Application of SELE in treatment of wet age-related macular degeneration

Abstract

The invention discloses an application of SELE in treatment of wet age-related macular degeneration. The research finds that the SELE can obviously promote the proliferation, migration and tube forming ability of vascular endothelial cells. By inhibiting SELE gene expression or activity, choroidal neovascularization in a wAMD mouse model can be significantly inhibited, and abnormal vascular leakage is reduced; meanwhile, the eye inflammatory response is effectively reduced, the inflammation-mediated vascular injury is relieved, the vascular endothelial cell biological behavior is intervened in a targeted manner, and the key link of pathological angiogenesis is blocked. The invention provides a brand new molecular target for wAMD treatment, and breaks through 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 object of the present application is to provide the use of a SELE inhibitor in the preparation of a medicament 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 a substance that inhibits SELE gene expression or inhibits the activity of SELE protein.

[0008] Preferably, the substance that inhibits SELE gene expression includes an agent that inhibits or silences the gene by means of RNA interference, gene knockout, chemical inhibition, miRNA-mediated silencing, transcription factor inhibition or epigenetic modification. SELE

[0009] Preferably, the agent of RNA interference includes siRNA, dsRNA or shRNA against SELE gene.

[0010] Preferably, the sequence of shRNA against SELE gene is shown in SEQ ID NO. 1; and the sequence of siRNA against SELE gene is shown in SEQ ID NO. 8.

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

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

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

[0014] More preferably, the ophthalmic preparation is an implant dosage form selected from ophthalmic microspheres or ocular implant tablets, and the implant dosage form is a sustained-release preparation.

[0015] The second object of the present application is to provide a medicament for treating wet age-related macular degeneration, which comprises a SELE inhibitor as an active ingredient.

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

[0017] The present application has found that SELE can promote the proliferation, migration and tube formation of vascular endothelial cells. Inhibition of SELE can reduce choroidal neovascularization and inflammation in a wAMD mouse model, and can be used as a potential target for treating wAMD.

[0018] ​The beneficial effects of this invention are: 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.

[0019] 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

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Figure 4Effects of SELE knockdown on THP-1 cell adhesion and transendothelial migration. Among them, (A) is the adhesion experiment to observe the adhesion of THP-1 cells to retinal vascular endothelial cells after SELE knockdown, scale 100 μm. (B) is the quantitative result of the number of adherent cells. (C) is the Transwell experiment to observe the transendothelial migration of THP-1 cells after SELE knockdown, scale 100 μm. (D) is the quantitative result of the number of transendothelial migration cells. All data are presented as mean ± standard deviation (SD). *: p<0.05, **: p<0.01.

[0024] Figure 5 Effects of SELE knockdown on laser-induced choroidal neovascularization and inflammatory response. Among them, (A) is the Western blot to detect the protein expression level of SELE in choroidal tissue. (B) is the IB4 (green) and F4 / 80 (red) immunofluorescence image of choroid in the CNV model. Scale: 100 μm. (C) is the quantitative result of IB4 positive area. (D) is the quantitative result of F4 / 80 positive area. All data are presented as mean ± standard deviation (SD). ***: p<0.001. DETAILED DESCRIPTION

[0025] The following examples are further illustrations of the application and are not intended to limit the same.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents and materials used are commercially available unless otherwise specified.

[0027] The materials involved in the following examples are as follows: (1) Plasmid The shRNA targeting SELE was designed by using pLKO.1 as the vector through the sigma website.

[0028] The designed sequence was synthesized by Tsingke Biotechnology Company and connected with the pLKO.1 vector using T4 DNA ligase. All plasmids were transformed using DH5α and extracted using a plasmid extraction kit (TIANGEN, DP117TA).

[0029] Table 1 shRNA sequence information .

[0030] (2) Protein and antibody Table 2 Protein and antibody .

[0031] (3) Primer Table 3 Primers .

[0032] (4) siRNA sequences Table 4 siRNA oligo sequences .

[0033] Example 1: Knockdown of SELE inhibits the proliferation of retinal vascular endothelial cells Cell proliferation assay (1) Human retinal endothelial cells (HRECs) treated with siRNA for 48 h were seeded in a 96-well plate, with 3 replicates.

[0034] (2) 10 μL of CCK-8 reagent was added to each well, and incubated at 37°C for 4 hours.

[0035] (3) The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader.

[0036] 2. Results To determine whether SELE has an effect on endothelial cell proliferation, we first constructed a human siRNA (siSELE) and detected its knockdown efficiency in HRECs using western blot (A in Figure 1 ). At the same time, CCK-8 showed that knocking down SELE could reduce the proliferation of HRECs (B in Figure 1 ). These results indicate that knockdown of SELE inhibits the proliferation of retinal vascular endothelial cells.

[0037] Example 2: Knockdown of SELE inhibits the migration of retinal vascular endothelial cells 1. Endothelial cell migration test (1) HRECs treated with siRNA for 48 h were seeded in a 24-well plate.

[0038] (2) The cells were scratched with a 200 μL tip perpendicular to the cells, with at least three wells per group.

[0039] (3) The cells were washed with extracellular matrix (ECM) three times, and then photographed under a microscope.

[0040] (4) The cells were placed in an incubator, and photographed again after 12 h.

[0041] 2. Results We used a scratch test to detect the effect of SELE on the migration ability of retinal vascular endothelial cells, and the results showed that knocking down SELE could inhibit the migration ability of retinal vascular endothelial cells ( Figure 2 ).

[0042] Example 3: Knockdown of SELE inhibits the tube formation ability of retinal vascular endothelial cells 1. Tube formation experiment (1) Add 50 μL Matrigel in each well of 96-well plate and polymerize at 37°C for 30 min.

[0043] (2) Seed siRNA-transfected HREC at a density of 10,000 cells per well on the gel.

[0044] (3) After 6 h of incubation, image the tube-like structures and then use ImageJ software for quantitative analysis.

[0045] 2. Results To determine whether SELE has an impact on the angiogenic ability of retinal vascular endothelial cells, we performed a tube formation experiment, and the results showed that knockdown of SELE in retinal vascular endothelial cells reduced the number of endothelial cell angiogenic branches and shortened the length of angiogenic branches (Fig. 3B and 3C). Figure 3 These results indicate that knockdown of SELE inhibits the tube formation ability of retinal vascular endothelial cells.

[0046] Example 4: Knockdown of SELE inhibits the adhesion and transendothelial migration ability of THP1 cells to endothelial cells 1. Cell adhesion experiment (1) Seed siRNA-treated HREC (8.0 × 10 4 ) in the upper chamber; (2) Add 1 μg / mL LPS and incubate for 4 h, then wash with PBS for 3 times; (3) Treat THP-1 (5.0 × 10 5 ) monocytes with 2.5 μM calcein AM (Beyotime) for 30 min; (4) Incubate AM-labeled THP-1 cells with HREC for 2 h; (5) Wash with PBS for 3 times; (6) Capture adherent THP-1 cells from 12 randomly selected visual fields using a fluorescence microscope (Zeiss, Germany) and analyze by Image.

[0047] 2. THP-1 cell transendothelial migration experiment (1) Seed siRNA-infected HREC (5 × 10 4 ) in each upper chamber and incubate to allow the cells to form a monolayer (2) Seed THP-1 cells (4 × 10 5Add to the upper chamber; (3) Add 1640 medium containing 5% FBS and 50 ng / mL CCL2 to the lower chamber; (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. (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.

[0048] 3. Results 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).

[0049] 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 4 These results collectively indicate that SELE plays a crucial role in endothelial cell-mediated THP1 adhesion and migration.

[0050] Example 5: SELE knockdown inhibits laser-induced choroidal angiogenesis and inflammation 1. Laser-induced CNV mouse model (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; (2) After laser photocoagulation, inject a complex containing shSELE or shCTRL plasmid-PEI (1µg / µL) into the vitreous cavity. (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).

[0051] (4) Rinse the choroid-RPE complex with PBS and lay it flat with the RPE side up; (5) Take images using a fluorescence microscope and analyze the CNV region using image analysis software.

[0052] 2. Retinal detachment, treatment, and staining 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.

[0053] 3. RNA isolation and extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR) 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.

[0054] 4. Western blot experiment (1) Preparing liquids: ①10×Running Buffer: add 144 g glycine, 10 g SDS powder, 30.3 g Tris powder in sequence, ddH2O as solvent, constant volume to 1 L, prepared as a storage solution. Diluted into 1×Running Buffer to prepare electrophoresis working solution for use; ②5×SDS Loading Buffer: add SDS powder 4 g, bromophenol blue 20 mg, DTT 3.085 g, Tris-HCL (1M pH 6.8) 10 mL, glycerol 20 mL in sequence, ddH2O as solvent, constant volume to 40 mL; ③10×Transfer Buffer: add 30.3 g Tris powder, 144 g glycine in sequence, ddH2O as solvent, constant volume to 1 L, prepared as a storage solution. Diluted into 1×Transfer Buffer to prepare transfer working solution for use according to the formula of 10×Transfer Buffer 100 mL + methanol 200 mL + ddH2O 700 mL; (2) Preparation of separation gel and concentrated gel: ①Preparation of 10% separation gel (10 mL): Table 5 Preparation of 10% separation gel .

[0055] ②Preparation of 5% concentrated gel (5 mL): Table 6 Preparation of 5% concentrated gel .

[0056] (3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis liquid to the groove, and detect whether there is a leakage phenomenon. Pull out the comb, and add the marker and sample to the gel hole in sequence. Add electrophoresis liquid to the electrophoresis tank, and set the program to 80 V for constant voltage electrophoresis for 30 min, and then to 120 V for constant voltage electrophoresis for 1 h; (4) Membrane transfer: First, activate the PVDF membrane with methanol, and take out the gel in step 3). Assemble the membrane transfer clamp in the order of "sandwich" structure, and pay attention to not leaving air bubbles during assembly. Assemble the membrane transfer device, add the transfer working solution, and set the program to 250 mA constant current for 2 h, and place it on ice for membrane transfer; (5) Blocking: Take out the membrane after membrane transfer, first add 1×TBST and place it on the shaking bed at room temperature for 5 min, discard, and add 5% skim milk as blocking solution and place it on the shaking bed at room temperature for blocking. The blocking time is 1 h; (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. (7) Washing the membrane: Wash the membrane three times with 1×TBST, 10 min each time; (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; (9) Same as step (7); (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.

[0057] 5. Results 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.

[0058] 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. Application of SELE inhibitors in the preparation of drugs for the treatment of wet age-related macular degeneration.

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, characterized in that, The SELE inhibitor is an inhibitor that inhibits... SELE Substances that inhibit gene expression or the activity of SELE protein.

4. The application according to claim 3, characterized in that, 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.

5. The application according to claim 4, characterized in that, The RNA interference reagents include those targeting SELE The gene's siRNA, dsRNA, or shRNA.

6. The application according to claim 5, characterized in that, 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.

7. The application according to any one of claims 1-6, characterized in that, The drug is an ophthalmic preparation.

8. The application according to claim 7, characterized in that, The ophthalmic preparation is a topical administration preparation, selected from eye drops, ophthalmic gels, or ophthalmic sprays.

9. The application according to claim 7, characterized in that, The ophthalmic preparation is an injectable dosage form, selected from intraocular injections or periocular injections.

10. The application according to claim 7, characterized in that, The ophthalmic preparation is an implantable formulation, selected from ophthalmic microspheres or ophthalmic implants, and the implantable formulation is a sustained-release formulation.

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