Application of clostridium praeparatum and / or ackermann mucilaophilum

Abstract

The invention relates to the technical field of microorganisms, in particular to an application of clostridium praeparatum and / or ackermann mucilaophilum. The invention provides an application of clostridium praeckii and / or Ackermann mucilaophilum in inhibition of inflammatory response and neovascularization after body injury, and provides a non-drug and probiotic biological treatment method based on clostridium praeckii and Ackermann mucilaophilum, and the application of clostridium praeckii and Ackermann mucilaophilum in inhibition of inflammatory response and neovascularization after body injury. The invention aims to provide a new solution for inflammatory response and abnormal angiogenesis problems in body injury (such as retinopathy, retinal detachment and the like).

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to the application of Faecalibacterium prausnitzii and / or Akkermansia muciniphila. Background Art

[0002] Retinal inflammatory and neovascular diseases, such as diabetic retinopathy and wet age-related macular degeneration, are one of the main causes of vision loss. Existing treatment methods mostly rely on intraocular drug interventions (such as anti-vascular endothelial growth factor (VEGF) drugs: including intravitreal injection of monoclonal antibodies (ranibizumab, bucizumab), fusion proteins (aflibercept, bevacizumab), and newly launched dual-target drugs (farximab), laser photocoagulation or surgical treatment. Although anti-VEGF treatment can stabilize vision and even improve visual function, 30-50% of patients still have no response or very poor response to anti-VEGF treatment. In addition, these methods have problems such as high cost, large side effects, and poor patient compliance.

[0003] Research on the intestinal flora and human health has revealed that microorganisms in the body are an important part of our ecosystem, supporting many physiological functions, such as being essential for the digestion of food and the absorption of nutrients, helping to train the immune system to distinguish between pathogens and harmless entities, and reducing the risk of autoimmune diseases and allergies. Maintaining a diverse and balanced microbiome is essential for human health. Gut dysbiosis is implicated in a range of diseases, including diabetes, autoimmune diseases, neurodegenerative diseases (including retinal degeneration), cardiovascular disease, and cancer. Mechanistically, dysbiosis can lead to intestinal barrier dysfunction, the release of microbial metabolites (such as short-chain fatty acids (SCFAs), trimethylamine N-oxide), pathogens, and gas-associated molecular patterns (DAMPs), and ultimately inflammatory disorders and tissue damage.

[0004] Akkermansia mucinensis and Faecalibacterium prausnitzii are both known as the new generation of probiotics. They can not only maintain intestinal health and promote the balance of flora, but also regulate metabolism and immune response, and prevent and treat a variety of chronic diseases. Akkermansia mucinensis has also been reported to be associated with a variety of neurodegenerative diseases and can improve amyotrophic lateral sclerosis and Alzheimer's disease. Oral Akkermansia mucinensis or probiotic products containing Akkermansia mucinensis are expected to become a new method for treating metabolic diseases such as obesity and diabetes. Faecalibacterium prausnitzii is one of the most important bacteria in the human intestinal flora, accounting for 5-15% of the total number of bacteria detected in fecal samples of healthy people. It is one of the important producers of butyric acid, which helps to maintain the stability of the intestinal environment, has good anti-inflammatory effects, can maintain the activity of bacterial enzymes, and protect the digestive system from intestinal pathogens.

[0005] The prior art has the following disadvantages:

[0006] Invasive: The treatment method uses surgery or intravitreal injection, and the surgical risks and drug side effects are high.

[0007] Safety is not high enough: It utilizes the intestinal microorganisms that naturally exist in the human body, which has good safety and strong patient tolerance.

[0008] Limitations of effect: 30-50% of patients have no response or very poor response to anti-VEGF treatment.

[0009] Inconvenient to use: Every time you take the medicine, you need to go to the hospital, which is very inconvenient for patients who live in remote areas or have difficulty in moving. Summary of the invention

[0010] In view of this, the present invention provides the use of Faecalibacterium prausnitzii and / or Akkermansia muciniphila. The present invention provides the use of Faecalibacterium prausnitzii and / or Akkermansia muciniphila in inhibiting inflammatory response and neovascularization after body injury. The non-drug, probiotic biotherapy method based on Faecalibacterium prausnitzii and Akkermansia muciniphila provided by the present invention aims to provide a new solution to the problem of inflammatory response and abnormal angiogenesis in body injury (such as retinopathy, retinal detachment, etc.).

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides the use of Faecalibacterium prausnitzii and / or Akkermansia muciniphila in any of the following items:

[0013] (I) preparing products for inhibiting inflammatory responses caused by damage to the body; and / or

[0014] (II) preparing products for inhibiting angiogenesis.

[0015] In some specific embodiments of the present invention, the body damage includes one or more of light damage, retinal inflammation, neovascular disease or retinal degeneration.

[0016] In some specific embodiments of the present invention, the retinal degenerative lesions include one or more of age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa or retinal damage.

[0017] In some specific embodiments of the present invention, the Faecalibacterium prausnitzii and / or Akkermansia muciniphila are used in the form of one or more of live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

[0018] In some specific embodiments of the present invention, the inhibition of inflammatory response caused by body damage includes reducing the number of inflammatory cells.

[0019] In some specific embodiments of the present invention, the inhibition of angiogenesis comprises inhibiting the area of ​​neovascularization.

[0020] The present invention also provides a microbial agent, including Faecalibacterium prausnitzii, Akkermansia muciniphila or their culture solution.

[0021] In some specific embodiments of the present invention, the dosage form of the microbial agent includes any one of a suspension, a suppository, a tablet, a capsule, a soft capsule, a preformed injection, an effervescent tablet and a gel.

[0022] In some specific embodiments of the present invention, the culture fluid comprises a pre-platform supernatant of Faecalibacterium prausnitzii and / or Akkermansia muciniphila.

[0023] The present invention also provides a method for inhibiting retinal inflammatory response and neovascularization for non-disease treatment purposes, comprising administering any of the following to a receptor:

[0024] (I), Faecalibacterium prausnitzii and / or Akkermansia muciniphila; and / or

[0025] (II) the microbial agent.

[0026] In some specific embodiments of the present invention, the administration method includes oral administration.

[0027] In some specific embodiments of the present invention, the administered dose includes 2.5×10 10 ~10 11 CFU / day / kg.

[0028] In some specific embodiments of the present invention, the administered dose includes 5×10 8 ~10 9 CFU / 200 μL / 20 g mouse.

[0029] In some specific embodiments of the invention, the administration cycle comprises ≥ 2 weeks.

[0030] In some specific embodiments of the present invention, the volume of the initial culture supernatant of the vitreous cavity injection of Faecalibacterium prausnitzii platform includes 1 μL~20 μL (mouse, rat, rabbit).

[0031] The method and microbial agent provided by the present invention have the following beneficial effects:

[0032] Non-invasive: Oral administration is possible, avoiding the surgical risks and drug side effects of traditional treatments.

[0033] High safety: It utilizes the naturally existing intestinal microorganisms in the human body, which is safe and well tolerated by patients.

[0034] Wide-ranging effects: It not only targets retinopathy itself, but also improves overall health by regulating the systemic immune and metabolic systems.

[0035] Low cost: Compared with the high cost of drugs, the production cost of microbial preparations is low, which helps to reduce the financial burden on patients.

[0036] Convenient and fast: Existing intravitreal injection and surgical methods require patients to go to the hospital for treatment regularly. Patients can carry medication and take it orally anytime and anywhere, which is of great convenience for patients with limited mobility and in remote areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0038] Figure 1 The relative abundance of Faecalibacterium prausnitzii and Akkermansia muciniphila in the retina, RPE / choroid, and feces after retinal injury; Figure A shows a fundus photograph of a mouse eye taken immediately after retinal laser injury, and Figure B shows a Venn diagram analyzing the common bacteria in the three tissues at three time points, R: retina group; C: RPE / choroid group; F: feces group;

[0039] Figure 2The figure shows the improvement effect of intravitreal injection of Faecalibacterium prausnitzii culture supernatant in the early and late stages of the proliferation platform on angiogenesis and inflammatory cells in mice with laser-induced retinal injury; A shows the experimental design diagram. After laser-induced retinal injury, the culture supernatant of bacteria was injected into the vitreous cavity immediately, and the eyeballs were collected 7 days later for RPE / choroid flat sheet and retinal flat sheet staining; B shows confocal images showing RPE / choroid flat sheet Collagen-1 (red), CD31 (green) and DAPI staining; CNV: simple laser-induced retinal injury; CM+CNV: blank culture medium group; Pre+CNV: early stage group; Late+CNV: late stage group; C shows the area of ​​collagen-1 and CD31 in the lesion on the seventh day after laser-induced retinal injury in different treatment groups. D. Confocal images showing retinal flat mounts stained with IBA-1 (red) and DAPI; E shows the number of IBA+ microglia per mm2 in the retinal equatorial region and lesion area on the seventh day after laser-induced retinal injury in different treatment groups; expressed as mean±SD, one-way ANOVA, n=40 / group, * P<0.05, **P<0.01, *** P<0.001;

[0040] Figure 3 The figures show the improvement effect of oral administration of single bacterial solution and mixed bacterial solution of Faecalibacterium prausnitzii and Akkermansia muciniphila on angiogenesis and inflammatory cells in laser-induced retinal injury; wherein, A shows the experimental design diagram; laser-induced retinal injury was induced after oral administration of single bacterial solution and mixed bacterial solution for 14 consecutive days, and the eyeballs were collected for RPE / choroid flat mount and retinal flat mount staining 7 days later; B shows confocal images showing RPE / choroid flat mount IBA-1 (red), CD31 (green) and DAPI staining (left), and the bar graph shows the area of ​​CD31 in the lesion and the number of IBA+ microglia on the seventh day after laser-induced retinal injury in different treatment groups (right); C shows confocal images showing IBA-1 (red) in retinal flat mount (left), and the bar graph shows the number of IBA+ microglia per unit area on the seventh day after laser-induced retinal injury in different treatment groups (right);

[0041] Figure 4 The results show the in vitro verification of the effect of Clostridium prausnitzii culture supernatant on the M1 / M2 polarization of peritoneal macrophages; A shows the flow cytometry gating of peritoneal macrophages; B shows the co-culture of low-concentration Clostridium prausnitzii culture fluid, high-concentration Clostridium prausnitzii culture fluid and peritoneal macrophages, and the expression of genes such as VEGF under no stimulation, LPS+IFN-γ stimulation, and IL-4 stimulation; expressed as mean±SD, one-way ANOVA, *P<0.05, **P<0.01, *** P<0.001, Blank=cell culture medium; FP hi=bacteria:cells=10:1; FP low=bacteria:cells=1:1. DETAILED DESCRIPTION

[0042] The present invention discloses the application of Faecalibacterium prausnitzii and / or Akkermansia muciniphila. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] The purpose of the present invention is to provide a new method for inhibiting retinal inflammatory response and neovascularization by using a mixed bacterial liquid of Faecalibacterium prausnitzii DSM 17677 and Akkermansia muciniphila ATCC BAA-835. The method regulates the intestinal microecological balance by oral probiotics, thereby affecting the local microenvironment of the retina, thereby achieving the purpose of treating and preventing retinopathy.

[0044] The present invention provides a new method for inhibiting retinal inflammatory response and neovascularization, using Faecalibacterium prausnitzii and Akkermansia muciniphila as active ingredients, regulating the balance of intestinal microecology through oral administration, non-invasive administration, and oral administration at any time and place, with high patient compliance.

[0045] The present invention uses key microorganisms screened out in the process of retinal damage to prepare a mixed microbial preparation to regulate local inflammatory reactions and the formation of new blood vessels in the damaged area, providing a new solution to the inflammatory reactions and abnormal angiogenesis problems of retinal inflammatory and neovascular diseases, retinal degenerative diseases such as age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, retinal damage (trauma, retinal detachment), etc.

[0046] The new method for inhibiting inflammatory response and angiogenesis caused by body damage is applicable to diseases including retinal inflammatory and neovascular diseases, retinal degenerative lesions such as age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, inflammation and angiogenesis caused by retinal damage (trauma, retinal detachment), etc.

[0047] The oral microbial dosage range includes 10 8 -10 9 CFU / 200 μL / 20 g mouse.

[0048] In some specific embodiments of the present invention, the microbial agent should include oral administration of 2.5×10 10 ~10 11 CFU / day / kg.

[0049] The oral administration of the microorganisms can be continuous or intermittent, and the time period can be from 2 weeks to long term.

[0050] The volume of the initial culture supernatant of the intravitreal injection of Faecalibacterium prausnitzii platform ranges from 1 μL to 20 μL (for mice, rats, and rabbits).

[0051] The microbial preparations for regulating local inflammatory response and angiogenesis in injured areas include Clostridium prausnitzii bacterial liquid and pre-platinum supernatant, Akkermansia muciniphila bacterial liquid and pre-platinum supernatant, and a mixed microbial preparation of Clostridium prausnitzii bacterial liquid and Akkermansia muciniphila bacterial liquid.

[0052] Unless otherwise specified, the raw materials and reagents used in the application of Faecalibacterium prausnitzii and / or Akkermansia muciniphila provided by the present invention can be purchased from the market.

[0053] The present invention will be further described below in conjunction with embodiments:

[0054] Example 1 Changes in the relative abundance of Faecalibacterium prausnitzii and Akkermansia muciniphila in the retina, RPE / choroid, and feces after retinal injury

[0055] 1. Animals

[0056] C57BL / 6J mice (6-8 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and housed in a standard experimental facility with a 12-h light / dark cycle and free access to food and water. The procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the protocol was approved by the Animal Welfare Ethics Committee of the Aier Eye Research Institute (reference number: AEI20230048).

[0057] 2. Induction of Retinal Damage

[0058] We used a retinal injury model using retinal laser burns. Retinal laser injuries were produced in mice using a previously described protocol. Briefly, mice were anesthetized with isoflurane, and pupils were dilated using drops of 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceutical Co., Ltd., Osaka, Japan). The ocular surface was moistened with sodium carboxymethylcellulose (Allergan Pharmaceuticals Ltd., Dublin, Ireland). Four laser burns were made around the optic nerve head using a Topcon photocoagulator (Topcon, Tokyo, Japan, wavelength 532 nm, power 100 mV, spot size 60 μm).

[0059] 3. Tissue Collection

[0060] Feces, retina, and RPE / choroid were collected from laser-injured mice, 1 h and 24 h (n = 8) under strict aseptic conditions in a Class II laboratory clean bench. After euthanasia, the contents of the rectum and colon were transferred to cryovials, snap-frozen in liquid nitrogen, and stored at −80°C for further analysis. Eyeballs were removed under aseptic conditions in a Class II laboratory clean bench. After enucleation, the eyeballs were rinsed with 10% iodine for 3 min, then thoroughly cleaned with 70% ethanol and rinsed with sterile saline. The surface of the eyeballs was sampled with a sterile cotton swab for 16S rRNA analysis as a procedural control. Eyeballs were dissected using autoclaved instruments. The anterior segment was removed, and the retina and RPE / choroid were separated with sterile ophthalmic microscissors, placed in nuclease-free EP tubes, snap-frozen in liquid nitrogen, and stored at −80°C for subsequent 16S rRNA sequencing. In addition, 16S rRNA sequencing was performed on cotton swabs used to wipe the surface of the eyeball after cleaning as an environmental control, while sterile swabs that did not touch any surface served as a negative control.

[0061] 4. DNA Extraction and Polymerase Chain Reaction Amplification

[0062] DNA was extracted using the TIANamp Stool DNA Kit (Qiagen, China) according to the manufacturer's instructions. The 16S rDNA hypervariable V3-V4 region was amplified using primers 341F: CCTACGGGNGGCWGCAG (SEQ ID NO: 1) and 806R: GGACTACHVGGGTATCTAAT (SEQ ID NO: 2). Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Silicon Valley, USA) according to the manufacturer's instructions. DNA was quantified using QuantiFluorTM-ST Micro-Fluor (Promega, USA). Sequencing libraries were constructed using the Nextera XTDNA Sample Preparation Kit (Illumina, USA). The constructed libraries were quantified using Qubit and Q-PCR. After the libraries were qualified, PE250 paired-end sequencing was performed on the Illumina NovaSeq 6000 sequencing platform.

[0063] 5. Bioinformatics and Statistical Analysis of 16S-rDNA Sequencing Data

[0064] Reads containing more than 10% unknown nucleotides (N) and reads with a base content of less than 80% with a q value greater than 20 were removed. Paired end clean reads were merged into raw tags using FLSAH (V.1.2.11) with a minimum overlap of 10 bp and a mismatch error rate of 2%. Noise sequences in Raw Tags were filtered using QIIME (V.1.9.1) to generate high-quality clean tags. Amplicon sequence variants (ASVs) were generated by DADA2. Briefly, the raw sequence data were demultiplexed using the demux plugin, followed by primer cutting using the cutadapt plugin. The sequences were then quality filtered, denoised, merged, and chimeras removed using the DADA2 plugin. Species annotation was performed using QIIME2 software. The annotation databases for 16S, 18S, and ITS were Greengenes_13_8, SIVLA_138, and Unite databases, respectively. Abundance statistics for each taxonomy were generated using a Perl script and visualized using Scalable Vector Graphics.

[0065] The results are as follows Figure 1 As shown in Table 1: We have successfully established laser-induced classical choroidal neovascularization (CNV), with four laser spots evenly distributed at 1-2 PD away from the optic disc (A); Faecalibacterium prausnitzii and Akkermansia muciniphila are common bacteria in the retina, RPE / choroid, and feces. One hour after retinal laser injury, the relative abundance of Faecalibacterium prausnitzii in the retina increased rapidly, and then significantly decreased 24 hours after retinal laser injury; 24 hours after retinal laser injury, the relative abundance of Akkermansia muciniphila in the feces increased significantly; therefore, we screened these two bacteria as key bacteria for body injury repair treatment.

[0066] Table 1 Common bacteria in three tissues at three time points

[0067]

[0068] Example 2 Effect of intravitreal injection of culture supernatants of Faecalibacterium prausnitzii in the early and late growth stages on angiogenesis and inflammatory cells in mice with laser-induced retinal injury

[0069] 1. Strain culture

[0070] Bacterial storage conditions: Slant cultures and freeze-dried cultures should be stored at 2~8℃.

[0071] Faecalibacterium prausnitzii:

[0072] Culture conditions:

[0073] 1. Culture medium: Improved fortified Clostridium broth culture medium (it is recommended to purchase finished culture medium on the market)

[0074] Medium formula (per liter):

[0075]

[0076] Instructions for use: Weigh 37.5 g of this product into 1 L of distilled water or deionized water, boil for more than 1 minute, divide into test tubes, sterilize at 121℃ under high pressure for 15 minutes, and set aside.

[0077] 2. Culture temperature: 37℃, culture under anaerobic environment.

[0078] 3. Cultivation time: 48~72h.

[0079] Akkermansia muciniphila:

[0080] Culture conditions:

[0081] 1. Culture medium: Thioglycollate liquid medium.

[0082] 2. Culture medium composition:

[0083]

[0084] 121℃, 15min, sterilize and set aside.

[0085] 3. Culture temperature: 37℃, anaerobic.

[0086] 4. Cultivation time: 5~7 days.

[0087] 2. Preparation of pre- and post-incubation supernatants of Clostridium prausnitzii DSM 17677

[0088] (a) Collection of supernatant in the early stage of platform (logarithmic growth phase)

[0089] Collection time: Determine the start and end time of the logarithmic growth phase by monitoring the growth curve of the strain. Collect the supernatant in the middle of the logarithmic growth phase (18-24 hours after inoculation of 5x10^7 CFU bacteria), when the strain grows vigorously, metabolizes actively, and the supernatant contains more components.

[0090] Centrifugation: Transfer the culture medium to a sterile centrifuge tube and centrifuge at 5000 r / min for 5 minutes. After centrifugation, carefully transfer the supernatant to a new sterile container to avoid mixing with cell debris.

[0091] (b) Collection of supernatant in the late platform phase (stable growth phase)

[0092] Collection time: After the strain enters the stable growth phase, observe the changes in its growth rate and metabolic activity. Collect the supernatant in the late stable growth phase (30 hours after inoculation), when the growth rate of the strain may slow down, but the accumulation of metabolites may reach a peak.

[0093] Centrifugation: Separation was performed using the same centrifugation method and conditions as in the previous stage of the platform, 5000 r / min, 5 min. Ensure the purity of the supernatant and avoid mixing with any cell debris or culture medium components.

[0094] (c) Subsequent processing and storage

[0095] Filtration and sterilization: The collected supernatant was filtered using a 23 μm filter to remove possible tiny particles and impurities.

[0096] (d) Storage: The supernatant was stored at -20°C to ensure its stability and usability.

[0097] 3. Intravitreal injection of bacterial culture supernatant

[0098] The laser burn retinal model was established in C57BL / 6J mice (6-8 weeks old) as in Example 1. After the mouse CNV model was established, intravitreal injection was performed immediately, and 1 μl of the pre-platform and late-platform bacterial culture supernatant of Faecalibacterium prausnitzii was injected respectively. The sham operation group was injected with blank bacterial culture medium, and the negative control group was only established with the laser burn retinal model without intravitreal injection.

[0099] IV. Immunofluorescence staining

[0100] 7 days after laser injury, the eyeballs were collected and fixed in 2% paraformaldehyde at room temperature for 2 h. After removing the cornea and lens, the retina and RPE / choroid-sclera were separated for retinal and RPE / choroidal flat mount staining. Subsequently, the flat mount was blocked with 10% goat serum and 2% BSA and permeabilized with 0.1% Tritonx-100 at room temperature for 2 h. The samples were incubated with rabbit anti-mouse collagen-1 (1:200, cat: AB34710, abcam); CD31 Monoclonal Antibody (ER-MP12) (1:200, cat: MA1-40074, Invitrogen); rabbit anti-mouse IBA-1 (1:200, cat: PAK6839, Wako) at 4°C overnight, and then incubated with goat anti-rabbit IgG (1:500, cat: 2616076, Invitrogen), goat anti-rat IgG (1:500, cat: A-11006, Invitrogen) and DAPI (1:500, cat: C0060, Solebro) at room temperature for 2 h. The samples were examined by confocal microscopy (Zeiss, Braunschweig, Germany).

[0101] The results are as follows Figure 2 , as shown in Table 2 and Table 3:

[0102] In order to understand the effect of Faecalibacterium prausnitzii on angiogenesis and inflammatory cells caused by body injury, we detected the fibrosis area and angiogenesis area of ​​RPE / choroid samples 7 days after laser injury ( Figure 2 A), we found that the supernatant of the pre-platform bacterial culture can reduce the fibrosis area and neovascularization area of ​​CNV lesions ( Figure 2 B~2D). In addition, in retinal flat mounts, the supernatant of pre-platform bacterial culture reduced the infiltration of microglia in the lesion area and the peripheral area in the retinal flat mounts ( Figure 2 E).

[0103] Table 2 The area of ​​collagen-1, fibrosis and angiogenesis in the lesions on the seventh day after laser-induced retinal injury in different treatment groups

[0104]

[0105] Table 3 Retinal equatorial area and lesion area on the seventh day after laser-induced retinal injury in different treatment groups (unit: mm) 2 Chinese IBA + The number of microglia

[0106]

[0107] Example 3 Effects of intragastric administration of Faecalibacterium prausnitzii and Akkermansia muciniphila alone or in combination on angiogenesis and inflammatory cells in laser-induced retinal injury

[0108] 1. Oral gavage

[0109] C57BL / 6J mice (6-8 weeks old) were gavaged with live Clostridium prausnitzii (5x10 10 CFU / ml) (purchased from Mingzhou Biotechnology DSM 17677); live bacterial liquid of Akkermansia muciniphila alone (5x10 10 CFU / ml) (purchased from ATCC BAA-835); mixed live bacterial solution of Faecalibacterium prausnitzii and Akkermansia muciniphila (2.5x10 10 CFU / day / kg); mixed live bacterial solution of Faecalibacterium prausnitzii and Akkermansia muciniphila (5x10 10 CFU / day / kg); sham operation group (PBS), gavage for 14 days, laser induced retinal injury on the 15th day (the method is the same as in Example 1), and the eyeballs were collected on the 22nd day. Immunofluorescence was used to detect the area of ​​RPE / choroidal angiogenesis and the changes in RPE / choroid and retinal microglia (the method is the same as in Example 2).

[0110] The results are as follows Figure 3 , as shown in Tables 4 to 9:

[0111] To investigate the effects of non-surgical administration of Faecalibacterium prausnitzii and Akkermansia muciniphila on angiogenesis and inflammatory cells in a laser-injured mouse model, we performed continuous intragastric administration of single bacterial solution and mixed bacterial solution for 14 days ( Figure 3 A) It was found that oral administration of Faecalibacterium prausnitzii and Akkermansia muciniphila alone or in combination could significantly reduce the area of ​​RPE / choroidal neovascularization and the number of retinal IBA-1+ microglia per unit area (P<0.01), and high-concentration mixed bacterial solution (5x10 10 CFU / ml) group had a more significant effect (P<0.001) ( Figure 3 B-3C); in addition, both low-concentration and high-concentration mixed bacterial solutions could significantly reduce the number of IBA-1+ microglia per unit area in RPE / choroid (P<0.05).

[0112] Table 4 The area of ​​CD31 lesions on the seventh day after laser-induced retinal injury in different treatment groups

[0113]

[0114] Table 5 Statistical analysis results of the CD31 lesion area on the seventh day after laser-induced retinal injury in different treatment groups

[0115]

[0116] Table 6 The number of IBA+ microglia in the lesions on the seventh day after laser-induced retinal injury in different treatment groups

[0117]

[0118] Table 7 Statistical analysis results of the number of IBA+ microglia in the lesions on the seventh day after laser-induced retinal injury in different treatment groups

[0119]

[0120] Table 8 The number of IBA+ microglia per unit area on the seventh day after laser-induced retinal injury in different treatment groups

[0121]

[0122] Table 9 Statistical analysis results of the number of IBA+ microglia per unit area on the seventh day after laser-induced retinal injury in different treatment groups

[0123]

[0124] Example 4 In vitro verification of the effect of Faecalibacterium prausnitzii on M1 / M2 polarization of peritoneal macrophages

[0125] 1. Flow cytometry to test the efficiency of mouse peritoneal macrophage extraction

[0126] C57BL / 6J mice (6-8 weeks old) were injected with 2 ml / mouse 3% thioglycolate broth (Sigma) in the lower abdomen. Peritoneal macrophages were obtained 3 days later. After lysis, 1x10^6 cells were seeded per well in a 12-well plate and incubated at 37°C. After 24 hours, the adherent cells were peritoneal macrophages. The extracted peritoneal macrophages were incubated with LD antibody (BD Biosciences, Cat: 564997) at room temperature for 15 min, and the samples were incubated with mouse FC receptor blocker (1:50, Cat: 551342) for 5 min. The samples were then incubated with a mixture of FITC conjugated anti-mouse F4 / 80 (BM8) (Cat: 100762, Biolegend, 1:100) and PerCP / Cyanine5.5 conjugated anti-mouse CD11b (Cat: 550993, BD Pharmingen, 1:100) for 30 min at 4°C. The samples were then fixed with 2% paraformaldehyde, washed, and resuspended in FACS buffer (Cat: 1029962; BD Biosciences, New Jersey), and detected by flow cytometry (BD FACSelesta, New Jersey).

[0127] 2. Co-culture of Clostridium prausnitzii with peritoneal macrophages and induction of M1 / M2 polarization of mouse peritoneal macrophages

[0128] Peritoneal macrophages that have adhered to the 12-well plate were co-cultured at a ratio of bacteria: cells = 10:1. M1 polarization stimulating factors (INF-γ (20 ng / ml) + LPS (100 ng / ml)) and M2 polarization stimulating factors (IL-4 (20 ng / ml)) were added after 6 hours, and the cells were harvested after 24 hours of culture. PCR was used to detect the VEGF gene to study the effect of Faecalibacterium prausnitzii on the angiogenesis of peritoneal macrophages.

[0129] The results are as follows Figure 4 , as shown in Table 10 to Table 15:

[0130] PCR results showed that compared with the control group, the bacterial solution could inhibit the expression of Vegf under no stimulation, INF-γ+LPS stimulation, and IL-4 stimulation.

[0131] The mechanism by which factors stimulate peritoneal macrophages is as follows:

[0132] The role of IFN-γ (interferon-γ):

[0133] IFN-γ is mainly produced by activated T lymphocytes and NK cells. It is an important cytokine that can activate macrophages and enhance their functions. When IFN-γ binds to receptors on the surface of peritoneal macrophages, it triggers a series of intracellular signal transduction pathways. For example, it can activate the JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway, phosphorylate STAT protein, and then enter the cell nucleus to regulate the expression of a series of genes.

[0134] These regulated genes include genes related to antigen presentation, such as MHCⅡ (major histocompatibility complex class II) genes. MHCⅡ molecules play a key role in the antigen presentation process. They can present exogenous antigen peptides to CD4⁺T lymphocytes, thereby initiating adaptive immune responses. After IFN-γ stimulation, the expression of MHCⅡ molecules on the surface of macrophages increases, enhancing their antigen presentation ability.

[0135] Role of LPS (lipopolysaccharide):

[0136] LPS is the main component of the cell wall of Gram-negative bacteria and is a typical pathogen-associated molecular pattern (PAMP). When peritoneal macrophages recognize LPS, they initiate an immune response through pattern recognition receptors (PRRs) on their surface, such as Toll-like receptor 4 (TLR4). After LPS binds to TLR4, it recruits a series of adaptor proteins, such as MyD88 (myeloid differentiation factor 88).

[0137] MyD88 can activate downstream signaling pathways, including the NF-κB (nuclear factor-κB) signaling pathway. NF-κB is an important transcription factor that binds to the inhibitory protein IκB in resting cells and is in an inactive state. When stimulated, IκB is phosphorylated and degraded, and NF-κB is released and enters the cell nucleus, initiating the expression of a variety of inflammation-related genes, such as TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β) and other cytokine genes. The secretion of these cytokines can attract more immune cells to the site of inflammation and participate in the inflammatory response and the clearance of pathogens.

[0138] Synergistic effect of IFN-γ and LPS:

[0139] When IFN-γ and LPS act on peritoneal macrophages at the same time, a synergistic effect occurs. IFN-γ can upregulate the expression of TLR4 on the surface of macrophages, making macrophages more sensitive to LPS. At the same time, the signaling pathway activated by LPS can also enhance the signaling pathway mediated by IFN-γ. For example, the activation of the NF-κB signaling pathway can promote the expression of genes related to the JAK-STAT signaling pathway. The activation of this signaling pathway will cause the phenotype of macrophages to differentiate toward the M1 type. This synergistic effect makes the activation of macrophages more complete, manifested as stronger phagocytic ability, higher antigen presentation efficiency and more secretion of inflammatory factors.

[0140] The mechanism of IL-4 stimulation of peritoneal macrophages:

[0141] IL-4 is mainly produced by the Th2 cell subset in activated T lymphocytes. When IL-4 binds to receptors on the surface of peritoneal macrophages, it activates the intracellular signal transduction pathway. It can activate the PI3K-Akt (phosphatidylinositol 3-kinase-protein kinase B) signaling pathway and phosphorylate the Akt protein.

[0142] Activation of this signaling pathway causes a change in the phenotype of macrophages, from classically activated macrophages (M1 type) to alternatively activated macrophages (M2 type). M2 macrophages have different functional characteristics, and the cytokine spectrum they secrete is different from that of M1 macrophages. M2 macrophages can secrete more cytokines such as IL-10 (interleukin-10) and TGF-β (transforming growth factor-β). IL-10 has anti-inflammatory effects and can inhibit inflammatory responses; TGF-β plays an important role in tissue repair and fibrosis.

[0143] We used two factors to induce peritoneal macrophages to differentiate into M1 and M2 types, creating an environment of cell inflammation and damage, and observed the effect of bacterial fluid on the expression of angiogenesis (VEGF) after peritoneal macrophages differentiated into M1 and M2 macrophages, so as to determine the effect of bacterial fluid on angiogenesis in vitro. Our results showed that bacterial fluid can inhibit the expression of angiogenesis factors caused by inflammatory conditions in vitro.

[0144] Table 10 Expression of VEGF and other genes in peritoneal macrophages co-cultured with low-concentration Clostridium prausnitzii and high-concentration Clostridium prausnitzii without stimulation

[0145]

[0146] Table 11 Statistical analysis results of the expression of genes such as VEGF in the absence of stimulation when low-concentration Clostridium prausnitzii and high-concentration Clostridium prausnitzii were co-cultured with peritoneal macrophages

[0147]

[0148] Table 12 Expression of VEGF and other genes in peritoneal macrophages co-cultured with low-concentration Clostridium prausnitzii and high-concentration Clostridium prausnitzii under LPS+IFN-γ stimulation

[0149]

[0150] Table 13 Statistical analysis results of the expression of genes such as VEGF in co-culture of low-concentration Clostridium prausnitzii and high-concentration Clostridium prausnitzii with peritoneal macrophages under LPS+IFN-γ stimulation

[0151]

[0152] Table 14 Expression of VEGF and other genes in peritoneal macrophages co-cultured with low-concentration Clostridium prausnitzii and high-concentration Clostridium prausnitzii under IL-4 stimulation

[0153]

[0154] Table 15 Statistical analysis results of the expression of genes such as VEGF under IL-4 stimulation in co-culture of peritoneal macrophages with low-concentration Clostridium prausnitzii and high-concentration Clostridium prausnitzii

[0155]

[0156] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Faecalibacterium prausnitzii and / or Akkermansia muciniphila in any of the following: (I) preparing products for inhibiting inflammatory responses caused by damage to the body; and / or (II) preparing products for inhibiting angiogenesis caused by body damage.

2. The use according to claim 1, characterized in that The body damage includes one or more of light damage, retinal inflammation, neovascular disease or retinal degenerative lesions.

3. The use according to claim 2, characterized in that The retinal degenerative lesions include one or more of age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa or retinal damage.

4. The use according to any one of claims 1 to 3, characterized in that The Faecalibacterium prausnitzii and / or Akkermansia muciniphila may be used in one or more forms including live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

5. A microbial agent, characterized in that: Including Faecalibacterium prausnitzii, Akkermansia muciniphila or their culture fluid.

6. The microbial agent according to claim 5, characterized in that The culture fluid includes the pre-platform supernatant of Faecalibacterium prausnitzii and / or Akkermansia muciniphila.

7. A method for inhibiting retinal inflammatory response and neovascularization for non-disease treatment purposes, characterized in that: This includes administering to a recipient any of the following: (I), Faecalibacterium prausnitzii and / or Akkermansia muciniphila; and / or (II) The microbial agent according to claim 5 or 6.

8. The method according to claim 7, characterized in that The administration method includes oral administration.

9. The method according to claim 7 or 8, characterized in that The dosages administered include 2.5×10 10 ~10 11 CFU / day / kg.

10. The method according to any one of claims 7 to 9, characterized in that: The dosing cycle comprises ≥ 2 weeks.

Images