Use of sele in the treatment of wet age-related macular degeneration

Abstract

The application discloses application of SELE in treatment of wet age-related macular degeneration. The application research finds that SELE can significantly promote proliferation, migration and tube formation ability of vascular endothelial cells. SELE By inhibiting gene expression or activity, the application can significantly inhibit choroidal neovascularization in a wAMD mouse model, reduce abnormal vascular leakage, effectively reduce ocular inflammatory response, reduce inflammation-mediated vascular damage, target intervention of biological behavior of vascular endothelial cells, and block a key link of pathological angiogenesis. The application provides a novel molecular target for wAMD treatment and breaks the limitation of the existing anti-VEGF therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of SELE in the treatment of wet age-related macular degeneration. Background Technology

[0002] Age-related macular degeneration (AMD), also known as age-related macular degeneration, primarily affects people over 60 years of age. AMD is a significant degenerative eye disease and a leading cause of irreversible blindness. With the increasing aging of the population, the problems caused by AMD are becoming increasingly prominent. AMD is divided into dry and wet types, with wet age-related macular degeneration (wAMD) being the leading cause of blindness due to AMD, accounting for over 90% of AMD-related blindness cases. wAMD is characterized by choroidal neovascularization (CNV), which can lead to retinal exudates, submaculatic hemorrhage, and subretinal fibrotic scarring. Studies have shown that vascular endothelial growth factor (VEGF) plays a crucial role in promoting choroidal neovascularization (CNV), which is one of the important contributing factors to the pathogenesis of wAMD. Currently, intravitreal injection of anti-VEGF drugs is the preferred treatment for wAMD, which can significantly improve patients' best-corrected visual acuity and maintain good visual acuity for a longer period. While anti-VEGF drugs effectively inhibit angiogenesis and vascular leakage, they fail to target inflammatory components and may even trigger intraocular inflammation. Inflammation is a key factor driving ocular neovascularization, thus increasing the risk of vision loss. Although steroids are commonly used to suppress intraocular inflammation, their application is limited by side effects. Therefore, there is an urgent need to delve deeper into the inflammatory mechanisms behind ocular neovascularization and to identify potential immunotherapeutic targets.

[0003] E-selectin (SELE) is the only adhesion molecule in the selectin family specifically expressed on activated endothelial cells, and its expression is typically induced by inflammatory factors such as TNF-α and IL-1β. In the vascular system, current research only suggests that SELE may promote adhesion interactions between endothelial progenitor cells and endothelial cells during ischemic tissue repair, thereby improving limb ischemia. The direct role of SELE in ocular neovascularization remains unclear, and there are no reported studies targeting SELE for the treatment of wAMD. Summary of the Invention

[0004] This invention is the first to discover inhibition or silencing. SELE The gene can inhibit ocular neovascularization and reduce ocular inflammation, suggesting that SELE may be a potential target for the treatment of wet age-related macular degeneration.

[0005] Therefore, the first objective of this invention is to provide the use of SELE inhibitors in the preparation of medicaments for treating wet age-related macular degeneration.

[0006] Preferably, the SELE inhibitor can inhibit ocular neovascularization and reduce ocular inflammatory response.

[0007] Preferably, the SELE inhibitor is an inhibitor of... SELE Substances that inhibit gene expression or the activity of SELE protein.

[0008] Preferably, the inhibition SELE Substances that suppress or silence gene expression include methods such as RNA interference, gene knockout, chemical repression, miRNA-mediated silencing, transcription factor repression, or epigenetic modification. SELE Gene reagents.

[0009] Preferably, the RNA interference reagent includes those targeting... SELE The gene's siRNA, dsRNA, or shRNA.

[0010] Preferably, the target SELE The shRNA sequence of the gene is shown in SEQ ID NO.1; the target SELE The sequence of the gene's siRNA is shown in SEQ ID NO.8.

[0011] Preferably, the drug is an ophthalmic preparation.

[0012] More preferably, the ophthalmic preparation is a topical administration preparation selected from eye drops, ophthalmic gels, or ophthalmic sprays.

[0013] More preferably, the ophthalmic preparation is an injectable dosage form, selected from intraocular injections or periocular injections.

[0014] More preferably, the ophthalmic preparation is an implantable formulation, selected from ophthalmic microspheres or ophthalmic implants, and the implantable formulation is a sustained-release formulation.

[0015] The second objective of this invention is to provide a drug for treating wet age-related macular degeneration, comprising a SELE inhibitor as the active ingredient.

[0016] Preferably, the medicament for treating wet age-related macular degeneration further comprises pharmaceutically acceptable excipients or carriers.

[0017] This invention has found that SELE can promote the proliferation, migration, and tube-forming ability of vascular endothelial cells. Inhibiting SELE can reduce choroidal angiogenesis and inflammation in wAMD mouse models, and may serve as a potential therapeutic target for wAMD.

[0018] The beneficial effects of this invention are:

[0019] This invention, through in-depth research into the role of SELE in the pathogenesis of wet age-related macular degeneration (wAMD), reveals for the first time that SELE gene expression levels are closely related to choroidal angiogenesis. The results indicate that SELE can effectively promote the proliferation, migration, and tube-forming capacity of vascular endothelial cells, playing a crucial regulatory role in the pathological progression of wAMD.

[0020] By inhibiting SELE Gene expression or activity, the present invention achieves the following significant technical effects: (1) significantly inhibits the formation of choroidal neovascularization in wAMD mouse model and reduces abnormal vascular leakage; (2) effectively reduces ocular inflammatory response and alleviates inflammation-mediated vascular damage; (3) targeted intervention in the biological behavior of vascular endothelial cells and blocks the key link of pathological angiogenesis; (4) provides a new molecular target for wAMD treatment and overcomes the limitations of existing anti-VEGF therapies. Attached Figure Description

[0021] Figure 1 The effect of SELE knockdown on retinal vascular endothelial cell proliferation was investigated. (A) Western blot analysis showed the SELE protein expression level in retinal endothelial cells. (B) CCK-8 assay was used to assess retinal endothelial cell proliferation after SELE knockdown. All data are presented as mean ± standard deviation (SD). **: p < 0.01.

[0022] Figure 2 The effect of SELE knockdown on the migration ability of retinal vascular endothelial cells. (A) shows typical images at 0 h and 6 h post-scratch, scale bar 50 μm. (B) shows the quantitative results of the ratio of migrating cells to the scratch area. All data are presented as mean ± standard deviation (SD). ***: p < 0.001.

[0023] Figure 3 The effect of SELE knockdown on the tube-forming ability of retinal vascular endothelial cells. (A) Tube-forming experiment showing the morphology of neovascularization in retinal vascular endothelial cells after SELE knockdown, scale bar 50 μm. (B) Quantitative results of the number of neovascular branches. (C) Quantitative results of the total length of neovascular tubes. All data are presented as mean ± standard deviation (SD). ***: p < 0.001.

[0024] Figure 4The effects of SELE knockdown on THP-1 cell adhesion and transendothelial migration were investigated. (A) shows the adhesion assay of THP-1 cells to retinal vascular endothelial cells after SELE knockdown, scale bar 100 μm. (B) shows the quantitative results of the number of adherent cells. (C) shows the transendothelial migration of THP-1 cells after SELE knockdown, scale bar 100 μm. (D) shows the quantitative results of the number of transendothelial cells. All data are presented as mean ± standard deviation (SD). *: p < 0.05, **: p < 0.01.

[0025] Figure 5 The effect of SELE knockdown on laser-induced choroidal angiogenesis and inflammatory response was investigated. (A) Western blot analysis of SELE protein expression levels in choroidal tissue. (B) Immunofluorescence images of IB4 (green) and F4 / 80 (red) in the choroid in a CNV model. Scale bar: 100 μm. (C) Quantitative results of IB4-positive area. (D) Quantitative results of F4 / 80-positive area. All data are presented as mean ± standard deviation (SD). ***: p < 0.001. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0028] The materials involved in the following embodiments are as follows:

[0029] (1) Plasmid

[0030] The shRNA targeting SELE was designed using pLKO.1 as a vector via the Sigma website.

[0031] The designed sequences were synthesized by Tsingke Biotechnology and ligated to the pLKO.1 vector using T4 DNA ligase. All plasmids were transformed with DH5α and extracted using a plasmid extraction kit (TIANGEN, DP117TA).

[0032] Table 1 shRNA sequence information

[0033] .

[0034] (2) Proteins and antibodies

[0035] Table 2 Proteins and Antibodies

[0036] .

[0037] (3) Primers

[0038] Table 3 Primers

[0039] .

[0040] (4) siRNA sequence

[0041] Table 4 siRNA oligo sequences

[0042] .

[0043] Example 1: SELE knockdown inhibits the proliferation of retinal vascular endothelial cells

[0044] Cell proliferation assay

[0045] (1) Human retinal endothelial cells (HREC) treated with siRNA for 48 h were seeded in 96-well plates with 3 replicates.

[0046] (2) Add 10 µL of CCK-8 reagent to each well and incubate at 37°C for 4 hours.

[0047] (3) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 450 nm.

[0048] 2. Results

[0049] To determine whether SELE has an effect on endothelial cell proliferation, we first constructed a human siRNA (siSELE) and used Western blot to detect its knockdown efficiency in HRECs. Figure 1 (A in the text). Meanwhile, CCK-8 studies showed that knocking down SELE reduced HREC proliferation (…). Figure 1 (B in the text). These results indicate that SELE knockdown inhibits the proliferation of retinal vascular endothelial cells.

[0050] Example 2: Knockdown of SELE inhibited the migration of retinal vascular endothelial cells

[0051] 1. Endothelial cell migration assay

[0052] (1) HRECs treated with siRNA for 48 h were seeded in 24-well plates.

[0053] (2) Use a 200 µL tip perpendicular to the cell to horizontally scratch the cell, with at least three wells per group.

[0054] (3) Wash the cells three times with extracellular matrix (ECM) and then take pictures under a microscope.

[0055] (4) Place the cells in an incubator and take another picture after 12 h.

[0056] 2. Results

[0057] We used a scratch assay to examine the effect of SELE on the migration ability of retinal vascular endothelial cells. The results showed that knocking down SELE could inhibit the migration ability of retinal vascular endothelial cells. Figure 2 ).

[0058] Example 3: Knockdown of SELE inhibited the tubular formation ability of retinal vascular endothelial cells.

[0059] 1. Tube forming experiment

[0060] (1) Add 50 µL of Matrigel matrix gel to each well of the 96-well plate and polymerize at 37°C for 30 minutes.

[0061] (2) HRECs transfected with siRNA for 48 hours were seeded on the gel at a density of 10,000 per well.

[0062] (3) After 6 hours of incubation, the tubular structure was imaged and then quantitatively analyzed using ImageJ software.

[0063] 2. Results

[0064] To determine whether SELE affects the angiogenesis capacity of retinal vascular endothelial cells, we conducted a tube formation experiment. The results showed that knocking down SELE in retinal vascular endothelial cells reduced the number of branches in the newly formed blood vessels and shortened the branch length of the new blood vessels. Figure 3 These results indicate that SELE knockdown inhibits the tube-forming ability of retinal vascular endothelial cells.

[0065] Example 4: Knockdown of SELE inhibited the adhesion of THP1 cells to endothelial cells and their transendothelial migration ability.

[0066] 1. Cell adhesion experiment

[0067] (1) HREC (8.0 × 10⁻⁶) treated with siRNA for 48 h 4 Inoculation was performed in the upper chamber;

[0068] (2) Add 1 µg / mL LPS and treat for 4 h, then wash 3 times with PBS;

[0069] (3) THP-1 (5.0 × 10⁻⁶) was treated with 2.5 µM calcein AM (Beyotime). 5 30 min for mononuclear cells;

[0070] (4) AM-labeled THP-1 cells were co-incubated with HREC for 2 h;

[0071] (5) Wash three times with PBS;

[0072] (6) Adherent THP-1 cells from 12 randomly selected visual regions were captured using a fluorescence microscope (Zeiss, Germany) and analyzed by Image.

[0073] 2. THP-1 cell transendothelial migration assay

[0074] (1) HRECs infected with siRNA (5 × 10⁻⁶) 4 The cells were inoculated into each upper chamber and cultured to form a monolayer.

[0075] (2) THP-1 cells (4 × 10⁻⁶) in a culture medium containing 1% FBS 5 Add to the upper chamber;

[0076] (3) Add 1640 medium containing 5% FBS and 50 ng / mL CCL2 to the lower chamber;

[0077] (4) After 12 hours, the THP-1 cells that had migrated in the lower chamber were transferred to a new well and treated with 150 ng / mL phorbol 12-myristate 13-acetate (PMA) for 12 hours to allow the THP-1 cells to attach.

[0078] (5) Adherent THP-1 cells were treated with 2.5 µm calcein AM and 12 randomly selected regions were captured using a fluorescence microscope and analyzed by Image.

[0079] 3. Results

[0080] To investigate the effect of SELE on retinal vascular endothelial cells on the inflammatory response, we performed a cell adhesion assay. After knocking down SELE in endothelial cells, we seeded THP1 mononuclear cells in a culture medium and then used a fluorescent dye to detect the adhesion of THP1 cells to endothelial cells. The results showed that knocking down SELE in endothelial cells significantly reduced the adhesion of fluorescently labeled THP1 cells to endothelial cells. Figure 4 (AB in the middle).

[0081] In addition, we performed a transendothelial migration assay. After knocking down SELE in endothelial cells, they were seeded into Transwell chambers, and THP1 was seeded outside the chambers. After starvation induction, THP1 migration and recruitment to endothelial cells were detected using fluorescent dyes. The results showed that knocking down SELE significantly reduced THP1 recruitment and migration. Figure 4These results collectively indicate that SELE plays a crucial role in endothelial cell-mediated THP1 adhesion and migration.

[0082] Example 5: SELE knockdown inhibits laser-induced choroidal angiogenesis and inflammation

[0083] 1. Laser-induced CNV mouse model

[0084] (1) Four laser photocoagulation spots (spot size 75 μm, power 90 mw, duration 75 ms) were applied to the eyes of adult mice within an area 1.5–2 mm from the diameter of the optical disc. Laser photocoagulation was performed at positions 12, 3, 6, and 9, avoiding large retinal vessels;

[0085] (2) After laser photocoagulation, inject a complex containing shSELE or shCTRL plasmid-PEI (1µg / µL) into the vitreous cavity.

[0086] (3) Eyes were collected on day 7. The eyeballs were fixed in 4% paraformaldehyde for 2 h, and the cornea, lens, lenticule, and vitreous body were removed to separate the choroid-RPE complex. The following experiments were performed: The choroid-RPE complex was washed with PBS and incubated overnight at 4°C in Alexa Fluor 488 (1:500) conjugated with 1% donkey serum, 1% BSA, and 0.1% Triton X-100; the choroid-RPE complex was rinsed with PBS and laid flat with the RPE side up; images were taken using a fluorescence microscope, and the CNV region was analyzed using image analysis software. The m5C level of mRNA in the choroid-RPE complex was detected using the MethylFlash™ 5-mC RNA methylation ELISA kit (Epigentek).

[0087] (4) Rinse the choroid-RPE complex with PBS and lay it flat with the RPE side up;

[0088] (5) Take images using a fluorescence microscope and analyze the CNV region using image analysis software.

[0089] 2. Retinal detachment, treatment, and staining

[0090] Eyeballs were collected, enucleated in PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min. The retina was dissected, softened in PBS containing 5% donkey serum and 0.5% Triton X-100 for 1 h, and then incubated overnight at 4°C with allogeneic proteins GS-IB4 and F4 / 80 (1:200) bound to primary antibody Alexa Fluor 488 (1:500). After rinsing with PBS, the retina was incubated with the corresponding secondary antibody at room temperature for 2 h. The retina was then analyzed using confocal fluorescence microscopy after being laid flat.

[0091] 3. RNA isolation and extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)

[0092] Retinal endothelial cells were collected, and total RNA was isolated from the cells using TRIzol reagent (TIANGEN). cDNA was synthesized from this RNA using the DNase-containing FastKing RT kit (TIANGEN), and qRT-PCR was performed using a SYBR Green (ROCHE) and ABI QuantStudio 6 Flex instrument (Life Technologies). Results were normalized to GAPDH transcripts. Relative folding changes in gene expression were calculated using the delta-delta Ct method.

[0093] 4. Western blot experiment

[0094] (1) Preparing liquids:

[0095] ①10×Running Buffer: Add 144 g glycine, 10 g SDS powder, and 30.3 g Tris powder sequentially, using ddH2O as the solvent, and bring the volume to 1 L to prepare a storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use.

[0096] ②5×SDS Loading Buffer: Add 4 g of SDS powder, 20 mg of bromophenol blue, 3.085 g of DTT, 10 mL of Tris-HCl (1M pH 6.8), and 20 mL of glycerol in sequence, and use ddH2O as a solvent to make up to 40 mL.

[0097] ③ 10× Transfer Buffer: Add 30.3 g Tris powder and 144 g glycine sequentially, using ddH2O as the solvent, and bring the volume to 1 L to prepare a storage solution. Dilute with 10× Transfer Buffer 100 mL + methanol 200 mL + ddH2O 700 mL to prepare 1× Transfer Buffer as the working solution for use.

[0098] (2) Preparation of separating gel and stacking gel:

[0099] ① Prepare 10% separating gel (10 mL):

[0100] Table 5 shows the preparation of 10% separating gel.

[0101] .

[0102] ②Prepare a 5% concentrate (5 mL):

[0103] Table 6 shows the preparation of 5% concentrated gel.

[0104] .

[0105] (3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to its tank, and check for leakage. Remove the comb and add the marker and sample into the gel well in sequence. Add electrophoresis solution to the electrophoresis tank and set the program to 80 V for constant voltage electrophoresis for 30 min, then switch to 120 V for constant voltage electrophoresis for 1 h.

[0106] (4) Transfer: First, activate the PVDF membrane with methanol, and remove the gel from step 3). Assemble the transfer clamp according to the "sandwich" structure, being careful not to leave air bubbles during assembly. Assemble the transfer apparatus, add the transfer working solution, set the program to 250 mA constant current for 2 h, and place it on ice for transfer.

[0107] (5) Blocking: Take out the membrane after the transfer is completed, add 1×TBST and place it on a shaker at room temperature for 5 min to clean it, discard it, and add 5% skim milk as the blocking solution and place it on a shaker at room temperature for blocking. The blocking time is 1 h.

[0108] (6) Primary antibody incubation: Discard the blocking solution, add 1×TBST and place on a shaker at room temperature to wash until the blocking solution is clean. Discard the liquid, add the primary antibody prepared with 5% BSA as antibody diluent, and incubate overnight on a shaker in a 4°C cold storage.

[0109] (7) Washing the membrane: Wash the membrane three times with 1×TBST, 10 min each time;

[0110] (8) Secondary antibody incubation: Discard the solution, add secondary antibody prepared with 5% skim milk at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 h;

[0111] (9) Same as step (7);

[0112] (10) Exposure and development: Prepare the developer (A solution: B solution = 1:1 ratio, prepare and use immediately). Place the film on the plate and drop the developer solution onto the film. Shake well, expose and develop on the machine, and save the results.

[0113] 5. Results

[0114] To investigate the role of SELE in in vivo on wAMD angiogenesis and inflammation, we constructed a SELE-targeting shRNA. After laser-induced choroidal neovascularization (CNV) modeling in wild-type mice, the shRNA was injected into the fundus of the CNV mice. Western blot analysis confirmed the knockdown efficiency of SELE in the choroid. Figure 5 (A) Simultaneously, staining of choroidal CNV lesions revealed that in the mouse CNV model, the absence of SELE significantly reduced the staining areas of IB4+ (endothelial cell marker) and F4 / 80+ (inflammatory factor marker) (BD in Figure 5), indicating that the SELE gene deletion inhibits the wAMD phenotype, and SELE knockdown inhibits laser-induced choroidal angiogenesis and inflammation.

[0115] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of SELE inhibitors in the preparation of drugs for treating wet age-related macular degeneration; wherein the SELE inhibitor is a drug targeting... SELE The shRNA or siRNA of the gene, which targets SELE The shRNA sequence of the gene is shown in SEQ ID NO.1; the target SELE The sequence of the gene's siRNA is shown in SEQ ID NO.

8.

2. The application according to claim 1, characterized in that, The SELE inhibitor can inhibit ocular neovascularization and reduce ocular inflammation.

3. The application according to claim 1 or 2, characterized in that, The drug is an ophthalmic preparation.

4. The application according to claim 3, characterized in that, The ophthalmic preparation is a topical administration preparation, selected from eye drops, ophthalmic gels, or ophthalmic sprays.

5. The application according to claim 3, characterized in that, The ophthalmic preparation is an injectable dosage form, selected from intraocular injections or periocular injections.

6. The application according to claim 3, characterized in that, The ophthalmic preparation is an implantable formulation, selected from ophthalmic implants, and the implantable formulation is a sustained-release formulation.

Images