ex on R28 cells: A: CCK-8 assay was used to detect the cell viability of R28 cells after treatment with different concentrations of Gas-Ex (1, 5, 10, 50 μg / mL) to determine the safe concentration range; B, C: CCK-8 assay was used to detect the cell viability of each treatment group (Control, Gas-Ex, GLU, GLU+Gas-Ex), showing that Gas-Ex significantly restored the decrease in cell viability caused by glutamate damage; D: Representative images of live / dead cell staining (green: live cells, Calcein-AM; red: dead cells, PI). The number of dead cells increased in the GLU group, and the number of dead cells decreased after Gas-Ex intervention; E: Quantitative results of lactate dehydrogenase (LDH) release, reflecting cell membrane integrity. Gas-Ex effectively reduced LDH release caused by glutamate damage; F: Quantitative results of intracellular ATP content, reflecting the cellular energy metabolism status. Gas-Ex significantly restored the decrease in ATP levels caused by glutamate damage; G: LDH measurement in cell supernatant, reflecting the degree of cell membrane integrity. Gas-Ex significantly restored cell membrane rupture caused by glutamate damage.

[0026] Figure 6 To illustrate the protective effect of Gas-Ex against NMDA-induced retinal damage in mice: A: Representative images of retinal patch Brn3a immunofluorescence staining, showing changes in the number of retinal ganglion cells (RGCs) in each group; B: Quantitative statistical analysis of RGC density, showing that Gas-Ex significantly reversed NMDA-induced RGC loss; C, D: HE-stained images of the retina (C) and statistical analysis of the number of ganglion cell layers (GCLs) (D), showing that Gas-Ex maintained retinal structural integrity and increased the number of GCL cells; E–G: Results of flash visual evoked potentials (F-VEP). E: Representative waveform; F: P1 wave latency; G: N1-P1 wave amplitude. Gas-Ex improved the prolonged latency and decreased amplitude caused by NMDA damage; H–J: Results of flash electroretinography (F-ERG). H: Representative waveform; I: a-wave amplitude; J: b-wave amplitude. Gas-Ex effectively reversed the decrease in a-wave and b-wave amplitude caused by NMDA damage; Figure 7Inhibitory effect of Gas-Ex on neuroinflammation: A: Representative images of cell morphological changes in the BV2 microglia OGD / R model; Gas-Ex alleviates OGD / R-induced cell activation (cell hypertrophy, increased branching); B: mRNA expression levels (qPCR) of pro-inflammatory factors (IL-1β, IL-6, TNF-α) in the BV2 cell OGD / R model; Gas-Ex significantly inhibits the expression of inflammatory factors; C: Representative images of retinal patch Iba1 immunofluorescence staining, showing morphological changes of microglia in each group. Microglia in the NMDA group were activated (cell hypertrophy, branching retraction); after Gas-Ex intervention, the morphology was closer to the resting state; D: Quantitative statistics of microglia activation status (e.g., number of branch nodes, total length, etc.); Gas-Ex significantly alleviates NMDA-induced microglia activation; E: mRNA expression levels (qPCR) of pro-inflammatory factors (IL-1β, IL-6, TNF-α) in retinal tissue; Gas-Ex inhibits NMDA-induced neuroinflammatory response. Detailed Implementation

[0027] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0028] Example 1: Preparation and characterization of extracellular vesicles (Gas-Ex) I. Preparation and Preservation of Gas-Ex 1. Weigh 150g of dried Gastrodia elata, slice it, grind it into powder (50-100 mesh), add 500ml of PBS (phosphate buffer solution), mix well, and place it on a shaker at 4℃ overnight (12-15h).

[0029] 2. Use a homogenate filter to collect the filtrate.

[0030] 3. The collected filtrate was subjected to gradient centrifugation at 4℃, and the supernatant was collected. The gradient centrifugation time and centrifugation force were as follows: 800×g, 10 minutes; 2000×g, 10 minutes; 10000×g, 20 minutes.

[0031] 4. After filtering the supernatant through a 0.45μm filter, the crude extract Gas-Ex was obtained by centrifuging at 4℃ and 130000×g for 70min in an ultra-high speed centrifuge and then resuspended in PBS.

[0032] 5. Centrifuge the resuspension at 4°C and 130,000 × g for 70 min in an ultracentrifuge. Resuspend the precipitate in 2 mL of PBS, filter sterilize using a 0.22 μm filter membrane, and aliquot and freeze at -80°C.

[0033] II. Characterization Analysis of Gas-Ex To confirm that the Gas-Ex prepared above are homogeneous and structurally intact extracellular vesicles, and to evaluate their purity, concentration, and basic physicochemical properties, we conducted a series of systematic characterization analyses: 1. Nanoparticle tracking and analysis technology ① Dilute the prepared Gas-Ex sample appropriately with PBS to ensure that the final detection concentration is within the optimal detection range of the instrument.

[0034] ② Turn on the instrument and its software, and use a 1 mL sterile syringe to inject sufficient PBS into the sample cell to perform a background check and ensure that the number of background particles in the optical field of view meets the requirements.

[0035] ③ Sample loading: Using a new 1 mL sterile syringe, draw up about 1 mL of diluted Gas-Ex sample and inject it into the sample cell at a constant flow rate.

[0036] ④ Data Acquisition: At 25℃, using the built-in camera at a height of 13, video was acquired under Brownian motion of the particles, and three 15-second video clips were collected.

[0037] ⑤ The acquired video was analyzed using NTA 3.4. The software automatically tracked the trajectory of each particle and calculated the hydration dynamic diameter of each particle. The final results were output as a particle size distribution map and particle concentration (particles / mL).

[0038] 2. Nanoflow cytometry and membrane structure staining ① Take an appropriate amount of Gas-Ex sample and mix it with a certain concentration of Membrane Red Stains dye working solution (nanofcm).

[0039] ②Incubate at 37℃ in the dark for 60 minutes.

[0040] ③ The dyed mixture was subjected to ultracentrifugation (130,000 g, 70 minutes, 10°C) to remove unbound free dye.

[0041] ④ Carefully discard the supernatant and resuspend the precipitated Gas-Ex particles in pre-filtered PBS.

[0042] ⑤ Turn on the instrument and its supporting software, and calibrate the nanoflow cytometer using standard fluorescent microspheres with known particle size and concentration.

[0043] ⑥ Dilute the stained and unstained Gas-Ex samples appropriately with PBS, load them sequentially for detection, and simultaneously collect the side-scattered light signal and fluorescence signal at a specific wavelength for each particle.

[0044] 3. Zeta potential measurement ① Dilute the Gas-Ex sample appropriately with sterile deionized water to reduce the sample conductivity and avoid interference with the measurement.

[0045] ② Sample cell loading: Slowly inject approximately 1 mL of diluted sample into the sample cell using a syringe, ensuring that no air bubbles are generated.

[0046] ③ Place the sample cell into the instrument's sample chamber. After equilibration, the instrument applies an electric field to induce electrophoretic movement of charged particles, detects their migration rate, and converts it into a zeta potential value. Each sample is measured three times.

[0047] 4. Observation using transmission electron microscopy ① Drop 10-20 μL of Gas-Ex sample onto the carbon film surface of the carbon support copper mesh, let it stand at room temperature for 5-10 minutes to adsorb, and then carefully absorb the excess liquid from the edge of the copper mesh with filter paper.

[0048] ② Add 10-20 μL of 2% phosphotungstic acid staining solution to the copper mesh containing the sample, and stain for 1-2 minutes. Blot the staining solution thoroughly with filter paper, and then let the copper mesh air dry at room temperature for at least 10 minutes.

[0049] ③ Place the completely dried copper mesh into the transmission electron microscope sample holder and observe it under a suitable accelerating voltage.

[0050] III. Determination of Protein Components in Gas-Ex The mass spectrometry scan range is divided into several windows based on the mass-to-charge ratio (m / z). Then, all precursor ions in each window are fragmented and detected, and fragment ion information of all precursor ions is collected for protein qualitative and quantitative analysis.

[0051] IV. Verification of Gas-Ex uptake effect 1. Fluorescent labeling and purification of Gas-Ex ① Take an appropriate amount of Gas-Ex sample and add Did dye working solution with a final concentration of 5-10 μM.

[0052] ② After gently mixing by pipetting, incubate at 37°C in the dark for 30 minutes. Then, centrifuge the mixture at high speed to separate the supernatant containing free dye. Resuspend the precipitated labeled Gas-Ex in sterile PBS and store it at -80°C in the dark.

[0053] 2. Cell Culture and Co-incubation ① R28 cells (rat retinal precursor cells) were seeded in confocal culture dishes and cultured in a 37°C incubator containing 5% CO2 until the cell confluence reached 60%-70%.

[0054] ② Replace the culture medium with fresh culture medium containing 10 μg / ml DId-labeled Gas-Ex.

[0055] ③ Place the cells back into the incubator and incubate (1 hour, 3 hours, 6 hours). Then discard the culture medium, rinse three times with pre-cooled culture medium, add 4% paraformaldehyde, and fix at room temperature for 30 minutes.

[0056] ④ After rinsing three times with PBS, add 0.5% Triton X-100 and permeabilize at room temperature for 15 minutes to increase cell membrane permeability.

[0057] ⑤ Add 5% BSA solution and seal for 30 minutes.

[0058] ⑥ Wash three times with PBS, add Actin-tracker Green-488 working solution, and incubate at room temperature in the dark for 30 minutes to label the cytoskeleton.

[0059] ⑦ After rinsing with PBS, add DAPI working solution and incubate at room temperature in the dark for 5-10 minutes to label cell nuclei.

[0060] ⑧ Finally, rinse three times with PBS and add anti-fluorescence quenching mounting medium to the confocal culture dish.

[0061] ⑨ Using a confocal laser scanning microscope, images of different fluorescence channels were acquired using appropriate lasers and superimposed to analyze the relative positions of the Did red fluorescence signal (Gas-Ex) with the cytoskeleton (Actin) and the cell nucleus (DAPI).

[0062] V. Experimental Results 1. Characterization of Gas-Ex The exosome-like extracellular vesicles isolated from Gastrodia elata were characterized, and the results are shown below: ① The particle size distribution and concentration of Gas-Ex were detected by nanoparticle tracking analysis: The prepared Gas-Ex particles were mainly concentrated in the range of about 80 nm to 200 nm, with a peak particle size of about 120 nm, which is consistent with the typical particle size characteristics of extracellular vesicles. Figure 2 (A and B). The number of vesicle particles per milliliter of the original sample was also determined to be approximately 5.94 × 10⁻⁶. 10 -4.58×10 12 particles / mL.

[0063] ② Nanoflow cytometry detection: To further confirm the vesicle size and assess sample purity at the single-particle level, we used nanoflow cytometry for detection. The results showed that the Gas-Ex particle size was mainly concentrated in the range of approximately 50 nm to 100 nm, with a peak particle size of around 70 nm, and the number of vesicle particles was approximately 1.22 × 10⁻⁶. 12 particles / mL ( Figure 2 (C). Simultaneously, by staining the samples with a membrane dye that specifically binds to the vesicular lipid bilayer, it was found that the vast majority (89.2%) of the detected particle signals exhibited membrane dye fluorescence signals, demonstrating that the Gas-Ex samples we prepared had high purity, with low levels of contaminated non-vesicular protein aggregates or impurities. Figure 2 (D).

[0064] ③ Surface charge measurement of Gas-Ex: We used a nanoparticle size analyzer and a Zeta potential meter to scan and measure the Gas-Ex sample at 25℃, and obtained an average Zeta potential value of -4.31mV for Gas-Ex. Figure 2 (E).

[0065] ④ Observation of the morphology and structure of Gas-Ex using transmission electron microscopy (TEM) Figure 2 (F, G): Under electron microscopy, numerous typical saucer-shaped or cup-shaped spherical vesicle structures can be clearly seen, with complete morphology and clear boundaries; the diameter is distributed in the range of tens to hundreds of nanometers. The results are in high agreement with the particle size analysis results of NTA and nanoflow cytometry mentioned above.

[0066] 2. Protein composition determination of Gas-Ex The Gas-Ex prepared in this invention was identified at the molecular level, and the Coomassie Brilliant Blue staining results were obtained after gel electrophoresis as follows: Figure 3 Image A shows that it contains a certain amount of protein, and we performed proteomics analysis on it. For example... Figure 3 As shown in Figure B, subcellular localization analysis of the identified proteins revealed that their composition includes multiple types such as cytoplasmic proteins (22.95%), nuclear proteins (18.03%), chloroplast proteins (16.39%), endoplasmic reticulum proteins (13.11%), and mitochondrial proteins (9.84%).

[0067] The high proportion of chloroplast proteins confirms that Gas-Ex is a plant-derived substance.

[0068] Furthermore, the compositional characteristics of Gas-Ex proteins fully meet the identification requirements of the International Society for Extracellular Vesicles (MISEV) 2018 guidelines.

[0069] ① The presence of a large number of transmembrane or membrane-bound proteins (such as proteins derived from the cell membrane, endoplasmic reticulum, and mitochondria) proves that Gas-Ex has a complete lipid bilayer membrane structure. ② The presence of abundant cytoplasmic proteins demonstrates that Gas-Ex possesses an internal chamber capable of carrying bioactive molecules; ③ It contains a high proportion of organelle proteins (such as nuclear and chloroplast proteins) that are not derived from the plasma membrane, confirming that Gas-Ex is a typical extracellular vesicle (exosome) formed through a complex intracellular biogenetic pathway.

[0070] Simultaneously, GO and KEGG analyses were performed on the proteomics sequencing results, such as... Figure 3 As shown in Figures C and D, Gas-Ex proteins are primarily enriched in the "Global and Overview Map" and multiple core metabolic pathways, including carbohydrate metabolism, amino acid metabolism, lipid metabolism, energy metabolism, and cofactor and vitamin metabolism. Simultaneously, some proteins are also annotated to pathways involving translation, transcription, transport and catabolism, and signal transduction. This indicates that Gas-Ex naturally carries a large number of proteins involved in cellular basal metabolic processes and the flow of genetic information. These endogenous proteins, as part of its bioactive components, constitute the material basis for Gas-Ex's ability to engage in complex biological interactions with recipient cells and potentially influence their metabolic state.

[0071] 3. Gas-Ex was extracted and verified. Figure 4 The confocal microscopy images clearly show that after co-incubation with Did-labeled Gas-Ex, R28 cells exhibited numerous punctate red fluorescent signals in the cytoplasm. The red fluorescent signals co-localized with the green cytoskeleton network, indicating that Gas-Ex was internalized and distributed within the cytoplasm.

[0072] Similarly, in mouse retinal tissue, we further observed the cellular uptake effect of Gas-Ex using immunofluorescence multiplex staining. The results showed that the DiD-labeled Gas-Ex red fluorescence signal significantly co-localized with the immunofluorescence signals of retinal ganglion cell marker Brn3a, astrocyte marker GFAP, and microglia marker IBA-1. Figure 4 (B~D). These results collectively demonstrate that Gas-Ex can be effectively taken up by various important cell types in the retina (including ganglion cells, astrocytes, and microglia) both in vivo and in vitro, providing direct morphological evidence for its regulatory role in retinal tissue.

[0073] Example 2: Protective effect of extracellular vesicles (Gas-Ex) on rat retinal ganglion-like cell line R28 cells I. Experimental Materials 1. Cell source and culture Cell line: The experiment used the rat retinal ganglion cell-like cell line R28.

[0074] Culture conditions: Cells were cultured in DMEM low-glucose complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a constant temperature incubator with 5% CO2, using conventional methods.

[0075] 2. Gas-Ex was prepared according to the method in Example 1.

[0076] II. Experimental Methods 1. Experimental Grouping To systematically evaluate the cytotoxicity and protective effects of Gas-Ex, the following groups were established: Control group: Routine culture, without any damage or drug treatment; Gas-Ex alone control group: Gas-Ex alone was added to assess its biocompatibility and its effects on cells; Glutamate injury model group (GLU): Cells were treated with sodium glutamate (10mM) to establish an in vitro neuroexcitotoxic injury model; Gas-Ex intervention group (GLU + Gas-Ex): Gas-Ex was added during monosodium glutamate treatment.

[0077] 2. Evaluation of protective effects against cytotoxicity and glutamate injury in a model (CCK-8 assay) Determine the safe working concentration range and optimal protective concentration of Gas-Ex for R28 cells: R28 cells in logarithmic growth phase were planted at a density of 1 × 10⁻⁶ cells per well. 4 Ingredients were seeded at a density of [number] cells / wells in 96-well plates and cultured for 24 hours until adherence. The old culture medium was discarded, and the experimental groups were replaced with normal medium containing different concentrations of Gas-Ex (1, 5, 10, 50 μg / mL) and damaged medium containing a specific concentration of glutamate (10 mM), respectively. The control group was replaced with an equal volume of complete medium. Each group had 6 replicates. After another 24 hours of culture, serum-free medium containing 10% CCK-8 reagent was added to each well, and the plates were incubated in the dark for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader.

[0078] With the cell viability of the control group as 100%, the relative cell viability of each concentration group was calculated, dose-toxicity curves were plotted, and the safe concentration for subsequent experiments was determined.

[0079] 3. Cell survival / death observation Cells were seeded into 24-well plates. After grouping and treatment, the culture medium was discarded, and the cells were washed with PBS. The cells were then incubated for 30 minutes with Calcein-AM (labeling live cells, showing green fluorescence) and propidium iodide (labeling dead cells, showing red fluorescence). After washing with PBS, the cells were observed and photographed under a fluorescence microscope.

[0080] 4. Cellular energy metabolism assessment (intracellular ATP content measurement) After grouping and intervention, the cell culture medium was discarded, and the cells were lysed using ATP detection-specific lysis buffer. The lysate was mixed with the ATP detection working solution in a specific ratio, and the luminescence signal was immediately detected using a chemiluminescent microplate reader. The total protein concentration of the same lysed sample was determined using a BCA protein quantification kit for standardization.

[0081] 5. Assessment of cell membrane integrity (lactate dehydrogenase release assay) After processing the cells into groups, the cell culture supernatant was collected and centrifuged at 1000 g for 5 minutes at 25°C. The supernatant was then mixed with the reaction solution according to the LDH detection kit instructions and incubated at room temperature in the dark for 30 minutes. The absorbance was measured at 490 nm using a microplate reader. Wells with complete cell lysis were also included as a maximum release control.

[0082] 6. Real-time quantitative PCR, qPCR Cells were lysed using TRIzol reagent, and total RNA was extracted according to standard procedures. RNA concentration and purity were determined using a micro spectrophotometer (an A260 / A280 ratio between 1.8 and 2.0 was considered acceptable). An equal volume of high-quality RNA (1 µg) was used to synthesize cDNA using a reverse transcription kit.

[0083] qPCR amplification was performed using the SYBR Green fluorescent dye method. Amplification was conducted on a real-time quantitative PCR instrument, with the following reaction program: pre-denaturation; followed by 40 cycles of denaturation, annealing / extension; and finally, melting curve analysis.

[0084] Using β-actin as an internal reference, the relative expression levels of target pro-inflammatory factors (TNF-α, IL-6, IL-1β) mRNA were calculated using the 2^(-ΔΔCt) method. Changes in gene expression in each treatment group were analyzed using the control group as a baseline.

[0085] III. Experimental Results To investigate the protective effect of Gas-Ex against glutamate-induced excitotoxicity of retinal neurons and to evaluate its therapeutic effect at the cellular level, this invention systematically compared and validated the Gas-Ex treatment group with the glutamate-damaged group.

[0086] 1. Gas-Ex improves cell viability and rescues cell death. Cell viability was assessed using the CCK-8 assay, and it was found that, Figure 5 As shown in Figure B, compared with the control group, the cell viability of the glutamate model group (GLU) was significantly decreased. However, in the GLU + Gas-Ex treatment group, cell viability was significantly restored. Quantitative analysis further confirmed that Gas-Ex can effectively reverse the glutamate-induced decrease in cell viability. Figure 5 (C) Results of double staining of live and dead cells ( Figure 5 The images (D and E) clearly show that a large amount of red fluorescence (dead cells) appeared in the GLU group, while the red fluorescence in the GLU + Gas-Ex treatment group was significantly reduced and the green fluorescence (live cells) was significantly increased, directly confirming the protective effect of Gas-Ex against cell death.

[0087] 2. Assess energy metabolism status by detecting intracellular ATP levels. Figure 5 The results showed that, compared with the control group, the ATP level in the GLU model group was significantly reduced, indicating cellular energy depletion. However, in the GLU + Gas-Ex treatment group, intracellular ATP levels significantly recovered. Simultaneously, cell membrane integrity was assessed using a lactate dehydrogenase release assay, revealing a significant increase in LDH activity in the supernatant of the GLU model group, while Gas-Ex treatment effectively reduced LDH release. Figure 5 The presence of G indicates that Gas-Ex can alleviate the damage of glutamate to the cell membrane.

[0088] 3. Gas-Ex itself has good biocompatibility. In all tests, the cell viability of the Gas-Ex control group alone was the highest. Figure 5 The proportion of cell death, ATP content, LDH release, and ROS levels in the control group (A) were not significantly different from those in the normal control group, demonstrating that at the concentration used in this experiment, Gas-Ex itself has no negative impact on cell growth, metabolism, and state, and has good biosafety.

[0089] Example 3: Protective effect of extracellular vesicles (Gas-Ex) on RGCs (retinal ganglion cells) and retinal function in a glaucoma model I. Experimental Materials 1. Laboratory animals Male C57BL / 6J mice aged 6-8 weeks (purchased from Hunan Silek Jingda Laboratory Animal Co., Ltd.) were housed in an SPF-grade environment with a 12-hour light-dark cycle and free access to food and water. All animal experimental procedures were approved by the Animal Ethics Committee of Xiangya Hospital, Central South University, and followed the guidelines for laboratory animal welfare.

[0090] 2. Gas-Ex was prepared according to the method in Example 1.

[0091] II. Experimental Methods 1. Experimental Grouping Mice were randomly divided into the following four groups: Control group: Intravitreal injection of an equal volume of PBS; Gas-Ex control group: Gas-Ex was injected intravitreally only to assess the safety of the drug itself; NMDA model group: intravitreal injection of NMDA (N-methyl-D-aspartate). NMDA + Gas-Ex treatment group: Gas-Ex was injected into the vitreous cavity simultaneously with NMDA injection.

[0092] 2. Establishment of animal models Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (100 mg / kg), and their pupils were dilated with 0.5% tropicamide. Using a 5-μL Hamilton microsyringe with a 32G needle, 1.5 μL of the drug was slowly injected intravitreally 1 mm posterior to the corneal limbus. Tobramycin-dexamethasone eye ointment was applied post-injection to prevent infection. Three days after NMDA injection, the mice were sacrificed and their eyes were enucleated.

[0093] 3. Retinal function testing Flash visual evoked potentials (F-VEP) and flash electroretinography (F-ERG): To assess visual pathway function, F-VEP testing was performed 3 days after modeling. After anesthesia, the recording electrode was placed subcutaneously in the occipital bone, the reference electrode in the frontal bone, and the ground electrode in the tail. The average waveform of 100 consecutive flash stimuli was recorded. The first positive peak in the F-VEP waveform was designated P1, and the first negative peak was designated N1. The latency of the P1 wave and the amplitude of the N1-P1 wave were analyzed.

[0094] Prior to F-ERG recording, mice underwent 12 hours of dark adaptation, and a dark testing environment was maintained using red light illumination. A gold wire loop electrode was placed on the corneal surface. The ground electrode was also placed in the tail, and the reference electrode was inserted subcutaneously on both sides of the nose. Stimulation and detection followed ISCEV standards. After testing, the amplitudes of the a and b waves in each group were analyzed.

[0095] 4. Assessment of retinal ganglion cell survival Eyeballs were fixed with 4% paraformaldehyde for 1 hour. Retinal slides were prepared, permeabilized with 0.5% Triton X-100 for 15 minutes, and blocked with 5% BSA for 30 minutes. RGCs were then labeled by incubation overnight at 4°C with Brn3a antibody (1:500). After incubation at room temperature for 1 hour with Alexa Fluor488-conjugated secondary antibody (1:500), the eyes were observed and photographed under a fluorescence microscope.

[0096] 5. Retinal tissue structure analysis (HE staining) After the eyeball was fixed with FAS fixative and embedded in paraffin, coronal sections (5 μm thick) were prepared along the optic nerve and stained with hematoxylin and eosin. Sections containing the optic nerve stump were selected for observation of the retinal structure under an optical microscope, especially the cell count and pathological changes such as vacuoles in the ganglion cell layer, and scanning micrographs were taken.

[0097] 6. Analysis of retinal microglia activation The morphology, number, and distribution of microglia, including the microglial marker Iba1, were observed on retinal slides using confocal microscopy.

[0098] III. Experimental Results 1. To investigate the protective effect of Gas-Ex against NMDA-induced retinal ganglion cell damage and to evaluate its potential efficacy in glaucoma treatment, this invention systematically compared and verified the therapeutic effect of Gas-Ex on a mouse acute glaucoma model.

[0099] (1) Gas-Ex improves the survival rate of retinal ganglion cells: such as Figure 6 As shown in Figure A, compared with the control group, the number of Brn3a-positive cells in the retinal patch of the NMDA model group was drastically reduced. However, in the NMDA + Gas-Ex treatment group, the number of Brn3a-positive cells was significantly restored, and their density was significantly higher than that of the model group. Quantitative statistical analysis of RGC density further confirmed that Gas-Ex can effectively reverse NMDA-induced RGC loss. Figure 6 (B)

[0100] (2) HE staining Figure 6 The retinal morphological changes shown in C and D also confirm the above conclusions. Compared with the pathological state of sparse and disordered ganglion cell layer in the NMDA model group, the retinal structural integrity of the NMDA + Gas-Ex treatment group was well maintained, the number of GCL layer cells was significantly greater than that in the model group, and the structure of each layer was clearer.

[0101] (3) Further F-VEP and F-ERG tests confirmed the protective effect of Gas-Ex on visual function recovery. Figure 5 As shown, the F-VEP in the NMDA model group was characterized by a significantly prolonged P1 wave latency and a significantly reduced N1-P1 wave amplitude, while both of these key indicators were significantly improved in the NMDA + Gas-Ex treatment group. Furthermore, in F-ERG testing, the decrease in a-wave and b-wave amplitude caused by NMDA damage was effectively reversed by Gas-Ex treatment. Figure 6 (China EJ).

[0102] Meanwhile, in all tests, the Gas-Ex control group alone showed no significant difference from the control group, proving that Gas-Ex itself has no negative impact on visual function.

[0103] 2. To further explore the intrinsic mechanism by which Gas-Ex exerts its neuroprotective effect, this invention further evaluated its effect on retinal neuroinflammation triggered by NMDA damage.

[0104] In the mouse microglia cell line BV2, an oxygen-glucose deprivation / reoxygenation (OGD / R) model was used to simulate ischemia-reperfusion injury. Cell morphological changes were observed, and the expression of pro-inflammatory phenotype-related genes was detected by qPCR. Figure 7 As shown in Figure A, OGD / R treatment significantly increased cell area and synapses; in the OGD / R + Gas-Ex treatment group, cell hypertrophy was significantly reduced and branching was less. Simultaneously, qPCR results of IL-1β, IL-6, and TNF-α also confirmed that the mRNA expression of cellular inflammatory factors significantly decreased after Gas-Ex treatment. Figure 7 (B)

[0105] Immunofluorescence staining of the microglial cell marker Iba1 was performed using retinal retinal slides, followed by high-resolution imaging observation using confocal microscopy.

[0106] The results are as follows Figure 7 As shown in Figure C, in the control group, Iba1-positive cells exhibited a typical resting-state morphology, with small cell bodies and slender, complex primary and secondary branches. The NMDA model group, however, showed strong microglial activation, characterized by an increased number of Iba1-positive cells, significant cell hypertrophy, branch retraction or even disappearance, and a transformation into an amoeboid morphology, indicating that the microglia had transitioned to a pro-inflammatory state. In the NMDA + Gas-Ex treatment group, the activated phenotype of microglia was significantly reversed. Compared to the model group, although the number of microglia in this group increased, their cell hypertrophy was significantly reduced, more cell branches were preserved, and their morphology was closer to the resting state. Figure 7Quantitative and statistical analysis of D also confirmed the role of Gas-Ex in alleviating microglial activation in the NMDA model. Furthermore, qPCR analysis confirmed that the mRNA expression of inflammatory factors in the NMDA model significantly decreased after Gas-Ex treatment. Figure 7 (E).

[0107] In summary: 1. This invention is the first to successfully prepare extracellular vesicles using dried Gastrodia elata as raw material. This not only effectively expands the range of raw material sources for extracellular vesicles, but also fully explores and utilizes the medicinal potential of Gastrodia elata, realizing the efficient development and high-value utilization of traditional Chinese medicine resources.

[0108] 2. The extracellular vesicle preparation method provided by this invention is simple to operate and has mild reaction conditions. It does not rely on complex experimental equipment and expensive reagents, which effectively reduces production costs. At the same time, this method can efficiently extract high-purity and highly bioactive extracellular vesicles with good reproducibility and high stability, which meets the technical requirements of large-scale industrial production and has good prospects for industrial application.

[0109] 3. The extracellular vesicles provided by this invention exhibit no significant cytotoxicity and good biocompatibility. Studies have shown that these extracellular vesicles can significantly inhibit apoptosis of R28 cells and loss of Brn3a⁺ cells in the mouse retina in a glutamate excitotoxicity model. Furthermore, these extracellular vesicles effectively protect the survival of retinal ganglion cells (RGCs) and retinal function in a glaucoma model, and significantly reduce retinal neuroinflammatory responses in the glaucoma model. In summary, the extracellular vesicles provided by this invention have a clear anti-glaucoma-related retinal damage effect and possess great potential for development into a glaucoma treatment drug.

[0110] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. An extracellular vesicle, characterized in that: The extracellular vesicles were prepared using dried Gastrodia elata as raw material.

2. A method for preparing extracellular vesicles according to claim 1, characterized in that, Including the following steps: (1) Pulverize the dried Gastrodia elata, add buffer salt solution, mix well and place on a low temperature shaker overnight; (2) Filter and collect the filtrate; (3) The filtrate was centrifuged at low temperature and the supernatant was collected; (4) After filtering the supernatant, centrifuge at low temperature to precipitate crude extracellular vesicles, and resuspend in buffer salt solution; (5) Centrifuge the resuspension at low temperature, and the precipitate is the target extracellular vesicle.

3. The preparation method according to claim 2, characterized in that: The buffer salt solution in steps (1) and (4) is PBS phosphate buffer solution.

4. The preparation method according to claim 2, characterized in that: The low temperature in steps (1), (3), (4), and (5) is 4°C.

5. The preparation method according to claim 2, characterized in that, The parameters for gradient centrifugation in step (3) are: 800×g, 10 minutes; 2000×g, 10 minutes; 10000×g, 20 minutes.

6. The preparation method according to claim 2, characterized in that: In step (4), a 0.45 μm filter is used for filtration.

7. The preparation method according to claim 2, characterized in that: In steps (4) and (5), the centrifugation parameters for low-temperature centrifugation are 130000×g for 70 min.

8. Use of the extracellular vesicles of claim 1 in the preparation of a glaucoma treatment drug.

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