Application of Bacteroides fragilis in the preparation of drugs for treating optic nerve damage

Abstract

A microorganism containing Bacteroides fragilis genes is used as an active ingredient in the preparation of pharmaceuticals, medical devices, or food for treating optic nerve damage. This delivers Bacteroides fragilis (or its functional components) into the gastrointestinal tract, which helps maintain the number of retinal ganglion cells and the thickness of the retinal nerve fiber layer, inhibits the activation of retinal microglia, inhibits the activation of retinal inflammatory pathways and the expression of inflammatory mediators, thereby protecting visual function.

Description

Technical Field

[0001] This invention relates to a new use of a known product, and more particularly to the biological effects of a microorganism on retinal ganglion cell protection, visual function protection, inhibition of microglia activation, inhibition of retinal inflammatory pathway activation and inflammatory mediator expression, and its application as an active ingredient in pharmaceutical manufacturing. Background Technology

[0002] Glaucoma is a group of diseases characterized by optic nerve atrophy and visual field defects, with pathologically elevated intraocular pressure being the main risk factor. Statistics show that glaucoma is the leading cause of irreversible blindness worldwide, and the number of people suffering from glaucoma globally is projected to reach 111.8 million by 2040, imposing a severe medical and economic burden on patients. Therefore, breakthroughs in its treatment are urgently needed.

[0003] Numerous compounds, proteins, and nucleic acids have been successfully developed and applied to the treatment of glaucoma, and many medical devices are also used in the clinical diagnosis and treatment of glaucoma.

[0004] The gut-brain axis plays a crucial role in various neurodegenerative diseases. Given the continuity between the optic nerve and the central nervous system, exploring the role of the gut-eye axis in ophthalmic diseases is particularly essential. Increasing research indicates significant changes in the gut microbiota of glaucoma patients, but the specific mechanisms and the relationship between these microbiota and disease treatment remain unclear. Summary of the Invention

[0005] One objective of this invention is to provide a microorganism as an active ingredient for protecting visual function, maintaining the number of retinal ganglion cells and the thickness of the retinal nerve fiber layer, inhibiting the activation of retinal microglia, inhibiting the activation of retinal inflammatory pathways and the expression of inflammatory mediators, and facilitating the treatment of optic nerve damage.

[0006] Another objective of this invention is to provide a Bacteroides fragilis as an active ingredient for the repair of optic nerve damage, which is beneficial for the treatment of glaucoma.

[0007] Another object of the present invention is to provide a medicine, including Bacteroides fragilis, for the treatment of glaucoma.

[0008] Another object of the present invention is to provide a medical device, including Bacteroides fragilis, for the treatment of glaucoma.

[0009] Bacteroides fragilis ( Bacteroides fragilisBelonging to the phylum Bacteroidetes, class Bacteroidia, order Bacteroidales, family Bacteroidaceae, and genus Bacteroides, it is a type of Gram-negative anaerobic bacillus that plays an important role in the human intestinal microbiota.

[0010] A microorganism, Bacteroides fragilis, was purchased from MicroBiotechnology (Shanghai) Co., Ltd., accession number: ATCC25285.

[0011] Another type of microorganism has been genetically edited to contain foreign genetic material.

[0012] Another type of microorganism includes plasmids, which contain foreign genetic material.

[0013] In one specific implementation, the exogenous genetic material is a Bacteroides fragilis gene.

[0014] In another specific implementation, the exogenous genetic material is more than 97% homologous to the Bacteroides fragilis gene.

[0015] In another specific implementation, the exogenous genetic material is more than 95% homologous to the Bacteroides fragilis gene.

[0016] In another specific implementation, the exogenous genetic material is more than 93% homologous to the Bacteroides fragilis gene.

[0017] In another specific implementation, the exogenous genetic material is more than 90% homologous to the Bacteroides fragilis gene.

[0018] In another specific implementation, the exogenous genetic material is more than 85% homologous to the Bacteroides fragilis gene.

[0019] In another specific implementation, the exogenous genetic material is more than 80% homologous to the Bacteroides fragilis gene.

[0020] In another specific implementation, the exogenous genetic material is more than 75% homologous to the Bacteroides fragilis gene.

[0021] In another specific implementation, the exogenous genetic material is more than 70% homologous to the Bacteroides fragilis gene.

[0022] In another specific implementation, the exogenous genetic material is more than 60% homologous to the Bacteroides fragilis gene.

[0023] In another specific implementation, the exogenous genetic material is more than 50% homologous to the Bacteroides fragilis gene.

[0024] It has been verified that Bacteroides fragilis can reduce the loss of retinal ganglion cells in glaucoma model mice, inhibit the activation of retinal microglia in glaucoma model mice, and inhibit the production of retinal nerve inflammation in glaucoma model mice. The visual function of mice treated with Bacteroides fragilis is protected.

[0025] The microorganisms of the present invention are used to make drugs that deliver Bacteroides fragilis (or its functional components) to the gastrointestinal tract to alleviate retinal damage and loss of retinal ganglion cells, including but not limited to maintaining the number of retinal ganglion cells, maintaining the thickness of the retinal nerve fiber layer, improving visual function, reducing retinal microglia activation, reducing retinal inflammatory pathway activation and inflammatory mediator expression.

[0026] Medical devices made from the microorganisms of the present invention deliver Bacteroides fragilis (or its functional components) to the gastrointestinal tract to alleviate retinal damage and loss of retinal ganglion cells, including but not limited to maintaining the number of retinal ganglion cells, maintaining the thickness of the retinal nerve fiber layer, improving visual function, reducing retinal microglia activation, reducing retinal inflammatory pathway activation and inflammatory mediator expression.

[0027] The Bacteroides fragilis (lyophilized powder) shown in this invention is mixed with other excipients to prepare a drug (formulation) for treating optic nerve damage.

[0028] These pharmaceutical excipients can be those commonly used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for use to be compatible with the substances in the formulation, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving metabolism in the body and enhancing the drug delivery effect.

[0029] Excipients used in the preparation of oral formulations generally include solvents, as well as necessary flavoring agents, antibacterial agents, emulsifiers, and coloring agents.

[0030] Excipients used in tablet manufacturing generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium), and lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate).

[0031] The excipients used to produce granules are similar to those used for tablets, but the granulation process is different. Depending on the requirements, the produced granules are mixed with a gliding agent and then encapsulated to obtain capsules.

[0032] Drug-containing medical devices combining drugs and medical devices are now quite common, such as microspheres containing Bacteroides fragilis, which can reside in the gastrointestinal tract. Common scaffold materials include PLA, PLGA, and γPGA. Microspheres or gels can also be made by mixing drugs with biocompatible and biodegradable materials, adhering to mucous membranes, and providing a microenvironment for Bacteroides fragilis growth. Attached Figure Description

[0033] Figure 1 This image shows the protection of retinal ganglion cells in mice on day 28 after glaucoma modeling. In figure a, a typical field of view of a mouse retinal patch under a fluorescence confocal microscope (scale bar = 30 μm), with β-III tubulin (red) labeling retinal ganglion cells; figure b, a schematic diagram of the field of view location in figure a; and figure c, a statistical graph of retinal ganglion cell survival rates in each group. *** indicates a statistically significant difference between the two groups (p < 0.001).

[0034] Figure 2 This image shows the protective effects of retinal nerve fiber layer thickness and retinal ganglion cell count on day 28 after glaucoma modeling in mice. Figure a shows typical field of view of hematoxylin-eosin stained retinal sections from each group (scale bar = 50 μm); figure b shows the cell count of the retinal ganglion cell layer (GCL) in each group; figure c shows the thickness of the retinal nerve fiber layer (RNFL); and figure d shows the thickness of the inner plexiform layer (IPL). * indicates a statistically significant difference between the two groups (p < 0.05), ** indicates a statistically significant difference between the two groups (p < 0.01), and *** indicates a statistically significant difference between the two groups (p < 0.001).

[0035] Figure 3The graph shows the results of Bacteroides fragilis's effect on visual function improvement in glaucoma model mice. In the visual cliff test, a is the statistical graph of the number of mice in each group who first stepped onto the cliff side, and b is the statistical graph of the total time that mice in each group stayed on the cliff side in the visual cliff test. *** indicates that there is a statistical difference between the two groups, p<0.001.

[0036] Figure 4 Figure 1 shows the results of Bacteroides fragilis inhibiting the activation of retinal microglia in a mouse glaucoma model. Figure 2 shows a typical field of view of a mouse retinal patch under a fluorescence confocal microscope (scale bar = 50 μm), with Iba1 (green) labeling microglia. Figure 3 shows the relative number of Iba1-positive cells in each group. Figure 4 shows the relative cumulative optical density of Iba1. ** indicates a statistically significant difference between the two groups (p < 0.01), and *** indicates a statistically significant difference between the two groups (p < 0.001).

[0037] Figure 5 Figure 1 shows the results of Bacteroides fragilis's inhibition of retinal neuroinflammatory pathway activation and inflammatory mediator expression in a mouse model of glaucoma. Specifically, a) is the Western blot map of iNOS, p-NFκB, and NFκB in each group; b) is the quantitative statistical graph of p-NFκB protein expression level normalized to NFκB in each group; c) is the quantitative statistical graph of iNOS protein expression level normalized to β-tubulin in each group; d) is the Western blot map of TNF-α, IL-1β, and IL-6 in each group; and e) is the quantitative statistical graph of TNF-α protein expression level normalized to β-tubulin in each group. f is a quantitative statistical graph of IL-1β protein expression level in each group after β-tubulin normalization; g is a quantitative statistical graph of IL-6 protein expression level in each group after β-tubulin normalization; h is a quantitative statistical graph of relative mRNA expression of TNF-α, IL-1β, IL-6 and iNOS in each group; i is an immunofluorescence image (scale bar = 50 μm) showing the localization and relative expression of TNF-α, IL-1β, IL-6 and iNOS in GCL (ganglionic cell layer), IPL (inner plexiform layer) and INL (nuclear layer) of mouse retinal sections in each group. * indicates statistical difference between the two groups, p < 0.05; ** indicates statistical difference between the two groups, p < 0.01; *** indicates statistical difference between the two groups, p < 0.001. Detailed Implementation

[0038] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0039] Unless otherwise specified, all other reagents used in the embodiments of this invention were purchased from Beyotime Corporation.

[0040] The mice used in this embodiment were 6-8 week old wild-type C57BL / 6J mice, which were raised under standard conditions.

[0041] 1) Establishing a mouse model of glaucoma

[0042] Male C57BL / 6J mice, aged 6-8 weeks and weighing 18-25g, were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. The mice were randomly divided into four groups: control group (Control group), glaucoma model group (MB group), Bacteroides fragilis colonization group (Bf group), and glaucoma model combined with Bacteroides fragilis colonization group (MB+Bf group). The glaucoma model was established as follows: After mixed anesthesia, 2 μl (approximately 5.0 × 10⁻⁶ g / L) of the glaucoma model was injected into the mice using a 30G needle. 6 Intraocular pressure (IOP) was measured using an Icare TonoLab (Icare, Finland) on postoperative days 1, 3, 7, 14, 21, and 28. The microbeads (15 μm in diameter, with a molecular weight per ml) were injected into the anterior chamber.

[0043] 2) Bacteroides fragilis intervention

[0044] Prior to colonization, mice were given an antibiotic mixture via drinking water for 14 days, consisting of ampicillin 1 g / L, metronidazole 1 g / L, neomycin 1 g / L, and vancomycin 0.5 g / L, followed by a 2-day washout period. From day 16 onwards, mice were administered 0.2 mL of PBS or a solution containing 10 g / L of antibiotics via gavage daily. 8 CFU / mL of Bacteroides fragilis ATCC25285 (non-toxigenic strain) in PBS suspension. A glaucoma model was established after 14 days, and the patient was continuously treated by gavage three times a week until the end of the experiment.

[0045] 3) Histological and morphological analysis

[0046] Fourteen or twenty-eight days after glaucoma modeling, retinal slides or ocular sections were harvested for histological and morphological analysis. For sampling, the heart was first perfused with physiological saline and 4% paraformaldehyde. After perfusion, the mouse eyeballs were removed and placed overnight in FAS (Servicebio) fixative. For retinal slide preparation, the mouse retina was completely removed, flattened, and fixed in 4% paraformaldehyde for 15 minutes. It was then blocked for 1 hour with 5% goat serum mixed with PBST and 0.3% Triton-X100. The retina was incubated overnight at 4°C with primary antibody β-III tubulin (1:1000, Abcam), followed by incubation with Alexa Fluor 594-labeled secondary antibody (1:200, Abbkine) for 2 hours. Images were taken using a fluorescence confocal microscope (Nikon). Eight fields of view were selected from the central and peripheral regions of the retina, and β-III tubulin-positive cells were counted using ImageJ. For the eyeballs used for sectioning, after OCT embedding, sections were prepared using a cryostat (10 μm) and hematoxylin-eosin staining was performed to analyze retinal structure. For each section, the number of retinal ganglion cell layer (GCL) cells, the thickness of retinal nerve fiber layer (RNFL), and the thickness of inner plexiform layer (IPL) were counted in 5 consecutive visual fields, and the average values ​​were calculated and normalized to the control group.

[0047] 4) Visual function testing in mice

[0048] The visual cliff test was used to assess visual function in mice. The experimental setup consisted of a transparent glass box (60cm long, 60cm wide, and 15cm high), with one half suspended above the edge of the experimental platform, forming the "cliff" side (90cm above the ground); the other half remained on the platform, forming the "platform" side. A transparent platform (10×7cm, 2cm high) was placed in the center of the box. Both the platform and the ground were covered with alternating black and white patterns (2cm long) to help mice develop depth vision. Light sources were placed 30cm above and 30cm below the experimental area to illuminate it. At the start of the experiment, mice were placed on the transparent platform. The initial direction chosen by each mouse (cliff side or platform side) and the number of mice that initially stepped towards the cliff side were recorded. Additionally, the total time each mouse spent in the "cliff" area during the first two minutes was recorded. Each mouse was tested only once, and the platform and box were thoroughly cleaned after each experiment.

[0049] 5) Retinal immunofluorescence

[0050] Mouse retinal sections were prepared following the steps described above. Lysis was performed at room temperature using 0.3% Triton X-100 for 15 minutes, followed by blocking with PBS containing 5% goat serum (Boster) and 0.3% Triton X-100 for 1.5 hours. The sections were then incubated overnight at 4°C with primary antibodies against TNFα (1:200, Proteintech), IL-1β (1:200, Proteintech), IL-6 (1:200, Proteintech), iNOS (1:200, Abclonal), RAGE (1:200, Abmart), and Iba1 (1:100, Servicebio). Afterward, the sections were incubated at room temperature for 1 hour with secondary antibodies against Alexa Fluor 488 (anti-rabbit) or Alexa Fluor 594 (anti-mouse) (1:200, Abbkine). Cell nuclei were stained with DAPI (Invitrogen). Finally, images were taken using a confocal microscope (Nikon). Each group included at least three samples. To assess the degree of microglia activation, five visual fields were randomly selected from each retina, and the number of Iba1-positive cells and the involved optical density (IOD) were calculated and averaged; each group included six samples. Results were analyzed using ImageJ software and standardized using a control group.

[0051] 6) RNA extraction and real-time quantitative PCR (qPCR)

[0052] Total RNA was extracted from retinal tissue using TRIzol reagent. RNA concentration and purity were determined using Nanodrop (THERMOFISHER). Reverse transcription was performed using the PrimeScript RT Reverse Transcription Kit (Takara). Primers used in this invention were referenced from PrimerBank and synthesized by Qingke Biotechnology (Beijing). In gene expression analysis, β-actin was used as an internal control, and fold change in expression was calculated using the 2^−Δ(ΔCT) relative quantification method.

[0053] 7) Western blot

[0054] Total proteins were extracted from retinal tissue using RIPA lysis buffer (Sanger Biotech). Protein samples were separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% skim milk in Tris-buffered saline containing Tween-20. The membrane was incubated overnight at 4°C with the following primary antibodies: TNFα (1:1000, Proteintech), IL-1β (1:1000, Proteintech), IL-6 (1:1000, Proteintech), iNOS (1:1000, Abclonal), NFκB (1:1000, Proteintech), and p-NFκB (1:1000, Abcam), followed by incubation at room temperature for 1 hour with anti-rabbit or anti-mouse secondary antibodies (1:1000, Beyotime). The membrane was developed using a Tanon imaging system (Shanghai) and BeyoECL Moon developing solution (Beyotime), and the images were quantitatively analyzed using ImageJ software.

[0055] 8) Statistical methods

[0056] Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software. t-tests were used for comparisons between two groups. When there were more than two groups, one-way ANOVA was used, followed by Turkey's test. A p-value < 0.05 was considered statistically significant. Example 1

[0057] This embodiment verifies the effect of Bacteroides fragilis on alleviating retinal damage and loss of retinal ganglion cells in a glaucoma model mouse.

[0058] Retinal smears, immunofluorescence staining, and confocal microscopy were performed on mice in each group. Typical field-of-view images are shown below. Figure 1 As shown in a. The β-III tubulin-positive cells in each field of view were counted and statistically analyzed. Figure 1 As shown in bc), compared with the control group (Control, white), the percentage of retinal ganglion cells in glaucoma model mice (MB, blue) was significantly reduced, while after gavage administration of B. fragilis (MB+B. fragilis, red), the percentage of retinal ganglion cells was significantly increased.

[0059] Eye sections and retinal hematoxylin-eosin staining were performed on mice in each group. Typical visual field images are shown below. Figure 2As shown in Figure a. The cell density of the retinal ganglion cell layer (GCL), the thickness of the retinal nerve fiber layer (RNFL), and the thickness of the inner plexiform layer (IPL) in each group of mice were statistically analyzed. It was found that compared with the control group (white), there were no significant differences in GCL cell density, RNFL thickness, and IPL thickness in the B. fragilis simple gavage group (B. fragilis, orange). In glaucoma model mice (MB, blue), both GCL cell density and RNFL thickness were significantly decreased. However, after B. fragilis gavage (MB+B. fragilis, red), both GCL cell density and RNFL thickness were significantly increased. Figure 2 b–d).

[0060] The above results indicate that Bacteroides fragilis can maintain the number of retinal ganglion cells and the thickness of the retinal nerve fiber layer, demonstrating that it can alleviate retinal damage and loss of retinal ganglion cells. Example 2

[0061] This embodiment verifies the effect of Bacteroides fragilis on visual function indicators in the visual cliff test of glaucoma model mice.

[0062] Tests showed that, compared with the control group, the number of mice in the glaucoma model mouse (MB) who first stepped onto the cliff side (blue portion) was increased, and the total time spent on the cliff side (blue portion) was significantly increased. Meanwhile, the visual function of glaucoma mice treated with Bacteroides fragilis (MB+B. fragilis) was protected, as evidenced by a decrease in the number of mice that first stepped onto the cliff side. Figure 3 a) and the time spent on the cliff side was shortened ( Figure 3 b).

[0063] This demonstrates that Bacteroides fragilis effectively improves visual function in glaucoma model mice. Example 3

[0064] This embodiment verifies the effect of Bacteroides fragilis on inhibiting the activation of retinal microglia in a mouse model of glaucoma.

[0065] To evaluate the effect of *Bacteroides fragilis* on the activation of retinal microglia in a mouse glaucoma model, we performed immunofluorescence staining on mouse retinal slices using Iba1 primary antibody (1:100, Servicebio) and Alexa Fluor 488 anti-rabbit secondary antibody. Typical confocal microscopy field of view images are shown below. Figure 4 As shown in figure a. Compared with the control group, glaucoma model mice (MB, blue) exhibited a higher number of Iba1-positive cells and a higher cumulative optical density (IOD) of Iba1, while supplementation with Bacteroides fragilis significantly reduced the number of Iba1-positive cells and the Iba1 cumulative optical density (IOD). Figure 4b–c).

[0066] The above results indicate that Bacteroides fragilis can inhibit the activation of retinal microglia in glaucoma model mice. Example 4

[0067] This embodiment verifies the effect of Bacteroides fragilis on inhibiting the activation of retinal inflammatory pathways and the expression of inflammatory mediators in a glaucoma model mouse.

[0068] This study used real-time quantitative PCR, Western blotting, and immunofluorescence to detect changes in the expression of key proteins and inflammatory mediators in the retinal inflammation pathway in glaucoma model mice. Compared with the control group, the expression level of iNOS and the phosphorylated NFκB / NFκB ratio in glaucoma model mice (MB) were significantly higher than those in the control group (Control). Supplementation with Bacteroides fragilis (MB+B. fragilis) significantly alleviated its expression. Figure 5 ac). Compared with the control group, TNF-α, IL-1β and IL-6 in glaucoma model mice (MB) showed lower protein levels (ac). Figure 5 d–g) and mRNA levels ( Figure 5 h) were all upregulated, and the results of immunofluorescence quantification further confirmed this. Figure 5 i), while supplementation with Bacteroides fragilis significantly inhibited the expression of the aforementioned inflammatory factors (i). Figure 5 d–i).

[0069] The above experimental results indicate that Bacteroides fragilis can inhibit the activation of retinal inflammatory pathways and the expression of inflammatory mediators in glaucoma model mice.

[0070] The above results indicate that Bacteroides fragilis can be used as a optic nerve protectant for the treatment of glaucoma or optic nerve damage.

Claims

1. Use of Bacteroides fragilis, having accession number ATCC 25285, for the preparation of a medicament for treating glaucoma.

2. Use according to claim 1, characterized in that The medicament delivers Bacteroides fragilis to the gastrointestinal tract.

3. Use according to claim 1, characterized in that The medicament improves visual function or inhibits the expression of inflammatory mediators, such as TNF-α, IL-1β and IL-6.

4. Use according to claim 1, characterized in that The medicament further comprises an excipient, encapsulates Bacteroides fragilis, and adheres to the mucosa.

Images