Use of probiotic lactobacillus reuteri Y7 for protecting eyes

Abstract

The invention provides an application of a probiotic lactobacillus reuteri Y7 for protecting eyes. The probiotic lactobacillus reuteri Y7 can be used for preventing or treating eye inflammatory response, cell apoptosis and retina injury of mammals caused by blue light exposure; the damage of blue light exposure to the retina is retinal atrophy or retinal thickness thinning caused by blue light, macular choroidal neovascularization and retinal photosensitive function disability; the damage of the blue light exposure to the retina can cause macular lesion, especially late dry macular lesion. Besides human beings, the Chinese medicinal composition also has curative effects on dogs, cats and other mammals.

Description

Technical Field

[0001] The invention relates to a probiotic Lactobacillus reuteri Y7, which is used for protecting eyes and preventing blue light-induced damage to retina. Background Art

[0002] According to epidemiological statistics, neurodegenerative diseases will replace cancer and become the second leading cause of death in North America by 2040. Neurodegeneration often occurs unconsciously, so it is also called the "Silent Epidemic". It usually occurs in the elderly. Since patients are gradually isolated from society during the onset of the disease, coupled with the current lack of relevant medical knowledge, the general public rarely understands and pays attention to it. With the aging of the population structure, the number of patients with neurodegeneration is also increasing. For family members, patients and the general public, the impact on the economy, psychology and lifestyle is really difficult to estimate and bear.

[0003] Age-related macular disease (AMD) is one of the major eye diseases that cause blindness in adults or the elderly. According to the World Health Organization (WHO), AMD has become the main cause of poor vision, and the number of people suffering from vision loss and blindness due to age-related macular degeneration is also on the rise.

[0004] In the early stages of AMD, extracellular deposits of Drusen are found to be deposited locally in the macula, and focal geographic Y7horioretinal thinning is observed, affecting the central or peripheral visual field. When the disease progresses to choroidal neovascularization (CNV), subretinal fluid accumulation and localized leaky retinal detachment or subretinal hemorrhage are caused.

[0005] In recent years, there has been an increasing amount of attention paid to eye-related light damage and its attendant issues. UV damage has long been a concern, as the incidence of damage from sunlight (sunburn, skin cancer) has been increasing. Today, the widespread use of light bulbs and digital devices that emit blue light (mobile phones, computers) has aroused our concern about blue light, and there is a consensus that more research and discussion is needed. The mammalian retina is extremely susceptible to oxidative stress. In fact, photoreceptors (PR) and retinal pigment epithelium (RPE) are prone to produce high concentrations of reactive oxygen species (ROS) due to the following factors: (1) PR contains many different photosensitive molecules and is often continuously exposed to light; (2) their metabolism and function require high oxygen conditions. The retina is characterized by extremely high oxygen consumption; (3) photoreceptor discs (PRs) are highly oxygenated and have a high oxygen content. The photoreceptor discs are rich in polyunsaturated fatty acids and are easily damaged by oxidative stress. The recycling of oxidatively damaged photoreceptor discs is through phagocytosis of the RPE. After the photoreceptor discs are oxidatively damaged, a large amount of ROS is easily accumulated in the pigment epithelial cell layer. These processes bring great risks to the retina, especially in the elderly, where retinal neurons and RPE cells are particularly susceptible to oxidation, causing damage to aging tissues.

[0006] Oxidative damage to the retina is usually caused by prolonged exposure of the eye to light pollution. It can be divided into natural or artificial light sources, which can lead to light / photic maculopathy. The long-term accumulation of light-induced damage is considered to be the main cause of AMD, which leads to vision deterioration and blindness in the elderly. Scientists have found that oxidative retinal damage in experimental animals exposed to strong light has consistent pathological characteristics with retinal damage in patients with advanced AMD. In fact, the basic characteristics of human dry (non-neovascular) AMD have been confirmed to be reproduced in animal models of light-induced retinal damage. Histological studies have shown that the retinas of animals exposed to various doses of light illumination will show AMD characteristics, such as PR apoptosis, RPE cell migration to the neural retina, interference in the blood-retinal barrier (BRB), and inflammatory infiltration. Studies using these animal models may provide important information about the general mechanisms of retinal damage underlying AMD and other degenerative retinal diseases. In addition, the current rapid progress of ophthalmic surgery and ophthalmic care instruments has increased the incidence of heterologous photochemical damage. It also represents another relevant issue in modern ophthalmology. Studies have shown that short-term exposure to blue light has adverse effects on retinal morphology, including degeneration of the pigment epithelium, deformation of the outer nuclear layer (ONL), PR loss, crypt formation and even induction of geographic atrophy (GA) and CNV in albino mice. Retinal cells show increased terminal deoxyribonucleic acid transferase dUTP nick end labeling, as well as increased Apoptosis-inducing factors and necrosis-promoting receptor-interacting protein (RIP) kinases. Albino Wistar rats were irradiated with white and blue LED lights (750 lux) for 28 days, and it was found that the amount of free radical generation increased and the thickness of the ONL decreased in the LED exposure group. Albino Wistar rats and pigmented Long Evans rats were exposed to 500-6000 lux (cold white, blue and green) LEDs for 1 to 28 days, and it was found that the blue component of white LEDs would increase PR loss and cause retinal toxicity. In addition, high-brightness light can cause deformation and destruction of RPE tight junction proteins such as Zonula-occludin 1 (ZO-1) and have an immediate impact on BRB. There have been some reports describing the molecular mechanisms involved in blue light-induced phototoxicity, but the widespread use of smartphones has made it more urgent for us to further understand the long-term effects of blue light exposure on the eyes.

[0007] Y7 probiotics are mainly Lactobacillus reuteri, which exists in the digestive system, breast milk, skin and urinary tract. Lactobacillus reuteri can produce antimicrobial molecules such as organic acids, ethanol and reuterin. Due to its antimicrobial activity, reuteri can inhibit the colonization of pathogenic microorganisms and reshape the composition of the symbiotic microbial community in the host, and benefit the host immune system by reducing the production of pro-inflammatory factors and promoting the development and function of regulatory T cells. It has the function of enhancing the integrity of the intestinal barrier. The colonization of Lactobacillus reuteri may reduce the displacement of microorganisms from the intestinal cavity to the tissue. Inflammatory diseases, including those located in the intestine and distant tissues, are improved by increasing the colonization of Lactobacillus reuteri.

[0008] With the popularization of 3C products, the incidence of AMD caused by blue light damage has increased. The current treatment for AMD requires laser or intraocular injection, and patients are prone to not actively receive treatment due to psychological fear.

[0009] In view of this, how to provide a probiotic that can be ingested to protect the eyes and effectively prevent or treat blue light-induced macular degeneration has become an important issue to be solved by the present invention. Summary of the invention

[0010] The present invention aims to provide a strain of Lactobacillus reuteri Y7, which is deposited in the Food Industry Development Research Institute of Taiwan Province, China, and has a deposit number of BCRC911162. In addition, the strain of Lactobacillus reuteri Y7 is also deposited in the National Institute of Advanced Industrial Science and Technology, International Patent Organisms Depository (IPOD, NITE) of Japan (Room 120, 2-5-8, Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan Postal Code 292-0818) according to the provisions of the Budapest Treaty, and has a deposit number of NITE BP-04078.

[0011] Another object of the present invention is to provide a probiotic composition comprising a therapeutically effective dose of Lactobacillus reuteri Y7 strain.

[0012] To achieve the aforementioned object of the invention, the probiotic composition is a medicine, a food or a food additive.

[0013] To achieve the aforementioned object of the invention, the Lactobacillus reuteri Y7 is a live bacterium.

[0014] To achieve the aforementioned object of the invention, the dosage form of the composition includes solution, suspension, emulsion, powder, lozenge, pill, syrup, buccal lozenge, tablet, chewing gum, syrup, colloid or external preparation.

[0015] Another object of the present invention is to provide a use of Lactobacillus reuteri Y7 for preparing a pharmaceutical composition for protecting eyes.

[0016] To achieve the aforementioned purpose of the invention, the eye protection is to prevent or treat eye inflammation, cell apoptosis and retinal damage in mammals caused by blue light exposure.

[0017] In order to achieve the aforementioned purpose of the invention, the damage to the retina caused by the blue light exposure is retinal atrophy or thinning of retinal thickness, macular choroidal neovascularization, and retinal photosensitivity dysfunction caused by blue light.

[0018] To achieve the aforementioned object of the invention, the damage to the retina caused by the blue light exposure may cause macular degeneration.

[0019] To achieve the aforementioned object of the invention, the macular degeneration is advanced dry macular degeneration.

[0020] To achieve the aforementioned object of the invention, the mammal is a human, pig, dog, cat, cow, horse, donkey, deer, goat, sheep, rabbit, mouse, rat, guinea pig or monkey.

[0021] To achieve the aforementioned object of the invention, the wavelength of the blue light is 380nm to 530nm.

[0022] To achieve the aforementioned object of the invention, the eye protection is achieved by maintaining the integrity of the intestinal barrier to alleviate eye inflammation, cell apoptosis and retinal damage.

[0023] In summary, compared with the existing technology, the new bacteria screened out by the present invention is Lactobacillus reuteri Y7. FP proves that Y7 probiotics can effectively alleviate the symptoms of AMD caused by blue light; SD-OCT and tissue section results show that low-dose and high-dose Y7 probiotics are effective in maintaining retinal thickness to avoid retinal atrophy caused by long-term blue light exposure; ERG shows that low-dose and high-dose Y7 probiotics intervention can significantly maintain its a, b, and c waves, and high-dose is the most effective, proving that Y7 probiotics can effectively avoid the effect of retinal photosensitivity disability caused by blue light.

[0024] ELISA analysis revealed that Y7 probiotics intervention can effectively increase antioxidant enzyme activity. q-PCR analysis revealed that Y7 probiotics can reduce eye inflammation and cell apoptosis. In addition, it was found that Y7 probiotics intervention has the effect of maintaining tight connections in the colon and thus maintaining barrier integrity.

[0025] The results of this experiment show that Y7 probiotics can protect the retinal structure from damage and photosensitivity degradation caused by blue light by increasing the activity of antioxidant enzymes in the body and reducing eye inflammation. Therefore, Y7 probiotics has the effect of preventing and treating AMD. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is an experimental flow chart of the present invention.

[0027] Figure 2 is the result of intraocular pressure measurement, NC is the control group, BL is the blue light group, LU is the blue light + lutein group, Y7L is the Y7 probiotic low-dose group, and Y7H is the Y7 probiotic high-dose group; # indicates p<0.05 compared with the NC group at the same time point; * indicates p<0.05 compared with the BL group at the same time point; the data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, and p<0.05 was considered statistically significant.

[0028] Figure 3 (Top) The fundus, retinal status and vascular distribution of mice in the fifth week (35th day) of blue light intervention, fundus photography (FP), fundus fluoresceinangiography (FFA), and spectral-domain optical coherencetomography (SD-OCT) in the fifth week (35th day); Figure 3 (Bottom) is the quantitative analysis of each layer of the OCT retina; data are expressed as mean ± SEM; NC: control group; BL: blue light group; Y7L: Y7 probiotic low-dose group; CM: Y7 probiotic medium-dose group; Y7H: Y7 probiotic high-dose group; LU: blue light + lutein group, n = 6 in each group; * represents a significant difference from the NC group (p < 0.05), # represents a significant difference between the BL groups (p < 0.05).

[0029] Figure 4Electroretinography (ERG) analysis after 35 days of blue light exposure (Day 35), data are expressed as mean ± SEM; NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; W1-W5: intervention with whole coffee fruit extract from Day 0 to Day 35; * indicates p < 0.05 compared with the NC group at the same time point; # indicates p < 0.05 compared with the BL group at the same time point; $ indicates p < 0.05 compared with the LU group at the same time point; data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, p < 0.05 was considered statistically significant.

[0030] Figure 5 (a) The fundus, retinal status and vascular distribution of mice before blue light intervention (day 0), fundus photography (FP), fundus fluoresceinangiography (FFA), and spectral-domain optical coherence tomography (SD-OCT) at week 0 (day 0); Figure 5 (b) is the quantitative analysis of each layer of the retina by OCT; no lesions or atrophy were found among the groups; data are expressed as mean ± SEM; NC: control group; BL: blue light group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; LU: blue light + lutein group, n = 6 in each group.

[0031] Figure 6 (a) is a H&E-stained retinal tissue section. It is a tissue section stained with H&E after sacrifice of each group. The upper picture is taken at the middle position of the area vertically below the pupil and close to the optic plexus. Figure 6 (b) Image J was used to analyze the retinal thickness of each group; data are expressed as mean ± SEM; NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; * indicates p < 0.05 compared with the NC group at the same time point; # indicates p < 0.05 compared with the BL group at the same time point; $ indicates p < 0.05 compared with the LU group at the same time point; data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, p < 0.05 was considered statistically significant.

[0032] Figure 7(a) is an analysis of the activity of the antioxidant enzyme glutathione peroxidase (GPX). One unit is defined as the amount of enzyme required to oxidize 1.0 μmol NADPH to NADP+ per minute at 25°C. Figure 7 (b) is the analysis of the antioxidant enzyme catalase activity (CAT), and the amount of CAT that decomposes 1 μmol H2O2 per minute in 1 mL of serum or plasma at 37°C is defined as 1 unit. Data are expressed as mean ± SEM; NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; * indicates p<0.05 compared with the NC group at the same time point; # indicates p<0.05 compared with the BL group at the same time; $ indicates p<0.05 compared with the LU group at the same time; data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, and p<0.05 was considered statistically significant.

[0033] Figure 8 (a) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene MCP-1 was measured by q-PCR; Figure 8 (b) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene IL-18 was measured by q-PCR; Figure 8 (c) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene NLRP3 was measured by q-PCR; Figure 8 (d) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene NF-KB p65 was measured by q-PCR; Figure 8 € RNA was extracted from the eyes of the test animals, and the expression of the inflammation-related gene Caspase1 was measured by q-PCR; Figure 8 (f) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene TNF-α was measured by q-PCR; Figure 8 (g) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene IL-6 was measured by q-PCR; Figure 8(h) RNA was extracted from the eyes of the test animals, and q-PCR was used to analyze the expression of the inflammation-related gene IL-1β. The data are expressed as mean ± SEM. NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; * indicates p < 0.05 compared with the NC group at the same time point; # indicates p < 0.05 compared with the BL group at the same time point; $ indicates p < 0.05 compared with the LU group at the same time point; the data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, and p < 0.05 was considered statistically significant.

[0034] Fig. 9 (a) RNA was extracted from the eyes of the test animals, and the expression of apoptosis-related gene Bax was analyzed by q-PCR; Fig. 9 (b) RNA was extracted from the eyes of the test animals and the expression of apoptosis-related gene Bcl-2 was analyzed by q-PCR; Fig. 9 (c) RNA was extracted from the eyes of the test animals, and the expression of apoptosis-related gene Caspase3 was analyzed by q-PCR; data are expressed as mean ± SEM; NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; * indicates p < 0.05 compared with the NC group at the same time point; # indicates p < 0.05 compared with the BL group at the same time point; $ indicates p < 0.05 compared with the LU group at the same time point; data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, p < 0.05 was considered statistically significant.

[0035] Fig.10 (a) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene Occludin was measured by q-PCR; Fig.10 (b) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene Cludin-1 was measured by q-PCR; Fig.10 (c) RNA was extracted from the eyes of the test animals and the expression of the inflammation-related gene ZO-1 was measured by q-PCR; Fig.10 (d) RNA was extracted from the buccal mucosa of the test animals and the expression of the inflammation-related gene Occludin was measured by q-PCR; Fig.10 (e) RNA was extracted from the buccal mucosa of the test animals and the expression of the inflammation-related gene Cludin-1 was measured by q-PCR; Fig.10 (f) RNA was extracted from the buccal mucosa of the test animals and the expression of the inflammation-related gene ZO-1 was measured by q-PCR; Fig.10 (g) RNA was extracted from the colon of the test animals and the expression of the inflammation-related gene Occludin was measured by q-PCR; Fig.10 (h) RNA was extracted from the colon of the test animals and the expression of the inflammation-related gene Cludin-1 was measured by q-PCR; Fig.10 (i) RNA was extracted from the colon of the test animals, and the expression of the inflammation-related gene ZO-1 was analyzed by q-PCR; the data are expressed as mean ± SEM; NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; * indicates p < 0.05 compared with the NC group at the same time point; # indicates p < 0.05 compared with the BL group at the same time point; $ indicates p < 0.05 compared with the LU group at the same time point; the data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, and p < 0.05 was considered statistically significant. DETAILED DESCRIPTION

[0036] Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by persons of ordinary skill in the art. Unless otherwise specified, the materials used in the present invention are commercially available and easily accessible.

[0037] [Definition of terms]

[0038] The following are definitions of terms used in this specification and the patent applications. Unless the language requires a different meaning, these definitions should be used to determine the meaning of the individual terms.

[0039] The term "a" or "an" does not exclude a plurality, that is, unless the language clearly indicates or requires otherwise, the single forms "a", "an" and "the" should be understood to include plural objects. In other words, unless otherwise expressly stated or the language of the reference clearly implies the opposite meaning, references to a single characteristic or limitation throughout the present invention should include the corresponding plural characteristics or limitations and vice versa. Therefore, unless otherwise defined, the terms "a", "an" and "the" have the same meaning as "at least one" or "one or more". For example, when referring to "an ingredient", it includes a mixture of multiple ingredients, etc.

[0040] The term "about" or "approximately" compensates for the variability allowed by the pharmaceutical industry and inherent in drug products, such as differences in content due to manufacturing variations and / or product degradation over time. The term allows for any variation in the actual drug product to be considered in the biological properties of the evaluated product in the subjects to be equivalent to the enumerated advantages of the claimed product.

[0041] The term "composition" refers to any type of composition into which a plurality of specified ingredients may be optionally incorporated together with any further ingredients.

[0042] The terms "include" and similar meanings should be interpreted in an open and inclusive manner to mean "including but not limited to".

[0043] The terms "one embodiment", "an embodiment", "a specific embodiment", etc., represent a unique feature, attribute or characteristic, or a group or set of unique features, attributes or characteristics combined with individual meanings and presented in at least one embodiment of the present invention. Such meanings appearing in various places in this patent specification do not necessarily refer to the same embodiment. In addition, these unique features, attributes or characteristics can be combined in any appropriate manner in one or more embodiments.

[0044] The term "pharmaceutically acceptable" refers to a compound or mixture that is used to prepare a pharmaceutical composition that is generally safe, non-toxic, and free of biological or other undesirable effects, including a compound or mixture that is suitable for human pharmaceutical use.

[0045] The term "prevent" refers to preventing a disease, condition, or symptom, as well as preventing further growth and spread of a disease, condition, or symptom after initial improvement or initial elimination of the cause of the disease, condition, or symptom.

[0046] The term "treat" refers to a therapeutic intervention that is effective in treating a disease, condition, or symptoms; however, treatment can also mean to ameliorate, improve, control, control progression, prevent progression, prevent recurrence, etc.

[0047] The term "subject" generally refers to a human being. However, the present invention is not limited to humans, but can be used for mammals as necessary; the term "mammal" means any animal classified as a mammal, including but not limited to humans, livestock, and farm animals, as well as zoo, sports, or pet animals, such as humans, primates, rodents, pigs, dogs, cats, cows, horses, donkeys, deer, goats, sheep, rabbits, mice, rats, guinea pigs, or monkeys.

[0048] The term "probiotic" refers to living microorganisms, that is, microorganisms capable of reproduction, which are recognized in the state of the art at the time of the patent application as microorganisms that confer a health benefit to the host when administered in appropriate amounts.

[0049] The term "therapeutically effective dose" refers to a dose sufficient to achieve the effect of treating or preventing a disease when the drug is administered to a subject for treating or preventing the disease.

[0050] [Experimental animals]

[0051] The present invention uses six-week-old male C57BL / 6 mice, which are placed in a breeding box with a temperature controlled at 22±2°C and a humidity of 55±5%, respectively, and a 12-hour light-dark cycle is maintained in the animal room. The mice are fed with animal feed for an adaptation period of one week. During the experiment, the control group and the adaptation week are given general solid feed for the experimental animals.

[0052] After the adaptation period, the mice were treated with the tested samples and exposed to blue light every other week. The weight, food and water intake of the mice were monitored during the experiment to observe the physiological changes of the mice during the experiment. Electroretinography (ERG), fundus photography (FP), fundus fluorescein angiography (FFA), spectral-domain optical coherence tomography (SD-OCT) and intraocular pressure (IOP) were collected on days 0, 21 and 35. The detailed experimental flow chart is shown in the figure. Figure 1 .

[0053] C57BL / 6 mice weighing 23-25g were randomly divided into seven groups. Six mice successfully induced by blue light were selected from each group for the experiment. The samples were dissolved in secondary water and given to the test mice by oral tube feeding once a day. The groups were:

[0054] Blank solvent group (NC): 0.2 ml of deuterium-depleted water (DDW) was administered by tube feeding;

[0055] Negative control group (blue light group, BL): 0.2 ml of secondary water was administered by tube feeding and irradiated with blue light;

[0056] Low-dose Y7 probiotics (Y7L): Low-dose Y7 probiotics were administered by tube feeding and irradiated with blue light, with a daily dose of 10 8 CFU was dissolved in 0.2 ml of secondary water;

[0057] High-dose Y7 probiotics (Y7H): High-dose Y7 probiotics were administered by tube feeding and irradiated with blue light, with a daily dose of 10 9 CFU was dissolved in 0.2 ml of secondary water;

[0058] Positive control group (Lutein, LU): Lutein was administered by tube feeding and irradiated with blue light. The recommended dose of lutein for humans is 30 mg. Based on the human surface area equivalent dose conversion ratio, for example, the equivalent dose coefficient of 20 g of mouse body weight to 60 kg of human surface area is 12.3, so the daily dose is 30 (mg) / 60 (kg) x 12.3 = 6.15 mg / kg bw.

[0059] Animal disease model - blue light illumination

[0060] A mouse cage (48×25×20cm) was placed in a stainless steel ventilation box, and blue light panels were installed around and above the mouse cage to provide a blue light source. When conducting animal blue light exposure experiments, 6-year-old mice were placed in a mouse cage in a blue light box and provided with drinking water and feed. During the experiment, they were exposed to 400Lux blue light with a 12-hour light-dark cycle. The purpose was to induce late-stage dry macular degeneration in mice caused by long-term chronic light damage, in order to simulate the macular degeneration caused by long-term exposure to short-wavelength blue light from 3C products in people's daily lives.

[0061] Fundus photography (FP) and fluorescein angiography (FFA)

[0062] Mice were anesthetized by intraperitoneal (IP) injection of Zoletil (40 mg / kg) and Xylazine (10 mg / kg). The deeply anesthetized mice were placed on an operating table and mydriatic was performed with Atropine (0.125%) eye drops. After 30 minutes of complete mydriasis, 2% Methocel gel was dropped as a corneal lubricant to prevent damage caused by excessive friction between the mouse eyes and the fundus lens. The vascular status of the retina was observed with a Micron III retinal imaging microscope (Phoenix Research Y7H Laboratories), and the mouse's fovea was placed in the center of the fundus microscope screen to capture retinal white light photographs (FP). Afterwards, retinal vascular fluorescence was developed with IP 10% sodium fluorescein aqueous solution and a 520 nm filter.

[0063] Spectral-domain optical coherence tomography (SD-OCT)

[0064] The OCT imaging system (Phoenix Research Laboratories, San Ramon, CA) used in this experiment is based on the principle of frequency-domain optical coherence tomography. The computed tomography is divided into five layers. The first layer is the distance from the ganglion cell layer (GY7L) to the outer plexiform layer (OPL), the second layer is the distance from the ONL, the third layer is the distance from the photoreceptor cell layer (inner segment / outer segment, IS / OS), and the fourth layer is the distance from the IS / OS to the RPE. Finally, the overall retinal thickness is measured from the nerve fiber layer (NFL) to the lower edge of the RPE layer. The above five layers were quantitatively analyzed using InSight XL (Phoenix Research Laboratories, San Ramon, CA).

[0065] Electroretinography (ERG)

[0066] Before the experiment, Atropine (0.125%) mydriatic eye drops were dripped into the mouse eyes to dilate the pupils, and then Zoletil (40 mg / kg) and Xylazine (10 mg / Kg) were injected intraperitoneally. After the mice were deeply anesthetized, they were placed on a support platform and then 2% Methocel gel lubricant was dripped on them. After the electrodes are placed, ERG analysis is started. The mice are stimulated with a single excitation light and the ERG spectrum is collected. The ERG spectrum obtained by the excitation light stimulation will produce a negative a-wave and a positive b-wave. In the spectrum analysis part, the time from the initial time of the excitation light to the peak time of the a-wave and the b-wave will be defined as the excitation time, the amplitude difference from the background electrogram value at the initial time of the excitation light to the peak electrogram value of the a-wave will be defined as the a-wave amplitude (amplitude of a-wave), and the amplitude difference from the peak electrogram value of the a-wave to the peak electrogram value of the b-wave will be defined as the b-wave amplitude (amplitude of b-wave). Observing the changes in the excitation time and amplitude indicates whether the function of the cells in the retina is normal or not. In electrophysiology, the a-wave generally indicates the health of the PR of the outer retina, the b-wave represents the health of the inner retinal nerve cells, such as bipolar cells and horizontal cells, and the c-wave indicates the health of the retinal RPE.

[0067] Real-time Quantitative Polymerase Chain Reaction (q-PCR)

[0068] After the animals were sacrificed, the eyes were collected, and RNA was extracted with Trizol and reverse transcribed into cDNA. Real-time quantitative polymerase chain reaction was used to detect gene expression of inflammatory hormones and apoptosis-related genes, such as TNF-α, IL-1β, IL-6, Bax, Bcl-2, Caspase1, Caspase3, NLRP3, etc., and compared with the endogenous levels of β-actin or GAPDH or 18S rRNA. The primer sequences are shown in Table 1.

[0069] Table 1. Gene sequence list

[0070]

[0071]

[0072] Statistical analysis

[0073] All data are expressed as mean ± standard error of the mean (SEM), and statistical analysis was performed using one-way ANOVA and Newman-Keuls test, with p < 0.05 considered statistically significant. The statistical analysis of this experiment will be performed using the statistical software Graph pad Prism (v9.2.0). Data on food intake, drinking water, body weight, organ weight, and clinical examinations were analyzed using one-way ANOVA.

[0074] Example 1 Physiological effects of Y7 probiotic supplementation and blue light exposure on mice

[0075] The results show the weight changes of mice during the experiment, and the food and water intake. The weight of the Y7H group was significantly higher than that of the NC group on the 7th, 28th, and 35th days, as shown in Table 2. On the 35th day of the experiment, the food intake of the Y7H group was significantly increased compared with that of the BL group, as shown in Table 3. There was no difference in water intake between the groups, as shown in Table 4. The IOP was monitored by the ICARETONOMETER TAOli (ICAREFINLANDOY, Vantaa, Finland) tonometer, and it was observed that the intraocular pressure of the blue light group (BL) increased in the fifth week. Therefore, the blue light exposure during the experiment did not cause high intraocular pressure in mice. Figure 2 , the results indicate that the blue light exposure animal model of this experiment will not affect the physiology of mice.

[0076] Table 2. Weight of mice during the experiment

[0077]

[0078]

[0079] The weight of mice in Y7H group was significantly higher than that in NC group on days 7, 28 and 35. Data are expressed as mean ± SEM; NC: control group; BL: blue light group; LU: blue light + lutein group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group. * indicates p<0.05 compared with NC group at the same time point; # indicates p<0.05 compared with BL group at the same time; $ indicates p<0.05 compared with LU group at the same time; data were analyzed by one-way ANOVA and post hoc test by Newman-Keuls test, p<0.05 was considered statistically significant.

[0080] Table 3. Food intake of mice during the experiment

[0081] Day 7 Day 14 Day 21 Day 28 Day 35 NC 5.46±0.29 4.70±0.56 4.59±0.20 4.35±0.64 4.47±0.13 BL 5.74±0.28 4.07±0.78 4.32±0.81 4.58±0.95 4.20±0.73 LU 5.71±1.17 4.17±1.15 5.04±1.82 5.33±2.17 4.43±2.12 Y7L 4.16±0.30 3.73±0.20 4.56±0.04 3.63±0.46 4.54±0.13 Y7H 4.46±0.52 5.31±0.77 5.61±1.25 <![CDATA[6.46±1.47 # ]]> <![CDATA[5.89±0.40 # ]]>

[0082] The food intake of mice during the experiment, the food intake of Y7H group was significantly higher than that of BL group on the 35th day; data are expressed as mean ± SEM; NC: control group; BL: blue light group; Y7L: low-dose Y7 probiotics group; Y7H: high-dose Y7 probiotics group; LU: blue light + lutein group, n = 6 in each group.

[0083] Table 4. Drinking water status of mice during the experiment

[0084]

[0085]

[0086] There was no significant difference in the feeding status of mice among the groups during the experiment; data are expressed as mean ± SEM; NC: control group; BL: blue light group; Y7L: Y7 probiotic low-dose group; Y7H: Y7 probiotic high-dose group; LU: blue light + lutein group, n = 6 in each group.

[0087] Example 2 Establishment of the blue light-induced macular degeneration animal model in mice

[0088] Continuous exposure to blue light for five weeks successfully induced AMD in mice. The fundus photograph (FP) of the blue light group (BL) showed an enlarged deposition of Drusen in the upper right corner. SD-OCT showed that the atrophy of the retina on the right side of the optic nerve was aggravated. The thickness of the ONL layer, IS-OS layer, and RPE layer was significantly thinned, indicating the loss of PR and RPE cells. The FFA results showed that there was no sign of CNV, a symptom of late wet AMD. Figure 3(Top); ERG observation showed that the amplitudes of a, b, and c waves in the blue light group were significantly decreased compared with those in the control group and the intervention group. Figure 4 , which means that the activity of PR, bipolar cells, horizontal cells, RPE and other retinal cells is damaged. Combined with the above, this disease model of blue light-induced late dry AMD is successful.

[0089] Example 3 Effects of Y7 probiotic supplementation and blue light exposure on fundus and retinal structure

[0090] SD-OCT can observe whether the retinal structure is affected by blue light exposure and causes retinal atrophy, edema and other lesions. Through quantitative analysis of SD-OCT, the changes of each layer of the retina during the experiment can be more clearly observed. Before the experiment, no atrophy, edema and other lesions were observed in the retina of each group. Figure 5 On the 35th day of the experiment, ONL layer atrophy was observed at the arrow position in the blue light group. Figure 3 (Top), quantitative analysis showed that the overall retinal thickness of BL was significantly atrophied compared with NC (p<0.05); quantitative analysis of the ONL layer showed that BL was significantly atrophied compared with NC (p<0.05), and Y7L, Y7H were significantly different from BL (p<0.05); quantitative analysis of the RPE layer showed that BL was significantly atrophied compared with NC (p<0.05), and LU, Y7L, and Y7H were significantly higher than BL (p<0.05), indicating that Y7 probiotic intervention can effectively slow down retinal atrophy caused by blue light. Figure 3 (Down).

[0091] From the above results, it can be seen that blue light exposure will cause significant atrophy of the overall retina, ONL layer and RPE layer thickness, and the PR cell nuclei are mainly located in the ONL layer, which means that the PR responsible for light and color reception and the RPE responsible for supplying retinal nutrients and maintaining the integrity of the retinal structure are lost, leading to retinal atrophy; low and high doses of Y7 probiotics and lutein intervention can effectively maintain retinal thickness and avoid retinal atrophy and functional damage caused by long-term blue light exposure. Among them, the high-dose group of Y7 probiotics is closer to the control group than lutein.

[0092] FFA can observe the distribution of fundus blood vessels to determine whether the late-stage wet AMD symptom - choroidal neovascularisation (CNV) - has appeared. No CNV was found in any of the groups on day 0 and day 35 before the experiment, indicating that this animal induction model can effectively control late-stage dry AMD.

[0093] Example 4 Effect of blue light exposure on retinal photosensitivity

[0094] This experiment uses the dark field (scotopic) electroretinogram (ERG) to observe whether the photosensitive function of the mouse retina is affected by blue light exposure. When the mouse receives light, the opsins on the photoreceptor cells combine with 11-cisretinal to carry out the visual cycle, thereby generating a membrane potential difference and transmitting electrical signals sequentially through the retinal neuron layer. ERG is to detect the electrophysiological function of the retina by recording the potential difference peaks generated by different neurons. This experiment mainly counts the amplitude difference between the negative a wave generated by the photoreceptor cells and the positive b wave generated by other retinal visual neurons to compare the differences between the groups. The a wave represents the activity of the photoreceptor cells, the b wave represents the activity of the bipolar cells and horizontal cells in the retina, and the c wave represents the activity of the RPE.

[0095] The a wave in ERG represents the activity of PR. According to the amplitude quantification diagram, on the 35th day, BL, Y7L, and Y7H were significantly decreased compared with NC (p<0.05), Y7L was significantly decreased compared with LU (p<0.05), and Y7H was significantly increased compared with BL (p<0.05), indicating that maintaining a high dose of Y7 probiotics can effectively maintain the activity of PR in the retina. Figure 4 The a wave in ERG represents the activity of bipolar cells and horizontal cells in the retina. The amplitude quantification diagram shows that on the 35th day, BL decreased significantly compared with NC (p<0.05), while BL decreased significantly compared with LU and Y7H (p<0.05), indicating that high doses of Y7 probiotics can effectively maintain the activity of bipolar cells and horizontal cells in the retina. Figure 4 The b wave in ERG represents the quantification of RPE activity amplitude. The BL group showed a significant decrease compared with the NC group (p<0.05). The LU, Y7L, Y7H group showed significant differences compared with the BL group (p<0.05). This indicates that lutein and Y7 probiotics intervention can effectively maintain RPE activity in the retina. Figure 4 The c wave.

[0096] The above results show that the amplitudes of a, b, and c waves of BL were significantly reduced compared with those of NC and the intervention group, indicating that its PR, bipolar cells, horizontal cells, and RPE were damaged, leading to the degeneration of photosensitivity; while the Y7 probiotic intervention group was able to maintain its a, b, and c waves, among which the high dose had the greatest protective effect, indicating that this interventional substance can effectively protect the retinal photosensitivity function.

[0097] Example 5 Retinal tissue sections supplemented with Y7 probiotics and exposed to blue light

[0098] The animals were sacrificed after the experiment was completed on the 35th day. The mouse eyes were removed for H&E staining and retinal tissue sections were observed to observe the changes in the morphology of each layer of the mouse retina after the experiment. At the same time, it was possible to verify whether the SD-OCT results were consistent with the tissue sections. The section results were used to capture the retinal part closest to the optic nerve at the vertical pupil at a magnification of 200 times. Figure 6 (a), followed by quantitative analysis of overall retinal thickness using Image J, e.g. Figure 6 (b) Compared with NC, the overall retinal thickness of BL, Y7L, Y7H, and LU was significantly reduced (p<0.05). However, there were significant differences between Y7L, Y7H, and LU and BL (p<0.05), indicating that Y7 probiotics and lutein intervention can effectively maintain retinal thickness. Figure 6 (b) Meanwhile, Y7 probiotics maintained retinal thickness in a dose-dependent manner.

[0099] The above results are consistent with the SD-OCT results at week 5, such as Figure 3 (superior), Figure 3 (below), thus once again verifying that Y7 probiotics have a more protective effect on retinal damage caused by blue light exposure.

[0100] Example 6 Antioxidant enzyme analysis after Y7 probiotic supplementation and blue light exposure

[0101] ELISA was used to analyze the expression of serum antioxidant-related enzymes, such as Figure 7 There was no significant difference in glutathione peroxidase (GPX) among the groups; catalase (CAT) showed a downward trend compared with NC in BL, while the Y7 probiotic intervention group showed a significant increase compared with BL.

[0102] Example 7 Effects of Y7 probiotic supplementation and blue light exposure on the expression of inflammation-related genes in the eye

[0103] Analysis of genes related to ocular inflammation in mice, including MCP-1 ( Figure 8 (a) 、IL-18( Figure 8 (b))、NLRP3( Figure 8 (c))、NF-KB p65( Figure 8 (d))、Caspase1( Figure 8 (e))、TNF-α( Figure 8 (f)), IL-6( Figure 8 (g)), IL-1β( Figure 8(h)) In NF-KB p65 and MCP-1, BL significantly increased expression compared with NC (p<0.05), while Y7L and Y7H significantly reduced expression compared with BL (p<0.05), and reduced the expression of inflammation-related genes in a dose-dependent manner, such as Figure 8 (a) Figure 8 (d), but only NF-kB p65 gene showed statistically significant difference between BL and NC, while Y7 probiotics showed statistical difference between BL and NC, such as Fig. 9 (a) Fig. 9 (b) As shown; IL-18, NLRP3, Caspase1 gene expression was found to increase in BL, while the expression of Y7L and Y7H showed a downward trend in the later stage of intervention, as shown in Figure 8 (b) Figure 8 (c) Figure 8 (e) In summary, the intervention of Y7 probiotics has a protective effect on the ocular inflammation response induced by blue light.

[0104] Example 8 Effects of Y7 probiotic supplementation and blue light exposure on the expression of apoptosis-related genes in the eye

[0105] Analysis of apoptosis gene expression in eye cells of the tested mice, including Bax ( Fig. 9 (a)), Bcl-2( Fig. 9 (a))、Caspase3( Fig. 9 (c)) In Bax, BL was found to significantly increase expression compared with NC (p<0.05), and Y7 probiotic intervention could significantly reduce gene expression (p<0.05), and Y7L was closer to NC than LU, such as Fig. 9 (a); In Bcl-2, Y7H was found to have significantly increased expression compared with BL, such as Fig. 9 (b); In Caspase3, BL also showed an upward trend compared with NC, while the Y7 intervention group showed a trend close to NC. Fig. 9 (c) Y7 probiotics can effectively reduce blue light-induced apoptosis in mouse eye cells.

[0106] Example 9 Effects of Y7 probiotic supplementation and blue light exposure on tight junction-related gene expression in various tissues

[0107] q-PCR analysis of blue light-induced tight junction-related gene expression of Occludin in the mouse eye, buccal mucosa, and colon Fig.10 (a) Fig.10 (d) Fig.10 (g)), Claudin-1 (in order Fig.10 (b) Fig.10 (e) Fig.10 (h))、ZO-1(in order Fig.10 (c) Fig.10 (f) Fig.10 (i)) Among the eye tight junction genes, BL showed a downward trend compared with NC, but in Occludin, the gene expression of Y7L and Y7H was significantly reduced compared with LU (p<0.05). In Claudin-1 and ZO-1, the gene expression of Y7L and Y7H showed a downward trend compared with LU. Fig.10 (a)-(c); Among the buccal mucosa tight junction genes, BL showed a downward trend compared with NC, and only Y7H in the ZO-1 gene was significantly higher than NC, BL, and LU (p<0.05). Fig.10 (d)-(f); Among the tight junction genes of colon, BL showed a downward trend compared with NC, while Y7L and Y7H significantly increased their gene expression in ZO-1 (p<0.05). In Occludin and Claudin-1, Y7L and Y7H showed an upward trend. Fig.10 (g)-(i).

[0108] In summary, Y7 probiotics have a better effect in protecting the barrier integrity in the intestine than in the eyes and cheek mucosa.

[0109] In summary, this study successfully induced late-stage dry AMD using an animal induction model, including the appearance of Drusen, significant atrophy of overall retinal thickness, retinal ONL layer and RPE layer thickness, and damage to photosensitivity function. In addition, FFA showed that blue light exposure did not cause CNV in the animal fundus.

[0110] Through FP, it is proved that Y7 probiotics can effectively slow down the symptoms of AMD caused by blue light; through SD-OCT and tissue section results, it can be known that low-dose and high-dose Y7 probiotics can effectively maintain retinal thickness and avoid retinal atrophy caused by long-term blue light exposure; through ERG, it can be known that low-dose and high-dose Y7 probiotics intervention can significantly maintain its a, b, and c waves, and the high dose is the most effective, proving that Y7 probiotics can effectively avoid the effect of retinal photosensitivity dysfunction caused by blue light.

[0111] ELISA analysis revealed that Y7 probiotics intervention can effectively increase antioxidant enzyme activity. q-PCR analysis revealed that Y7 probiotics can reduce eye inflammation and cell apoptosis. In addition, it was found that Y7 probiotics intervention has the effect of maintaining tight connections in the colon and thus maintaining barrier integrity.

[0112] The results of this experiment show that Y7 probiotics can protect the retinal structure from damage and photosensitivity degradation caused by blue light by increasing the activity of antioxidant enzymes in the body and reducing eye inflammation. Therefore, Y7 probiotics has the effect of preventing and treating AMD.

[0113]

Biological Material Deposit

[0114] Food Industry Development Institute, Taiwan Province, China, the preservation date is November 29, 2022, and the preservation number is BCRC911162.

[0115] The deposit date is February 9, 2024, and the deposit number is NITE BP-04078.

Claims

1. A Lactobacillus reuteri Y7 strain, characterized in that: The deposit number of the Lactobacillus reuteri Y7 strain is NITE BP-04078.

2. A probiotic composition, characterized in that: The invention comprises a therapeutically effective dose of the Lactobacillus reuteri Y7 strain as claimed in claim 1.

3. The probiotic composition according to claim 2, characterized in that The probiotic composition is a medicine, a food or a food additive.

4. The probiotic composition according to claim 2, characterized in that The Lactobacillus reuteri Y7 is a living bacterium.

5. The probiotic composition according to claim 2, characterized in that The dosage form of the composition includes solution, suspension, emulsion, powder, lozenge, pill, syrup, buccal lozenge, tablet, chewing gum, syrup, colloid or external preparation.

6. A use of Lactobacillus reuteri Y7 as claimed in claim 1, characterized in that: The Lactobacillus reuteri Y7 is used for preparing a medical composition for protecting eyes.

7. The use according to claim 6, characterized in that The eye protection is to prevent or treat eye inflammation, cell apoptosis and retinal damage in mammals caused by blue light exposure.

8. The use according to claim 7, characterized in that The damage to the retina caused by blue light exposure is retinal atrophy or thinning of retinal thickness, macular choroidal neovascularization, and retinal photosensitivity dysfunction caused by blue light.

9. The use according to claim 7, characterized in that: Damage to the retina caused by the blue light exposure may cause macular degeneration.

10. The use according to claim 9, characterized in that The macular degeneration is advanced dry macular degeneration.

11. The use according to claim 7, characterized in that: The mammal is a human, pig, dog, cat, cow, horse, donkey, deer, goat, sheep, rabbit, mouse, rat, guinea pig or monkey.

12. The use according to claim 7, characterized in that: The wavelength of the blue light is 380nm to 530nm.

13. The use according to claim 7, characterized in that: The eye protection is achieved by maintaining the integrity of the intestinal barrier to slow down ocular inflammation, cell apoptosis and retinal damage.

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