Use of lactobacillus plantarum in the preparation of a medicament for the prevention or treatment of dry eye

Abstract

The application discloses a use of lactobacillus plantarum in preparation of a medicine for preventing or treating dry eye, and belongs to the technical field of microbial medicine. The application aims to explore the improvement effect of lactobacillus plantarum from an ocular surface and metabolites thereof on dry eye. Through in-depth research on the influence of lactobacillus plantarum and metabolites thereof on ocular surface tissues, the key role substances of the lactobacillus plantarum and metabolites thereof in the treatment of post-refractive dry eye are revealed, and a new target for the treatment of dry eye is provided. Meanwhile, by locally giving the probiotics, the probiotic properties are maximally reserved, a theoretical basis for the clinical application of the probiotics in the treatment of post-refractive dry eye is provided, and a new thought and strategy for clinical practice are provided.

Description

Technical Field

[0001] This invention relates to the field of microbial pharmaceutical technology, specifically to the use of *Lactobacillus plantarum* in the preparation of medicaments for the prevention or treatment of dry eye. Background Technology

[0002] Dry eye syndrome (DES) is a disease caused by multiple factors, accompanied by increased tear film permeability and ocular surface inflammation. With the increasing prevalence of corneal refractive surgery worldwide, the number of patients with DES is rising daily. DES is a common complication after corneal refractive surgery, affecting a large number of patients and placing a significant burden on their visual function, daily life, professional work, and quality of life. Although the safety and efficacy of refractive surgery for myopia have been continuously reported and confirmed, refractive surgery-induced DES directly impairs patients' functional vision and can also cause epithelial hyperplasia and stromal remodeling leading to refractive regression, severely impacting patient satisfaction after surgery.

[0003] Currently, commonly used clinical treatments for dry eye include artificial tears, meibomian gland massage, and warm compresses. However, these methods only temporarily relieve symptoms and do not address the root cause of the problem. Therefore, finding a new treatment that can improve dry eye symptoms is urgently needed. With the development of biotechnology, some biological agents have come into focus due to their good efficacy and few adverse reactions, and are widely used in the relief and treatment of various diseases, among which probiotics have shown outstanding performance.

[0004] Although most studies on the probiotic effects of probiotics have observed their effects in gut tissue, multiple studies have shown that the potential impact of these probiotics is not limited to the gut, and topical administration is the most suitable approach for ocular treatment. Studies have found that eye drops containing cell-free supernatant of *Lactobacillus plantarum* significantly reduced the secretion of ocular inflammatory parameters, inflammatory cell infiltration, and clinical symptoms of inflammation in animal models of uveitis. Simultaneously, in vitro studies have also found that *Lactobacillus plantarum* supernatant significantly reduced the production of pro-inflammatory cytokines and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated human retinal pigment epithelial cell lines. Previous studies have found that the structure of the normal ocular surface flora in dry eye patients differs from the distribution of the flora in healthy ocular surfaces. These changes may be associated with severe ocular surface abnormalities, including epithelial damage and reductions in the hydroprotein and mucin layers, suggesting a higher proportion of potentially pathogenic microorganisms. Summary of the Invention

[0005] To address the issue that topical administration of probiotics in existing technologies fails to alleviate dry eye symptoms and promote corneal nerve regeneration by improving the ocular surface flora, this invention provides the use of *Lactobacillus plantarum* in the preparation of drugs for the prevention or treatment of dry eye. This invention aims to explore the ameliorative effects of *Lactobacillus plantarum* and its metabolites derived from the ocular surface on dry eye. Through in-depth research on the effects of *Lactobacillus plantarum* and its metabolites on ocular surface tissues, the aim is to reveal its key components in the treatment of dry eye after refractive surgery, providing new targets for the treatment of dry eye. Simultaneously, by preserving the probiotic properties to the greatest extent possible through topical administration of probiotics, this provides a theoretical basis for the clinical application of probiotics in the treatment of dry eye after corneal refractive surgery, offering new ideas and strategies for clinical practice.

[0006] To achieve the above objectives, the present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum The *Lactobacillus plantarum* described therein is named *Lactobacillus plantarum* NCU-40, and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34104. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is April 7, 2025.

[0007] Furthermore, the 16S rDNA sequence of the *Lactobacillus plantarum* NCU-40 is shown in SEQ ID NO: 1.

[0008] A second aspect of the present invention provides a metabolite of *Lactobacillus plantarum* NCU-40, wherein the metabolite is a cell-free fermentation supernatant of *Lactobacillus plantarum* NCU-40, or an ethanol extract of the fermentation broth of *Lactobacillus plantarum* NCU-40, an ethyl acetate extract of the fermentation broth, or a lyophilized powder thereof.

[0009] A third aspect of the present invention provides an ophthalmic composition for improving dry eye, comprising live bacteria of *Lactobacillus plantarum* NCU-40 as described in the present invention and / or metabolites as described in the present invention, and a pharmaceutically acceptable ophthalmic carrier.

[0010] Furthermore, the viable bacterial concentration of the *Lactobacillus plantarum* NCU-40 is 1 × 10⁻⁶. 6 CFU / mL to 1×10 7 CFU / mL; the mass concentration of GABA (γ-aminobutyric acid) in the metabolite is 1 μM to 10 μM.

[0011] Furthermore, the pharmaceutically acceptable ophthalmic carrier is selected from one or more of sodium hyaluronate, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, sodium chloride, and phosphate buffer.

[0012] Furthermore, the dosage form of the composition is eye drops, ophthalmic gel, ophthalmic cream, ophthalmic spray, or ophthalmic implant.

[0013] A fourth aspect of the present invention provides the use of the *Lactobacillus plantarum* NCU-40 or its metabolites in the preparation of a medicament for the prevention or treatment of dry eye.

[0014] Furthermore, the dry eye includes post-refractive dry eye and neuropathic dry eye.

[0015] Furthermore, the refractive postoperative dry eye includes dry eye that occurs after laser-assisted in situ keratomileusis (LASIK), femtosecond laser small incision lenticule extraction (SMILE), and laser-assisted subepithelial keratomileusis (LASEK).

[0016] Furthermore, the drug exerts its effect through all of the following mechanisms: (a) Reversing ocular surface microbial remodeling after refractive surgery and restoring the ocular surface microecological balance; (b) Protect corneal epithelial cells from damage caused by hyperosmolar stress; (c) Suppressing acute postoperative inflammatory response.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: (1) For the first time, *Lactobacillus plantarum* NCU-40 was isolated from the conjunctival sac microenvironment of healthy human eyes. As an inherent commensal bacterium of the human eye surface, it has natural compatibility with the ocular microenvironment compared to exogenous probiotics from the gut, breast milk, and fermented foods in existing technologies. It can survive stably on the ocular surface for a long time and continue to play a role without causing ocular surface flora imbalance, allergic reactions, or immune rejection, thus fundamentally solving the safety concerns of exogenous probiotics in ocular applications.

[0018] (2) This strain can simultaneously intervene in the three core pathological processes of post-refractive surgery dry eye: rapidly promoting corneal epithelial cell proliferation and barrier repair, upregulating conjunctival goblet cell mucin secretion to stabilize the tear film, and inhibiting NF-κB-mediated postoperative acute inflammatory cascade. Cell experiments confirmed that it can significantly inhibit hyperosmolar stress-induced apoptosis of HCECs, reducing the apoptosis rate by ≥60%. Animal experiments confirmed that 36 h after injury, the wound healing rate of the *Lactobacillus plantarum* treatment group reached 71.33%, which was much higher than the 57.67% in the model group, filling the gap in the existing technology for the treatment of postoperative acute ocular surface injury.

[0019] (3) This strain and its metabolites can be prepared into various commonly used clinical dosage forms such as eye drops, ophthalmic gels, and ophthalmic sprays to meet clinical storage and use needs. They are suitable for the prevention and treatment of dry eye syndrome of different severity and types, and have broad prospects for clinical translation. Attached Figure Description

[0020] Figure 1 This indicates the changes in corneal nerve parameters between the DE and NDE groups. (A) CNFD, corneal nerve fiber density; (B) CNBD, corneal nerve branch density; (C) CNFL, corneal nerve fiber length; (D) CTBD, total corneal branch density; (E) CNFW, corneal nerve fiber width; (F) CNFA, ​​corneal nerve fiber area; (G) CfracDim, corneal fractal dimension. ns: No statistically significant difference, *p<0.05, **p<0.01, ****p<0.0001.

[0021] Figure 2 This study analyzed the diversity and structural characteristics of the ocular surface microbiome in patients with dry eye and non-dry eye after FS-LASIK surgery. (A) Sparse curve; (B) Chao1 index; (C) Venn diagram; (D) Principal coordinate analysis of β diversity (PCoA); (E) Effect size of linear discriminant analysis (LEfSe). *p< 0.05.

[0022] Figure 3 This study demonstrates the differences in ocular surface microbiome structure between dry-eye and non-dry-eye patients after FS-LASIK surgery. (A) Stacked bar chart of species composition at the phylum level; (B–C) Comparison of the relative abundance of Proteobacteria and Bacteroidetes in the dry-eye and non-dry-eye groups; (D) Species distribution at the genus level; (E–I) Comparison of the relative abundance of Pseudomonas, Veillonella, Streptococcus, Coagulobacterium, and Lactobacillus; (J) Heatmap of differences in ocular surface microbiota between the dry-eye and non-dry-eye groups. *p< 0.05, **p< 0.01.

[0023] Figure 4 This study investigated the correlation between ocular surface microbiota and dry eye and corneal neurological parameters. Spearman correlation analysis showed the correlation between genus-level microbial abundance and key postoperative clinical indicators, including dry eye-related indicators and corneal neurological parameters. Blue indicates a positive correlation, and red indicates a negative correlation. *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 5This study compared the ocular surface microbiota before and after surgery in patients with dry eye and those without dry eye following FS-LASIK. (A) Stacked bar chart of species before and after FS-LASIK surgery in the dry eye group; (B–H) Relative abundance of Staphylococcus, Pseudomonas, Corynebacterium, Helicobacter, Veillonella, Lawrence, and Coagulobacter before and after surgery; (I) Stacked bar chart of species before and after FS-LASIK surgery in the non-dry eye group; (J–L) Relative abundance of Corynebacterium, Veillonella, and Lawrence before and after surgery. Post_DE: Patients with DE after FS-LASIK surgery; Post_NDE: Patients with NDE after FS-LASIK surgery. *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 6 This study provides a comprehensive evaluation of the growth characteristics, in vitro antioxidant activity, and hemolytic activity of *Lactobacillus plantarum* NCU-40. (A) Growth curve of *Lactobacillus plantarum* NCU-40; (B) Evaluation of the in vitro antioxidant capacity of *Lactobacillus plantarum* NCU-40; (C) Hemolytic results of *Lactobacillus plantarum* NCU-40.

[0026] Figure 7 This study demonstrates the susceptibility of *Lactobacillus plantarum* NCU-40 to antibiotics. (A) Antibiotic susceptibility assay, antibiotic abbreviations: E, erythromycin; CTR, ceftriaxone; AMP, ampicillin; TET, tetracycline; PEN, penicillin; SXT, sulfamethoxazole-trimethoprim; C, chloramphenicol; MY, lincomycin; GEN, gentamicin; CIP, ciprofloxacin; (B) Quantitative analysis of inhibition zone diameter in the drug susceptibility test.

[0027] Figure 8 This demonstrates the evaluation of the antibacterial properties of *Lactobacillus plantarum*. (A–B) Schematic diagram of the antibacterial experiment and quantitative analysis of the diameter of the inhibition zone.

[0028] Figure 9 This study evaluated the cell adhesion properties of *Lactobacillus plantarum* NCU-40. (A) Adhesion experiment of *Lactobacillus plantarum* NCU-40 on human conjunctival epithelial cells; (B) Adhesion experiment of *Lactobacillus plantarum* NCU-40 on human corneal epithelial cells; (C) Quantification of colony-forming units (CFU) adhering to epithelial cells, expressed as CFU / 100 cells.

[0029] Figure 10This study evaluated the intervention effect of ocular lactobacillus in a corneal nerve transection dry eye model. (A) Schematic diagram of experimental design; (B) Representative images of corneal fluorescein staining in each group; (C) Corneal fluorescein staining scores in each group; (D) Tear film breakup time in each group; (E) Tear secretion in each group. The experimental groups included: C, normal control group (n = 3), daily PBS instillation; M, corneal nerve transection dry eye model group (n = 3), daily PBS instillation; ML, dry eye model (n = 3), daily instillation of *Lactobacillus plantarum*; MS, dry eye model mice (n = 3), daily instillation of *Lactobacillus salivarius*. ns, no statistical significance, *p<0.05, ****p<0.0001.

[0030] Figure 11 This study evaluated the efficacy of ocular Lactobacillus strains in repairing corneal epithelial damage in mice. (A) Schematic diagram of experimental design; (B) Corneal fluorescein staining images of each group; (C) Quantitative analysis of wound area at different time points; The experimental groups included: CP group (n = 3), PBS instilled after corneal epithelial injury; CL group (n = 3), Lactobacillus plantarum eye drops instilled after modeling; CS group (n = 3), Lactobacillus salivarius eye drops instilled after modeling. ns, no statistical significance, *p<0.05, ****p<0.0001.

[0031] Figure 12 This study evaluated the therapeutic effect of *Lactobacillus plantarum* NCU-40 in a corneal neurectomy dry eye model. (A) Schematic diagram of the experimental design; (B) Representative images of corneal fluorescein staining in each group; (C) Corneal fluorescein staining score; (D) Tear film breakup time in each group; (E) Tear secretion volume in each group. The experimental groups included: C, normal control group (n = 3), daily PBS instillation; M, corneal neurectomy dry eye model group (n = 3), daily PBS instillation; ML, dry eye model (n = 3), daily *Lactobacillus plantarum* instillation. No statistically significant difference was observed (p < 0.0001).

[0032] Figure 13 This study investigated the effects of *Lactobacillus plantarum* NCU-40 on the expression of *Lactobacillus* spp. on the ocular surface and corneal inflammatory factors. (A) Relative copy number of *Lactobacillus* spp. in mice; (BC) Relative mRNA expression levels of II1b and Tnf in corneal tissue. **p<0.01, ***p<0.001, ****p<0.0001.

[0033] Figure 14This study investigated the effects of *Lactobacillus plantarum* NCU-40 on the expression of corneal tight junction proteins and nerve-related proteins. (A) Western blot analysis of the expression of tight junction-related proteins and corneal nerve-related proteins in each group; (BF) Quantitative analysis of ZO-1, Occludin, Claudin-1, BDNF, and β-tubulin III (Tuj1). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0034] Figure 15 This study demonstrates the effects of *Lactobacillus plantarum* NCU-40 on the pathological changes of mouse corneal tissue and AQP5 in the lacrimal gland. (A) Representative image of corneal HE staining (scale bar: 50 μm); (B) Immunofluorescence staining of corneal β-tubulin III (Tuj1) (green, scale bar: 50 μm); (C) Immunofluorescence staining of lacrimal gland AQP5 (red, scale bar: 100 μm).

[0035] Figure 16 This study investigated the effects of cell-free supernatant and inactivated cells of *Lactobacillus plantarum* NCU-40 on hyperosmolar-induced HCEC damage. (A) Schematic diagram of the experimental design; (B) HCEC viability assay results after treatment with different concentrations of MLS; (C) HCEC viability assay results after treatment with different concentrations of MHL; (D) Comparison of cell viability among groups at the optimal intervention concentration; (E) Fluorescence illustration of live and dead cells using Calcein AM / PI; Scale bar: 50 μm; (F) Quantitative analysis of cytotoxicity. No statistically significant differences were found in the values ​​of ns, ***p<0.001, ****p<0.0001.

[0036] Figure 17 This study investigated the effects of different components of *Lactobacillus plantarum* NCU-40 on the migration, proliferation, and inflammatory response of HCECs in each group. (A) Representative images from the scratch assay, scale bar: 200 μm; (B) Quantitative analysis of scratch migration rate; (C) Representative fluorescence images from the EdU assay (red indicates EdU-positive cells, blue indicates DAPI-labeled cell nuclei, scale bar: 50 μm); (D) Statistical analysis of the proportion of EdU-positive cells; (E–F) Levels of IL-1β (E) and IL-6 (F) in the cell culture supernatant of each group. No statistically significant differences were observed (**p<0.01, ***p<0.001, ****p<0.0001).

[0037] Figure 18This study investigated the effects of different components of *Lactobacillus plantarum* NCU-40 on the expression of tight junction proteins in each group of HCECs. (A) Western blot analysis of tight junction-related protein expression in each group; (B–D) Quantitative analysis of ZO-1, Occludin, and Claudin-1. No statistically significant differences were found in the expression of tight junction proteins in each group; *p<0.05, **p<0.01.

[0038] Figure 19 This study compared the metabolomics of *Lactobacillus plantarum* NCU-40 cell-free supernatant with the control medium. (A) Chemical classification and distribution of identified metabolites; (B) Volcano plot analysis of differentially abundant metabolites; (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot; (D) Hierarchical clustering heatmap of differentially abundant metabolites; (E) Screening and importance ranking analysis of marker metabolites based on machine learning; (F) ROC curve analysis of GABA; (G) Comparison of relative GABA abundance among groups. Groups: Ctrl, MRS medium group (n = 6); Sup, *Lactobacillus plantarum* NCU-40 cell-free supernatant group (n = 6). *ns*, no statistically significant difference; *p<0.05.

[0039] Figure 20 This study investigated the effects of *Lactobacillus plantarum* NCU-40 on GABA levels in the conjunctiva, cornea, and tears of mice. (A) GABA levels in mouse conjunctiva; (B) GABA levels in mouse cornea; (C) GABA levels in mouse tears. ***p<0.001; ****p<0.0001. Detailed Implementation

[0040] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0041] This invention, based on clinical research background and existing research gaps, takes the differences in ocular surface microecology as its starting point and systematically conducts screening and evaluation of ocular surface lactobacilli from a functional perspective. By isolating and identifying lactobacillus strains from the ocular surface of healthy volunteers, and then using an animal dry eye model, the repair effects of target strains on clinical symptoms of dry eye and corneal tissue damage are verified. Simultaneously, the probiotic properties of the strains are comprehensively evaluated in vitro. This invention aims to establish a complete research system encompassing "clinical relevance analysis, strain functional screening, and in vivo experimental verification." On the one hand, it lays a solid experimental foundation for further in-depth exploration of the molecular mechanisms by which ocular surface lactobacilli exert their effects; on the other hand, it provides new theoretical support and research ideas for microecological targeted intervention in dry eye disease.

[0042] The in vitro functional evaluation results of *Lactobacillus plantarum* NCU-40 in this invention show that the strain has outstanding in vitro proliferation capacity. Simultaneously, the strain exhibits high levels of free radical scavenging, metal ion chelation, and reducing abilities, demonstrating its ability to effectively reduce cell damage caused by oxidative stress. This conclusion is similar to the protective effect shown by the strain in a dry eye model. Drug sensitivity testing results verified that *Lactobacillus plantarum* NCU-40 is sensitive to most commonly used clinical antibiotics, exhibiting resistance to only a very small number of antibiotics. The overall safety of the strain meets the standards for probiotic application. Antibacterial experiments confirmed that the strain has a significant inhibitory effect on various opportunistic pathogens on the ocular surface, suggesting that it maintains the stability of the ocular surface microecology through two pathways: competitive exclusion of pathogens and secretion of active metabolites. Cell adhesion experiments showed that *Lactobacillus plantarum* NCU-40 has strong adhesion to human conjunctival epithelial cells and also exhibits some adhesion to corneal epithelial cells, laying the foundation for the strain to achieve short-term colonization and sustained biological function on the ocular surface.

[0043] This invention, combining the results of previous functional strain screening, focuses on verifying the in vivo therapeutic effects of *Lactobacillus plantarum* NCU-40. The study comprehensively analyzed multiple aspects, including dry eye clinical phenotype, ocular surface microecological regulation, local inflammatory response, corneal barrier integrity, nerve repair, and lacrimal gland function. The combined experimental results show that *Lactobacillus plantarum* NCU-40 not only improves dry eye symptoms in mice but also effectively increases the abundance of *Lactobacillus* species on the ocular surface, downregulates the expression of pro-inflammatory factors, promotes the recovery of corneal tight junction protein and neural structural marker protein expression, and also increases the expression of AQP5 in the lacrimal gland. Therefore, *Lactobacillus plantarum* NCU-40 has a strong protective effect against the progression of dry eye pathology and provides a reference experimental basis for subsequent research on probiotic treatment of dry eye.

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0045] Main reagent / consumable names and sources: Tear test filter strips, Tianjin Jingming New Technology Development Co., Ltd.; Fluorescein sodium ophthalmic test strips, Tianjin Jingming New Technology Development Co., Ltd.; Promecaine hydrochloride, ALCON-COUVREUR NV, Belgium; Sterile sampling swabs, Xinghe Biotechnology; Genomic DNA extraction kit, MP, USA. Biomedicals, Inc.; PBS, Beyotime Biotechnology Co., Ltd., China; MRS medium, Shanghai Hongshun Biotechnology Co., Ltd.; MEM medium, Beina Biotechnology Co., Ltd.; Phenol red cotton thread for tear detection, Tianjin Jingming New Technology Development Co., Ltd.; Glycerol, Beijing Solarbio Biotechnology Co., Ltd.; Fetal bovine serum (FBS), Shenzhen Biocare Biotechnology Co., Ltd.; Penicillin / streptomycin mixture, Shenzhen Biocare Biotechnology Co., Ltd.; 0.25% trypsin, Gibco, USA; Antimicrobial susceptibility testing discs, Thermo Fisher Scientific, USA; Crystal violet staining solution, Wuhan Sewell Biotechnology Co., Ltd., China; Levofloxacin eye drops, Santen Pharmaceutical Co., Ltd., Japan; Tobramycin eye drops, Novartis Pharmaceuticals, Belgium; Isoflurane, Shenzhen Reward; DMEM medium, Beijing Solarbio Biotechnology Co., Ltd.; RIPA lysis buffer, Solarbio Biotechnology Co., Ltd., China; PMSF, Solarbio Biotechnology Co., Ltd., China; Western blot blot primary antibody dilution buffer, Wuhan Sewell Biotechnology Co., Ltd., China; 5× protein loading buffer, Solarbio Biotechnology Co., Ltd., China; ECL chemiluminescence solution, Wuhan Sewell Biotechnology Co., Ltd., China; BCA protein quantification kit, Solarbio Biotechnology Co., Ltd., Beijing; glycine, Solarbio Biotechnology Co., Ltd., Beijing; ammonium persulfate powder, Solarbio Biotechnology Co., Ltd., Beijing; sodium dodecyl sulfate (SDS), Solarbio Biotechnology Co., Ltd., Beijing; tris(hydroxymethyl)aminomethane, Solarbio Biotechnology Co., Ltd., Beijing; Skim Milk skim milk powder, Solarbio Biotechnology Co., Ltd., Beijing; 1M Tis-HCl pH = 6.8, Wuhan Sewell Biotechnology Co., Ltd., China; 1M Tis-HCl pH = 8.8. Wuhan Sewell Biotechnology Co., Ltd., China; Color pre-stained protein molecular weight standard, Shanghai Yamei Biotechnology Co., Ltd.; Transfer filter paper, Nanjing Yucheng Experimental Equipment Co., Ltd.; 30% Acr-Bis (29:1), Wuhan Sewell Biotechnology Co., Ltd., China; PVDF membrane, Millipore; Tween-20, Beijing Solarbio Biotechnology Co., Ltd.; Anhydrous methanol, Xilong Scientific Co., Ltd.; Hifair® AdvanceFast 1st Strand cDNA Synthesis Kit, Yisheng Biotechnology Co., Ltd.; anti-ZO-1 antibody, Wuhan Sanying Biotechnology Co., Ltd.; anti-Occludin antibody, Wuhan Sanying Biotechnology Co., Ltd.; anti-Claudin-1 antibody, Wuhan Sanying Biotechnology Co., Ltd.; anti-BDNF antibody, Abcam, USA; anti-Tuj1 antibody, Beyotime Biotechnology Co., Ltd., China; Secondary antibody anti-rabbit IgG, Wuhan Sanying Biotechnology Co., Ltd.; Secondary antibody anti-mouse IgG, Wuhan Sanying Biotechnology Co., Ltd.; RNO7-EASY Spin tissue / cell RNA extraction kit, Beijing Adley Biotechnology Co., Ltd.; anti-AQP5 antibody, Wuhan Sewell Biotechnology Co., Ltd., China; QIAamp DNA hostless microbiome kit, Qiagen Enterprise Management (Shanghai) Co., Ltd.

[0046] Example 1: A study on the causal relationship between dry eye syndrome and ocular surface microbial remodeling after FS-LASIK surgery 1.1 Clinical cohort study Thirty patients who underwent FS-LASIK surgery were included in this study. All enrolled patients completed standardized dry eye assessments before and 3 months after surgery. During subsequent data processing, 31 patients who were unable to complete the full course of testing due to loss of contact during the follow-up period, and 2 patients who developed other postoperative complications were excluded. Based on pre-established evaluation criteria, the remaining patients were divided into two groups: (1) FS-LASIK postoperative dry eye group (DE group); (2) non-dry eye group (NDE group). To ensure the comparability of baseline data between the two groups, only the first 30 patients meeting the inclusion criteria were selected from each group.

[0047] The invention has been approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University (Approval No.:

[2024] No. 34). The research process strictly follows the principles of the Declaration of Helsinki and has been registered on the ClinicalTrials.gov platform (Registration No.: NCT06448468).

[0048] 1.2 Sample Collection Ocular surface swab samples were collected in a sterile preparation room and performed entirely by professionally trained ophthalmologists. The physician disinfected their hands, wore sterile gloves, and then instilled promecaine hydrochloride into the patient's eye for surface anesthesia. After 1–3 minutes of anesthesia taking effect, the patient was instructed to look upwards, and the physician gently swabbed the lower conjunctival fornix twice in a circular motion with a sterile swab. After sample collection, the swabs were immediately sealed in cryovials free of DNA / RNA contamination, transported on dry ice, and stored at -80°C for centralized nucleic acid extraction.

[0049] 1.3 Confocal Microscopy Detection and Image Analysis Corneal nerve status was assessed using in vivo confocal microscopy (IVCM) performed by a Heidelberg HRT3-RCM microscope following a standardized procedure. Experienced operators ensured accurate eye localization. Two observers conducted the subsequent analysis in a blinded manner. Three high-quality images were selected based on image sharpness, contrast, and the absence of artifacts. The selected images were then analyzed using ACCMetrics software developed by the University of Manchester, UK. The software automatically quantifies the following parameters: (1) corneal nerve fiber density (CNFD, unit: roots / mm²); (2) corneal nerve fiber length (CNFL, unit: mm / mm²); (3) corneal nerve branch density (CNBD, unit: roots / mm²); (4) corneal total branch density (CTBD, unit: roots / mm²); (5) corneal nerve fiber area (CNFA, ​​unit: mm² / mm²); (6) corneal nerve fiber width (CNFW, unit: mm / mm²); (7) corneal fractal dimension (CFracDim), reflecting the complexity and integrity of the spatial distribution of nerves. The average of three images is taken as the final measurement result.

[0050] 1.4 DNA extraction and 16S rRNA sequencing Total genomic DNA was extracted from the samples using the MagBeads FastDNA Kit for Soil (catalog number: 116564384; MP Biomedicals, USA) according to the manufacturer's instructions and stored at -20°C for later use. Subsequent sequencing was performed by Shanghai Paisenuo Biotechnology Co., Ltd., using the Illumina NovaSeq sequencing platform with a NovaSeq 6000 SP reagent kit (500 cycles) for 2 × 250 base pairs.

[0051] 1.5 Statistical Analysis This embodiment uses SPSS 26.0 and R 4.2.1 software for statistical analysis of the data. The Shapiro-Wilk test was used to determine whether all continuous variables conformed to a normal distribution. Normally distributed measurement data were expressed as mean ± standard deviation; non-normally distributed data were presented using the median (interquartile range). Between-group data comparisons were performed using either the independent samples t-test or the Mann-Whitney U test, depending on the actual data distribution. Within-group paired-samples difference analysis used the Wilcoxon signed-rank test. Categorical variables were uniformly expressed as percentages, and between-group comparisons used the chi-square test or Fisher's exact test. Spearman correlation analysis was used to determine the degree of association between the variables. This invention defines a two-sided p-value < 0.05 as statistically significant.

[0052] 1.6 Results (1) Morphology of corneal basal nerve: The comparison of postoperative corneal nerve parameters between the DE group and the NDE group also showed that the values ​​of multiple indicators in the DE group were significantly lower than those in the NDE group, involving multiple parameters such as CNFD, CNBD, CNFL, CTBD and CfracDim (see details). Figure 1 The AD and G values ​​in the group were significantly higher than those in the NDE group, while the CNFW index value was significantly higher. Figure 1 In the E group, the CNFA index value is slightly lower than that of the NDE group ( Figure 1 In the F-means, there were statistically significant differences among the groups for all indicators (all p < 0.05). Overall, the corneal nerve damage was more severe in patients with postoperative dry eye.

[0053] (2) Differential microbiome characteristics of DE and NDE groups after FS-LASIK To further explore the microbiological mechanisms of dry eye after FS-LASIK surgery, this invention conducted 16S rRNA sequencing analysis on ocular surface samples from patients in the DE and NDE groups after surgery. The sequencing results show that the sparsity curve indicates sufficient sequencing depth, and the gene richness of both groups reached saturation. Detailed results can be found in [link to detailed results]. Figure 2 In comparison, group DE showed significantly higher α diversity, and the Chao1 index also showed a significant upward trend (p = 0.02). Figure 2 (B in the original text) The results indicated that the ocular surface microbial richness was increased in group DE. Subsequent principal coordinate analysis (PCoA) based on Jaccard distance showed that there were differences in β-diversity between the two groups. Figure 2 The D in the figure indicates that the two groups of ocular surface microbial community structures are different.

[0054] After quality control screening, a total of 2054 operational taxonomic units (OTUs) were identified in this experiment, of which 277 OTUs were common to both groups. Figure 2 The C in the DE group showed that the total number of OTUs and the microbial load were higher than those in the NDE group. Subsequent LEfSe analysis further confirmed the difference in microbial composition between the two groups. Figure 2 (E in the text).

[0055] In summary, the results indicate that the α- and β-diversity of the ocular surface microbiota were significantly altered in patients with dry eye after FS-LASIK surgery, supporting the important role of microbial dysbiosis in postoperative dry eye.

[0056] (3) Differences in the taxonomic composition of microorganisms Phylum-level analysis showed that Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the dominant phyla. Figure 3 (A) Compared to the NDE group, the abundance of Proteobacteria decreased while the abundance of Bacteroidetes increased in the DE group. Figure 3 BC in the genus). At the genus level, Staphylococcus ( Staphylococcus ), Pseudomonas spp. Pseudomonas Corynebacterium spp. Corynebacterium ) and Acinetobacter spp. Acinetobacter ) is the dominant genus ( Figure 3 D). Veillonella spp. in group DE ( Veillonella Streptococcus ( Streptococcus ) and Aggregates ( Aggregatibacter The abundance increased significantly. Figure 3 FH in the middle), while Pseudomonas spp. ( Pseudomonas ) and Lactobacillus genus ( Lactiplantibacillus The abundance was significantly reduced. Figure 3 E and I in the diagram). Heatmap analysis clearly shows the severe dysbiosis in group DE (E, I). Figure 3The J in the study further confirms that specific community shifts occur in ocular surface microbes in dry eye patients after FS-LASIK surgery.

[0057] (4) Correlation between bacterial genus abundance and dry eye and corneal neurological indicators Further validation using Spearman correlation analysis showed that the postoperative ocular surface microbiota was significantly correlated with various key clinical indicators at the genus level. Figure 4 The abundance of *Staphylococcus* was positively correlated with the severity of dry eye symptoms (correlation coefficient ρ = 0.26, p < 0.05). Simultaneously, the abundance of this genus was significantly negatively correlated with several corneal neural parameters, including nerve fiber length (CNFL) ρ = -0.38, tortuous nerve density (CTBD) ρ = -0.30, nerve fiber area (CNFA) ρ = -0.32, and fractal dimension (CFracDim) ρ = -0.46 (all p values ​​< 0.05). The association pattern between *Corynebacterium* and clinical indicators was similar to that of *Staphylococcus*, also showing a clear negative correlation trend.

[0058] In addition, the abundance of Lactobacillus was increased and negatively correlated with OSDI score, with a correlation coefficient ρ = -0.28, p<0.05; at the same time, the abundance of Lactobacillus was also positively correlated with improved tear secretion function, with a corresponding Schirmer test value ρ = 0.26, p<0.05; and also positively correlated with corneal nerve fiber density (CNFD), a nerve regeneration-related indicator, ρ = 0.33, p<0.05.

[0059] (5) Longitudinal dynamic evolution characteristics of ocular surface microbiomes in DE group and NDE group A longitudinal dynamic analysis of the ocular surface microbiota after FS-LASIK surgery revealed that the overall phylum-level microbial structure remained stable in both groups of patients; however, the genus-level microbial composition underwent changes. Figure 5 Postoperatively, the abundance of multiple bacterial genera fluctuated significantly in the DE group. The abundance of Staphylococcus, Rolstonia, Veillonella, and Agrobacterium all increased substantially (p<0.05); meanwhile, the abundance of Pseudomonas, Corynebacterium, and Helicobacter decreased significantly. Figure 5 (AH in the middle). This special manifestation of bacterial imbalance also suggests that surgery may induce ocular surface microecological dysbiosis in DE patients, thereby affecting local ocular immune homeostasis.

[0060] The NDE group showed no significant changes in the phylum-level microbial community structure, and there was no statistically significant difference between groups (p>0.05). However, unique changes were observed at the genus level: the abundance of Corynebacterium decreased significantly (p<0.01), while the abundance of Cynospirillum and Ralstonia increased postoperatively (p<0.05). Figure 5 (IL in the text). Comparing the changes in the postoperative microbiota between the two groups, it was found that the decrease in the abundance of Pseudomonas spp. only occurred in the DE group. This suggests that host-specific factors such as tear composition, ocular surface epithelial repair capacity, and preoperative baseline neuroimmune status are likely the core mechanisms regulating the remodeling of the postoperative ocular surface microbiota.

[0061] 1.7 Based on the above results, this invention is the first to demonstrate that FS-LASIK surgery induces changes in the ocular surface microbiota, with a decrease in the abundance of Lactobacillus species. This decrease is highly correlated with various dry eye indicators and the degree of corneal nerve damage, suggesting that this microbiota may be a key symbiotic organism for maintaining ocular surface homeostasis. Based on this conclusion, this invention will further screen for Lactobacillus strains with protective potential on the ocular surface and investigate the effects of these strains on tear film stability, corneal repair, and nerve regeneration using animal dry eye models. This will provide an experimental basis and theoretical foundation for the subsequent development of probiotic-based dry eye prevention and treatment programs.

[0062] Example 2: Isolation and identification of Lactobacillus plantarum NCU-40 from ocular surface.

[0063] 2.1 Conjunctival sac sample collection This experiment selected five healthy volunteers as research subjects, and collected microbial samples from their conjunctival sacs. The sampling process followed aseptic techniques and strictly adhered to medical ethics requirements; all subjects signed written informed consent forms in advance; individuals who had recently used antibiotics or probiotic preparations were excluded before enrollment. The sampling procedure was strictly performed according to the aseptic collection standards for conjunctival sacs described above. After sample collection, the swabs were placed in centrifuge tubes containing 1 mL of sterile PBS buffer and vortexed for 1 minute to allow the microorganisms to be fully eluted into the buffer, preparing a conjunctival sac microbial suspension. After the suspension was prepared, it underwent serial dilution and plate separation. The sample settling time was strictly controlled throughout the process to avoid microbial inactivation affecting subsequent separation efficiency. Before formal dilution and plating, the centrifuge tubes were inverted 3-5 times to ensure uniform suspension within the tubes, preventing microbial precipitation, which could lead to uneven plating concentrations, experimental bias, and other problems, thus ensuring the stability and reproducibility of the experimental results.

[0064] 2.2 Isolation and purification of microorganisms from the conjunctival sac Take 1 mL of the prepared conjunctival sac microbial suspension and perform aseptic serial dilutions in a clean bench, using PBS buffer as the dilution matrix, and dilute stepwise to 10⁻⁶. -1 10 -2 10 -3Three concentration gradients were used, with three parallel samples for each dilution gradient. Strict aseptic technique was maintained throughout the process to prevent cross-contamination. After each gradient dilution, 0.1 mL of the suspension was evenly added to the surface of MRS solid medium (DeMan-Rogosa-Sharpe Medium, MRS). Using a sterile spreader, the suspension was gently spread to ensure complete and even distribution on the medium surface, avoiding localized droplet aggregation or colony clumps. After spreading, the petri dishes were inverted and placed in a 37°C constant temperature aerobic incubator for 48-72 hours. Colony growth was observed daily, and colony morphology, size, color, and density were recorded in detail. After the culture is completed, select a single colony that meets the typical characteristics of Lactobacillus (the colony is round, with a smooth, raised surface, regular edges, and a milky white or pale yellow color, with a diameter controlled between 0.5 and 1.0 mm). Use a sterile inoculation loop to transfer it to 5 mL of MRS liquid medium and incubate at 37°C for 18-24 h to finally obtain a pure culture solution, which is then stored for later use.

[0065] 2.3 Genomic DNA extraction and strain identification (1) Cultivation and preservation of pure bacterial culture Inoculate the pure bacterial culture into 5 mL of liquid MRS medium and incubate at 37°C for 18-24 h for expansion culture. After expansion culture, use the glycerol preservation method: take an appropriate amount of bacterial culture and mix it thoroughly with 30% glycerol, and store it in a low temperature environment for later use.

[0066] (2) Genomic DNA extraction The experimental procedure is as follows: ① Take the expanded bacterial culture, centrifuge at 8000 rpm for 2 min, discard the supernatant, retain the bacterial precipitate, and add 600 μL of lysis buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.0, 50 mM EDTA, 4% SDS), 200 μL of Tris-saturated phenol, and 0.3–0.4 g of glass beads. Vortex for 30 s, repeating three times to ensure adequate suspension and lysis of the bacterial cells; ② Centrifuge at 8000 rpm for 1 min, carefully transfer the supernatant to a new centrifuge tube, avoiding contamination with the precipitate and organic phase, and add 250 μL of 10 M ammonium acetate. Gently mix and incubate on ice for 10 min, then centrifuge again at 8000 rpm for 1 min; ③ Slowly add the supernatant above the organic layer to the DNA adsorption column, centrifuge to adsorb DNA onto the column surface, and wash the adsorption column twice with 600 μL of 75% ethanol, centrifuging at 8000 rpm for 1 min each time. 4. Transfer the adsorption column to a new sterile EP tube, air dry at room temperature for about 30 minutes, add 50 μL TE buffer (pH 8.0), let stand for a while to elute DNA, and take 25 μL of elution buffer to a high-throughput sequencing platform to complete sequencing.

[0067] (3) Identification and preservation of strains The gene sequences of the strains obtained through high-throughput sequencing were submitted to the NCBI database for homology comparison analysis, and the species were determined based on the comparison results. The results showed that one strain was related to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The strain showed 99.8% homology with *Lactobacillus plantarum*, and was named *Lactobacillus plantarum* NCU-40. Its 16S rDNA gene sequence is shown in SEQ ID NO: 1. *Lactobacillus plantarum* NCU-40 was inoculated into MRS liquid medium and cultured anaerobicly at 37°C for 18 h. The cells were collected by centrifugation, resuspended in 20% glycerol saline, and stored at -80°C. This strain was also deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 7, 2025, with accession number CGMCC NO. 34104 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] The 16S rDNA sequence is as follows: CATGGGGGCCGTTAGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAAT (SEQ ID NO: 1).

[0069] 2.4 Strain biological characteristic experiment (1) Growth curve determination The -40℃ frozen bacterial culture was revived in 5 mL of liquid MRS medium and cultured aerobically at 37℃ for 24 h. Afterwards, it was transferred to fresh MRS medium. The bacterial culture was collected every 2 h, and the optical density (OD) was measured. 600 ), and plot the growth curve.

[0070] (2) Evaluation of antioxidant performance Antioxidant performance was evaluated using cell-free supernatant of the bacteria, and a comprehensive assessment was made based on five core indicators, including DPPH free radical scavenging rate and hydroxyl free radical scavenging ability. The DPPH free radical scavenging rate was measured under light-protected conditions. 2 mL of bacterial supernatant was thoroughly mixed with an equal volume of 0.2 mmol / L DPPH methanol solution, reacted for 30 min in the dark, and the absorbance was measured at 517 nm. The scavenging rate was then calculated using the corresponding formula. For hydroxyl free radical scavenging ability, 1 mL of supernatant was added to the Fenton reaction system, reacted for 30 min, and the absorbance was measured at 510 nm. Superoxide anion scavenging rate was assessed using the pyrogallol autoxidation method, with reaction kinetics monitored at 330 nm in a buffer system containing pyrogallol and Tris-HCl. Fe... 2+ Chelating ability was determined by the phenanthroxazine colorimetric method. The supernatant was added to the mixture with ferrous sulfate, NaOH and VC, and the absorbance was measured at 546 nm after the reaction was terminated. Total reducing power was determined by the potassium ferricyanide reduction method. The supernatant was reacted with PBS and K3[Fe(CN)6], and the reaction was terminated with trichloroacetic acid. Then, 0.1% FeCl3 was used for color development. The absorbance value at 700 nm was positively correlated with the reducing power.

[0071] (3) Hemolysis test The hemolytic performance test of the bacterial strain was performed as follows: Activated *Lactobacillus plantarum* and *Staphylococcus aureus* bacterial suspensions were centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the bacterial pellet was collected. The pellet was then resuspended in sterile PBS buffer to prepare a bacterial suspension. 10 μL of the bacterial suspension was inoculated dropwise onto a BHI agar plate supplemented with 10% defibrinated sheep blood. After inoculation, the plate was placed in a 37℃ incubator and incubated statically for 24 h. Close observation and recording were performed throughout the incubation process.

[0072] (4) Drug sensitivity evaluation Collect bacterial cells from the logarithmic growth phase by centrifugation at 6000 rpm for 10 min. Resuspend the cells in sterile physiological saline and adjust to a suitable concentration. Spread the bacterial suspension evenly on the surface of MRS solid medium and let it stand for 15 min until the bacterial suspension is completely absorbed. Then, attach various antibiotic sensitivity test strips to the surface of the plates. Ten antibiotics were selected for this study: erythromycin (E), penicillin (PEN), ciprofloxacin (CIP), ampicillin (AMP), lincomycin (MY), ceftriaxone (CRO), gentamicin (GEN), chloramphenicol (C), tetracycline (TE), and trimethoprim-sulfamethoxazole (SXT). Incubate the plates at 37℃ for 24 h. Measure the diameter of the inhibition zone in mm using calipers. Each experiment was repeated three times.

[0073] (5) Evaluation of anti-pathogen activity Before conducting in vitro antibacterial experiments on the strain, Staphylococcus aureus was activated ( Staphylococcus aureus Staphylococcus epidermidis ( Staphylococcus epidermidis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Escherichia coli ( Escherichia coli ), Proteus mirabilis ( Proteus mirabilis Five common ocular surface pathogens were identified. Indicator bacterial suspensions were evenly spread onto their respective agar plates, and sterile Oxford cups were then placed stably on the plate surface. 250 μL of cell-free supernatant from *Lactobacillus plantarum* NCU-40 was slowly added to each Oxford cup. A control group was also included, with an equal volume of sterile MRS liquid medium added to the control Oxford cups. All plates were incubated at 37°C for 6–8 h. After incubation, the diameter of the inhibition zone was measured using calipers to determine the antibacterial ability of the strains.

[0074] (6) Evaluation of cell adhesion ability Cell adhesion ability was evaluated using human conjunctival epithelial cells (HCOnEpiCs) and corneal epithelial cells (HCECs), cultured in DMEM or MEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. After trypsin digestion, cells in logarithmic growth phase were cultured at 5 × 10⁻⁶ cells / year. 6 Cells were seeded per well in a six-well plate. After approximately 50% confluence of cells, the cells were washed twice with PBS. 1.5 mL of fresh culture medium and 1 mL of bacterial suspension were added to each well, and the cells were incubated for 2 hours. After incubation, the culture medium was aspirated, and the cells were washed five times with PBS to remove any unattached bacteria. The cell layer was fixed with methanol for 30 minutes, stained with crystal violet, and observed and photographed under an optical microscope.

[0075] 2.5 Evaluation of the probiotic properties of Lactobacillus plantarum NCU-40 (1) Growth curve of Lactobacillus plantarum NCU-40 This experiment used a combination of spectrophotometry and plate counting to dynamically monitor the growth characteristics of *Lactobacillus plantarum* NCU-40 and to plot its growth curve. The final results showed that *Lactobacillus plantarum* NCU-40 entered the logarithmic growth phase after 4 hours of culture, and reached a stationary phase after 12 hours. During this stage, the OD value of the strain... 600 The value reached a peak of 2.559, corresponding to a viable bacteria concentration that remained approximately 5 × 10⁻⁶. 9 CFU / mL (see details) Figure 6 (A) The overall growth curve of this strain conforms to the typical S-shaped proliferation pattern, and the growth stages of the strain are clearly defined, with 0-4 h being the lag phase and 4-12 h being the logarithmic growth phase; after entering the stationary phase, the cell density remains in a high range. This result confirms that *Lactobacillus plantarum* NCU-40 has extremely strong in vitro proliferation ability.

[0076] (2) Evaluation of the in vitro antioxidant capacity of Lactobacillus plantarum NCU-40 Probiotics can alleviate oxidative stress by scavenging free radicals and maintaining the body's redox balance. Based on this mechanism, this experiment conducted a comprehensive in vitro antioxidant performance evaluation of *Lactobacillus plantarum* NCU-40, analyzing the strain's antioxidant capacity through multiple index measurements. The results showed that *Lactobacillus plantarum* NCU-40 effectively resisted DPPH free radicals, hydroxyl radicals (-OH), and superoxide anion radicals (O2). - The scavenging abilities of the three ions were 50.22%, 50.37%, and 78.58%, respectively, targeting ferrous ions (Fe²⁺, Fe³ ... 2+ The chelation rate reached 48.24%, and the absorbance corresponding to the total reducing power of the strain was measured to be 0.772 at a wavelength of 700 nm (see details). Figure 6 (B in the text). Comprehensive analysis revealed that *Lactobacillus plantarum* NCU-40, isolated from the ocular surface, exhibited excellent in vitro antioxidant activity and possesses potential cytoprotective effects.

[0077] (3) Evaluation of the hemolytic properties of Lactobacillus plantarum NCU-40 To further verify the safety of the strain, a hemolysis experiment was conducted. The strain was incubated at a constant temperature of 37℃ for 24 h, and the hemolytic reaction around the colonies on blood agar plates was observed. Staphylococcus aureus was used as a positive control; a clear hemolytic ring formed around the colonies in this group, and red blood cells showed complete lysis, proving the effectiveness of the hemolysis experiment system. In contrast, the Lactobacillus plantarum NCU-40 group did not show any hemolytic ring around the colonies, and the blood agar plates remained a uniform bright red color, without any red blood cell lysis or plate discoloration (see details). Figure 6 (C in the text). Combined with the hemolysis test results, it was confirmed that *Lactobacillus plantarum* NCU-40 does not have hemolytic activity, which meets the basic standards for the safe application of probiotics. This provides solid data support for subsequent in vivo experimental studies of this strain and further exploration of its application value.

[0078] (4) Sensitivity analysis of Lactobacillus plantarum NCU-40 to antimicrobial agents Antibiotic susceptibility is a core reference indicator for evaluating the safety of probiotic applications and exploring the application potential of strains. To further improve the strain safety evaluation system, the susceptibility of *Lactobacillus plantarum* NCU-40 to various commonly used antibiotics was determined using antimicrobial susceptibility testing. The results showed that *Lactobacillus plantarum* NCU-40 exhibited varying degrees of susceptibility to erythromycin (E), penicillin (PEN), ciprofloxacin (CIP), ampicillin (AMP), sulfamethoxazole / trimethoprim (SXT), ceftriaxone (CRO), chloramphenicol (C), and tetracycline (TE), with corresponding average inhibition zone diameters of 2.70 cm, 0.67 cm, 0.13 cm, 2.50 cm, 1.03 cm, 2.33 cm, 2.83 cm, and 1.60 cm, respectively. The strain showed resistance to only lincomycin (MY) and gentamicin (GEN) (see details). Figure 7 (AB in the text). This shows that Lactobacillus plantarum NCU-40 exhibits good overall drug sensitivity, combining strain safety with excellent functional characteristics.

[0079] (5) Evaluation of the antibacterial properties of Lactobacillus plantarum NCU-40 The antibacterial activity of probiotics is an important indicator of a strain's ability to antagonize pathogenic microorganisms and maintain host microbial homeostasis. Based on this research direction, this experiment systematically evaluated the in vitro antagonistic ability of *Lactobacillus plantarum* NCU-40 against common ocular surface pathogens. The antibacterial experiment results showed that *Lactobacillus plantarum* NCU-40 exhibited strong inhibitory effects against various common opportunistic pathogens of the ocular surface, including *Escherichia coli*, *Staphylococcus epidermidis*, *Proteus mirabilis*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*, with corresponding inhibition zone diameters of 28.00 mm, 26.00 mm, 28.67 mm, 28.67 mm, and 29.00 mm (see details). Figure 8 (AB in the text). Combined with experimental data, it was confirmed that *Lactobacillus plantarum* NCU-40 can effectively inhibit the growth and reproduction of various ocular surface pathogens, exhibiting excellent in vitro antibacterial properties and possessing great potential for further development and application.

[0080] (6) Evaluation of the cell adhesion effect of Lactobacillus plantarum NCU-40 Cell adhesion ability is a core prerequisite for probiotics to colonize the corresponding tissues of the host and subsequently exert their physiological functions. To investigate this indicator, an in vitro cell adhesion experiment was conducted on *Lactobacillus plantarum* NCU-40. The results showed that after co-incubating *Lactobacillus plantarum* NCU-40 with HCOnEpiCs and HCECs for 2 hours, the adhesion effect of the strain on the surface of HCOnEpiCs was very prominent, while the adhesion effect on HCECs was relatively weak. Figure 9 (AB in the text). After quantitative analysis, the corresponding NCU-40 adhesion amount on every 100 conjunctival epithelial cells was approximately 9000 CFU, while on corneal epithelial cells, the adhesion amount was only 2700 CFU ( ). Figure 9 (C in the text). In summary, *Lactobacillus plantarum* NCU-40 exhibits excellent overall cell adhesion ability in vitro, and during the adhesion incubation process, it does not negatively affect the normal growth of conjunctival and corneal epithelial cells, demonstrating good biocompatibility.

[0081] Example 3: The therapeutic effects of NCU-40 in improving core symptoms of dry eye and promoting corneal epithelial repair.

[0082] 3.1 Experimental Animals and Model Establishment Female Balb / c mice aged 6-8 weeks were selected as experimental subjects for this animal experiment. All animal procedures were strictly performed in accordance with the approval protocol of the Laboratory Animal Ethics Committee of Nanchang University, with the corresponding ethics approval number NCULAE-20221031190. To prevent postoperative ocular infection, all mice were administered levofloxacin and tobramycin eye drops three days prior to modeling. All subsequent experimental procedures were conducted under inhalation anesthesia in mice, with isoflurane induction concentration set at 3% and maintenance concentration controlled at 1.5%-2% during surgery.

[0083] (1) Corneal nerve transection dry eye model Under a surgical microscope, mice were fixed in place, and a 2.5 mm diameter corneal trephine was vertically inserted into the center of the cornea and gently rotated 5 times to sever the nerve fibers within the corneal stroma, thus establishing a dry eye model. In the sham surgery group, no nerve cutting was performed; only the corneal surface was gently pressed with a sterile cotton swab. On the third postoperative day, mice conforming to the dry eye phenotype were selected and randomly assigned to groups for intervention: PBS and *Lactobacillus plantarum* bacterial solution (1 × 10⁻⁶) were instilled into the eyes of each group of mice. 6CFU / mL, 5 μL / eye), Lactobacillus salivarius culture (1 × 10⁻⁶). 6 CFU / mL, 5 μL / eye), 3 times daily, for 7 consecutive days.

[0084] (2) Corneal epithelial injury model Under a surgical microscope, using a sterile corneal epithelial curette, the corneal epithelium within a 2 mm diameter area in the center of the cornea was gently scraped away, starting from the center (avoiding damage to the limbus and sclera). Immediately postoperatively, patients were randomly assigned to one of two groups for intervention: PBS and *Lactobacillus plantarum* bacterial suspension (1 × 10⁻⁶). 6 CFU / mL, 5 μL / eye, every 6 h / time) or Lactobacillus salivarius culture (1 × 10⁻⁶). 6 CFU / mL, 5 μL / eye, every 6 h / time. Corneal epithelial healing area was quantitatively assessed at 0, 12, 24, and 36 h postoperatively using slit-lamp microscopy combined with sodium fluorescein staining and photography (analysis using ImageJ software).

[0085] 3.2 Evaluation of ocular surface function (1) Tear break-up time (TBUT) was measured on days 0, 3, and 10. 5 μL of 0.25% sodium fluorescein solution was instilled into the conjunctival sac of mice, and excess fluid was absorbed from the ocular surface using sterile filter paper. After standing for 10 seconds, tear film breakup was observed under slit-lamp cobalt blue light. The time from the mouse's last blink to the appearance of the first dark spot on the corneal surface was defined as the tear film breakup time. Each mouse underwent three repeated measurements in one eye, and the average value was used for subsequent statistical analysis.

[0086] (2) Corneal fluorescein staining (CFS) score Corneal fluorescein staining was performed on days 0, 3, and 10. 5 μL of 0.25% sodium fluorescein solution was instilled into the conjunctival sac of mice, and after 30 seconds, corneal staining images were captured using a slit-lamp with cobalt blue light. Scoring criteria: The mouse cornea was evenly divided into four quadrants, and scores ranging from 0 to 3 were assigned based on the extent of staining and the degree of damage in each quadrant. The final score was the sum of the scores from all four quadrants, with a maximum total score of 12 points per eye.

[0087] For mice with corneal epithelial agenesis, the corneal epithelial healing area was observed at 0 h, 12 h, 24 h, and 36 h. 5 μL of 0.25% sodium fluorescein solution was dropped into the conjunctival sac, and corneal staining images were taken under cobalt blue light under a slit lamp 30 s later. The healing area was calculated using ImageJ software.

[0088] (3) Measurement of tear secretion Tear secretion was measured on days 0, 3, and 10 using the phenol red cotton thread method. A phenol red cotton thread was placed in the lower conjunctival sac of mice for 3 minutes, and the wet length (mm) of the cotton thread was measured after removal. This was used as an evaluation index for tear secretion.

[0089] 3.3 Statistical Analysis All experimental data were statistically analyzed using GraphPad Prism version 9.5.1 software. Differences between two groups were assessed using a two-tailed t-test; pairwise comparisons between multiple groups were performed using one-way ANOVA; and repeated measures ANOVA was used if the data involved repeated measures. All data were presented as mean ± standard deviation. A p-value < 0.05 was considered statistically significant. The significance level was defined as follows: ns, indicating no statistical difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0090] 3.4 Results (1) Therapeutic effect of ocular lactobacillus in corneal nerve transection dry eye model Based on clinical trials, the abundance of lactobacilli is positively correlated with tear secretion and corneal nerve fiber density. Therefore, this invention isolated two lactobacilli strains from the conjunctival sac of healthy volunteers: *Lactobacillus plantarum* NCU-40 and *Lactobacillus salivarius* (which had no therapeutic effect on the dry eye model and was therefore not preserved), and conducted in vivo functional screening.

[0091] The results showed that in the corneal nerve transection dry eye model (experimental design see...), Figure 10 A in the model group, compared with the model group ( Figure 10 In the BE (Brain-Earning Injection) study, topical instillation of *Lactobacillus plantarum* significantly improved the clinical phenotype of dry eye, including corneal fluorescein staining score (10.00 ± 1.00 vs 2.67 ± 1.16, p<0.0001), tear secretion (3.33 ± 0.58 mm vs 6.33 ± 1.16 mm, p<0.0001), and tear film breakup time (2.37 ± 0.25 s vs 4.73 ± 0.32 s, p<0.0001). In contrast, no significant improvement was observed in the *Lactobacillus salivarius* intervention group, and there were no statistically significant differences in any of the indicators compared to the model group (p>0.05). These results indicate that *Lactobacillus plantarum* has a significant effect in improving dry eye signs, while the improvement effect of *Lactobacillus salivarius* is limited.

[0092] (2) Evaluation of the efficacy of ocular Lactobacillus strains in repairing corneal epithelial damage in mice To further evaluate the efficacy of candidate lactobacillus strains in repairing corneal epithelial damage, the epithelial repair-promoting effects of two target lactobacillus strains were compared and analyzed (experimental design see...). Figure 11 A in the middle). The results show ( Figure 11 In the case of corneal cysts (BC), 36 hours after injury, the wound healing rate in the NCU-40 treatment group reached 71.33%, significantly higher than the 57.67% in the model group, and the difference between the groups was statistically significant (p<0.0001). This result confirms that NCU-40 can effectively accelerate the corneal epithelial healing process. In contrast, the wound healing rate in the Lactobacillus salivarius intervention group was only 60.67%, similar to the model group, with no statistically significant difference between the groups (p = 0.307).

[0093] The study found that the combined results of improved clinical phenotypes of dry eye and corneal epithelial repair showed that *Lactobacillus plantarum* had more prominent in vivo efficacy and more stable effects.

[0094] Example 4: Evaluation of the efficacy of Lactobacillus plantarum NCU-40 in an animal dry eye model.

[0095] 4.1 Construction of experimental animal models and intervention protocols For the construction and intervention of experimental animal models, please refer to the corneal nerve transection dry eye model in Example 3.

[0096] 4.2 Evaluation of ocular surface function For specific operating procedures of TBUT measurement, CFS score, and tear secretion measurement, please refer to Example 3.

[0097] 4.3 Extraction and qPCR analysis of ocular surface microbial DNA (1) Collection of microbial samples from mouse ocular surface After the experimental procedures were completed, microorganisms were collected from the ocular surface of mice under sterile conditions. Sterile ultrafine fiber swabs were used for sampling, gently wiping along the conjunctival sac and corneal surface of the mice. Each eye was wiped 3-5 times to collect secretions and attached microorganisms from the ocular surface. Throughout the sampling process, extra care was taken to avoid the eyelid skin and hair areas to minimize interference from external factors on the experimental samples. All mouse samples were pooled, with samples from both eyes of a single mouse collected together, and samples from every three mice grouped together. After sampling, the swabs were immediately placed in sterile centrifuge tubes and rapidly stored at -80°C for subsequent DNA extraction and analysis.

[0098] (2) Microbial DNA extraction The QIAamp DNA Microbiome Kit (Qiager Enterprise Management (Shanghai) Co., Ltd.) was used to extract ocular surface microbial DNA. The core advantage of this kit is its ability to selectively lyse host cells while efficiently enriching microbial DNA, making it suitable for low biomass samples. The procedure was as follows: ① The sample swab was placed in lysis buffer and thoroughly shaken before performing host cell lysis to reduce interference from mouse DNA; ② Proteinase K and microbial lysis buffer were added sequentially to ensure complete lysis of bacterial cells in the sample; ③ The DNA was recovered by purification using a silica gel column, centrifugation, and washing; ④ 30-50 μL of nuclease-free water was used to elute the purified DNA. After DNA extraction, the concentration and purity of the sample DNA were measured using a NanoDrop 2000 spectrophotometer. Samples that passed the tests were stored at -20℃ for later use.

[0099] (3) Lactobacillus-specific qPCR detection ① Primer design: Primer sequences were designed and synthesized by Shanghai Sangon Biotech. The primer sequence list is detailed in Table 1. Table 1 Primer sequence listing ②Preparation of RT-qPCR reaction system: According to the kit instructions, the system preparation is shown in Table 2; ensure that the process is carried out under nuclease-free conditions, add each reagent in the order of dosage in the table, mix and put into the RT-qPCR instrument.

[0100] Table 2 RT-qPCR reaction system ③ Perform amplification according to the procedure in the instruction manual (see Table 3 for details).

[0101] Table 3 RT-qPCR reaction procedure ④Results Analysis: After homogenization of the internal reference gene 16S rRNA, 2... -△△Ct Statistical analysis was conducted using the method.

[0102] 4.4 Detection of mRNA expression in mouse corneal tissue (1) Corneal tissue sampling After the experiment, mice were anesthetized and euthanized, and their eyeballs were removed under aseptic conditions. Corneal tissue was dissected under a dissecting microscope, removing any residual tissue from the limbus and iris to ensure the purity of the corneal tissue. Each group of corneal tissue was combined according to the experimental protocol (3 mice were combined into one group). Immediately after corneal sampling, the tissue was placed in liquid nitrogen and then cryopreserved at -80°C for later use to prevent RNA degradation and ensure the accuracy of subsequent experiments.

[0103] (2) Extraction and concentration determination of total RNA from corneal tissue Total RNA was extracted from corneal tissue using the Adley RNA Extraction Kit, strictly following the instructions to avoid exogenous RNase contamination. The specific steps are as follows: ① Take 15-25 mg of fresh corneal tissue, add 500 μL of lysis buffer (RLT), and homogenize the sample thoroughly using a tissue homogenizer until the tissue is completely broken down and homogeneous; ② Add 0.5 times the volume of anhydrous ethanol to the homogenate, vortex thoroughly to mix, and transfer the mixture to an RNA adsorption column. Incubate at room temperature for 1 min to allow nucleic acids to fully adsorb onto the silica membrane; ③ Centrifuge at 13,000 rpm for 30 s, discard the filtrate, and return the adsorption column to a clean collection tube; ④ Add 700 μL of protein removal buffer (RW1) to the adsorption column, incubate at room temperature for 30 s, and centrifuge again at 13,000 rpm for 30 s. Discard the waste liquid after centrifugation; ⑤ Add 500 μL of wash buffer (RW), centrifuge at 13,000 rpm for 15 seconds. 6. Discard the filtrate and repeat the rinsing step once to remove residual impurities in the sample; 7. After washing, place the adsorption column into an empty collection tube and centrifuge at 13,000 rpm for 2 min to thoroughly remove residual rinsing solution and ethanol; 8. Transfer the adsorption column to a brand new 1.5 mL RNase-free centrifuge tube, slowly add 30-50 μL of RNase-free ddH2O to the center of the adsorption membrane, let it stand at room temperature for 1 min, and then centrifuge at 13,000 rpm for 1 min to complete RNA elution and collection. The concentration and purity of the extracted RNA sample were determined using a NanoDrop 2000 spectrophotometer. An A260 / A280 ratio within the range of 1.8-2.0 was considered to be of acceptable purity and could be used for subsequent experiments.

[0104] (3) Reverse transcription ①Preparation: Thaw the reverse transcription kit and add the appropriate volume of RNase-free water according to the total RNA sample.

[0105] ② Reverse transcription synthesis: Prepare the synthesis system according to the reverse transcription kit instructions. The system configuration is shown in Table 4. The reverse transcription program settings are shown in Table 5. After the reaction, store the samples at -20℃ for later use.

[0106] Table 4. Preparation of reverse transcription reaction system Table 5 Reverse transcription reaction program settings (4) Real-time quantitative PCR (RT-qPCR) The mouse-derived primer sequences were designed and synthesized by Shanghai Sangon Biotech. The primer sequence list is detailed in Table 6.

[0107] Table 6 Primer sequence listing (5) Preparation of RT-qPCR reaction system According to the kit instructions, the system preparation is shown in Table 2. Ensure the process is performed under nuclease-free conditions, adding each reagent in the order listed in the table, mixing, and then placing the mixture into the RT-qPCR instrument.

[0108] (6) Perform amplification according to the procedure in the instruction manual (see Table 3 for details).

[0109] (7) Results Analysis By homogenizing the internal reference gene Gapdh, and using 2 -△△Ct The results obtained from this experiment were statistically analyzed.

[0110] 4.5 Protein extraction, quantification, and Western Blot analysis of corneal tissue (1) Protein extraction and concentration determination Take approximately 100 mg of fresh or -80°C frozen corneal tissue, gently rinse with pre-chilled PBS to remove blood and impurities, blot dry the surface liquid, and cut into small pieces; add 1 mL of pre-chilled RIPA lysis buffer and 10 μL PMSF (100 mM), and homogenize thoroughly on ice using a tissue homogenizer; after homogenization, briefly shake (approximately 15 s) and incubate on ice for 15 min to promote protein release, repeat 3 times; centrifuge at 12,000 rpm for 10 min at 4°C, and collect the clear supernatant as the total protein extract; protein concentration is determined using the BCA method: mix an appropriate amount of sample with BCA working solution in a 96-well plate, incubate at 37°C for 30 min, measure the absorbance at 562 nm, and calculate the concentration according to the standard curve. Add 5×SDS loading buffer to the protein sample at a 4:1 ratio, heat at 95°C for 10 min for denaturation, and store at -20°C for later use.

[0111] (2) SDS-PAGE electrophoretic separation Select a separating gel of appropriate concentration based on the molecular weight of the target protein, and add equal amounts of protein and pre-stained molecular weight to the sample wells. Initially, pass the stacking gel at 80 V. After the bromophenol blue enters the separating gel, adjust the voltage to 120-150 V until electrophoresis is complete.

[0112] (3) Protein transfer After electrophoresis, the gel and PVDF membrane were assembled into a wet transfer system and placed in an ice bath to prevent the temperature from rising. The transfer conditions were set according to the protein molecular weight to complete the protein transfer.

[0113] (4) Blocking and antibody incubation After transfer, the PVDF membrane was placed in 5% skim milk blocking solution and incubated at room temperature for 1 h to reduce nonspecific binding; then, primary antibody working solution was added and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST buffer for 10 min each time, and then HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. The membrane was washed 3 more times with TBST buffer.

[0114] (5) Color development and imaging The working solution was prepared according to the ECL chemiluminescence reagent instructions, and the solution was uniformly added to the membrane surface for color development. Images were then captured and saved using a gel imaging system for subsequent analysis.

[0115] 4.6 Histopathological examination (1) Tissue sampling On day 10 after modeling, the mice were general anesthetized and euthanized by cervical dislocation. The optic nerve was cut along the optic nerve, and the intact eyeball was removed. The periorbital tissue was separated, the lacrimal gland was removed, and the tissue was immediately placed in pre-cooled PBS for temporary storage.

[0116] (2) Preparation of paraffin sections and HE staining The samples were fixed in 4% paraformaldehyde for 24 h; then subjected to gradient dehydration: ① treated sequentially in 70%, 80%, 90%, and 95% ethanol for 30 min each, followed by 100% ethanol I and II for 20 min each; ② after dehydration, cleared twice in xylene for 20 min each time; ③ soaked sequentially in paraffin I, II, and III for 45 min each; ④ after paraffin embedding, the tissue was embedded and prepared into continuous sections with a thickness of 5 μm using a paraffin microtome. HE staining procedure: ① sections were dewaxed in xylene, hydrated with gradient ethanol, and rinsed with running water; ② hematoxylin stained for 5 min, rinsed with tap water, differentiated with hydrochloric acid ethanol, and rinsed again; ③ placed in a weakly alkaline solution for blue reversion for 30 s, and rinsed with running water for 10 min; ④ then eosin stained for 5 min, dehydrated with gradient ethanol, cleared in xylene, mounted with neutral resin, observed and imaged under an optical microscope.

[0117] (3) Immunofluorescence staining The immunofluorescence staining procedure was performed as follows: ① Cut the prepared paraffin block into thin slices, attach them to a special anti-detachment glass slide, and bake them at 60℃ for 2 hours. After baking, allow the slides to fully return to room temperature before proceeding with subsequent operations; ② After baking, dewax the slides twice with xylene, each time for no more than 15 minutes; ③ Perform gradient rehydration, immersing the slides in anhydrous ethanol twice for 5 minutes each, followed by treatment with 95%, 90%, 80%, and 70% ethanol solutions for 5 minutes each. After rehydration, rinse the slides twice with distilled water; ④ Select citrate antigen retrieval solution at pH 6.0 and perform antigen retrieval on the slides using a water bath for 15 minutes. After retrieval, allow the slides to cool to room temperature, then wash them three times with PBS buffer for 5 minutes each; ⑤ Add 5% goat serum blocking solution to the slides and block them at room temperature for 1 hour. h. After blocking, discard excess blocking solution. No additional washing of the sections is required; proceed directly to antibody incubation. ⑥ Dilute the antibody 1:500 and add the diluted rabbit anti-Tuj1 antibody to the corneal sections and rabbit anti-AQP5 antibody to the lacrimal gland sections. Then place the sections in a humidified chamber and incubate overnight at 4°C. ⑦ The next day, remove the sections, discard the surface primary antibody solution, and wash the sections three times with PBST buffer containing 0.05% Tween-20 for 5 min each time to remove unbound free antibodies. Dilute the secondary antibody 1:500 and add Alexa Fluor® 488-labeled secondary antibody to the corneal sections and Alexa Fluor® 594-labeled secondary antibody to the lacrimal gland tissue sections. Incubate at room temperature in the dark for 2 h. ⑧ After incubation, wash the sections three more times with 0.05% Tween-20 PBST buffer for 5 min each time. After 5 min, rinse gently with distilled water to remove residual buffer solution; add DAPI staining solution to the slide and stain in the dark for 5 min, then rinse three times with PBS buffer for 5 min each time. After rinsing, add anti-fluorescence quencher and mount the slide. Place the prepared slide under a fluorescence microscope to observe the tissue morphology and collect the corresponding experimental images.

[0118] 4.7 Statistical Analysis For specific steps of statistical analysis, please refer to Example 3.

[0119] 4.8 Results (1) Evaluation of the efficacy of Lactobacillus plantarum NCU-40 in a corneal nerve transection dry eye model Based on the preliminary screening results of functional strains, this invention further conducts a systematic evaluation of the in vivo therapeutic effects of *Lactobacillus plantarum* NCU-40. Detailed experimental results are available in [link to relevant documentation]. Figure 12The results showed that, compared with group M mice, the dry eye-related phenotypes in mice were significantly improved after intervention with *Lactobacillus plantarum* NCU-40. Specific results for each experiment are as follows: the corneal fluorescein staining score of mice decreased, with group M at 9.67 ± 0.58 and group ML at 4.33 ± 1.16, and the difference was statistically significant (p<0.0001). Figure 12 The TBUT was significantly prolonged, increasing from 2.50 ± 0.10 s to 5.43 ± 0.15 s, with significant differences between groups (p < 0.0001). See the relevant results below. Figure 12 The tear secretion of mice in the D group also increased significantly, from 2.33 ± 0.29 mm to 5.50 ± 0.50 mm, p < 0.0001. Figure 12 (E in the text).

[0120] The results show that topical application of Lactobacillus plantarum NCU-40 can effectively reduce corneal fluorescein staining scores and prolong tear film breakup time in mice, while also increasing tear secretion. This confirms that the strain has good in vivo therapeutic effects in a corneal nerve transection dry eye model.

[0121] (2) Effects of Lactobacillus plantarum NCU-40 on the abundance of Lactobacillus plantarum and expression of inflammatory factors on the ocular surface of mice To further explore the underlying mechanism by which *Lactobacillus plantarum* NCU-40 exerts its therapeutic effect in vivo, this invention detected the abundance of *Lactobacillus* spp. on the ocular surface of mice and the mRNA expression levels of inflammatory factors. qPCR analysis revealed a significant decrease in the abundance of *Lactobacillus* spp. on the ocular surface of mice in group M, which recovered after local intervention with *Lactobacillus plantarum* NCU-40. Figure 13 (A) Meanwhile, the mRNA expression levels of II1b and Tnf in the corneal tissue of mice in group M showed a significant upregulation trend. Compared with group M, the expression levels of the above two inflammatory factors in the treatment group mice were significantly decreased, and the differences between the groups were statistically significant (p<0.05). Figure 13 (BC in the middle).

[0122] The results show that Lactobacillus plantarum NCU-40 can restore the level of Lactobacillus in the dry eye model and reduce the expression of IL-1β and TNF-α in corneal tissue, consistent with previous results showing improvement in functional indicators.

[0123] (3) Regulation of corneal barrier function and nerve-related proteins by Lactobacillus plantarum NCU-40 in mice To further determine the regulatory effects of *Lactobacillus plantarum* NCU-40 on corneal barrier function and neurotransmitter expression in mice, this invention used Western blot to detect the expression levels of tight junction proteins and neurotransmitters in the corneal tissue of each group of mice. Figure 14 (A) The results showed that, compared with group C, the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the corneal tissue of mice in group M were significantly downregulated, and the differences between groups were significant (p<0.05). After local intervention with Lactobacillus plantarum NCU-40, the expression levels of tight junction proteins were significantly restored, and the differences compared with group M were also significant (p<0.05). For details of the corresponding protein expression results, please refer to [link to relevant documentation]. Figure 14 In addition, the expression levels of brain-derived neurotrophic factor (BDNF) and the neuronal-specific class III β-tubulin (Tuj1), a neurostructural marker, were also decreased in the corneal tissue of mice in group M (p<0.05), while in group ML, the expression levels of these two neurotransmitter-related proteins were effectively restored, and the differences between the groups were statistically significant (p<0.05). The results are as follows... Figure 14 As shown in EF.

[0124] The combined results suggest that, compared with the M group, intervention with Lactobacillus plantarum NCU-40 can increase the expression levels of corneal tight junction protein and nerve-related proteins.

[0125] (4) Effects of Lactobacillus plantarum NCU-40 on pathological changes in mouse corneal tissue and expression of lacrimal gland functional proteins To further evaluate the effects of *Lactobacillus plantarum* NCU-40 on corneal tissue structure, corneal Tuj1 expression, and lacrimal gland functional protein expression in different groups of mice, this invention used corneal HE staining, corneal Tuj1 immunofluorescence staining, and lacrimal gland aquaporin 5 (AQP5) immunofluorescence analysis. HE staining results showed that in group C, the corneal epithelial structure was intact, cells were regularly arranged, and the boundaries between layers were clear; in group M, the corneal epithelial layer was thinner, cells were disordered, collagen fibers in the stromal layer were loose, gaps were widened, some stromal cells were smaller and fewer in number, and mild edema was present. After intervention with *Lactobacillus plantarum* NCU-40, the corneal epithelial cell arrangement was more orderly than in the model group, the collagen fiber distribution in the stromal layer was denser, the degree of edema was reduced, and the morphology and distribution of stromal cells were somewhat restored. Figure 15 (A in the middle).

[0126] Tuj1 immunofluorescence staining showed that the corneal nerve fiber density was decreased and the fluorescence signal was weakened in group M; while the corneal nerve fiber distribution was increased and the fluorescence intensity was enhanced in group ML. Figure 15 (B in the text). Immunofluorescence assay of AQP5 in the lacrimal gland showed that AQP5 expression decreased in group M, while AQP5 fluorescence signal increased in group ML, showing a recovery trend compared to group M. Figure 15 (C in the middle).

[0127] This shows that, compared with the M group, intervention with Lactobacillus plantarum NCU-40 can improve corneal tissue structure, increase the expression of the corneal neural structural marker Tuj1 protein, and increase the level of lacrimal gland AQP5.

[0128] Example 5: Screening of key substances for the protection of HCECs from hyperosmolar stress by Lactobacillus plantarum NCU-40 5.1 Introduction Based on Examples 1-4 above, this example uses *Lactobacillus plantarum* NCU-40 to prepare corresponding inactivated bacterial cells and cell-free supernatant. Simultaneously, an in vitro dry eye model of human corneal epithelial cells induced by hyperosmolar stress is constructed to systematically investigate the regulatory effects of two different components on corneal epithelial cell damage. This example mainly explores the ocular surface protective potential of this strain from two dimensions: bacterial cell structural components and secreted metabolites, hoping to provide a reasonable experimental approach for research on probiotic intervention in ocular surface diseases. This experiment analyzes the characteristics of different components of probiotics at the molecular and cellular levels, further clarifying the functional basis of their ocular surface protective effects. It also provides theoretical support and experimental methods for subsequent screening of active metabolites and development of novel dry eye interventions, possessing certain academic research value and clinical application potential.

[0129] 5.2 Experimental Materials and Methods 5.2.1 Experimental Materials Main reagents and consumables The main reagents and consumables used in this invention are listed in Table 7, and the other reagents and consumables are as described in the foregoing embodiments.

[0130] Table 7 Main Reagents and Consumables 5.2.2 Experimental Methods 5.2.2.1 Cell Culture This invention uses human corneal epithelial cells (HCECs) for in vitro experiments. These cells were purchased from Beina Biotechnology Co., Ltd., and the cell number is BNCC337876. Cell culture was performed in MEM medium supplemented with a mixture of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator using standard procedures. When the cells reached 80%–90% confluence, they were passaged according to standard protocols. All subsequent experiments were conducted using cells in the logarithmic growth phase.

[0131] 5.2.2.2 Construction of a hyperosmolar dry eye cell model To simulate the hyperosmolar environment of dry eye, the osmotic pressure of the culture medium was adjusted to 500 mOsm / L (NaCl adjustment) and continuously treated for 24 h to establish a model group (M group), while normal culture medium served as the control group (C group). Appropriate interventions were then implemented.

[0132] 5.2.2.3 Preparation of inactivated Lactobacillus plantarum NCU-40 cells and cell-free supernatant After NCU-40 cells were cultured in MRS liquid medium to the logarithmic growth phase, the cells were collected by centrifugation and washed twice with PBS. The cells were then inactivated by incubating in a 95°C water bath for 15 min. After centrifugation and washing, the cells were resuspended in sterile PBS for later use. The supernatant was collected after centrifugation of the cultured bacterial solution, filtered through a 0.22 μm filter to obtain a cell-free supernatant, and stored at 4°C for later use.

[0133] 5.2.2.4 Optimal intervention concentration determined by CCK-8 assay This experiment used the CCK-8 assay to screen the optimal intervention concentrations of inactivated *Lactobacillus plantarum* NCU-40 cells and cell-free supernatant. HCECs were first seeded in 96-well plates. After full cell adhesion, they were subjected to hypertonic stress treatment. Then, gradient concentrations of inactivated cells (1:1000, 1:100, 1:10, 1:1, 10:1) and different volume fractions of cell-free supernatant (10%, 20%, 30%, 40%, 50%, 60%) were added, and the cells were continuously treated for 24 h. After the intervention, 10 μL of CCK-8 assay reagent was added to each well, and the cells were incubated at 37℃ for 1-2 h. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm, and cell viability was calculated. Subsequent experiments will select the intervention concentration that best improves cell viability without cytotoxicity. Based on the concentration screening results, the samples were grouped as follows: normal culture medium group (C group), hypertonic culture medium group (M group), hypertonic culture medium combined with optimal concentration of inactivated bacteria group (MLS group), and hypertonic culture medium combined with optimal concentration of supernatant group (MHL group).

[0134] 5.2.2.5 Calcein AM / PI double staining for live and dead cells The experimental procedure is as follows: (1) Cell digestion, seeding, plating and grouping are consistent with the CCK-8 method experimental procedure described above; (2) Remove the original culture medium of each group of cells, wash the cells with PBS buffer 2-3 times to thoroughly remove residual esterase in the wells and avoid interfering with subsequent staining results; (3) Add an appropriate amount of serum-free culture medium to each well, then add the prepared Calcein-AM / PI working solution, 100 µL per well, gently shake the culture plate to mix the liquid, and then place it in a cell culture incubator to incubate in the dark for 30 min; (4) After incubation, observe the cell status of each group with a fluorescence microscope and take pictures, record the number of live cells and dead cells in each group and perform analysis.

[0135] 5.2.2.6 Cell Scratch Assay This invention investigates the effects of different components of *Lactobacillus plantarum* on the migration ability of HCECs after hyperosmolar stress treatment through a cell scratch assay. The procedure is as follows: (1) HCECs were seeded into 6-well cell culture plates at a density of 100,000 cells per well and cultured until the cells reached 100% confluence. Then, a 200 μL sterile pipette tip was used to make a straight scratch evenly in the center of each well. The cell layer was then gently rinsed with PBS buffer to remove free cells detached from the scratches. (2) Hyperosmolar medium containing different components of *Lactobacillus plantarum* NCU-40 was added to each well to treat the cells in groups. (3) After scratch modeling, the cell status was observed and images were collected at three time points: 0 h, 24 h, and 48 h. Subsequently, ImageJ software was used to mark the scratch boundaries and calculate the scratch area. The cell migration rate was calculated uniformly according to the following formula: Migration rate = (0 h scratch area – scratch area at each corresponding time point) / 0 h scratch area × 100%.

[0136] 5.2.2.7 EdU cell proliferation experiment The proliferation capacity of HCECs in each group was evaluated using the EdU cell proliferation assay kit. The experimental steps were as follows: (1) Cells were seeded in 96-well plates and cultured at 37°C in a 5% CO2 incubator until adherence was achieved. After the confluence reached 70%–80%, hypertonic treatment and corresponding interventions were performed. (2) Two hours before the end of the treatment, EdU working solution with a final concentration of 10 μM was added to each well and incubated for another 2 hours to allow the cells undergoing DNA synthesis to incorporate EdU. (3) The culture medium was discarded, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde for 15 min. The cells were then washed three more times with PBS. (4) 0.5% Triton X-100 was added for permeation for 10 min and then washed again. (5) Click reaction solution was prepared according to the kit instructions and added to each well (200 µL / well) and incubated in the dark for 30 min to allow EdU to react specifically with the fluorescent label. (6) After the reaction, wash the cells three times with PBS and then add DAPI staining solution and incubate at room temperature in the dark for 15 min. (7) Observe and take pictures using a fluorescence microscope. EdU positive cells show red fluorescence, and DAPI-labeled cell nuclei show blue fluorescence.

[0137] 5.2.2.8 ELISA detection of inflammatory factors in cell supernatant Collect the supernatant from each group of cell cultures, centrifuge at 4℃ and 3000 r / min for 10 min, and immediately transfer the supernatant to -80℃ for cryopreservation to avoid repeated freeze-thaw cycles affecting the test results. This invention uses a double-antibody sandwich ELISA kit to detect the content of IL-1β and IL-6 in the supernatant. All operation steps are strictly performed in accordance with the kit instructions. The simplified process is as follows: (1) The kit standard is serially diluted according to the instructions for subsequent standard curve plotting; (2) The diluted standard and the cell supernatant sample to be tested are added to the wells of the enzyme-labeled plate coated with specific monoclonal antibody. Multiple replicates are set for each sample to ensure accurate detection results; (3) Biotin-labeled secondary antibody working solution is added to each well. After incubation at room temperature, the plate is washed multiple times to remove unbound excess components; (4) HRP-labeled avidin is added, and incubation is completed in the dark. The plate is then washed again; (5) The chromogenic substrate is added, and the sample is allowed to stand at room temperature until it is fully chromogenic. After chromogenic development, the stop solution is added to terminate the reaction. Finally, the OD value of each well was detected at a wavelength of 450 nm using an ELISA reader. A standard curve was plotted with the standard concentration as the x-axis and the corresponding OD value as the y-axis, and regression fitting was performed. Then, the final concentration of cytokines (pg / mL) was calculated based on the OD value of the sample to be tested.

[0138] 5.2.2.9 Non-targeted metabolomics analysis Non-targeted metabolomics analysis was commissioned to Shanghai Paisennong Biotechnology Co., Ltd. *Lactobacillus plantarum* NCU-40 was inoculated into MRS liquid medium. After reaching the plateau phase, the supernatant was collected by centrifugation. A blank control was also set up, with sterile MRS liquid medium centrifuged under the same conditions. All samples were uniformly processed and metabolite analysis was performed using liquid chromatography-mass spectrometry (LC-MS / MS). LC-MS was performed using a Thermo Vanquish Flex liquid chromatograph coupled with a Thermo Orbitrap Exploris 120 mass spectrometer. Data acquisition, metabolite identification, differential metabolite screening, and annotation were all performed according to the company's standard experimental procedures and quality control requirements to ensure stable and reliable final results.

[0139] 5.2.2.10 ELISA detection of GABA content in conjunctiva, corneal tissue, and tear film. The conjunctiva and corneal tissues of mice obtained in the previous animal experiment were separated, ground thoroughly to prepare tissue homogenate, and the supernatant was collected after centrifugation for later use. The tear samples were also from the previous animal experiment. After collection, they were frozen at -80℃. When ready for testing, they were taken out, thawed, centrifuged to remove impurities, and then tested. The experimental procedure is as follows: (1) Add 50 μL of standard or test sample to each reaction well, then add 50 μL of biotinylated detection antibody working solution, incubate at 37℃ for 30 min, and wash the plate 3 times; (2) Add 100 μL of HRP conjugate working solution, continue to incubate at 37℃ for 30 min, and wash the plate 3 times again; (3) Add 100 μL of substrate solution, incubate at 37℃ in the dark for 15 min; (4) Finally, add 50 μL of stop solution, and immediately use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm. Using the concentration of the standard as the x-axis and the corrected OD value as the y-axis, a standard curve was plotted using four-parameter logical fitting (4PL). Based on this, the GABA concentration in each sample was calculated, with tissue GABA content expressed in pg / mg and tear GABA content expressed in pg / mL.

[0140] 5.2.2.11 Western blot detection of tight junction protein expression For specific procedures on protein extraction, concentration determination, and Western blotting of human corneal epithelial cells, please refer to Example 4.

[0141] 5.2.3 Statistical Analysis For specific steps of statistical analysis, please refer to Example 3.

[0142] 5.3 Results 5.3.1 Screening of optimal concentrations of cell-free supernatant and inactivated bacterial cells of Lactobacillus plantarum NCU-40 and evaluation of cell viability This invention constructs an in vitro dry eye model by inducing HCEC damage through hyperosmolarity, thereby investigating the effects of different components of *Lactobacillus plantarum* NCU-40 on cells under hyperosmolar stress. Experimental grouping details are provided below. Figure 16 The results of A. CCK-8 showed that cell viability decreased significantly after hyperosmolar stimulation. In the bacterial component concentration gradient screening experiment, intervention with 50% supernatant (MLS) resulted in the most significant increase in cell viability; while inactivated bacteria (MHL) at a concentration of 1:100 showed a relatively significant increase in cell viability. Figure 16 (B–C in the group). Therefore, the optimal concentration was selected for a cross-sectional comparison. The results showed that MLS significantly improved cell viability under hyperosmolar stress compared to the MHL group (B–C). Figure 16 (D in the middle).

[0143] To determine the degree of cell damage, Calcein AM / PI double staining was used to stain for live and dead cells in each group. After hypertonic treatment, the proportion of dead cells (PI-positive) increased to 16.62%, indicating that the hypertonic environment severely damaged the cell membrane integrity, leading to increased cell death. After MLS intervention, the proportion of PI-positive cells decreased to 1.83%, indicating that MLS can block cell death induced by hypertonic stimulation. However, the proportion of PI-positive cells in the MHL group was 14.07%, and the cell death rate was not significantly improved. Figure 16 (E–F in ​​the text). The findings suggest that the protective effect of *Lactobacillus plantarum* NCU-40 against HCECs under hyperosmolar conditions is primarily due to the cell-free supernatant of the strain; inactivated bacterial cells did not play a core protective role.

[0144] 5.3.2 Effects of different components of Lactobacillus plantarum NCU-40 on cell migration, proliferation, and inflammatory response Scratch assay results showed that, compared with group C, hyperosmolar stimulation significantly inhibited the migration ability of HCECs (67.19% vs 30.25%, p<0.0001). MLS treatment promoted cell migration, restoring the migration rate to 64.89% (p<0.0001), close to the normal level, while the improvement effect in the MHL group did not reach statistical significance. Figure 17 (A–B in the diagram) suggests that the supernatant plays an important role in improving cell migration under hypertonic conditions. EdU proliferation assay results showed that after 48 h of hypertonic stimulation, the cell proliferation rate decreased to 18.08%, while MLS intervention significantly increased the EdU-positive rate to 44.06%, compared to 21.43% in the MHL group. Figure 17 The C–D results in the study consistently support the view that MLS can promote epithelial repair.

[0145] ELISA results showed that hyperosmolar stimulation significantly induced increases in IL-1β and IL-6. After MLS treatment, IL-1β decreased from 232.7 ± 6.01 pg / mL to 165.1 ± 3.32 pg / mL (p<0.0001), and IL-6 decreased from 287.7 ± 12.4 pg / mL to 101.5 ± 11.32 pg / mL (p = 0.0001), while no significant changes were observed in the MHL group. Figure 17 The E–F values ​​suggest that MLS can alleviate hyperosmolar induced inflammatory responses.

[0146] 5.3.3 Effects of different components of Lactobacillus plantarum NCU-40 on the expression of tight junction proteins in HCECs Western blot analysis revealed that hyperosmolar stimulation significantly downregulated the expression of ZO-1, Occludin, and Claudin-1, suggesting that hyperosmolar stimulation leads to damage to the corneal epithelial barrier structure. After MLS intervention, the expression levels of these three tight junction proteins were significantly upregulated, while no significant improvement was observed in the MHL group. Figure 18 (A–D in the original text).

[0147] In summary, the protective effect of Lactobacillus plantarum NCU-40 against hyperosmolar-induced corneal epithelial damage mainly depends on its secreted soluble active substances, while the contribution of bacterial cell structural components is limited.

[0148] 5.3.4 Non-targeted metabolomics analysis of cell-free supernatant from Lactobacillus plantarum NCU-40 Based on the significant protective effects of NCU-40 cell-free supernatant in multiple in vitro functional experiments, this invention employs non-targeted metabolomics technology to analyze its chemical components in order to screen for metabolites that play a key role.

[0149] Metabolic profile comparisons showed significant metabolic differences between the supernatant of *Lactobacillus plantarum* NCU-40 and the control culture medium. Statistical analysis revealed that organic acids and their derivatives, heterocyclic compounds, benzenes, lipids and lipid molecules, organic oxygen-containing compounds, phenylpropanoids and polyketides, nitrogen-containing organic compounds, and alkaloids and their derivatives constituted a higher proportion of the differentially metabolites; the remaining substances accounted for less than 2%. Figure 19 (A in the middle).

[0150] The volcano plot results showed that a total of 419 metabolites were upregulated and 119 metabolites were downregulated, indicating significant metabolic remodeling in the supernatant of *Lactobacillus plantarum* NCU-40. Figure 19 (B in the original text). Meanwhile, PCA analysis showed that the two groups of samples were clearly separated in the metabolic space, and the clustering degree within each group was high, indicating that this experiment had good reproducibility. Figure 19 (C in the text). This metabolomics study detected a total of 19,693 metabolic characteristics, of which 15,414 did not meet the screening criteria for differential metabolites. Figure 19 (D in the middle).

[0151] Among all differentially expressed metabolites, γ-aminobutyric acid (GABA) was identified as a key metabolite with significant upregulation. Heatmap clustering analysis revealed that GABA was stably highly expressed in the supernatant of *Lactobacillus plantarum* NCU-40 and formed a clear cluster with the control group samples. Figure 19 (E in the text); Quantitative analysis of the bar chart further confirmed that the relative abundance of GABA was much higher than that of the control medium, and the difference was statistically significant. Figure 19 The ROC curve analysis of GABA showed that its sensitivity and specificity were both 1 (G); Figure 19 (F in the middle).

[0152] Based on the results of volcano map screening, heat map cluster analysis, and bar chart quantitative analysis, GABA showed a stable upward trend in multidimensional statistical analysis, suggesting that it may be an important biologically significant active ingredient in the cell-free supernatant of Lactobacillus plantarum NCU-40.

[0153] 5.3.5 Regulatory effect of Lactobacillus plantarum NCU-40 on GABA in ocular surface tissue and tear film of dry-eye mice To further verify the changes in the content of the key metabolite GABA in vivo, this invention detected the GABA content in conjunctival tissue, corneal tissue, and tear fluid in the first round of animal experimental samples. The results showed that after intervention with *Lactobacillus plantarum* NCU-40, GABA levels showed a significant increasing trend in different sites: the GABA content in conjunctival tissue increased from 38.20 ± 11.77 pg / mg in the model group to 394.3 ± 36.04 pg / mg; the GABA level in corneal tissue increased from 33.69 ± 2.34 pg / mg to 132.5 ± 17.95 pg / mg; and the GABA content in tear fluid also increased from 19.17 ± 5.91 pg / mL to 83.15 ± 8.86 pg / mL. All of these results were statistically significant. Figure 20 (A–C in the original text).

[0154] The above results are consistent with the results of in vitro metabolomics analysis, further suggesting that GABA may be the key active molecule that plays a protective role in the supernatant of Lactobacillus plantarum NCU-40, and also lay a solid foundation of in vivo experimental evidence for further in-depth functional verification and molecular mechanism research.

[0155] discuss Corneal nerve damage is also one of the core mechanisms inducing neurogenic dry eye. Tuj1, a commonly used neurofibrillary marker, directly reflects the distribution density of corneal nerves, while BDNF mainly participates in the regulation of nerve cell survival and regeneration, and is crucial for corneal nerve repair. NCU-40 can effectively upregulate the expression levels of Tuj1 and BDNF in corneal tissue, and immunofluorescence detection can also visually show an increase in corneal nerve fiber density. This result is corroborated by experimental data showing prolonged tear film breakup time and improved tear secretion. Simultaneously, this result also demonstrates that neurotrophic factors can improve tear secretion function in dry eye patients and alleviate dry eye symptoms by promoting corneal nerve regeneration.

[0156] Downregulation of AQP5 expression in the lacrimal gland directly hinders normal tear production, thereby exacerbating dry eye symptoms. NCU-40 can effectively restore the expression level of AQP5 in the lacrimal gland. Therefore, this invention hypothesizes that NCU-40 may intervene in the pathological process of dry eye from multiple dimensions by regulating the "corneal nerve-lacrimal gland functional axis".

[0157] In summary, this invention demonstrates the protective effect of NCU-40 on dry eye mice from multiple perspectives, including ocular surface microecological regulation, inflammatory response suppression, corneal barrier repair, and neuro-lacrimal axis regulation. The conclusions of this invention are consistent with existing related studies, providing solid and reliable experimental support for subsequent research on the local intervention of probiotics in dry eye.

[0158] This invention constructs an in vitro dry eye model by treating HCECs with a hypertonic culture medium, simulating typical pathological features such as decreased cell viability, suppressed migration and proliferation, excessive secretion of inflammatory factors, and decreased expression of tight junction proteins. This provides a stable and reliable dry eye model for screening key components of *Lactobacillus plantarum* NCU-40. GABA, as an inhibitory neurotransmitter, can also play a role in regulating inflammatory responses and alleviating oxidative stress in non-neuroepithelial tissues, consistent with the protective effects exhibited by the supernatant in in vivo and in vitro experiments.

[0159] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum NCU-40, characterized in that, The *Lactobacillus plantarum* NCU-40 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34104. The *Lactobacillus plantarum* NCU-40 was isolated from the microenvironment of the conjunctival sac of a healthy human eye.

2. The *Lactobacillus plantarum* NCU-40 according to claim 1, characterized in that, The 16S rDNA sequence of *Lactobacillus plantarum* NCU-40 is shown in SEQ ID NO:

1.

3. An ophthalmic composition for improving dry eye syndrome, characterized in that, It contains live bacteria of *Lactobacillus plantarum* NCU-40 as described in claim 1, and a pharmaceutically acceptable ophthalmic carrier.

4. The ophthalmic composition according to claim 3, characterized in that, The pharmaceutically acceptable ophthalmic carrier is selected from one or more of sodium hyaluronate, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, sodium chloride, and phosphate buffer.

5. The ophthalmic composition according to claim 3, characterized in that, The composition is in the form of eye drops, ophthalmic gel, ophthalmic cream, ophthalmic spray, or ophthalmic implant.

6. Use of the Lactobacillus plantarum NCU-40 of claim 1 in the preparation of a medicament for treating dry eye syndrome.

7. The use according to claim 6, characterized in that, The drug is a topical eye drop preparation.

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