Preparation method of exosome preparation based on gene-edited stem cells and its application in the treatment of dry eye syndrome

Abstract

This invention discloses a method for preparing a stem cell exosome preparation based on gene-edited stem cells and its application in treating dry eye syndrome, belonging to the field of biomedical technology. The preparation comprises stem cell exosomes overexpressing CREBH and *Nepeta cataria* extract, with a mass ratio of CREBH-overexpressing stem cell exosomes to *Nepeta cataria* extract of 1:(1-10). The combined use of these two components significantly improves cell survival and inhibits the release of inflammatory factors in a hyperpermeability-induced corneal epithelial cell model. Furthermore, in a rat model of dry eye syndrome, it significantly increases tear secretion, prolongs tear film breakup time, and restores the number of conjunctival goblet cells, demonstrating superior effects compared to single-component formulations. Therefore, this preparation, through multi-target synergistic action, can become an effective treatment for dry eye syndrome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing exosome preparations based on gene-edited stem cells and their application in treating dry eye syndrome. Background Technology

[0002] Dry eye disease (DED) is a common, multifactorial ocular surface disease characterized by loss of tear film homeostasis, accompanied by ocular discomfort, visual disturbances, tear film instability, and hyperosmolarity, which can damage the ocular surface. Its core pathological mechanism involves ocular surface inflammation and immune dysregulation. Hyperosmolarity of the tear film can directly damage corneal epithelial cells, activate the NLRP3 inflammasome and NF-κB signaling pathway, and induce the release of pro-inflammatory factors such as IL-1β, TNF-α, and MMP-9, forming a vicious cycle of inflammation. At the immune level, the innate immune system recognizes environmental stress through Toll-like receptors, while Th1 / Th17 cells and their secreted IFN-γ and IL-17 are key effector factors leading to chronic ocular surface inflammation and tissue damage in adaptive immunity. Furthermore, IFN-γ can induce endoplasmic reticulum stress (ER stress) and unfolded protein response (UPR) in conjunctival goblet cells, leading to impaired synthesis of the protective mucin Muc5AC, further disrupting tear film stability.

[0003] Currently, treatment strategies for dry eye mainly include artificial tears, corticosteroids, and immunosuppressants. However, artificial tears only provide temporary relief and cannot stop the inflammatory process; long-term use of corticosteroids carries risks of side effects such as increased intraocular pressure and cataract formation. Therefore, there is an urgent need to develop a new treatment approach that combines anti-inflammatory and repair-promoting effects while also having a higher safety profile.

[0004] In recent years, exosomes derived from mesenchymal stem cells (MSCs) have become a research hotspot in regenerative medicine due to their immunomodulatory, anti-inflammatory, and pro-regenerative properties. Exosomes carry bioactive molecules such as proteins, mRNA, and miRNA, which can mimic the therapeutic functions of parental stem cells while avoiding the tumorigenicity and immune rejection risks associated with live cell transplantation. Previous studies have shown that exosomes derived from umbilical cord MSCs can target and regulate inflammatory pathways by delivering miR-146a, reducing corneal epithelial cell damage under hyperosmolar conditions, and have demonstrated good therapeutic effects in dry eye animal models.

[0005] Existing research has confirmed that cAMP-responsive element-binding protein H (CREBH) is activated under endoplasmic reticulum stress and metabolic stress, participating in the regulation of the expression of acute-phase reactive proteins and lipid metabolism-related genes, playing a key role in restoring cellular homeostasis. Given the significant endoplasmic reticulum stress and inflammatory response on the ocular surface in dry eye, using gene editing technology to overexpress CREBH in stem cell exosomes is expected to enhance its ability to regulate inflammation and repair tissues. Meanwhile, the extract of the natural product *Nepeta bracteata* Benth. has been used in traditional medicine to treat inflammation-related diseases. Modern pharmacological studies have confirmed that its abundant diterpenoid compounds can effectively inhibit nitric oxide (NO) production in an LPS-stimulated macrophage inflammation model, exhibiting clear anti-inflammatory activity.

[0006] Based on this, the present invention combines dental pulp stem cell exosomes overexpressing CREBH with extracts of Nepeta macrocarpa, aiming to synergistically intervene in the complex pathological process of dry eye from multiple dimensions such as regulating intracellular stress response, mediating immune regulation and directly clearing inflammatory mediators, thereby providing a safer and more efficient new treatment option. Summary of the Invention

[0007] Therefore, this invention provides a method for preparing exosome preparations based on gene-edited stem cells and their application in treating dry eye syndrome, in order to overcome the shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a gene-edited stem cell exosome formulation is provided, comprising: Stem cell exosomes overexpressing CREBH, and Nepeta macrocarpa extract.

[0009] Furthermore, the mass ratio of stem cell exosomes overexpressing CREBH to Nepeta cataria extract was 1:(1-10).

[0010] Furthermore, the mass ratio of the stem cell exosomes overexpressing CREBH to the extract of Nepeta macrocarpa is 1:(2-5).

[0011] Furthermore, the mass ratio of the stem cell exosomes overexpressing CREBH to the extract of Nepeta macrocarpa is 1:4.

[0012] Furthermore, the CREBH-overexpressing stem cell exosomes are derived from dental pulp stem cells.

[0013] Furthermore, the method involves overexpressing exogenous CREBH on exosomes derived from gene-edited stem cells.

[0014] Furthermore, the stem cell exosomes overexpressing CREBH are obtained by integrating the CREBH gene into the AAVS1 safe harbor site of the stem cell genome.

[0015] Furthermore, it also includes pharmaceutically acceptable carriers.

[0016] According to a second aspect of the present invention, a method for preparing an exosome preparation based on gene-edited stem cells is provided, comprising the following steps: (1) Preparation of stem cell exosomes overexpressing CREBH; (2) Preparation of Nepeta macrocarpa extract; (3) The stem cell exosomes overexpressing CREBH are mixed with the extract of Nepeta macrocarpa in a certain proportion and a pharmaceutically acceptable carrier is added to prepare the stem cell exosome preparation.

[0017] Furthermore, the method for preparing stem cell exosomes overexpressing CREBH is as follows: (1) Gene editing of stem cells to stably overexpress CREBH, and screening to obtain stem cells that stably overexpress CREBH; (2) Culture the CREBH-overexpressing stem cells obtained in step (1) and collect the supernatant; (3) The supernatant collected in step (2) was subjected to exosome separation and purification to obtain stem cell exosomes overexpressing CREBH.

[0018] Furthermore, the gene editing tools include, but are not limited to, CRISPR / Cas9; and the methods for exosome isolation and purification include, but are not limited to, phosphatidylserine (PS) affinity assay.

[0019] Furthermore, the method for preparing the extract of Nepeta macrocarpa is as follows: Take the dried whole herb of *Nepeta macrocarpa*, pulverize it, add it to a 70-80% ethanol aqueous solution at a material-to-liquid ratio of 1g:(8-10)mL, heat and reflux to extract 2-3 times, each time for 1.5-2 hours; combine the extracts, concentrate under reduced pressure to obtain an extract; disperse the extract in water, extract with ethyl acetate 3-4 times, combine the ethyl acetate extracts, recover the solvent under reduced pressure, and dry to obtain *Nepeta macrocarpa* extract.

[0020] Furthermore, the pharmaceutically acceptable carriers include, but are not limited to, sodium hyaluronate and phosphate-buffered saline (PBS).

[0021] According to a third aspect of the invention, the use of the gene-edited stem cell exosome preparation obtained by the aforementioned preparation method in the preparation of a medicament for treating dry eye syndrome is provided.

[0022] Furthermore, the dosage form of the drug is eye drops, ophthalmic gel, or ophthalmic implant.

[0023] The present invention has the following advantages: This invention provides a stem cell exosome formulation for treating dry eye syndrome, comprising exosomes overexpressing CREBH dental pulp stem cells (DPSCs), extract of Nepeta macrocephala, and a pharmaceutically acceptable carrier. This formulation combines genetically modified stem cell exosomes with natural plant extracts to treat dry eye syndrome through a synergistic effect across multiple targets and pathways, specifically in the following aspects: (1) Improvement of core indicators of dry eye syndrome This stem cell exosome preparation significantly increased tear secretion, prolonged tear film breakup time, reduced corneal fluorescein staining scores, and promoted the recovery of conjunctival goblet cells in DED model rats. Its mechanism of action may be related to CREBH overexpression activating the SIRT3 / MnSOD pathway to scavenge mitochondrial reactive oxygen species and inhibiting NLRP3 inflammasome activation, thus helping to break the vicious cycle of oxidation and inflammation and protect the integrity of ocular surface tissues from the source.

[0024] (2) Suppress inflammatory response and reduce ocular surface damage This stem cell exosome preparation significantly reduced the levels of TNF-α and IL-1β in hyperosmolar corneal epithelial cells and in the corneal and conjunctival tissues of dry eye model rats. Its anti-inflammatory mechanism may be related to CREBH-mediated antioxidant regulation and the multi-target inhibitory effect of Nepeta macrocephala extract on inflammatory factors, with the two synergistically reducing the ocular surface immune inflammatory response and alleviating dry eye symptoms.

[0025] (3) Enhance corneal epithelial barrier function This stem cell exosome preparation can significantly improve the survival rate of corneal epithelial cells under hyperosmolar induction and reduce cell damage. Its protective effect may be related to the overexpression of CREBH, which upregulates tight junction proteins Claudin5 / 8 and downregulates Claudin2, thereby strengthening the corneal epithelial barrier function, reducing tear leakage, and stabilizing the tear film.

[0026] (4) Promotes the repair and regeneration of ocular surface tissues This stem cell exosome preparation can significantly increase the number of conjunctival goblet cells and promote the repair of damaged corneal epithelium. Its regeneration mechanism may be related to CREBH activating miR-143 / 145 / growth factor (GF) and its related signaling pathways, promoting epithelial cell proliferation and migration. At the same time, the nutritional factors carried by the DPSC-derived exosomes can synergistically play a role in tissue repair.

[0027] In summary, this invention combines CREBH DPSCs overexpressing exosomes with Nepeta cataria extract to achieve synergistic multi-target intervention with antioxidant, anti-inflammatory, barrier protection, and regeneration-promoting effects. This stem cell exosome preparation demonstrates superior efficacy compared to single-component formulations in cell protection, functional recovery, and tissue repair, providing a comprehensive new strategy for the treatment of dry eye. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention or the technical solutions in 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 merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0029] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0030] Figure 1 The results of CREBH protein level detection in exosomes overexpressing CREBH DPSCs in Example 1 of this invention; Figure 2 This is the cell viability test result of human corneal epithelial cells under high-osmotic induction in Example 2 of the present invention; Figure 3 The results show the detection of TNF-α and IL-1β levels in human corneal epithelial cells under high-osmotic induction in Example 2 of this invention; where A represents TNF-α and B represents IL-1β. Figure 4 The results of functional index detection in the dry eye (DED) model rats in Example 3 of this invention are shown; where A is tear secretion volume, B is tear film breakup time, and C is corneal fluorescein staining score. Figure 5 The results of conjunctival goblet cell count detection (PAS staining) in the dry eye (DED) model rat in Example 3 of this invention; Figure 6 The results show the detection of TNF-α and IL-1β levels in corneal and conjunctival tissue homogenates from rats with dry eye syndrome (DED) in Example 3 of this invention; where A represents TNF-α and B represents IL-1β. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] According to a first aspect of the present invention, a gene-edited stem cell exosome formulation is provided, comprising: Stem cell exosomes overexpressing CREBH, and Nepeta macrocarpa extract.

[0033] Furthermore, the mass ratio of stem cell exosomes overexpressing CREBH to Nepeta cataria extract was 1:(1-10).

[0034] Furthermore, the mass ratio of stem cell exosomes overexpressing CREBH to Nepeta macrocarpa extract was 1:(2-5).

[0035] Furthermore, the mass ratio of stem cell exosomes overexpressing CREBH to Nepeta macrocarpa extract was 1:4.

[0036] Furthermore, the stem cell exosomes overexpressing CREBH are derived from dental pulp stem cells.

[0037] Furthermore, exogenous CREBH was overexpressed on exosomes of gene-edited stem cells.

[0038] Furthermore, stem cell exosomes overexpressing CREBH were obtained by integrating the CREBH gene into the AAVS1 safe harbor site of the stem cell genome.

[0039] Furthermore, it also includes pharmaceutically acceptable carriers.

[0040] According to a second aspect of the present invention, a method for preparing an exosome preparation based on gene-edited stem cells is provided, comprising the following steps: (1) Preparation of stem cell exosomes overexpressing CREBH; (2) Preparation of Nepeta macrocarpa extract; (3) The stem cell exosomes overexpressing CREBH were mixed with the extract of Nepeta macrocarpa in a certain proportion and a pharmaceutically acceptable carrier was added to prepare the stem cell exosome preparation.

[0041] Furthermore, the method for preparing stem cell exosomes overexpressing CREBH is as follows: (1) Gene editing of stem cells to stably overexpress CREBH, and screening to obtain stem cells that stably overexpress CREBH; (2) Culture the CREBH-overexpressing stem cells obtained in step (1) and collect the supernatant; (3) The supernatant collected in step (2) was subjected to exosome separation and purification to obtain stem cell exosomes overexpressing CREBH.

[0042] Furthermore, gene editing tools include, but are not limited to, CRISPR / Cas9; methods for exosome isolation and purification include, but are not limited to, phosphatidylserine (PS) affinity methods.

[0043] Furthermore, the method for preparing the extract of Nepeta macrocarpa is as follows: Take the dried whole herb of *Nepeta macrocarpa*, pulverize it, add it to a 70-80% ethanol aqueous solution at a material-to-liquid ratio of 1g:(8-10)mL, heat and reflux to extract 2-3 times, each time for 1.5-2 hours; combine the extracts, concentrate under reduced pressure to obtain an extract; disperse the extract in water, extract with ethyl acetate 3-4 times, combine the ethyl acetate extracts, recover the solvent under reduced pressure, and dry to obtain *Nepeta macrocarpa* extract.

[0044] Furthermore, pharmaceutically acceptable carriers include, but are not limited to, sodium hyaluronate and phosphate-buffered saline (PBS).

[0045] According to a third aspect of the invention, the use of a gene-edited stem cell-based exosome preparation obtained by the preparation method in the preparation of a medicament for treating dry eye syndrome is provided.

[0046] Furthermore, the dosage form of the drug is eye drops, ophthalmic gel, or ophthalmic implant.

[0047] Preparation Example Preparation of extract from Nepeta macrocarpa Take 1g of dried whole herb of *Nepeta macrocarpa*, pulverize it, add 10mL of 75% ethanol aqueous solution, and heat under reflux to extract three times, each time for 1.5h; combine the extracts and concentrate under reduced pressure to obtain an extract; disperse the extract in water, extract three times with ethyl acetate, combine the ethyl acetate extracts, recover the solvent under reduced pressure, and dry to obtain *Nepeta macrocarpa* extract.

[0048] Example 1 Preparation of exosomes overexpressing CREBH DPSCs 1. Construction of CREBH overexpression DPSCs cell lines 1.1 Cell Culture Human primary dental pulp stem cells (DPSCs, purchased from Wuhan Huana Biotechnology Co., Ltd.) were harvested, thawed, and cultured in a 37°C, 5% CO2 incubator. α-MEM medium containing 10% exosome-free fetal bovine serum was used, with medium changes every 2-3 days. Validated third-generation DPSCs were selected for subsequent gene modification experiments.

[0049] 1.2 Construction of CRISPR / Cas9 Knock-in Vectors The nucleotide sequence of the highly efficient sgRNA targeting the human AAVS1 safe harbor site is as follows: 5'-GTCCCCTCCACCCCACAGTG-3' (SEQ ID No. 1); The cutting efficiency, as measured by T7EI, is 86.8 ± 5.1%, calculated using the following formula: ; The sequence was cloned into the pX330-U6-sgRNA-CBH-Cas9-P2A-EGFP plasmid (purchased from Wenzhou Kemiao Biotechnology Co., Ltd.) to construct the sgRNA-AAVS1 / Cas9 plasmid.

[0050] A DNA fragment containing the CREBH gene coding sequence shown in SEQ ID No. 2 (GenBank accession number: NM_001271995.2), the left homologous arm shown in SEQ ID No. 3 (GenBank accession number: NC_000019.10), and the right homologous arm shown in SEQ ID No. 4 (GenBank accession number: NC_000019.10) was synthesized and cloned into the pUC57 vector to construct the AAVS1-CREBH-Puro donor vector. Sequencing confirmed that the amplified product was completely identical to the reference sequence.

[0051] To ensure that the donor vector is not cleaved by the sgRNA / Cas9 complex, a synonymous mutation is performed on the recognition sequence in the donor vector corresponding to the sgRNA without altering the amino acid sequence, using point mutation technology.

[0052] 1.3 Cell transfection and stable clone screening The sgRNA-AAVS1 / Cas9 plasmid and the AAVS1-CREBH-Puro donor vector were co-transfected into P3 generation DPSCs. Forty-eight hours after transfection, 1 μg / mL puromycin was added for selection for 14 days, and single resistant clones were picked for expansion culture. Genomic DNA was extracted from the resistant clones, and positive clones were identified by PCR amplification across the 5' and 3' homologous arms and sequencing. Simultaneously, CREBH mRNA and protein expression levels were detected by qPCR and Western blot.

[0053] Finally, the three clones with the highest CREBH expression levels were selected for expanded culture and used for subsequent exosome preparation.

[0054] 2. Preparation and Identification of Exosomes 2.1 Collection of conditioned medium Overexpressing CREBH DPSCs and wild-type DPSCs were cultured in α-MEM medium containing 10% exosome-free FBS.

[0055] When the cell confluence reaches 80%, replace with serum-free α-MEM medium and continue culturing for 48 hours, then collect the cell culture supernatant.

[0056] 2.2 Exosome Isolation and Identification MagCapture TM Exosome extraction kit (PS affinity method) was used to isolate exosomes from the collected cell culture supernatant, strictly following the instructions. The resulting exosome precipitate was resuspended in an appropriate amount of PBS, aliquoted, and stored at -80°C for later use.

[0057] Transmission electron microscopy, nanoparticle tracking analysis, and Western blot identification revealed that the particles separated from overexpressing CREBHDPSCs and wild-type DPSCs exhibited a typical cup-shaped bilayer membrane structure with a main diameter peak in the range of 30-150 nm. The positive expression of exosome markers CD9, CD63, and CD81 was observed, while the negative marker Calnexin showed no signal, which meets the definition criteria of exosomes of the International Society for the Study of Extracellular Vesicles (MISEV).

[0058] Western blot analysis confirmed that the expression level of CREBH protein in CREBH-overexpressing DPSCs exosomes was significantly higher than that in DPSCs exosomes (e.g., ...). Figure 1 As shown in the figure, the engineered exosomes enriched with CREBH were successfully constructed, providing a biological agent with a clear effect for subsequent research on the treatment of dry eye syndrome.

[0059] Example 2 Dry eye cell model 1. Cell Culture and Model Establishment Human corneal epithelial cell lines (purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.) were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 incubator, with the medium changed every 2 days.

[0060] Once the cell confluence reached 80%, the original culture medium was discarded and replaced with fresh DMEM / F12 medium. Sterile saturated sodium chloride solution was added to adjust the osmotic pressure to 500 mOsm / L, while maintaining the pH at 7.2 ± 0.2. The cells were then cultured in an incubator for 24 hours to construct a hyperosmolarly induced dry eye cell model.

[0061] 2. Preparation and Identification of Exosomes Exosomes were extracted from DPSCs overexpressing CREBH and from the culture supernatant of DPSCs, and were used in subsequent experiments after being identified as qualified.

[0062] 3. Experimental grouping and intervention Human corneal epithelial cells were seeded into 96-well plates for intervention, and the groups were as follows: Table 1 Experimental Groups and Intervention Plans

[0063] Each group has 5 replicates. After gently shaking, continue culturing for 24 hours, then collect cells and supernatant for testing.

[0064] 4. Detection Indicators 4.1 Cell viability assay (CCK-8 assay) Add 10 μL of CCK-8 solution to each well, incubate at 37°C for 2 h, and then measure the absorbance (OD value) at 450 nm. Cell viability is calculated using the following formula: .

[0065] 4.2 Detection of inflammatory factors Collect cell supernatant and detect TNF-α and IL-1β concentrations according to the ELISA kit instructions.

[0066] 5. Statistical Analysis All experimental data are expressed as mean ± SD and were analyzed using GraphPad Prism 9.3.0 software. p < 0.05 was considered statistically significant.

[0067] 6 Results 6.1 Cell viability like Figure 2As shown, compared with the model control group, all intervention groups improved the cell survival rate of human corneal epithelial cells under hyperosmolarity-induced damage to varying degrees. Specifically, the cell survival rates of the CREBH-overexpressing DPSCs exosome group, the medium- and high-dose *Nepeta cataria* extract groups, the stem cell exosome preparations groups 1-4, and the positive control group (dexamethasone) were all significantly increased (p < 0.0001); the wild-type DPSCs exosome group also showed a significantly increased effect (p < 0.001); the low-dose *Nepeta cataria* extract group showed a significant increase (p < 0.05). Notably, the increase in cell survival rate of the CREBH-overexpressing DPSCs exosome group was significantly better than that of the wild-type DPSCs exosome group (p < 0.0001), indicating that CREBH overexpression can effectively enhance the repair capacity of DPSCs exosomes for hyperosmolarity-induced corneal epithelial cell damage. Furthermore, when CREBH DPSCs overexpressing exosomes were combined with Nepeta cataria extract, the cell survival rate of each stem cell exosome preparation group reached over 82%. Among them, the cell survival rate of the stem cell exosome preparation group 4 was not significantly different from that of the normal control group.

[0068] 6.2 Inflammatory Factors like Figure 3 As shown, compared with the model control group, all intervention groups significantly reduced the levels of TNF-α and IL-1β in human corneal epithelial cells induced by high osmosis. p <0.0001). Notably, the reduction effect of the CREBH overexpression DPSCs exosome group was significantly better than that of the wild-type DPSCs exosome group ( p <0.01), indicating that overexpression of CREBH can effectively enhance the anti-inflammatory effect of DPSC exosomes. Furthermore, the stem cell exosome preparations in groups 1-4 (1:2 to 1:5) all showed superior anti-inflammatory effects compared to the single component, with a dose-dependent increase. Among them, the TNF-α levels in groups 2-4 (1:3 to 1:5) of the stem cell exosome preparations were not statistically significantly different from those in the normal control group (e.g., <0.01). Figure 3 As shown in Figure A), there was no statistically significant difference in IL-1β levels between the stem cell exosome preparation groups 3 and 4 (1:4 to 1:5) and the normal control group (as shown in Figure A). Figure 3 (As shown in B).

[0069] Example 3 Dry eye (DED) rat model 1. Model Establishment SPF-grade male SD rats, 6-8 weeks old and weighing 230-250g, were selected. After one week of acclimatization, ophthalmic examination (tear secretion test and corneal fluorescein staining) confirmed the absence of ocular surface disease. Scopolamine hydrobromide was dissolved in 0.9% sterile saline at a concentration of 6 mg / mL and administered subcutaneously to the rats at 9:00, 12:00, 15:00, and 18:00 daily, 0.5 mL each time, for 28 consecutive days to establish the ocular model.

[0070] 2. Preparation of overexpression exosome formulations Based on the cell screening results in Example 2, 50 μg of CREBH-overexpressing stem cell exosomes prepared in Example 1 were mixed thoroughly with 200 μg of *Nepeta cataria* extract from the preparation example. Sodium hyaluronate (final concentration 0.2%) was added as an eye lubricant, and the mixture was brought to a final volume of 1 mL with PBS (pH=7.4) to obtain a high-dose stem cell exosome preparation. 25 μg of CREBH-overexpressing stem cell exosomes prepared in Example 1 were mixed thoroughly with 100 μg of *Nepeta cataria* extract from the preparation example. Sodium hyaluronate (final concentration 0.2%) was added as an eye lubricant, and the mixture was brought to a final volume of 1 mL with PBS (pH=7.4) to obtain a low-dose stem cell exosome preparation.

[0071] All preparations should be made and used immediately.

[0072] 3. Experimental grouping and dosing regimen Rats that successfully developed the model were randomly divided into 7 groups of 8 rats each, plus a normal control group (8 normally fed rats), for a total of 8 groups. The specific grouping and drug administration regimens are as follows: Normal control group: No modeling was performed; the patient was given PBS buffer containing only 0.2% sodium hyaluronate. Model control group: After modeling, the model was given PBS buffer containing only 0.2% sodium hyaluronate; Low-dose stem cell exosome preparation group: After modeling, the patient was given 25 μg / mL of CREBH DPSCs overexpressing exosomes and 100 μg / mL of Nepeta macrocarpa extract. High-dose stem cell exosome preparation group: After modeling, the patient was given 50 μg / mL of CREBH DPSCs overexpressing exosomes and 200 μg / mL of Nepeta macrocarpa extract. CREBH DPSCs overexpression exosome group: After modeling, 50 μg / mL of CREBH DPSCs overexpression exosome preparation was administered; Nepeta macrocarpa extract group: After modeling, 200 μg / mL Nepeta macrocarpa extract was administered; Wild-type DPSCs exosome group: After modeling, 50 μg / mL DPSCs exosome preparation was administered.

[0073] All groups were treated with eye drops 4 times daily (10 μL / eye each time, 4 hours apart) for 4 consecutive weeks.

[0074] 4. Detection Indicators 4.1 Functional Indicator Testing All rats in the experimental groups underwent the following tests on the second day after 4 weeks of intervention: Tear secretion (Schirmer I test, SIT): A phenol red cotton thread was placed on the outer 1 / 3 of the lower conjunctival sac of the rat. After the eyes were closed for 20 seconds, the thread was removed and the length (mm) of the thread changing from yellow to red was measured with calipers. Three measurements were taken for each eye and the average value was taken.

[0075] Tear break-up time (TBUT): The day after SIT test, 1 μL of 1% sodium fluorescein was instilled into the conjunctival sac. After blinking, the cornea was observed under cobalt blue light with a slit-lamp microscope, and the time (s) when the first black spot appeared was recorded. The experiment was repeated 3 times and the average value was taken.

[0076] Corneal fluorescence staining score (CFS): After TBUT, the corneal staining is observed under a slit lamp and scored according to the following criteria: 0 (no staining); 1 (scattered punctate distribution); 2 (dense punctate distribution); 3 (patchy staining). The average score is taken for each eye.

[0077] 4.2 Histological indicators After the indicators in section 4.1 were tested, the rats were euthanized, and conjunctival tissue from the fornix was collected. The tissue was fixed in 4% paraformaldehyde for 24 hours, then routinely embedded in paraffin and sectioned (4-5 μm thick). After dewaxing to water, the sections were stained with periodic acid-Schiff (PAS): following the PAS kit instructions, the cell nuclei were counterstained with hematoxylin, and the sections were dehydrated, cleared, and mounted. The morphology of conjunctival goblet cells (goblet cell cytoplasm was purplish-red) was observed under a light microscope. Three non-contiguous sections were randomly selected from each eye, and the number of PAS-positive cells in the superior and inferior conjunctiva was counted at 200x magnification. The average value was calculated, expressed as cells / section.

[0078] 4.3 Detection of inflammatory factors Corneal and conjunctival tissues were collected and added to pre-chilled RIPA lysis buffer (1:10 by weight / volume). The homogenate was then centrifuged, and the supernatant was collected. The concentrations of TNF-α and IL-1β were measured according to the ELISA kit instructions. Protein concentrations were expressed as pg / mg total protein.

[0079] 5. Statistical Analysis All experimental data are expressed as mean ± SD and were analyzed using GraphPad Prism 9.3.0 software. p < 0.05 was considered statistically significant.

[0080] 6 Results 6.1 Results of Functional Indicators Test like Figure 4 As shown, compared with the normal control group, the tear secretion (SIT) of the model control group rats was significantly reduced ( p <0.001), tear film breakup time (TBUT) was significantly shortened ( p <0.001), corneal fluorescein staining score (CFS) significantly increased ( p <0.001), indicating that the dry eye model was successfully established.

[0081] Compared with the model control group, the SIT of rats in each treatment group increased to varying degrees. Among them, the SIT of the low-dose and high-dose stem cell exosome preparation groups, the CREBH DPSCs overexpression exosome group, and the Nepeta cataria extract group all showed extremely significant increases. p <0.0001); SIT was also significantly increased in the wild-type DPSCs exosome group ( p <0.001) (e.g. Figure 4 As shown in A).

[0082] Compared with the model control group, TBUT and CFS were significantly increased in all treatment groups. p <0.0001). Among them, the high-dose stem cell exosome preparation group showed the most significant improvement (e.g., Figure 4 B and Figure 4 (As shown in C).

[0083] The above results indicate that the combined use of CREBH DPSCs overexpressing exosomes with Nepeta cataria extract, especially high-dose stem cell exosome preparations, can significantly improve tear secretion function, tear film stability, and corneal epithelial integrity in dry-eye rats. Its effect is better than that of single components, which suggests that the stem cell exosome preparations of the present invention have good application potential in the treatment of dry eye.

[0084] 6.2 Results of histological indicators like Figure 5 As shown, PAS staining results of conjunctival goblet cells revealed that the normal control group rats had an abundant number of conjunctival goblet cells, at (17.17±1.51) cells / section. The model control group showed a significant reduction in the number of goblet cells to (6.15±1.61) cells / section, indicating that the goblet cell injury model was successfully established.

[0085] Compared with the model control group, the number of goblet cells increased to varying degrees in all treatment groups. Specifically, the number of goblet cells in the low-dose stem cell exosome preparation group, the high-dose stem cell exosome preparation group, and the CREBH overexpression exosome group recovered to (12.26±1.23), (15.55±1.56), and (10.38±1.03) cells / section, respectively, all significantly higher than the model control group (p<0.0001). Notably, there was no significant difference between the high-dose stem cell exosome preparation group and the normal control group. The number of goblet cells in the *Nepeta cataria* extract group was (7.28±1.94) cells / section, significantly higher than the model control group (p<0.05). There was no statistically significant difference between the wild-type exosome group and the model control group.

[0086] The above results indicate that the combined use of CREBH DPSCs overexpression exosomes with Nepeta cataria extract, especially high-dose stem cell exosome preparations, can significantly improve the repair and regeneration of conjunctival goblet cells in dry-eye rats, and its effect is better than that of a single combination.

[0087] 6.3 Results of Inflammatory Factor Detection like Figure 6 As shown, compared with the normal control group, the levels of TNF-α and IL-1β in the corneal and conjunctival tissue homogenates of rats in the model group were significantly increased (p < 0.0001), indicating that the model was successfully established.

[0088] Compared with the model control group, all intervention groups significantly reduced the levels of TNF-α and IL-1β in corneal and conjunctival tissue homogenates of DED model rats (p < 0.0001). Notably, the levels of TNF-α and IL-1β in the high-dose stem cell exosome preparation group were not statistically different from those in the normal control group.

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A gene-edited stem cell exosome preparation, characterized in that, Include: Stem cell exosomes overexpressing CREBH, and Nepeta macrocarpa extract; The mass ratio of the stem cell exosomes overexpressing CREBH to the extract of Nepeta macrocarpa was 1:(2-5). The stem cell exosomes overexpressing CREBH were derived from human primary dental pulp stem cells; The preparation method of the Nepeta macrocarpa extract is as follows: Take the dried whole herb of *Nepeta macrocarpa*, pulverize it, add it to a 70-80% ethanol aqueous solution at a material-to-liquid ratio of 1g:(8-10)mL, heat and reflux to extract 2-3 times, each time for 1.5-2 hours; combine the extracts, concentrate under reduced pressure to obtain an extract; disperse the extract in water, extract with ethyl acetate 3-4 times, combine the ethyl acetate extracts, recover the solvent under reduced pressure, and dry to obtain *Nepeta macrocarpa* extract.

2. The exosome preparation based on gene-edited stem cells as described in claim 1, characterized in that, The stem cell exosomes overexpressing CREBH were obtained by integrating the CREBH gene into the AAVS1 safe harbor site of the stem cell genome.

3. The exosome preparation based on gene-edited stem cells as described in claim 1 or 2, characterized in that, It also includes pharmaceutically acceptable carriers.

4. A method for preparing a gene-edited stem cell exosome preparation, used to prepare the gene-edited stem cell exosome preparation as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of stem cell exosomes overexpressing CREBH Human primary dental pulp stem cells were obtained and cultured and passaged at 37°C and 5% CO2 to obtain third-generation human primary dental pulp stem cells. Design an sgRNA with the nucleotide sequence shown in SEQ ID No. 1 that targets the safe harbor site of human AAVS1, and construct the sgRNA-AAVS1 / Cas9 plasmid; The AAVS1-CREBH-Puro donor vector containing the CREBH gene coding sequence shown in SEQ ID No. 2, the left homologous arm shown in SEQ ID No. 3, and the right homologous arm shown in SEQ ID No. 4 was synthesized. Using CRISPR / Cas9 gene editing technology, the sgRNA-AAVS1 / Cas9 plasmid and the AAVS1-CREBH-Puro donor vector were co-transfected into the third-generation human primary dental pulp stem cells. After drug screening and molecular identification, stem cells overexpressing CREBH were obtained. Stem cells overexpressing CREBH were cultured, and the cell culture supernatant was collected. The cell culture supernatant was subjected to exosome isolation and purification to obtain stem cell exosomes overexpressing CREBH; (2) Preparation of Nepeta macrocarpa extract; (3) The stem cell exosomes overexpressing CREBH are mixed with the extract of Nepeta macrocarpa in a certain proportion and a pharmaceutically acceptable carrier is added to prepare the stem cell exosome preparation.

5. The use of the gene-edited stem cell exosome preparation obtained by the preparation method described in claim 4 in the preparation of a drug for treating dry eye syndrome.

6. The application as described in claim 5, wherein the dosage form of the drug is eye drops, ophthalmic gel, or ophthalmic implant.

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