Lactiplantybacillus plantarum subspecies plantarum strain, bacterial membrane vesicles of Lactiplantybacillus plantarum subspecies plantarum strain, and their use in the improvement of eye diseases.

Abstract

To provide pharmaceutical compositions for use to ameliorate eye diseases.SOLUTION: Provided herein are a strain of Lactiplantibacillus plantarum ssp. plantarum, membrane vesicles thereof, and uses thereof for ameliorating eye diseases. The Lactiplantibacillus plantarum ssp. plantarum EP21 and bacterial membrane vesicles are derived from the Lactiplantibacillus plantarum ssp. plantarum EP21. Both the Lactiplantibacillus plantarum ssp. plantarum strain EP21 and bacterial membrane vesicles derived therefrom can suppress inflammation by inhibiting inflammation factor thereby can ameliorate eye diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the Lactiplantybacillus plantarum subspecies plantarum strain, bacterial membrane vesicles (MVs) derived from the Lactiplantybacillus plantarum subspecies plantarum strain, and more particularly to compositions containing Lactiplantybacillus plantarum subspecies plantarum strain EP21, compositions containing bacterial MVs derived from Lactiplantybacillus plantarum subspecies plantarum strain EP21, and their use in improving eye diseases. [Background technology]

[0002] Myopia is increasing worldwide, and is particularly prevalent in East Asia. Recent publications estimate that by 2050, nearly half of the world's population (49.8% (95% CI 43.4-55.7)) will be myopic (≤-0.5D), and 9.8% (95% CI 5.7-19.4) will be highly myopic (≤-5D). High myopia is a significant risk factor for blindness, as it increases the risk of retinal detachment, macular and choroidal degeneration, premature cataracts, and glaucoma. The probability of neovascularization in highly myopic individuals is nine times higher than in healthy individuals. In Taiwan, the prevalence of myopia is 9.4% in 6-year-olds and reaches 75% in 15-year-olds. The prevalence rises to 80-90% in 18-year-olds, with 10-20% of people in this age group having high myopia. Therefore, myopia prevention to prevent blindness is a globally important issue.

[0003] Axial myopia is the most common type of myopia. Excessive elongation of the vitreous ventricle leads to an increase in axial length more than the increase in corneal refractive power. Scleral tissue remodeling is also associated with axial lengthening and myopia. This is because connective tissue synthesis decreases and the breakdown of type I collagen is accelerated. As myopia progresses, the composition and ductility of the sclera change. However, the exact pathogenesis of myopia remains unclear. For example, atropine, dopamine, pirenzepine, and 7-methylxanthine can inhibit myopia progression, but the underlying molecular mechanisms are still unknown. Both environmental and genetic factors are involved in the development of myopia. Recent studies have shown that environmental factors have a significant impact on myopia progression. Many hypotheses predict that genetic factors influence the molecular mechanisms of myopia development, while environmental factors (e.g., time spent outdoors) do not directly affect myopia. Current research indicates that retinal signaling plays a crucial role in eyeball growth and axial adjustment. Signals that accelerate scleral tissue remodeling originate from the retina and affect photoreceptors and retinal pigment epithelium (RPE). Myopia features include a longer axial length (longer eyeball), a deeper vitreous cavity, a thinner lens, and a flatter cornea.

[0004] In addition to environmental and genetic factors, inflammation is also known to be associated with the development of myopia. The first paper mentioning myopia and inflammation was published in 2011. This paper examined the correlation between myopia and intraocular inflammation. Several reports suggest a link between myopia and allergic conjunctivitis. Conventional techniques have shown that atropine is widely used clinically to slow the progression of myopia by reducing the expression levels of c-Fos, NFκB, IL-6, and TNF-α, which are involved in chronic inflammation. This paper, cited by the American Optometric Society, shows that atropine may suppress myopia progression by inhibiting inflammation. In animal models of allergic conjunctivitis, allergic inflammation (particularly allergic conjunctivitis) accelerates the progression of myopia. Also, direct application of PM2.5 to the surface of Syrian hamster eyes accelerates the progression of myopia. Therefore, exposure to PM2.5 not only increases eye inflammation but also induces myopia. Several studies have shown that retinal dopamine is recognized as a signal that stops the normal growth of the eye and plays a role in the development of myopia. Acupuncture reduces inflammation by activating dopamine decarboxylase and increasing dopamine levels. Dopamine can further suppress inflammation by inhibiting the expression of TNF-α and IL-6. In other words, acupuncture suppresses the progression of myopia by suppressing inflammation caused by the activation of the dopamine-D1R signaling pathway. Currently, there are several anti-inflammatory drugs that suppress the progression of myopia, such as resveratrol, dicumarol, and herbal formulations containing two types of herbs. Other researchers have also reported that administering bovine lactoferrin to mice prevents the development of lens-induced myopia in mice via the IL-6-MMP-2 axis. Furthermore, clinical trials have shown that crocetin, a potent anti-inflammatory compound, has an effect of suppressing the progression of myopia in children when used in the treatment of myopia. In 2021, inflammation was identified as one of the risk factors for myopia by the IMI (International Myopia Institute). Conventional technologies have provided clinical and experimental data demonstrating a link between myopia and inflammation, but the cause of the inflammatory response remains unknown. No other disease or environmental factor causes such a high incidence of myopia (in some regions, the incidence of myopia in those under 18 is over 90%).

[0005] Various mediators have been reported to be involved in the development of myopia. In myopia, transforming growth factor beta (TGF-β) and matrix metalloproteinase-2 (MMP2) levels increase, while type I collagen decreases. The transforming growth factor beta (TGF-β) signaling pathway involves several different reactions related to inflammatory responses, histofibrosis, and remodeling. Therefore, TGF-β is closely related to the development of myopia. Three different isomers of TGF-β exist in mammals. In ocular tissue, the expression level of TGF-β2 is highest. The expression of TGF-β1, TGF-2, and TGF-3 are all positively correlated with the optical axis.

[0006] Furthermore, matrix metalloproteinases (MMPs) are responsible for the degradation of ECM (extracellular matrix) proteins such as collagen, gelatin, fibronectin, and aggrecan, and also act as mediators of angiogenesis during inflammation. Therefore, cooperation among MMP families is thought to be involved in the pathogenesis of myopia. In chicken sclera and tree shrew models, increased MMP2 expression has been observed in morphological deprivation myopia (FDM) results. In human samples, myopic eyes with high MMP2 expression have also been identified. In addition, studies have shown that TGF-β affects MMP2 expression through NFκB activation.

[0007] The complement system is also involved in the development and mechanism of myopia. In patients with pathological myopia (-8D to -25D), the expression levels of C3 (p=0.004) and CH50 (p<0.001) are dramatically increased. In the sclera of guinea pigs with myopia that has a negative lens focus, the expression levels of C1q, C3, and C5b-9 are significantly increased. CD55 inhibits the activity of C3 and C5 convertases by inhibiting convertase formation and promoting their degradation. CD55 prevents the activation of the complement system, and increased CD55 expression levels can suppress the progression of myopia.

[0008] Microorganisms in different organs / tissues are different and constantly changing. Current research suggests that the composition and balance of microorganisms play a crucial role in our health and disease. In an autoimmune uveitis model, Horai et al. (2015) found that gut microbiota induce autoreactive T helper 17 (Th17) cells, triggering retinal-associated uveitis. Antibiotic use can alleviate uveitis and reduce the number of autoreactive Th17 cells. Supplementation with broad-spectrum antibiotics can not only upregulate regulatory T cells but also suppress autoreactive effector T cells. It is also known that human leukocyte antigen B27 affects the immune system by increasing intestinal permeability and is associated with autoimmune uveitis and gut microbiota abnormalities. Furthermore, disruption of the gut microbiota is one of the factors involved in the progression of type 2 diabetes (DM), increasing the concentrations of lipids, fatty acids, and glucose in the plasma, and altering immune cells to secrete inflammatory molecules (TNFα, IL-1β, IL-6, IFN-γ, inflammatory adipokines, and chemokines). These factors also affect diabetic retinopathy (DR). Chronic low levels of inflammation associated with metabolic disorders and oxidative stress are also linked to changes in the composition of the gut microbiota. The gut microbiota is associated with age-related macular degeneration (AMD). Compared to healthy individuals, patients with advanced AMD also exhibit abnormalities in their gut microbiota.

[0009] The mammalian gut is home to numerous symbiotic bacteria (common microbiota) that interact with the host through secreted proteins, metabolites, and bacterial membrane vesicles (BMVs). BMVs are produced at various stages of bacterial growth. Therefore, BMVs are considered to be a long-range communication and signaling system, involved in interactions between host and bacteria, or between bacteria themselves. Membrane vesicles released from the outer membrane of Gram-positive bacteria are called outer membrane vesicles (OMVs), and Gram-positive bacteria release membrane vesicles (MVs). Studies have suggested that BMVs are associated with microbial pathogenicity and disease. In the presence of antibiotics, BMVs increase bacterial viability and transmit antibiotic resistance genes or antibiotic-degrading enzymes to other bacteria, causing disease. BMVs transmit microbial-associated molecular patterns (MAMPs) to host cells, which can trigger inflammatory or anti-inflammatory responses. Generally, BMVs from pathogenic bacteria have pro-inflammatory effects on host cells, while probiotic BMVs have immunomodulatory effects. Furthermore, BMVs can deliver DNA or RNA into cells and induce cytoprotective mechanisms through the regulation of pattern recognition receptors or host cell gene expression. OMVs have been detected in cerebrospinal fluid, indicating that OMVs can cross the blood-brain barrier. Metagenomic analysis of BMVs can be used to determine bacterial-host interactions. Metagenomic analysis of serum extracellular vesicles has revealed differences in the gut microbiota between psoriasis patients and asthma patients. [Overview of the project]

[0010] Therefore, the main objective of the present invention is to provide the Lactiplantybacillus plantarum subspecies plantarum strain EP21, which is used to improve eye diseases and is deposited with the Food Industry Development Institute (FIRDI) under deposit number BCRC911210.

[0011] Preferably, the Lactiplantybacillus plantarum subspecies plantarum strain EP21 includes the sequence segment shown in Sequence ID No. 3.

[0012] Another object of the present invention is to provide Lactiplantibacillus plantarum subsp. plantarum strain EP21 for use in the preparation of a pharmaceutical composition for ameliorating ocular diseases, and to provide a pharmaceutical composition comprising Lactiplantibacillus plantarum subsp. plantarum strain EP21.

[0013] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

[0014] Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and analogs and substitutes thereof. A person skilled in the art can select appropriate excipients and adjust the ratios according to professional or common techniques.

[0015] Preferably, the carrier suitable for the present invention includes, but is not limited to, water, physiological saline, phosphate buffered saline (PBS), aqueous solutions containing alcohol, and combinations thereof.

[0016] Preferably, the food additive is a substance added to edible materials to prepare products that can be consumed by animals (including humans). The use and ratio of the food additive are determined by a person skilled in the art according to professional or common techniques. The food additive includes natural and artificial sweeteners, colorants, salting and acidulants, flavors, emulsifiers, fat substitutes, hardeners, leavening agents, lubricants, humectants, preservatives, stabilizers, and thickeners.

[0017] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, solutions, suspensions, capsules, tablets, lozenges, troches, powders, lyophilized powders, lotions, emulsions, suppositories, slurries, and ointments.

[0018] More preferably, by techniques mastered by those skilled in the art, an isolated strain of Lactiplantibacillus plantarum subsp. plantarum or a subculture thereof is prepared together with a pharmaceutically acceptable vehicle to form a dosage form suitable for oral administration.

[0019] Preferably, the effective dose of the pharmaceutical composition is 1×10 6 cfu / kg to 1×10 10 cfu / kg administered to the subject.

[0020] More preferably, the effective dose of the pharmaceutical composition is 5×10 8 cfu / kg to 5×10 9 cfu / kg administered to the subject.

[0021] Preferably, the aforementioned eye diseases include, but are not limited to, myopia, dry eye syndrome (DES), asthenopia, keratitis, macular degeneration, retinitis pigmentosa (RP), proliferative diabetic retinopathy (PDR), ischemic retinopathy, choroidal neovascularization, glaucoma, systemic lupus erythematosus (SLE), and Sjögren's syndrome (SS).

[0022] More preferably, the aforementioned ischemic retinopathy includes diabetic retinopathy (DR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO), and ischemic optic neuropathy (ION).

[0023] Another object of the present invention is to provide a composition comprising bacterial membrane vesicles for ameliorating eye diseases. The aforementioned bacterial membrane vesicles (MV) are derived from the aforementioned Lactiplantibacillus plantarum subsp. plantarum strain EP21.

[0024] Preferably, the vesicle diameter of the aforementioned MV is 100 nm to 300 nm.

[0025] Preferably, the vesicle diameter of the aforementioned MV is 120 to 255 nm.

[0026] Another object of the present invention is to provide a pharmaceutical composition comprising the membrane vesicles (MVs) for improving eye diseases.

[0027] After administering the Lactiplantybacillus plantarum subspecies EP21 and the MV of the Lactiplantybacillus plantarum subspecies EP21 to a subject, eye diseases can be improved by suppressing excessive increases in axial length and changes in refraction. The expression of activated p-NFκB, NFκB, NLRP3, IL1β, TNFα, and IL6 is also inhibited, and the expression of the anti-inflammatory target IL10 increases.

[0028] Preferably, the pharmaceutical composition containing the membrane vesicles (MVs) is prepared in a dosage form suitable for application to a subject as an injection or eye drop. Types of injections include intravenous injection (IV), intraperitoneal injection (IP), and tail vein injection.

[0029] Preferably, the effective dose of the pharmaceutical composition containing the MV is 1 × 10 per day. 5 From particles / kg to 1 × 10 11 This involves administering particles / kg to the subject.

[0030] More preferably, the optimal effective dose of the pharmaceutical composition containing MV is 2.7 × 10⁻⁶ per day. 8 5.3 × 10⁻¹⁴ particles / kg 9 This involves administering particles / kg to the subject.

[0031] The following describes in detail the preparation method of compositions containing Lactiplantibacillus plantarum subspecies plantarum strain EP21 and membrane vesicles (MVs) derived from Lactiplantibacillus plantarum subspecies plantarum strain EP21, the testing of pharmaceutical compositions prepared using Lactiplantibacillus plantarum subspecies plantarum strain EP21 or its membrane vesicles (MVs) for improving eye diseases, and the testing and analysis of the inhibitory effects of pharmaceutical compositions prepared using Lactiplantibacillus plantarum subspecies plantarum strain EP21 or its membrane vesicles (MVs) on inflammatory factors. Therefore, it has been proven that compositions containing Lactiplantibacillus plantarum subspecies plantarum strain EP21 or its membrane vesicles (MVs) can indeed improve eye diseases. Furthermore, the membrane vesicles (MVs) and their pharmaceutical compositions can cross the blood-retinal barrier (BRB) via the bloodstream, enhance the immunomodulatory properties of cells, reduce TGF-β2-induced retinal inflammation, and thereby further slow the progression of myopia caused by TGF-β2. [Brief explanation of the drawing]

[0032] [Figure 1A] This is a bar graph of the axial length of rats according to one embodiment (Embodiment 1) of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), EP21 is the treatment group administered EP21 orally, T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21.

[0033] [Figure 1B] This is a bar graph showing the relative change in refraction (right eye / left eye) of rats according to Embodiment 1 of the present invention. Here, NC is the negative control group (injected with equilibrium salt solution and eye wash buffer), EP21 is the treatment group administered EP21 orally, T2 is the positive control group (injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21.

[0034] [Figure 2A]This is an immunofluorescence (IF) image of TGF-β according to Embodiment 1 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), EP21 is the treatment group administered EP21 orally, T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21 (scale bar: 165 μm).

[0035] [Figure 2B] This bar graph in Figure 2A shows the quantification of the IF intensity of the groups.

[0036] [Figure 3A] This is an immunofluorescence (IF) image of MMP2 according to Embodiment 1 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), EP21 is the treatment group administered EP21 orally, T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21 (scale bar: 165 μm).

[0037] [Figure 3B] This bar graph in Figure 3A shows the quantification of the IF intensity of the groups.

[0038] [Figure 4A] This is an immunofluorescence (IF) image of COL1A1 according to Embodiment 1 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eyewash buffer), EP21 is the treatment group administered EP21 orally, T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21 (scale bar: 165 μm).

[0039] [Figure 4B] This is a bar graph showing the quantification of the IF intensity of the groups in Figure 4A.

[0040] [Figure 5A] This is an immunofluorescence (IF) image of NFκB according to Embodiment 1 of the present invention. NC represents the negative control group (the group injected with equilibrium salt solution and eyewash buffer), EP21 represents the treatment group administered EP21 orally, T2 represents the positive control group (the group injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 represents the group induced with TGF-β2 and treated with EP21 (scale bar: 165 μm).

[0041] [Figure 5B] This bar graph in Figure 5A shows the quantification of the IF intensity of the groups.

[0042] [Figure 6A] This is an immunofluorescence (IF) image of TNF-α according to Embodiment 1 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), EP21 is the treatment group administered EP21 orally, T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS orally for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21 (scale bar: 165 μm).

[0043] [Figure 6B] This is a bar graph showing the quantification of the IF intensity of the groups in Figure 6A.

[0044] [Figure 7A] This is a Western blotting analysis of NFκB according to Embodiment 1 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), T2 is the positive control group (the group injected with TGF-β2 and orally administered 1X PBS for 3 weeks), and T2+EP21 is the group induced with TGF-β2 and treated with EP21.

[0045] [Figure 7B] This is a bar graph showing the quantitative results for the groups in Figure 7A.

[0046] [Figure 8] This is a bar graph showing the cell viability of another embodiment (Embodiment 2) of the present invention. Here, the vertical axis represents different numbers (8 × 10⁸, 4 × 10⁸, 2 × 10⁸, and 1 × 10⁸) of EP21 MV used to treat retinal pigment epithelial cells RPE1 according to the present invention.

[0047] [Figure 9A] This is a Western blotting analysis of RPE1 cells according to another embodiment (Embodiment 3) of the present invention. Here, NC is the control group, IL-1β is the group treated with 1.25 ng / ml of IL-1β, and IL-1β+MV is the group pretreated with 2.2 × 10⁶ EP21 MV cells and then treated with IL-1β.

[0048] [Figure 9B] This is a bar graph showing the quantitative results for the groups in Figure 9A.

[0049] [Figure 9C] This is a Western blotting analysis of ARPE19 cells according to Embodiment 3 of the present invention. Here, NC is the control group, IL-1β is the group treated with 1.25 ng / ml of IL-1β, and IL-1β+MV is the group pretreated with 2.2 × 10⁶ EP21 MV cells and then treated with IL-1β.

[0050] [Figure 9D] This is a bar graph showing the quantitative results for the groups in Figure 9C.

[0051] [Figure 9E] This is a Western blotting analysis of hRPEpiC cells according to Embodiment 3 of the present invention. Here, NC is the control group, IL-1β is the group treated with 1.25 ng / ml of IL-1β, and IL-1β+MV is the group pretreated with 2.2 × 10⁶ EP21 MV cells and then treated with IL-1β.

[0052] [Figure 9F]This is a bar graph showing the quantitative results for the groups in Figure 9E.

[0053] [Figure 10A] This is a Western blotting analysis of RPE1 cells according to another embodiment (Embodiment 5) of the present invention. Here, NC is the control group, IL-1β is the group treated with 1.25 ng / ml of IL-1β, IL-1β+MV is the group administered with IL-1β and MV, IL-1β+MV+DNase I is the group administered with IL-1β, MV and DNase I, and IL-1β+MV+RNase A is the group administered with IL-1β, MV and RNase A. MV represents 2.2 × 10⁶ EP21 membrane vesicles, and the final concentrations of DNase I and RNase A are both 100 ng.

[0054] [Figure 10B] Figure 10A is a bar graph showing the quantitative results for each group.

[0055] [Figure 11A] This is a bar graph of IL6 expression in another embodiment (Embodiment 5) of the present invention. Here, cells are administered TNFα (2.5 ng / ml) and MV of TNFα+ strain EP21, respectively.

[0056] [Figure 11B] This is a bar graph showing IL6 expression in Embodiment 5 of the present invention. Here, cells are administered IL1β (1.25 ng / ml) and IL1β+ MV of strain EP21, respectively.

[0057] [Figure 12]This is a bar graph showing the expression levels of IL6 determined by the enzyme-linked immunosorbent assay (ELISA) according to Embodiment 5 of the present invention. Here, NC is the control group, TNFα is the group administered 2.5 ng / ml of TNFα, TNFα+EP21 RNA is the group administered 1.5 μg of TNFα+EP21 RNA, and TNFα+EP21 MV RNA is the group administered 1.5 μg of TNFα+EP21 MV RNA.

[0058] [Figure 13A] This is a bar graph of the axial length of rats in a further embodiment (Embodiment 6). Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS eye drops for 3 weeks), T2+EP21 MV(3.3×10⁹)ED is the group administered eye drops containing 3.3×10⁹ MV particles of strain EP21 for 3 weeks, and T2+EP21 MV(9.9×10⁸)ED is the group administered eye drops containing 9.9×10⁸ MV particles of strain EP21 for 3 weeks.

[0059] [Figure 13B] This is a bar graph showing the relative change in refraction (right eye / left eye) of rats in Embodiment 6. Here, NC is the negative control group (the group injected with equilibrium salt solution and eyewash buffer), T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS eye drops for 3 weeks), T2+EP21 MV(3.3×10⁹)ED is the group administered eye drops containing 3.3×10⁹ MV particles of strain EP21 for 3 weeks, and T2+EP21 MV(9.9×10⁸)ED is the group administered eye drops containing 9.9×10⁸ MV particles of strain EP21 for 3 weeks.

[0060] [Figure 14A]This is a bar graph of the axial length of rats in a further embodiment (Embodiment 7). Here, NC is the negative control group (injected with equilibrium salt solution and eye wash buffer) that received 1X PBS via tail vein injection, T2 is the positive control group (injected with TGF-β2 and 1X PBS via tail vein injection for 3 weeks), T2+EP21 MV(3.3×10⁹)IV is the group that received 1X PBS containing 3.3×10⁹ MV particles of strain EP21 via tail vein injection for 3 weeks, and T2+EP21 MV(3.3×10⁸)IV is the group that received 1X PBS containing 3.3×10⁸ MV particles of strain EP21 via tail vein injection for 3 weeks.

[0061] [Figure 14B] This is a bar graph showing the relative change in refraction (right eye / left eye) in rats of Embodiment 6. Here, NC is the negative control group (injected with equilibrium salt solution and eye wash buffer) that received 1X PBS via tail vein injection, T2 is the positive control group (injected with TGF-β2 and 1X PBS via tail vein injection for 3 weeks), T2+EP21 MV(3.3×10⁹)IV is the group that received 1X PBS containing 3.3×10⁹ MV particles of strain EP21 via tail vein injection for 3 weeks, and T2+EP21 MV(3.3×10⁸)IV is the group that received 1X PBS containing 3.3×10⁸ MV particles of strain EP21 via tail vein injection for 3 weeks.

[0062] [Figure 15A] This is a bar graph of the axial length of rats in Embodiment 7. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer) that received 1X PBS intraperitoneally, T2 is the positive control group (the group injected with TGF-β2 and 1X PBS intraperitoneally for 3 weeks), T2+EP21 MV(3.3×10⁹)IP is the group that received 1X PBS containing 3.3×10⁹ MV particles of strain EP21 intraperitoneally for 3 weeks, and T2+EP21 MV(3.3×10⁸)IP is the group that received 1X PBS containing 3.3×10⁸ MV particles of strain EP21 intraperitoneally for 3 weeks.

[0063] [Figure 15B]This is a bar graph showing the relative change in refraction (right eye / left eye) of rats in Embodiment 7. Here, NC is the negative control group (group injected with equilibrium salt solution and eye wash buffer) that received 1X PBS intraperitoneally, T2 is the positive control group (group injected with TGF-β2 and 1X PBS intraperitoneally for 3 weeks), T2+EP21 MV(3.3×10⁹)IP is the group that received 1X PBS containing 3.3×10⁹ MV particles of strain EP21 intraperitoneally for 3 weeks, and T2+EP21 MV(3.3×10⁸)IP is the group that received 1X PBS containing 3.3×10⁸ MV particles of strain EP21 intraperitoneally for 3 weeks.

[0064] [Figure 15C] This is a Western blotting analysis of Embodiment 7. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV(3.3×10⁹) is the group injected with 1X PBS containing 3.3×10⁹ EP21 MV particles for 3 weeks.

[0065] [Figure 16A] This is an immunofluorescence (IF) image of Embodiment 7. Here, the isotype group and NC are the negative control group (the group injected with equilibrium salt solution and eyewash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV is the group induced with TGF-β2 and treated with 3.3 × 10⁹ MV particles of strain EP21 according to the present invention (scale bar: 165 μm).

[0066] [Figure 16B] This is an immunofluorescence (IF) image of NFκB in Embodiment 7. Here, NC is the negative control group (the group injected with equilibrium salt solution and eyewash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV is the group induced with TGF-β2 and treated with 3.3 × 10⁹ MV particles of strain EP21 according to the present invention (scale bar: 165 μm).

[0067] [Figure 16C] Figure 16B is a bar graph showing the quantification of the IF intensity of the groups.

[0068] [Figure 16D] This is an immunofluorescence (IF) image of IL10 according to Embodiment 7 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eyewash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV is the group induced with TGF-β2 and treated with 3.3 × 10⁹ MV particles of strain EP21 (scale bar: 165 μm).

[0069] [Figure 16E] Figure 16D is a bar graph showing the quantification of the IF intensity of the groups.

[0070] [Figure 16F] This is an immunofluorescence (IF) image of TNFα according to Embodiment 7 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV is the group induced with TGF-β2 and treated with 3.3 × 10⁹ MV particles of strain EP21 (scale bar: 165 μm).

[0071] [Figure 16G] This bar graph shows the quantification of the IF intensity of the groups in Figure 16F.

[0072] [Figure 16H] This is an immunofluorescence (IF) image of NLRP3 according to Embodiment 7 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eye wash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV is the group induced with TGF-β2 and treated with 3.3 × 10⁹ MV particles of strain EP21 (scale bar: 165 μm).

[0073] [Figure 16I] This bar graph in Figure 16H shows the quantification of the IF intensity of the groups.

[0074] [Figure 16J] This is an immunofluorescence (IF) image of IL1β according to Embodiment 7 of the present invention. Here, NC is the negative control group (the group injected with equilibrium salt solution and eyewash buffer), T2 is the positive control group (the group injected with TGF-β2 and 1X PBS for 3 weeks), and T2+MV is the group induced with TGF-β2 and treated with 3.3 × 10⁹ MV particles of strain EP21 (scale bar: 165 μm).

[0075] [Figure 16K] Figure 16 is a bar graph showing the quantification of the IF intensity of the groups in J.

[0076] [Figure 17] This is an immunofluorescence (IF) image of Embodiment 8 of the present invention, showing that the MV of strain EP21 is taken up by DIL-labeled retinal pigment epithelial cells RPE1.

[0077] [Figure 18A] This is an immunofluorescence (IF) image of Embodiment 9 of the present invention, showing that the MV of strain EP21 crosses the blood-retinal barrier (BRB) through the bloodstream.

[0078] [Figure 18B] This is a bar graph showing the quantification of the IF intensity of the groups in Figure 18A.

[0079] [Figure 19A] The results for axial length of rats according to Embodiment 10 of the present invention are shown. Here, T2 is the positive control group (the group injected with TGF-β2 and administered 1X PBS enterally for 3 weeks), and the other three strains are Lactobacillus casei Shirota, Lactiplantilaptibacillus plantarum subspecies plantarum PS128, and Lactobacillus paracasei NTU101.

[0080] [Figure 19B] This is an immunohistochemistry (IHC) image (scale bar: 40 μm) of Embodiment 10 according to the present invention.

[0081] [Figure 19C] This is a Western blotting analysis of Embodiment 10 of the present invention. Here, T2 is the positive control group (the group that received TGF-β2 injections and 1X PBS orally for 3 weeks), and the remaining four groups were administered Lactobacillus casei Shirota, Lactobacillus paracasei NTU101, Lactiplantibacillus plantarum subspecies plantarum PS128, and strain EP21, respectively, after being injected with TGF-β2.

[0082] [Figure 19D] Figure 19C is a bar graph showing the quantification of the IF intensity of the groups. [Modes for carrying out the invention]

[0083] The following embodiments are for further illustration of the present invention and do not limit its scope. Those skilled in the art can make certain improvements and modifications without departing from the scope of the present invention. Unless otherwise specified, the following terms used herein and in each claim have the definitions set forth below.

[0084] The singular expression "a" used in the description and claims is intended to cover not just one item, but one or more items, such as at least one, at least two, or at least three. Furthermore, the terms "includes" and "have" used in the specification and claims of this invention are not limiting and do not exclude any additional or unnecessary elements or components described in the specification and claims. Also note that the term "or" usually includes "and / or" unless explicitly indicated otherwise in this specification. The terms "about" or "substantially" used in the specification and scope of this application are used to correct any possible minor differences, but such minor changes do not alter the nature of the invention.

[0085] Probiotics, or probiotic bacteria, are microorganisms whose cells, strains, extracts, or metabolites have beneficial effects on the health of the host. They are typically produced from the human body or supplements and are beneficial to humans.

[0086] In this specification, “pre-test measurement” means measuring the axial length and refractive error of each experimental animal. Pre-test measurement includes the following procedure: Intraperitoneal injection of 50-80 mg / kg of liquid anesthetic (Zoletil®-50) into the test animals and chemical fixation. Refractive error and axial length are measured using a handheld strip retinal detector and A-scan ultrasound biometrics, respectively. Based on the measurement results, the difference in axial length and refractive error between the two eyes is expected to be 0.02 mm and 2(D) or less, respectively. Mice found to have congenital eye defects during pre-test measurement will not be counted in the subsequent experiment because they may introduce errors into the experimental results.

[0087] The flowcharts and procedures for each embodiment comply with laboratory safety guidelines. The term “effective dose” as used herein refers to the dosage range obtained according to the experimental data and results of each embodiment of the present invention. Those skilled in the art can select appropriate receptors according to specialized or general techniques, adjust dosage ratios, and convert dosages depending on the type of receptor and dosage form applied.

[0088] Preparation Embodiment 1: Source and preparation of test strain

[0089] The bacterial strain according to the present invention was collected and isolated from the feces of hamsters given herbal medicine. This strain was inoculated onto De Man, Rogosa, Sharpe (MRS) agar (BD) using the four-quadrant streaking method and cultured for 16-18 hours. Then, the bacterial suspension was diluted 100-fold and cultured on 40 ml of MRS at 37°C for 16-18 hours. Next, the obtained bacterial suspension was diluted 100-fold and cultured on 1000 ml of MRS at 37°C for 24 hours. One probiotic colony was selected from the plate, and a portion of its 16S rRNA gene sequence was amplified using a bacterial-specific primer pair such as SEQ ID NO: 1 (forward primer: 8F primer) and SEQ ID NO: 2 (reverse primer: 1492R primer) to obtain nucleic acid segments such as the sequence shown in SEQ ID NO: 3. The method for total RNA extraction is well known to those skilled in the art and does not require further explanation. The sequence of the nucleic acid segment (SEQ ID NO: 3) was compared with six 16S rRNA gene sequences of the Lactiplantybacillus plantarum subspecies L. plantarum from the National Center for Biotechnology Information (NCBI) GenBank (accession numbers KX057658.1, ON506095.1, MK524162.1, MT613641.1, MT597711.1, and MT597692.1). The results showed a 99% similarity between the sequences. Therefore, the strain of the present invention is Lactiplantybacillus plantarum subspecies L. plantarum, and will be hereinafter abbreviated as strain EP21.

[0090] The following are the physiological characteristics of strain EP21. Growth temperature: 35°C to 40°C; Growth pH: 4.0-7.0; Oxygen requirement level: facultative anaerobic. The strain EP21 of the present invention shows a positive result in the Gram staining test.

[0091] This strain EP21 was deposited on November 21, 2023 at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (331 Shih-Pin Rd., Hsinchu 300, Taiwan) under the deposit number BCRC 911210.

[0092] Preparation Embodiment 2: Extraction of Membrane Vesicles (MV)

[0093] The bacterial solution of strain EP21 (hereinafter abbreviated as EP21) is centrifuged at 8000 rpm for 1 hour, and the supernatant is collected. The supernatant is filtered through a 0.22 μm filter cup, and concentrated to 10 times the volume with a centrifugal filter. Place in an ultracentrifuge and centrifuge at 28000 rpm, 4°C for 6 hours to collect the pellet. Next, the pellet is dissolved in 50% OptiPrep TM solution, and 45%, 40%, 35%, 30%, 25%, 20% OptiPrep TM solutions are sequentially added to an ultracentrifuge tube, and density gradient centrifugation (28000 rpm, 4°C, overnight) is performed to carry out density gradient separation. The isolated EP21 membrane vesicles (MV) are stored at -80°C. After preparation, the morphology of the MV is detected using a transmission electron microscope (JEM 1400 FLASH). EP21 MV is negatively stained using 1% PTA (phosphotungstic acid), applied to a 400-mesh copper grid (Model: 01754-F, TED PELLA) for 1 minute, then excess solution is removed with filter paper, and the grid is placed in a dryer before observation. The transmission electron microscope uses 120 KV to accelerate the electron beam passing through the MV. In this way, the morphology of different fractions (ODG fractions) after density gradient separation and EP21 MV is observed. The results show that the vesicle diameter of the EP21 MV of the present invention is about 177.95±32.6 nm.

[0094] Nanoparticle Tracking Analysis (NTA) (version NTA3.4 build 3.4.003) is used to detect the size and number of EP21 MV vesicles (particles). The instrument is set to SOP standard measurement for detection, and the number of EP21 MV vesicles is estimated by multiplying the obtained results by a dilution factor for application to the next experiment. The NTA results indicate that the vesicle diameter of EP21 MV in this invention is approximately 156.36 ± 25.7 nm.

[0095] The prepared EP21 MV was rapidly frozen with liquid nitrogen, and its form was captured on a 0.2 μm PC film (model: GTTP02500, Isopore). TM The EP21 MVs were fixed onto a surface. After applying a platinum coating to the surface, the vesicle size and morphology of the EP21 MVs were observed using a cryo-scanning electron microscope (JEOL JSM-7800F). The purified EP21 MVs were hydrophobically labeled with the 5 μm fluorescent dye DIL (CAS number: 41085-99-8, Alfa Aesar, Switzerland) (30 minutes, 37°C), unbound dye was removed by ultracentrifugation (28000 rpm, 4°C for 2 hours), and the cells were frozen at -80°C for subsequent phagocytosis of the MVs and tracking of the MVs in in vivo experiments. Cryo-scanning electron microscopy results showed that the vesicle diameter of the EP21 MVs was approximately 153.04 ± 17.6 nm, and the vesicle diameter of the isolated EP21 MVs was approximately 195.28 ± 59.5 nm.

[0096] Embodiment 1: Vitamin EP21 administration test for myopia suppression

[0097] Three-week-old Brown Norway rats were used as test animals. After pre-test measurements of the animals, 250 ng / ml of TGF-β2 (transforming growth factor beta-2) was injected into the upper eyelid of the right eye once a week to induce inflammation for three weeks. In the negative control group, a balanced salt solution (BSS, eyewash buffer used in the preparation of injectable drugs) was injected and administered enterally with 1X PBS for three weeks. In the positive control group, TGF-β2 was injected and administered enterally with 1X PBS for three weeks. In the treatment group, TGF-β2 was injected and the logarithmic phase strain EP21 (2.5-3 × 10⁻¹⁰) was administered. 9Administer CFU / 0.1 ml / day via tube for 3 weeks. Measure axial length and refraction before and after treatment to confirm changes between groups. After collecting the entire eyeball as a sample, perform Western blotting, immunohistochemical staining, immunohistochemical (IHC) analysis, and dopamine level analysis. Measure the 24-hour growth curve using INFINITE M NANO (TECAN), select strain EP21 in the logarithmic phase, and administer to animals. Collect colonies of the EP21 strain and culture in 6 ml of MRS (model: 288130, BD) at 37°C for 16-18 hours. Dilute the EP21 bacterial solution 100-fold daily and culture in 40 ml of MRS at 37°C for 6 hours until it reaches the logarithmic phase. Next, centrifuge the EP21 bacterial solution at 3500 rpm for 15 minutes and wash the EP21 pellet with 1X PBS. After centrifugation, dissolve in 1X PBS to form EP21, and administer 0.1 ml (2.5-3 × 10) to each rat. 9 Administer EP21 at a dose of CFU / ml.

[0098] As shown in Figures 1A and 1B, TGF-β2 significantly increases the axial length of the rat eye and alters its refractive error. Administration of EP21 suppresses the excessive increase in axial length and the excessive change in refractive error. In this embodiment, one-way analysis of variance (ANOVA) is used as the statistical method. * means p-value < 0.05. ** means p-value < 0.01. *** means p-value < 0.001. **** means p-value < 0.0001.

[0099] After inducing myopia in the eyes with TGF-β2, the effects of EP21 administration on TGF-β, MMP2 (matrix metalloproteinase-2), and COL1A1 (alpha-1 type I collagen) will be analyzed using immunofluorescence (IF). Fixed tissue will be embedded in paraffin, sectioned, deparaffinized, rehydrated, and boiled with epitope exposure reagent. Nonspecific binding sites on the tissue will be blocked at room temperature with 1% BSA. The primary antibody will be reacted overnight at 4°C, and the secondary antibody will be reacted at room temperature for 1 hour. Nuclear staining will be performed with DAPI (4',6-diamidino-2-phenylindole) (1:1000), and the tissue will be reacted in the dark for 5 minutes. After mounting with an anti-bleaching reagent, fluorescence excitation will be performed using a fluorescence microscope, and images will be taken. Quantitative analysis of the experimental results will be performed using ImageJ, and one-way ANOVA will be performed using GraphPad Prism (statistical software). A p-value < 0.05 (indicated by *) is generally considered statistically significant. The results are shown in Figures 2A-4B. TGF-β2 increases the expression of TGF-β, MMP2, and COL1A1, while EP21 administration inhibits TGF-β (Figures 2A-2B), MMP2 (Figures 3A-3B), and COL1A1 (Figures 4A-4B).

[0100] The expression of TGF-β2-induced transcription factor NFκB in the eyeball was analyzed using the immunofluorescence method described above. Quantitative analysis of the experimental results was performed using ImageJ, and the obtained values ​​were analyzed using one-way ANOVA with GraphPad Prism (statistical software). A P-value < 0.05 is generally considered statistically significant. The results are shown in Figures 5A-6B. While TGF-β2 promotes the expression of NFκB and TNF-α, administration of EP21 of the present invention inhibits NFκB (Figures 5A-5B) and TNF-α (Figures 6A-6B).

[0101] Next, the expression level of TGF-β2-inducible transcription factor p-NFκB in the eyeball is analyzed by Western blotting. Radioimmunoprecipitation (RIPA) lysis buffer (50 Mm Tris-HCl, 250 Mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor, and phosphatase inhibitor) is added to the eyeball or retinal tissue, and the tissue is finely chopped. Proteins in the tissue are extracted by sonication, and after centrifugation at 12000 rpm at 4°C for 15 minutes, the supernatant is collected. Sample proteins are quantified using Bradford reagent (Bio-Rad, #5000006). 2 mg / ml BSA is used as the standard solution, and the linear range of the calibration curve is 10 μg (micrograms) to 1 μg. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is used for protein separation. After electrophoresis, the protein sample is transferred from the gel to a 0.45 μm polyvinylidene fluoride (PVDF) membrane for 50 minutes. The transferred membrane is treated with 5% skim milk at room temperature for 1 hour to fill and block nonspecific binding sites on the membrane. After incubation with the primary antibody at 4°C overnight, the secondary antibody is added and reacted at room temperature for 1 hour. Next, an enhanced chemiluminescence (ECL) reagent is added, the results are recorded using a luminometer, and the results are quantified using Image J. The expression of the target protein is calibrated using the internal standard method with the quantified results. GAPDH is used as a loading control antibody, and quantitative analysis of the experimental results is performed using Image J. The obtained values ​​(p-NFκB / NFκB / GAPDH) are statistically analyzed, and one-way ANOVA is performed on the values ​​using GraphPad Prism. A p-value < 0.05 is usually considered statistically significant. The results are shown in Figures 7A-7B. While TGF-β2 activates NFκB expression, administration of EP21 of the present invention inhibits NFκB activation.

[0102] Embodiment 2: Cell viability test

[0103] Retinal pigment epithelial cells (RPE1) were placed in a 96-well plate in 3 × 10⁶ rows. 3Inoculate cells per well and culture for approximately 16-18 hours. After cell adhesion, 8 × 10 8 , 4×10 8 , 2×10 8 , 1 x 10 8 Different numbers of EP21 membrane vesicles (MVs) containing [specific compound] are added and treated for 24 hours. The cells are reacted with a cell viability reagent (20 μl PMS + 2 ml MTS) for 2 hours, and the absorbance is measured at 490 nm. The viability is (treatment / control) × 100%, and is used to evaluate the cytotoxicity of EP21 MVs against human retinal pigment epithelial cells (RPE1).

[0104] RPE1 cells were treated for 24 hours with EP21 MV having different numbers of vesicles obtained in Preparation Embodiment 2, and cell proliferation was observed. The results were analyzed using a cell viability reagent and GraphPad Prism. The results are shown in Figure 8. There was no significant change in the cell viability of RPE1 cells after treatment with EP21 MV having different numbers of vesicles (ns means no significant difference compared to the control group). Therefore, the EP21 MV of the present invention does not exhibit cytotoxicity.

[0105] Embodiment 3: Evaluation of the effect of EP21 MV on the activation of the transcription factor NFκB

[0106] Three types of human retinal pigment epithelial cells (RPE1, ARPE 19, hRPEtiC) were each placed in 6-well plates in 3 × 10⁶ rows. 5 Inoculate with individual cells per well and culture for approximately 16-18 hours. Next, EP21 MV (2.2 × 10⁻¹⁶) 6 The samples are pretreated with individual vesicles / particles, and then further treated with IL-1β (interleukin-1 beta) (1.25 ng / ml) for 10 minutes.

[0107] The expression level of the transcription factor p-NFκB was analyzed by Western blotting. GAPDH was used as a loading control antibody, and the experimental results were quantified using Image J. The obtained values ​​(p-NFκB / NFκB / GAPDH) were statistically analyzed, and a one-way ANOVA was performed using GraphPad Prism. A P value < 0.05 is usually considered statistically significant. The results are shown in Figures 9A-9F. All groups treated with EP21 MV showed inhibition of IL-1β-induced transcription factor NFκB activation.

[0108] Embodiment 4: Effect of nuclease on EP21 MV

[0109] EP21 MV treated with nucleases (DNase I and RNase A) was added to human retinal pigment epithelial cells (RPE1) at a final concentration of 100 ng and pretreated for 2 hours. Next, the inflammatory substance IL-1β (concentration 1.25 ng / ml) was added to confirm whether EP21 MV still exerted anti-inflammatory activity on human retinal pigment epithelial cells after nuclease treatment. The expression of the transcription factor p-NFκB was analyzed by Western blotting. GAPDH was used as a loading control antibody, and the experimental results were quantified using Image J. The obtained values ​​(p-NFκB / NFκB / GAPDH) were statistically analyzed, and a one-way ANOVA was performed using GraphPad Prism. A P value < 0.05 is usually considered statistically significant. The results shown in Figures 10A-10B indicate that the activation of the IL-1β-induced transcription factor NFκB was inhibited in the group administered with EP21 MV and the group administered with nucleases.

[0110] Embodiment 5: Cytokine inhibition by EP21 MV

[0111] To test whether the EP21 MV of the present invention can suppress the secretion of inflammatory cytokines over a long period, inflammation of retinal pigment epithelial cells RPE-1 was induced for 24 hours using 2.5 ng / ml TNFα or 1.25 ng / ml IL1β. 4.4 × 10⁶ treatment groups were assigned. 6Adding individual EP21 MV particles suppresses IL6 secretion induced by TNFα or IL1β. As shown in Figure 11A, the EP21 MV of the present invention can suppress IL6 secretion induced by TNFα. As shown in Figure 11B, the current EP21 MV can suppress IL6 secretion induced by IL1β.

[0112] To test whether the EP21 MV RNA of the present invention can suppress the secretion of inflammatory cytokines over a long period, retinal pigment epithelial cells RPE-1 were transfected with EP21 RNA strain and EP21 MV RNA strain (1.5 μg each), and inflammation was induced for 24 hours using 2.5 ng / ml TNFα. Next, enzyme-linked immunosorbent assay (ELISA) was used to detect whether the transfected cells could suppress TNFα-induced IL6 secretion. The ELISA procedure included the following steps: Dilute the antibody (IL6) in 1X PBS in an ELISA plate (4°C, overnight), add blocking buffer, and react for 1 hour. Add the sample and allow to bind for 2 hours, then add horseradish peroxidase (HRP) and react for 30 minutes. Next, add the 3,3'5,5'-tetramethylbenzidine (TMB) substrate to develop color. Between the two adjacent steps, wash the plate three times with 1X PBS. Depending on the color development of the standard at different dilution concentrations, 1N HCl is added to stop the reaction, and the absorbance at 450 nm is read. As shown in Figure 12, the EP21 RNA and EP21 MV RNA of the present invention can also suppress TNFα-induced IL6 secretion.

[0113] Embodiment 6: Effect of EP21 MV-containing eye drops on TGF-β2-induced myopic rats

[0114] Three-week-old Brown Norway rats were used as test animals. After pre-test measurements of the animals, 250 ng / ml of TGF-β2 was injected into the upper eyelid of the right eye once a week to induce inflammation for three weeks. The negative control group was injected with a balanced salt solution (BSS, eye wash buffer, used in the preparation of the injectable drug) and administered 1X PBS eye drops for three weeks. The positive control group was injected with TGF-β2 and administered 1X PBS eye drops for three weeks. The first treatment group was injected with TGF-β2 and administered EP21 MV-containing eye drops for three weeks (a total of 3.3 × 10⁶ doses twice a week for three weeks). 9 (High-concentration eye drops containing 10 particles were instilled.) The second treatment group received injections of TGF-β2 and administration of EP21 MV-containing eye drops for 3 weeks (7 times a week for 3 weeks, totaling 9.9 × 10⁶ doses). 8 Low-concentration eye drops containing a certain number of particles were instilled. Axial length and refraction were measured before and after treatment, and changes between groups were examined. As shown in Figures 13A and 13B, the results indicate that TGF-β2 significantly increases axial length and alters refraction in rats. In the treatment groups, instillation of eye drops containing EP21 MV with different particle counts suppressed excessive increases in axial length and excessive changes in refraction.

[0115] Embodiment 7: Effect of EP21 MV on TGF-β2-induced myopic rats

[0116] 1. Administer EP21 MV via tail vein.

[0117] Three-week-old Brown Norway rats were used as test animals. After pre-test measurements of the animals, 250 ng / ml of TGF-β2 was injected into the upper eyelid of the right eye once a week to induce inflammation for three weeks. In the negative control group, a balanced salt solution (BSS, eye wash buffer, used for preparing the injectable drug) was injected, and 1X PBS was injected into the tail vein for three weeks. In the positive control group, TGF-β2 was injected, and 1X PBS was injected into the tail vein for three weeks. In the first treatment group, TGF-β2 was injected, and EP21 MV was administered by tail vein injection for three weeks (high concentration twice a week for three weeks, totaling 3.3 × 10⁶ doses). 9(Inject individual particles). The second treatment group is injected with TGF-β2 and administered EP21 MV by tail vein injection for 3 weeks (at a low concentration twice a week for 3 weeks, totaling 3.3 × 10⁶ doses). 8 (Each particle is injected.) Axial length and refraction are measured before and after treatment, and changes between groups are examined. As shown in Figures 14A and 14B, the results show that TGF-β2 significantly increases axial length and alters refraction in rats. In treatment groups administered EP21 MV with different particle counts via tail vein injection, both excessive increases in axial length and changes in refraction are suppressed.

[0118] 2. Intraperitoneal administration of EP21 MV

[0119] Three-week-old Brown Norway rats were used as test animals. After pre-test measurements of the animals, 250 ng / ml of TGF-β2 was injected into the upper eyelid of the right eye once a week to induce inflammation for three weeks. In the negative control group, a balanced salt solution (BSS, eye wash buffer, used for preparing the injectable drug) was injected, and 1X PBS was administered intraperitoneally for three weeks. In the positive control group, TGF-β2 was injected, and 1X PBS was administered intraperitoneally for three weeks. In the first treatment group, TGF-β2 was injected, and EP21 MV was administered intraperitoneally for three weeks (high concentration twice a week for three weeks, totaling 3.3 × 10⁶ doses). 9 (Inject individual particles). The second treatment group is injected with TGF-β2 and EP21 MV is administered intraperitoneally for 3 weeks (at a low concentration twice a week for 3 weeks, totaling 3.3 × 10⁶ doses). 8 (Each particle is injected). Axial length and refraction are measured before and after treatment to check for changes between groups. The entire eyeball is taken as a sample and subjected to Western blotting and immunohistochemistry (IHC) analysis. As shown in Figures 15A and 15B, the results show that TGF-β2 significantly increases axial length and alters refraction in rats. In the treatment groups, excessive increases in axial length and excessive changes in refraction are suppressed by intraperitoneal injection of EP21 MV with different particle counts.

[0120] A sample (rat eyeball) of P21 MV administered intraperitoneally in Embodiment 7 was treated with RIPA lysis buffer (50 Mm Tris-HCl, 250 MNaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor, and phosphatase inhibitor), and the sample was finely chopped and Western blotting was performed. As shown in Figure 15C, TGF-β2 activates the expression of inflammatory targets including p-NFκB, NFκB, NLRP3, IL1β, and TNFα, and decreases the expression of the anti-inflammatory target IL10. Therefore, intraperitoneal injection of EP21 MV of the present invention can inhibit the activation of inflammatory targets including p-NFκB, NFκB, NLRP3, IL1β, and TNFα, and increase the expression of the anti-inflammatory target IL10.

[0121] Samples (rat eyeballs) of P21 MV administered intraperitoneally according to Embodiment 7 were analyzed by immunofluorescence and quantified using Image J. The resulting values ​​were then interpreted using one-way ANOVA with GraphPad Prism. A P-value < 0.05 is generally considered statistically significant. As shown in Figures 16A-16K, TGF-β2 activates the expression of inflammatory targets including p-NFκB, NFκB (Figures 16B-16C), NLRP3 (Figures 16H-16I), IL1β (Figures 16J-16K), and TNFα (Figures 16F-16G), while decreasing the expression of the anti-inflammatory target IL10 (Figures 16D-16E). Therefore, intraperitoneal injection of EP21 MV according to the present invention can inhibit the activation of inflammatory targets including p-NFκB, NFκB, NLRP3, IL1β, and TNFα, and increase the expression of the anti-inflammatory target IL10.

[0122] Embodiment 8: Phagocytosis assay

[0123] Retinal pigment epithelial cells RPE1 (1 × 10 4 The ) is inoculated onto a chamber slide (PEZGS0816, MilliporeSigma®) and cultured for approximately 16-18 hours. After cell adhesion, the EP21 MV (4 × 10) labeled with DIL obtained in Preparation Embodiment 2 is cultured. 8RPE1 cells are treated with ) for 24 hours. Next, fluorescence excitation is performed using a modular DMi8 inverted microscope (Leica / CCD, Andor ZYLA 4.2 Plus) and EP21 MVs taken up by the cells are observed. For in vivo MV tracking, Sprague-Dawley (SD) rats are used as test animals. DIL-labeled EP21 MVs (1 × 10⁻¹⁶) 10 The rats are injected into the tail. After 24 hours, the SD rats are euthanized, their eyes are collected, and the tissue is fixed in formalin. Next, the tissue is dehydrated in 30% sucrose solution for 2 hours, embedded in an optimal cutting temperature compound (OCT, FSC 22 Clear, 3801480, Leica), and frozen and stored at -80°C. Tissue sections are prepared using a tissue chopper with both the machine and carrier set to -20°C, and each section is 12 μm thick. The nuclear tissue is stained with DAPI (1:1000) and allowed to react in the dark for 5 minutes. After mounting with a bleaching inhibitor, fluorescence excitation is performed using a fluorescence microscope and images are taken.

[0124] As shown in Figure 17, the EP21 MV of the present invention is taken up by retinal pigment epithelial cells RPE1.

[0125] Embodiment 9: Test for EP21 MV to cross the blood-retinal barrier (BRB)

[0126] EP21 MV (1×10) labeled with DIL 10 The EP21 MV of the present invention is injected into the tail of an SD rat. After 24 hours, the SD rat is euthanized, the eyeball is collected, and the tissue is fixed in formalin. Next, it is dehydrated in a 30% sucrose solution for 2 hours, embedded in an optimal cutting temperature compound (OCT, FSC 22 Clear, 3801480, Leica), and cryopreserved at -80°C. Tissue sections are prepared using a tissue chopper with both the machine and carrier set to -20°C, and each section is 12 μm thick. The nuclear tissue is stained with DAPI (1:1000) and reacted in the dark for 5 minutes. After mounting with a bleaching inhibitor, fluorescence excitation is performed using a fluorescence microscope and images are taken. As shown in Figures 18A and 18B, the results show that the EP21 MV of the present invention can pass through the bloodstream into the BRB.

[0127] Example 10: Testing of other probiotics that suppress inflammation

[0128] Three-week-old Brown Norway rats were used as test animals. After pre-test measurements of the animals, 250 ng / ml of TGF-β2 was injected into the upper eyelid of the right eye once a week to induce inflammation for three weeks (days 1, 8, and 15, respectively). The negative control group was injected with a balanced salt solution (BSS, eye wash buffer, used for preparing the injectable drug) along with 1X PBS enteral nutrition for three weeks. The positive control group was injected with TGF-β2 and 1X PBS for three weeks. TGF-β2 was injected into the treatment group, and logarithmic probiotics (2.5-3 × 10⁶) were administered. 9 Administer CFU / 0.1 ml / day via enteral nutrition for 3 weeks. In addition to EP21 from Preparation Embodiment 1, the other three groups of probiotics are Lactobacillus casei Shirota, Lactobacillus paracasei NTU 101, and Lactiplantibacillus plantarum subspecies plantarum PS128. Measure the axial length and refraction before and after treatment to confirm the changes between each group. After collecting the entire eyeball as a sample, perform Western blotting and immunohistochemistry (IHC) analysis.

[0129] As shown in Figure 19A, TGF-β2 significantly increases axial length and alters refractive error in rats. In the EP21-treated group, excessive increases in axial length and refractive errors are suppressed. Furthermore, during the progression of myopia, the genes for two tissue remodeling proteins, TGF-β and MMP2, are upregulated. MMP2 is an extracellular matrix enzyme that breaks down type I collagen (collagen 1) on the sclera. As myopia progresses, the eyeball gradually lengthens, causing light to be projected in front of the retina. This leads to myopia. Figure 19B shows the results of immunohistochemistry (IHC) analysis used to detect the amounts of TGF-β, MMP2, and collagen 1 in the retina. The intensity of the color after color development represents the amount of target proteins on the retina. The control group shown in Figure 19B represents the group treated with TGF-β2. The results show that in the TGF-β2 induction group with myopia, the expression of TGF-β2 and MMP2 increased, while the expression of collagen 1 decreased. Administration of EP21 decreased the expression of TGF-β2 and MMP2, and increased the expression of collagen 1. These changes indicate that administration of EP21 improves TGF-β2-induced myopia. Western blotting results are shown in Figures 19C and 19D. Administration of EP21 according to the present invention significantly inhibits the activation of the transcription factor NFκB. As can be seen from the results of the above tests, the EP21 strain of the present invention has anti-inflammatory effects compared to other lactic acid bacteria (Lactobacillus) and can slow the progression of myopia.

Claims

1. Deposited under deposit number BCRC (Biosource Collection and Research Center) 911210, is Lactiplantybacillus plantarum subspecies EP21, used for the treatment of eye diseases.

2. The aforementioned strain comprises the sequence segment shown in Sequence ID No. 3, wherein the strain is the Lactiplantybacillus plantarum subspecies plantarum strain EP21 according to claim 1.

3. Use of Lactiplantybacillus plantarum subspecies plantarum strain EP21 according to claim 1 or 2 in the preparation of a pharmaceutical composition for improving eye diseases.

4. The use according to claim 3, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers, adjuvants, and food additives.

5. The use of the pharmaceutical composition according to claim 3, wherein the dosage form includes a solution, suspension, capsule, tablet, lozenge, troche, powder, freeze-dried powder, lotion, emulsion, suppository, slurry, and ointment.

6. The use of the pharmaceutical composition as described in claim 3, wherein the pharmaceutical composition is administered orally to a subject.

7. The effective dose of the pharmaceutical composition is 1 x 10 per day. ６ From cfu / kg to 1 × 10 １０ The use according to claim 6, wherein the value is cfu / kg.

8. The effective dose of the pharmaceutical composition is 5 × 10 per day. ８ cfu / kg to 5 × 10 ９ The use according to claim 7, wherein the value is cfu / kg.

9. The use according to claim 3, wherein the aforementioned eye disease includes myopia, dry eye syndrome (DES), asthenoplasty, keratitis, macular degeneration, retinitis pigmentosa (RP), proliferative diabetic retinopathy (PDR), ischemic retinopathy, choroidal neovascularization, and glaucoma.

10. The use according to claim 9, wherein the ischemic retinopathy includes diabetic retinopathy (DR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO), and ischemic optic neuropathy (ION).

11. A composition comprising bacterial membrane vesicles for use in improving eye diseases, comprising bacterial membrane vesicles (MV) derived from the Lactiplantybacillus plantarum subspecies plantarum strain EP21 as described in claim 1.

12. The composition according to claim 11, wherein the vesicle diameter of the MV is 100 nm to 300 nm.

13. The composition according to claim 12, wherein the vesicle diameter of the MV is 120 nm to 255 nm.

14. A composition comprising bacterial membrane vesicles for use in improving eye diseases, comprising bacterial membrane vesicles (MV) derived from the Lactiplantybacillus plantarum subspecies plantarum strain EP21 as described in claim 2.

15. The composition according to claim 14, wherein the vesicle diameter of the MV is 100 nm to 300 nm.

16. The composition according to claim 15, wherein the vesicle diameter of the MV is 120 nm to 255 nm.

17. Use of a composition comprising bacterial membrane vesicles according to any one of claims 11 to 16 in the preparation of a pharmaceutical composition for improving eye diseases.

18. The use according to claim 17, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers, adjuvants, and food additives.

19. The use according to claim 17, wherein the dosage form of the pharmaceutical composition includes a solution, suspension, capsule, tablet, lozenge, troche, powder, freeze-dried powder, lotion, emulsion, suppository, slurry, and ointment.

20. The use of the pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is applied to a subject by injection or eye drops.

21. The use according to claim 20, wherein the injection includes intravenous injection (IV), intraperitoneal injection (IP), and tail vein injection.

22. The effective dose of the pharmaceutical composition is 1 × 10 per day. ５ From particles / kg to 1 x 10 １１ The use according to claim 20, wherein the particle density is per kg.

23. The effective dose of the pharmaceutical composition is 2.7 × 10 per day. ８ From particles / kg to 5.3 × 10 ９ The use according to claim 22, wherein the particle density is per kg.

24. The use according to claim 17, wherein the eye disease includes myopia, dry eye syndrome (DES), asthenoplasty, keratitis, macular degeneration, retinitis pigmentosa (RP), proliferative diabetic retinopathy (PDR), ischemic retinopathy, choroidal neovascularization, and glaucoma.

25. The use according to claim 24, wherein the ischemic retinopathy includes diabetic retinopathy (DR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO), and ischemic optic neuropathy (ION).

Images