Use of indole-3-lactic acid in reducing retinal or choroidal neovascularization and reducing retinal inflammation

Abstract

The application discloses application of indole-3-lactic acid in reducing retinal or choroidal neovascularization and reducing retinal inflammation, and belongs to the technical field of biological medicine. The application provides a new medical application of a microbial metabolite, indole-3-lactic acid, in particular, application of the microbial metabolite in preparation of a medicine for preventing and / or treating an eye disease related to choroidal neovascularization (CNV), and the effect is achieved by activating an aromatic hydrocarbon receptor channel, inhibiting inflammation and angiogenesis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of indole-3-lactic acid in reducing neovascularization of the retina or choroid and alleviating retinal inflammation. Background Technology

[0002] Choroidal neovascularization (CNV) is the main type of subretinal neovascularization (SRNV), primarily causing neovascular age-related macular degeneration (nAMD). As a common eye disease affecting vision, nAMD mainly affects the elderly. nAMD is an important subtype of AMD, causing severe central vision loss. Current treatments for AMD include anti-VEGF (vascular endothelial growth factor) drugs, which can significantly improve AMD and have become the first-line treatment. The principle behind AMD treatment is: VEGF is a signaling protein that, under normal circumstances, is responsible for promoting angiogenesis and maintaining vascular health. In patients with wet AMD, due to factors such as age, oxidative stress, and inflammation, retinal pigment epithelial cells experience ischemia and hypoxia, leading to VEGF overexpression. The core principle of anti-VEGF drugs is to "neutralize" or "block" excessive VEGF, preventing it from binding to receptors on vascular endothelial cells.

[0003] The specific role and mechanism of indole-3-lactic acid in CNV remain unclear. Aromatic hydrocarbon receptors are key transcription factors that sense environmental and endogenous signals, play a central role in immune regulation, and are potential receptors for various microbial metabolites. Summary of the Invention

[0004] The purpose of this invention is to provide a novel pharmaceutical use for indole-3-lactic acid (ILA), namely its use in the preparation of medicaments for improving and / or treating retinal and choroidal neovascularization diseases, including but not limited to age-related macular degeneration, retinopathy of prematurity, pathological neovascular high myopia, polypoid choroidal retinopathy, retinal vasculitis, and diabetic retinopathy with vision loss; particularly its use in the preparation of medicaments for the prevention and / or treatment of ocular diseases related to choroidal neovascularization (CNV), the effects of which are achieved by activating the aryl hydrocarbon receptor (AHR) pathway, inhibiting inflammation, and angiogenesis.

[0005] Technical solution Based on the above objectives, this invention reveals and confirms for the first time through in vivo and in vitro experiments that indole-3-lactic acid (ILA) can effectively inhibit CNV both in vivo and in vitro: intraperitoneal injection of ILA can significantly reduce the area of ​​laser-induced CNV lesions in mice and improve retinal structure; at the cellular level, ILA can directly inhibit the tube-forming ability of human retinal microvascular endothelial cells (HRMEC).

[0006] Indole-3-lactic acid (ILA) works by inhibiting inflammation and angiogenesis-related factors: ILA treatment significantly downregulates pro-inflammatory factors (such as...) in the retina and HRMEC cells of CNV mice. IL-6, TNF-α, IL-1β, Spp1, B2m, C3 ) and angiogenic factors (such as Fgf2 / FGF2 The expression of ).

[0007] The core mechanism of action of indole-3-lactic acid (ILA) depends on the activation of the AHR pathway: ILA inhibits the release and migration of inflammatory factors in microglia (BV2) stimulated by lipopolysaccharide (LPS), an effect that can be completely reversed by the AHR-specific inhibitor CH223191. The inhibitory effect of ILA on the tube-forming ability of HRMEC cells can also be reversed by CH223191. This indicates that ILA, acting as a ligand for AHR, activates this pathway, thereby inhibiting neuroinflammation (microglia) and angiogenesis (endothelial cells), ultimately improving CNV.

[0008] Based on the above findings, this invention proposes that indole-3-lactic acid (ILA), its pharmaceutically acceptable salt, or its precursors can be used to prepare drugs that regulate inflammatory responses and / or inhibit angiogenesis by activating the AHR pathway, said drugs for the prevention and / or treatment of choroidal neovascularization diseases.

[0009] This invention provides the use of indole-3-lactic acid in the preparation of medicaments for improving and / or treating retinal and choroidal neovascularization diseases, including but not limited to age-related macular degeneration, retinopathy of prematurity, pathological neovascular high myopia, polypoid choroidal retinopathy, retinal vasculitis, and visual impairment due to diabetic retinopathy.

[0010] In one embodiment of the invention, the drug is used for at least one of (a) to (g): (a) Reduces the area of ​​laser-induced CNV lesions in mice and improves retinal structure; (b) Inhibits the tube-forming ability of human retinal microvascular endothelial cells (HRMECs); (c) Downregulation of pro-inflammatory factors in CNV mouse retina and HRMEC cells IL-6, TNF-α, IL-1β, Spp1, B2m, C3 The level of expression; (d) Downregulation of pro-angiogenic factors Fgf2 / FGF2 The level of expression; (e) Inhibits the release and migration of inflammatory factors in microglia BV2 stimulated by lipopolysaccharide.

[0011] In one embodiment of the present invention, the drug contains indole-3-lactic acid as the active ingredient, and also contains a drug carrier and pharmaceutically acceptable excipients.

[0012] In one embodiment of the present invention, the drug further includes pharmaceutically acceptable pharmaceutical excipients; In one embodiment of the present invention, the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers: starch and sucrose; humectants: glycerol; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters: quaternary ammonium compounds; surfactants: polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red; lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients may also be added to the composition: flavoring agents and sweeteners.

[0013] In one embodiment of the present invention, the drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0014] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pellets, granules, injections, powder injections, or aerosols.

[0015] In one embodiment of the present invention, the indole-3-lactic acid has the chemical formula C3. 11 H 11 NO3 has the following structural formula: .

[0016] The present invention also provides a medicament for use in the treatment and / or relief of retinal and choroidal neovascularization diseases, wherein the retinal and choroidal neovascularization diseases include, but are not limited to, age-related macular degeneration, retinopathy of prematurity, pathological neovascular high myopia, polypoid choroidal retinopathy, retinal vasculitis, and visual impairment due to diabetic retinopathy. The drug is any pharmaceutically acceptable dosage form made with indole-3-lactic acid as the active ingredient and pharmaceutically acceptable excipients.

[0017] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pills, granules, injections, powder injections, or aerosols. In one embodiment of the present invention, the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers: starch and sucrose; humectants: glycerol; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters: quaternary ammonium compounds; surfactants: polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red; lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients may also be added to the composition: flavoring agents and sweeteners.

[0018] In one embodiment of the present invention, the indole-3-lactic acid has the chemical formula C3. 11 H 11 NO3 has the following structural formula: .

[0019] Beneficial effects 1. For the first time, indole-3-lactic acid (ILA) and its AHR pathway have been clearly linked to CNV treatment, providing a direct basis for developing therapies based on gut-eye axis regulation.

[0020] 2. Clear mechanism and synergistic effect of multiple targets: Indole-3-lactic acid ILA acts on both immune cells (microglia) and vascular endothelial cells. It plays a synergistic therapeutic role in two key links of inhibiting inflammation and angiogenesis through the AHR pathway, which may result in a more comprehensive therapeutic effect.

[0021] 3. High safety potential: Indole-3-lactic acid (ILA) is an endogenous microbial metabolite, which theoretically has better biocompatibility and may reduce side effects.

[0022] 4. Diverse applications: Indole-3-lactic acid (ILA) can be used as a single active ingredient, or in combination with other anti-VEGF drugs, or as an effector substance in probiotic therapy (such as AKK). Attached Figure Description

[0023] Figure 1 Indole-3-lactic acid (ILA) intervention in mice reduced the CNV area of ​​the retina and choroid and inhibited the activation of retinal microglia.

[0024] Figure 2Spermine treatment in CNV mice inhibited the expression of inflammation- and angiogenesis-related genes.

[0025] Figure 3 HRMECs treated with indole-3-lactic acid (ILA) inhibited the expression of inflammation- and angiogenesis-related genes and suppressed their tube formation.

[0026] Figure 4 Spermine inhibits the formation of HRMECs and the expression of inflammation-related genes.

[0027] Figure 5 Indole-3-lactic acid (ILA) inhibits inflammation in microglia by activating the anti-inflammatory response (AHR).

[0028] Figure 6 Indole-3-lactic acid (ILA) inhibits microglial migration and HRMEC tube formation by activating AHR. Detailed Implementation

[0029] The present invention is further illustrated by the following examples, but is not intended to limit the scope of the invention.

[0030] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0031] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0032] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0033] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0034] About the model: This invention uses a binocular laser photocoagulation model, which is related to retinal and choroidal neovascularization diseases, including one or more of the following: age-related macular degeneration, retinopathy of prematurity, pathological neovascular high myopia, polypoid choroidal retinopathy, retinal vasculitis, and diabetic retinopathy with vision loss.

[0035] The chemical formula of indole-3-lactic acid involved in the following examples is C 11 H 11 NO3, CAS number: 1821-52-9, C8H6N-CH2-CH(OH)-COOH, its structural formula is as follows: .

[0036] The experimental materials involved in the following embodiments are as follows: The instruments and equipment used in calculating the neovascularization area of ​​the retina and choroid in the following examples are as follows: upright fluorescence microscope, Zeiss, Germany.

[0037] The methods for euthanizing mice and removing eyeballs involved in the following examples are as follows: The mice were immobilized with pins. Simultaneously, cotton balls were inserted into 15 ml centrifuge tubes, and 500 µl of isoflurane was injected into the cotton balls to anesthetize the mice through inhalation. The mice were then euthanized by perfusion with physiological saline. Both eyeballs were harvested. Eyeballs prepared for slide preparation were placed in 4% (w / v) paraformaldehyde solution for subsequent retinal and choroidal smear staining. Eyeballs prepared for molecular verification were placed in physiological saline, and the retinas were immediately removed and placed in RZ solution (purchased from Tiangen Biotech (Beijing) Co., Ltd.) for subsequent qPCR experiments. In the quantitative experiments described below, each group had 3 biological replicates and 5 technical replicates, and the results were averaged.

[0038] The detection methods involved in the following embodiments are as follows: HE staining of the retina and IBA1 immunofluorescence staining Immunofluorescence staining of paraffin sections of the retina requires first embedding the eyeball in paraffin and sectioning it after it has been fully fixed in paraformaldehyde. After dewaxing in xylene and rehydration in a gradient of ethanol, the sections are subjected to high-pressure heat repair or protease repair to expose the masked antigen sites. Then, the sections are blocked with serum, incubated with specific primary antibodies (IBA1 antibody, GFAP antibody), labeled with fluorescent secondary antibodies, and counterstained with DAPI. Finally, the sections are mounted with an anti-fluorescence quencher and observed under a fluorescence microscope.

[0039] HRMEC's ​​management capabilities Indole-3-lactic acid (ILA) was used at a concentration of 20 μM to intervene in the growth of HRMEC cells to a density of 70%-80% over 48 h. Consumables used in the tube formation assay (24-well plates, pipette tips, sterile centrifuge tubes, etc.) were pre-chilled at -20°C. The aliquoted matrix gel (Vazyme) was placed in an ice box overnight at 4°C to thaw.

[0040] At the start of the experiment, the matrix gel was diluted to the required concentration using DMEM complete medium. 250 μl was added to each well of a 24-well plate, taking care to avoid air bubbles, and a ring of PBS was placed around the edge of the matrix gel. The plate was then incubated at 37°C for 45 min to allow gelation.

[0041] HRMECs treated with indole-3-lactic acid (ILA) and their control group (untreated HRMECs) had cell counts of 8 × 10⁻⁶. 4 -20×10 4 The cell suspension was added to the surface of the matrix gel at a volume of 500 μl per well.

[0042] The 24-well plate was placed in an incubator and cultured for 6-8 hours. A vascular network was observed, and images were taken using a Nikon microscope. The vascular pathways were analyzed using ImageJ software.

[0043] Detection of pro-inflammatory genes, inflammatory factors, and pro-angiogenic genes in the retina This method uses real-time quantitative PCR (qPCR) to quantitatively detect gene expression levels, and consists of three steps: 1. Total RNA extraction from cells: Tiangen Total RNA Extraction Kit, core steps: after lysing cells, add chloroform, centrifuge to separate the aqueous phase, add anhydrous ethanol to precipitate, purify by adsorption column, wash with protein removal solution and rinsing solution, and then elute RNA with RNase-free water.

[0044] 2. Novizan HiScript III RT SuperMix RNA Reverse Transcription (cDNA Synthesis) Kit, core steps: Incubate total RNA and gDNA removal mixture (42℃, 2 min); add reverse transcriptase mixture for reverse transcription (50℃, 15 min; 85℃, 5 sec); obtain cDNA and store at -20℃ for later use.

[0045] 3. qPCR gene expression quantification: Novizan ChamQ Universal SYBR Green qPCR Master Mix kit Instrument: Light Cycler 480 II (Roche); Detected genes: Pro-inflammatory genes: IL-6, TNF-α, IL-1β Spp1, B2m, C3 .

[0046] Internal control gene: ACTB (β-actin). Reaction program: 95℃ pre-denaturation for 30 sec; 40 cycles (95℃ for 5 sec, 60℃ for 10 sec). Data processing: Relative expression levels were calculated using the ΔΔCt method with ACTB as the internal control, with 3 biological replicates per group.

[0047] 4. Statistical analysis: Software: GraphPad Prism 9.0; Presentation: mean ± standard error (SEM); Test method: one-way ANOVA, P < 0.05 was considered statistically significant.

[0048] Example 1: Therapeutic effects of indole-3-lactic acid (ILA) and spermidine on a mouse CNV model The animals used in this experiment were 6-8 week old male C57BL / 6 mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The animal housing was provided by the Animal Facility of Wuxi Medical College, Jiangnan University. During the rearing process, the mice were provided with alternating light and dark lighting for 12 hours each day, with an ambient temperature of 20-26℃, and unlimited food and water. All animal research complied with the regulations of the American Academy of Visual Ophthalmology (ARVO) and the Animal Experiment Ethics Committee of Jiangnan University.

[0049] The specific steps are as follows: Blank control group: From day 0 to day 7, mice were injected intraperitoneally with PBS buffer at a volume of 200 μl daily. After one week, bilateral laser photocoagulation (CNV modeling) was performed. The mice were fasted on the day of modeling and continued to be injected intraperitoneally with PBS buffer for three consecutive days after modeling. ILA group: From day 0 to day 7, mice were injected intraperitoneally with ILA solution (prepared with PBS buffer) at a concentration of 20 mg / kg per day, with an injection volume of 200 μl. One week later, bilateral laser photocoagulation modeling (CNV modeling) was performed. The mice were fasted on the day of modeling and continued to be injected intraperitoneally with ILA for three consecutive days (20 mg / kg per day). Spermine group: Mice were fasted on the same day and underwent bilateral laser photocoagulation modeling (CNV modeling). Immediately after modeling, 20 μM sterile spermine solution (prepared with PBS buffer) was injected into the vitreous cavity of the eye, with an injection volume of 2 μl. Each group was sacrificed 3 days after modeling, and the retinas of both eyes were harvested for subsequent experiments.

[0050] The method for establishing the laser-induced CNV mouse model is as follows: Six- to eight-week-old male C57BL / 6 mice were anesthetized 5-10 minutes before the experiment by intraperitoneal injection of tribromoethanol (0.2 ml / 10 g body weight). The pupils were dilated with compound tropicamide eye drops (1 drop). Photocoagulation was performed using a 532 nm laser (Quantelmedical, VITRA, France) and a slit-lamp microscope (Haag-Streit, BQ900, Germany). Four photocoagulations were performed at two optic disc diameters around the optic disc, at the 3, 6, 9, and 12 o'clock positions, with the following parameters: energy 120 mW, exposure time 0.1 s, and spot diameter 0 nm. The presence of bubbles during photocoagulation was considered a successful result.

[0051] Characterization of modeling results: The choroid lining showed acute inflammation due to laser stimulation, resulting in the growth of new blood vessels. The Brush membrane was destroyed, and new blood vessels grew into the retina.

[0052] 2. Experimental Results (1) Phenotypic assessment: Mice were euthanized, and their eyeballs were harvested for retinal and choroidal smear preparation. Isolectin B4 (IB4) staining was used to visualize CNV lesions and to calculate their area.

[0053] The results show that ( Figure 1 ): The CNV areas of the retina and choroid in the blank control group were 6 μm. 2 (retina) and 5μm 2 (choroid); The CNV areas of the retina and choroid in the indole-3-lactic acid (ILA) treatment group were 2.5 μm. 2 (Retina), 3.5μm 2 (choroid); The CNV areas of the retina and choroid in the spermidine treatment group were 2 μm. 2 (Retina), 2.8μm 2 (Choroid).

[0054] It is evident that the CNV area of ​​mice treated with indole-3-lactic acid (ILA) and spermidine was significantly reduced after intervention. Furthermore, SAT1, a key enzyme in polyamine metabolism, was significantly upregulated when ILA was used to treat human colonic epithelial cells (HCE). This suggests that ILA may work synergistically with the host's polyamine metabolism to further inhibit CNV.

[0055] 3. Histological analysis: (1) Retinal HE staining showed: The indole-3-lactic acid (ILA) treatment group showed less damage to the interlaminar structure of the retina and reduced inflammatory cell infiltration.

[0056] Model group (blank control group) retina: Brush membrane structure ruptured, new blood vessels penetrated the choroid and grew into the retina, resulting in retinal discontinuity, accompanied by inflammatory cell infiltration, reduction of ganglion cells in some areas, and thinning of the nerve fiber layer.

[0057] The spermidine group's retina showed a relatively intact interlayer structure, no breakage in the outer nuclear layer, a minimal decrease in cell number, and a higher cell number in the nerve fiber layer compared to the model group.

[0058] (2) IBA1 immunofluorescence staining showed: The number of activated microglia in the retinal lesion area was reduced in the indole-3-lactic acid (ILA) treatment group.

[0059] Model group (blank control group) retina: A large number of microglia were observed to aggregate in the lesion area.

[0060] Retinal tissue of spermidine group: The number of IBA1+ cells was significantly reduced in the spermidine-treated group.

[0061] Example 2: Inhibitory effect of ILA on the expression of inflammation and angiogenesis-related genes 1. In vivo molecular verification: RNA and protein were extracted from mouse retinal tissue in Example 1 and detected by qPCR and Western Blot.

[0062] The results of indole-3-lactic acid (ILA) treatment in CNV mice inhibiting the expression of inflammation and angiogenesis-related genes are as follows: Figure 2 As shown in Table 1.

[0063] Table 1: Experimental Results

[0064] The results showed that pro-inflammatory genes (IOLs) in the retina of the indole-3-lactic acid (ILA) treatment group were significantly reduced. Spp1, B2m, C3 ) and pro-angiogenic genes ( Fgf2 The expression of ) was significantly downregulated at both the mRNA and protein levels.

[0065] 2. In vitro cell validation of the therapeutic effect of indole-3-lactic acid (ILA): Tube formation experiment using human retinal microvascular endothelial cells (HRMEC): (1) Experimental preparation: Indole-3-lactic acid (ILA) was used at a concentration of 20 μM to intervene in the growth of HRMEC cells to a density of 70%-80% for 48 h. Consumables used in the tube formation experiment (24-well plates, pipette tips, sterile centrifuge tubes, etc.) were pre-cooled in a -20℃ freezer in advance. The aliquoted matrix gel (Vazyme) was placed in an ice box overnight and placed in a 4℃ freezer to thaw overnight.

[0066] (2) At the beginning of the experiment, use DMEM complete medium to dilute the matrix gel to the required concentration, add 250 μl to each well of the 24-well plate, avoid generating air bubbles, and add a ring of PBS around the edge of the matrix gel. Incubate at 37°C for 45 min to allow it to gel.

[0067] (3) HRMECs treated with 20 μM indole-3-lactic acid (ILA) and their control group (untreated HRMECs) had cell counts of 14 × 10⁻⁶. 4 ~15×10 4 The cell suspension was added to the surface of the matrix gel at a volume of 500 μl per well.

[0068] (4) Place the 24-well plate in an incubator and incubate for 6-8 hours to observe the formation of a vascular network. Take pictures with a Nikon microscope and use ImageJ software to analyze the tube formation path.

[0069] Untreated HRMEC cells were used as a control group. After the experiment, the tube-forming ability and inflammatory factors of HRMEC cells were detected. The results showed that ( Figure 3 ): After 6 hours of treatment with indole-3-lactic acid (ILA), the tube-forming ability of HRMECs was significantly inhibited, with the number of tube-forming nodes decreasing by 51% after ILA treatment. Simultaneously, qPCR detection showed that HRMECs in the indole-3-lactic acid (ILA)-treated group... B2M and FGF2 Gene expression is downregulated.

[0070] 3. In vitro cell validation of the therapeutic effect of spermidine: Tube formation experiment using human retinal microvascular endothelial cells (HRMEC): (1) Experimental preparation: Spermidine was used at a concentration of 20 μM to treat HRMEC cells grown to a density of 70%-80% for 48 h. Consumables used in the tube formation experiment (24-well plates, pipette tips, sterile centrifuge tubes, etc.) were pre-cooled in a -20℃ freezer. Vazyme was aliquoted and placed in an ice box overnight in a 4℃ freezer to thaw.

[0071] (2) At the beginning of the experiment, use DMEM complete medium to dilute the matrix gel to the required concentration, add 250 μl to each well of the 24-well plate, avoid generating air bubbles, and add a ring of PBS around the edge of the matrix gel. Incubate at 37°C for 45 min to allow it to gel.

[0072] (3) HRMECs treated with 20 μM spermidine and their control group (untreated HRMECs) had cell counts of 14 × 10⁻⁶. 4 ~15×10 4 The cell suspension was added to the surface of the matrix gel at a volume of 500 μl per well.

[0073] (4) Place the 24-well plate in an incubator and incubate for 6-8 hours to observe the formation of a vascular network. Take pictures with a Nikon microscope and use ImageJ software to analyze the tube formation path.

[0074] Untreated HRMEC cells were used as a control group. After the experiment, the tube-forming ability and inflammatory factors of HRMEC cells were detected. The results showed that ( Figure 4 ): The results showed that, under the same conditions, the therapeutic effect of indole-3-lactic acid (ILA) was better than that of spermidine.

[0075] 4. Microglial cell inflammation model Using the mouse microglia (BV2) cell line, control group, LPS stimulation group, LPS+ILA co-treatment group and CH223191 inhibitor group were established.

[0076] The specific method is as follows: BV2 cells were revived and passaged. When the cells grew to 70%-80%, experiments were conducted. LPS+ILA+CH223191 co-treatment group: Indole-3-lactic acid (ILA) and CH223191 (AHR antagonist, concentration 10 µM) were added to BV2 cells; the concentration of indole-3-lactic acid was 1000 µM, and the cells were cultured at 37℃ and 5% CO2 for 4 h. After culturing, LPS at a concentration of 100 ng / ml was added, and the cells were cultured at 37℃ and 5% CO2 for another 6 h. Blank control group: Untreated BV2 cells were cultured at 37°C and 5% CO2 for 10 h; LPS treatment group (LPS): BV2 cells were cultured at 37℃ and 5% CO2 for 4 h, then lipopolysaccharide (LPS, concentration 100 ng / mL) was added, and the cells were cultured at 37℃ and 5% CO2 for another 6 h. LPS+ILA co-treatment group: Indole-3-lactic acid (ILA) was added to BV2 cells; the concentration of indole-3-lactic acid was 1000µM, and the cells were cultured at 37℃ and 5% CO2 for 4 h. After 4 h of culture, LPS at a concentration of 100 ng / ml was added, and the cells were cultured at 37℃ and 5% CO2 for another 6 h. Six hours after LPS intervention, cells were collected for qPCR experiments. Cells were scratched upon LPS addition during migration, and microscopic images were taken at 0 and 12 hours. ImageJ software was used to separate the scratched areas from the cellular areas by setting a threshold. Subsequently, the pixel area of ​​the scratched areas was measured, and the percentage of scratches caused by cell migration was calculated.

[0077] The results are shown in Table 3:

[0078] qPCR testing showed: LPS significantly increased IL-6, TNF-α, IL-1β Expression; co-treatment with ILA significantly inhibited this upregulation; however, the inhibitory effect of ILA was reversed upon the addition of the AHR inhibitor CH223191.

[0079] Cell migration experiments showed that: When cells were untreated, the proportion of cells in the channel was 28%, which increased to 44% after LPS stimulation, decreased to 35% after LPS+ILA treatment, and recovered to 43% after CH223191 intervention. Indole-3-lactic acid (ILA) can inhibit LPS-induced BV2 cell migration, and this effect is also reversed by CH223191.

[0080] Verification of the endothelial cell tube-forming mechanism: In HRMEC tube formation experiments, the AHR inhibitor CH223191 was added. The results showed that CH223191 could partially reverse the inhibitory effect of ILA on HRMEC tube formation, demonstrating that the anti-angiogenic effect of ILA is partially dependent on the AHR pathway.

[0081] in conclusion The above examples fully demonstrate that indole-3-lactic acid (ILA), a key metabolite of the gut symbiotic bacterium AKK, can reach ocular tissues via blood circulation and effectively improve choroidal neovascularization (CNV) by activating the aryl hydrocarbon receptor (AHR) signaling pathway and inhibiting microglia-mediated neuroinflammation and abnormal retinal vascular endothelial cell regeneration. Therefore, ILA has a clear application prospect in the preparation of drugs for the treatment of CNV and related fundus diseases.

[0082] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The use of indole-3-lactic acid and / or spermidine in the preparation of drugs for improving and / or treating retinal and choroidal neovascularization diseases, characterized in that, The neovascular diseases of the retina and choroid include, but are not limited to, age-related macular degeneration, retinopathy of prematurity, pathological neovascular high myopia, polypoid choroidal retinopathy, retinal vasculitis, and visual impairment due to diabetic retinopathy.

2. The application according to claim 1, characterized in that, The drug is used for at least one of (a) to (g): (a) Reduce the area of ​​laser-induced individual choroidal neovascularization (CNV) lesions and improve retinal structure; (b) Inhibits the tube-forming ability of human retinal microvascular endothelial cells (HRMECs); (c) Downregulate the expression levels of pro-inflammatory factors IL-6, TNF-α, IL-1β, Spp1, B2m, and C3 in the retina and HRMEC cells of individual choroidal neovascularization (CNV) mice; (d) Downregulate the expression level of pro-angiogenic factors Fgf2 / FGF2; (e) Inhibits the release and migration of inflammatory factors in microglia BV2 stimulated by lipopolysaccharide.

3. The application according to claim 1 or 2, characterized in that, The drug contains indole-3-lactic acid and / or spermidine as active ingredients, and also contains a drug carrier and pharmaceutically acceptable excipients.

4. The application according to any one of claims 1 to 3, characterized in that, The drug also includes pharmaceutically acceptable excipients; Optionally, the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; Other excipients may also be added to the composition: flavoring agents and sweeteners.

5. The application according to claim 3, characterized in that, The drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

6. The application according to any one of claims 1 to 5, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pellets, granules, injections, powder for injection, or aerosols.

7. The application according to any one of claims 1 to 6, characterized in that, The chemical formula of the indole-3-lactic acid is C. 11 H 11 NO3 has the following structural formula: 。 8. A drug, characterized in that, The drug is used to treat and / or alleviate retinal and choroidal neovascularization diseases, including but not limited to age-related macular degeneration, retinopathy of prematurity, pathological neovascular high myopia, polypoid choroidal retinopathy, retinal vasculitis, and diabetic retinopathy with vision loss; the drug is any pharmaceutically acceptable dosage form made with indole-3-lactic acid and / or spermidine as active ingredients and pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release tablets, capsules, soft capsules, pellets, granules, injections, powder for injection, or aerosols. Preferably, the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; Other excipients may also be added to the composition: flavoring agents and sweeteners.

10. The medicament according to claim 8 or 9, characterized in that, The chemical formula of the indole-3-lactic acid is C. 11 H 11 NO3 has the following structural formula: 。

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