Application of ganoderma lucidum exosome in preparation of corneal injury repair preparation

Abstract

The invention discloses application of ganoderma lucidum exosomes in preparation of corneal injury repair preparations, and belongs to the technical field of biological medicines. The ganoderma lucidum exosome realizes corneal injury repair by promoting corneal endothelial cell proliferation and migration ability, promotes corneal endothelial proliferation and cell migration and accelerates corneal endothelial injury repair through an EGFR / ERK signal channel, can be used for treatment of corneal epithelium defect related ocular surface diseases, corneal lesion and other diseases, and has a wide application prospect. And a new thought is provided for treatment of corneal endothelial diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Ganoderma lucidum exosomes in the preparation of corneal damage repair agents. Background Technology

[0002] Reishi mushroom, a famous medicinal and edible fungus in my country, exhibits multiple benefits due to its active ingredients, including immune regulation, cardiovascular protection, liver protection, blood sugar regulation, and sleep aid. It is widely used in daily health maintenance. Reishi is rich in active ingredients and has been found to possess strong anti-tumor activity in in vitro and in vivo experiments. Its exosomes, as naturally derived extracellular vesicles, typically have a diameter between 30-150 nm and can carry various proteins, RNA, DNA, and other substances, participating in intercellular communication.

[0003] CN116875526A discloses a Ganoderma lucidum exosome, its preparation method, and applications, disclosing its use in the preparation of antioxidant drugs, medical foods, health foods, functional foods, or skincare products, but these uses only relate to its antioxidant effects. CN119055688A discloses the application, extraction method, and composition of Ganoderma lucidum exosomes in the treatment of glioma. The exosomes can be taken up by glioma cells and accumulated intracellularly; they can inhibit the proliferation of glioma cells, have no significant cytotoxicity to normal microglia, and exhibit anti-glioma activity and the ability to induce apoptosis in glioma cells. CN120361065A discloses the application of Ganoderma lucidum exosomes or their compositions in the preparation of anti-breast cancer products, clarifying the anti-breast cancer activity of Ganoderma lucidum exosomes and providing a development direction as an active ingredient for anti-breast cancer treatment. It is evident that the applications of Ganoderma lucidum exosomes are mainly concentrated in the fields of anti-tumor and anti-oxidation.

[0004] The cornea is a crucial component of the eye's refractive system. Because it is exposed on the ocular surface, it is easily damaged, providing an opportunity for pathogenic factors to invade. Corneal damage repair is vital for maintaining normal corneal physiological function. The decreased ability of limbal stem cells to replenish and repair corneal epithelium due to aging or other factors leads to a decline in the body's ability to repair corneal damage. Currently, corneal epithelial repair agents are mainly used clinically for corneal epithelial repair, but the repair effect is unsatisfactory. Modern medicine has conducted extensive research on the biochemical indicators of Ganoderma lucidum in brain tissue, liver, and blood, but research on its effects on the eye is scarce. Guo Shuguang et al. from Heilongjiang University of Traditional Chinese Medicine conducted a study on the protective effect of Ganoderma lucidum spore eye drops on radiation-induced rabbit eyes, finding that it can prevent oxidative damage caused by radiation, achieving an anti-aging effect on the ocular surface. Su Qinghua et al. from the Institute of Biomedical Materials at Taipei Medical University in China studied the micronized cell wall component SACCHACHITIN as a corneal epithelial damage repair material, demonstrating a rapid recovery function for corneal epithelial damage. However, to date, there have been no reports on the effects of Ganoderma lucidum exosomes on the eyes, especially on corneal damage repair. Summary of the Invention

[0005] To address the problems in the existing technology, this invention provides an application of Ganoderma lucidum exosomes in the preparation of corneal damage repair agents. Ganoderma lucidum exosomes promote corneal endothelial proliferation and cell migration through the EGFR / ERK signaling pathway, accelerating the repair of corneal endothelial damage and providing a new approach for the treatment of corneal endothelial diseases.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides the application of Ganoderma lucidum exosomes in the preparation of corneal damage repair agents.

[0007] Furthermore, the aforementioned Ganoderma lucidum exosomes promote corneal damage repair by enhancing the proliferation and migration of corneal endothelial cells. The Ganoderma lucidum exosomes accelerate corneal endothelial repair through the EGFR / ERK signaling pathway, promoting corneal endothelial cell proliferation and migration.

[0008] Furthermore, the corneal damage repair preparation can be any clinically used dosage form, including tablets, capsules, oral solutions, injections, eye drops, or ointments.

[0009] Furthermore, the corneal damage repair preparation further includes at least one pharmaceutically inactive ingredient; the pharmaceutically inactive ingredient includes a pharmaceutically acceptable carrier. According to the general understanding in the art, "pharmaceutically acceptable" means that the carrier is chemically and / or physically compatible with other components in the composition, or even has a beneficial effect, and is physiologically compatible with the subject of the composition or has the effect of preventing, improving or even treating diseases.

[0010] The pharmaceutically acceptable carrier may include, but is not limited to, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous mediators, non-aqueous mediators (e.g., non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents, buffers, antioxidants, suspending / dispersing agents, chelating agents, emulsifiers, diluents, adjuvants, excipients, auxiliary reagents, or combinations of one or more of the above components; examples of the auxiliary reagents include fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, or emulsifiers.

[0011] Furthermore, the Ganoderma exosomes mentioned herein are derived from one or more of Ganoderma lucidum, including Ganoderma sinense, Ganoderma purpurea, Ganoderma lucidum var. chinensis ... and Ganoderma lucid

[0012] Furthermore, the method for extracting Ganoderma lucidum exosomes includes the following steps: (1) Grind the soaked Ganoderma lucidum to obtain a grinding liquid; (2) The grinding liquid is coarsely filtered to obtain coarse filtrate; (3) The supernatant after centrifugation of the coarse filtrate is mixed with the enzyme preparation and enzymatically hydrolyzed to obtain the enzymatic hydrolysate; (4) After the enzymatic hydrolysate is purified, filtered and concentrated, Ganoderma exosomes are obtained.

[0013] Further, the soaking in step (1) involves soaking Ganoderma lucidum in PBS buffer solution; the soaking time is 2-24 hours, preferably 6-8 hours; the enzyme preparation in step (3) includes one or more of pectinase, cellulase, β-1,3-glucanase and ligninase.

[0014] Further, the mass of the enzyme preparation used in step (3) is 0.1-3% of the mass of Ganoderma lucidum; the mass fractions of each component in the enzyme preparation are pectinase 10-30%, cellulase 30-50%, β-1,3-glucanase 5-15%, and ligninase 20-40%.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: This invention discloses the application of Ganoderma lucidum exosomes in the preparation of corneal damage repair agents, and finds that Ganoderma lucidum exosomes achieve corneal damage repair by promoting the proliferation and migration of corneal endothelial cells; This invention discloses that Ganoderma lucidum exosomes promote corneal endothelial cell proliferation and migration through the EGFR / ERK signaling pathway, accelerate the repair of corneal endothelial damage, and can be used to treat corneal epithelial defect-related ocular surface diseases and corneal lesions, providing a new approach to the treatment of corneal endothelial diseases. Attached Figure Description

[0016] Figure 1 Image of Ganoderma lucidum exosomes taken up by human corneal epithelial cells in Example 2; Figure 2 This is a diagram showing the cell proliferation results of human corneal epithelial cells after treatment with Ganoderma lucidum exosomes in Example 3, as detected by CCK8 assay. Figure 3 This is a diagram showing the BrdU staining results of human corneal epithelial cells from the control group in Example 3; Figure 4 This is a diagram showing the BrdU staining results of human corneal epithelial cells from the experimental group (Ganoderma exosomes) in Example 3. Figure 5 This is a statistical chart showing the proliferation of human corneal epithelial cells in the experimental and control groups of Example 3. Figure 6 This is a graph showing the detection results of human corneal epithelial cell migration in Example 4; Figure 7 This is a diagram showing the protein changes in the EGFR / ERK signaling pathway in human corneal epithelial cells, as described in Example 5. Figure 8 The image shows the fluorescein staining of the corneas of mice after treatment with Ganoderma lucidum exosomes in Example 6. A: Corneal injury control group; B: Ganoderma lucidum exosome repair group; 1A, 1B: 0h after corneal injury; 2A, 2B: 24h after corneal injury; 3A, 3B: 48h after corneal injury; 4A, 4B: 72h after corneal injury. Figure 9 This is a comparative analysis diagram of the area of ​​fluorescein sodium staining in the cornea of ​​mice in Example 6. Detailed Implementation

[0017] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0019] Example 1 Ganoderma exosomes were extracted from Ganoderma lucidum according to the method disclosed in Example 1 of patent CN120361065 A for subsequent experiments.

[0020] Example 2 Human corneal epithelial cells take up Ganoderma lucidum exosomes (1) Culture human corneal epithelial cells (HCECs) in DMEM / 10% FBS medium and incubate in a 5% CO2 incubator; (2) Exosome staining: Ganoderma lucidum exosomes were stained according to the instructions of the PHK26 dye kit; (3) Intake experiment: After dilution by 1000X, the concentration of Ganoderma lucidum exosomes was 8.7 x 10⁻⁶. 7 Particles / mL. The stained Ganoderma lucidum exosomes were added to HCECs (cultured to adherent state) for 6 hours. Corneal cells were stained for membrane structure and nucleus according to the instructions for Dio live cell dye and Hoechst nuclear dye. The staining was observed under a confocal microscope. Results are as follows: Figure 1 As shown (PKH26 (red): Ganoderma lucidum exosomes, DIO (green): cell membrane, Hochest33342 (blue): cell nucleus, Merge (merge): merging of various channels); by Figure 1 It is known that Ganoderma lucidum exosomes are internalized in human corneal epithelial cells.

[0021] Example 3 Human corneal epithelial cell proliferation experiment (1) CCK8 experiment: Human corneal epithelial cells (HCECs) in the logarithmic growth phase were seeded into 96-well plates and cultured until adherent. Experimental and control groups were set up. The experimental group received Ganoderma lucidum exosomes, while the control group received an equal volume of PBS. Cell proliferation was assessed at 24h, 48h, 72h, 96h, and 120h using the CCK8 assay kit. The results are as follows: Figure 2 As shown, by Figure 2 It is known that Ganoderma lucidum exosomes can promote the proliferation of human corneal epithelial cells.

[0022] (2) BrdU experiment: Human corneal epithelial cells (HCECs) in the logarithmic growth phase were seeded into 96-well plates and cultured until adherent. Experimental and control groups were set up. Ganoderma lucidum exosomes were added to the experimental group, while an equal volume of PBS was added to the control group. Cell proliferation was detected using a BrdU assay kit after 24 hours. The cell proliferation status of the experimental and control groups is shown below. Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 The image shows three replicate independent experiments (each row represents one independent experiment). In each column, DAPI represents staining of cell nuclei (blue), BrdU represents staining of proliferating cells (red), and merge indicates that DAPI and BrdU stained images are overlapped at the same location. The image shows the proliferation of human corneal epithelial cells. Figure 5 As shown, by Figures 3-5It is known that Ganoderma lucidum exosomes can promote the proliferation of human corneal epithelial cells.

[0023] Example 4 Human corneal epithelial cell migration assay Human corneal epithelial cells (HCECs) in logarithmic growth phase were seeded into 6-well plates, with separate experimental and control groups. The experimental group received Ganoderma lucidum exosomes, while the control group received an equal volume of PBS. After 24 hours of growth, when the cells reached 70-80% maturity, a scratch was made along the central portion of the cell line. Cell growth and migration were observed under a microscope at 0h, 24h, and 48h. The results are shown below. Figure 6 As shown, by Figure 6 It is known that Ganoderma lucidum exosomes can promote the migration of human corneal epithelial cells.

[0024] Example 5 Research on the mechanism by which Ganoderma lucidum exosomes promote corneal cell proliferation and migration Human corneal epithelial cells (HCECs) in logarithmic growth phase were seeded into 6-well plates, with experimental and control groups respectively. The experimental group received Ganoderma lucidum exosomes, while the control group received an equal volume of PBS. After 48 hours, cells were collected, protein lysis buffer was added, and proteins were extracted. Western blot analysis was performed on the protein samples to detect EGFR (total protein) and p-EGFR, ERK (total protein) and p-ERK, and β-acting in the EGFR / ERK signaling pathway. The results are shown below. Figure 7 As shown, by Figure 7 It was found that after treatment with Ganoderma lucidum exosomes, p-EGFR expression was significantly increased, while EGFR (total protein) showed no significant change; p-ERK expression was significantly increased, while ERK (total protein) showed no significant change; indicating that exosomes increase the phosphorylation level (i.e., activation state) of EGFR and ERK, suggesting that they promote cell proliferation and cell migration through the EGFR / ERK signaling pathway.

[0025] Example 6 Animal experiments (1) Establishment of a mouse corneal epithelial injury model Thirty healthy 6-week-old male C57BL / 6J mice were anesthetized by intraperitoneal infiltration with 5% sodium pentobarbital to fully expose the eyeballs. Promecaine hydrochloride eye drops were used for surface anesthesia. A 2mm corneal trephine was used to gently press on the mouse cornea to ensure that the trephine was located in the center of the cornea. The central corneal epithelium of the mouse was gently scraped off with a corneal epithelial knife. (2) Corneal fluorescein staining Immediately after the mouse corneal epithelial injury model was established, sodium fluorescein staining agent was dropped into the conjunctival sac, evenly spread on the cornea, and then observed and photographed under a slit-lamp microscope. The uniform staining of the central 2mm area of ​​the mouse cornea with sodium fluorescein proved that the corneal epithelial injury model was successfully established. (3) Grouped exosome drip treatment The mice that successfully developed the model were divided into an experimental group and a control group. The experimental group was given Ganoderma lucidum exosomes four times a day (20 μL each time, with an exosome concentration of 1 x 10⁻⁶). 8 (Particles / mL), control eyes were treated with PBS eye drops four times a day (20uL each time), and corneal epithelial repair was observed daily for three days; (4) Slit lamp observation After eye drops were applied for 24 h, 48 h, and 72 h, the staining results were observed under a slit-lamp microscope. The images of fluorescein sodium staining of mouse corneas after treatment with Ganoderma lucidum exosomes are shown below. Figure 8 As shown, A: Corneal injury control group; B: Ganoderma lucidum exosome repair group, 1A, 1B: 0h after corneal injury; 2A, 2B: 24h after corneal injury; 3A, 3B: 48h after corneal injury; 4A, 4B: 72h after corneal injury; Figure 8 The repair status of the eyes in the experimental and control groups at 0h, 24h, 48h, and 72h (the area stained with sodium fluorescein in the mouse cornea) was statistically analyzed. A smaller green area indicated better repair. Figure 8 and Figure 9 It is known that Ganoderma lucidum exosomes can significantly promote the repair of damaged corneas.

Claims

1. Application of Ganoderma lucidum exosome in preparation of corneal injury repair preparation.

2. Use according to claim 1, characterized in that, The Ganoderma lucidum exosome promotes the proliferation and migration of corneal endothelial cells to achieve corneal injury repair.

3. Use according to claim 1, characterized in that, The corneal injury repair preparation is any clinically used dosage form, including tablets, capsules, oral liquids, injections, eye drops or eye ointments.

4. Use according to claim 1, characterized in that, The corneal injury repair preparation further comprises at least one non-active pharmaceutical ingredient.

5. The use according to claim 1, characterized in that, The Ganoderma lucidum exosome is derived from one or more of Ganoderma lucidum, Ganoderma sinense, Ganoderma applanatum, Ganoderma tsugae, Ganoderma boninense and Ganoderma atrum.

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