Application of BCL3 inhibitors in the preparation of drugs for treating glaucoma

Abstract

The present invention discloses the use of a BCL3 inhibitor in the preparation of a drug for treating glaucoma. The present invention discovers the role of the BCL3 / NF-κB / NLRP3 signaling pathway in glaucoma and demonstrates that BCL3 inhibitors (such as JS-6) have never been reported to reduce retinal ganglion cell apoptosis and pyroptosis, and are therefore useful in treating glaucoma-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of a BCL3 inhibitor in the preparation of a drug for treating glaucoma. Background Art

[0002] Glaucoma is the leading cause of irreversible blindness in the world. It is mainly caused by pathologically high intraocular pressure (ph-IOH), which leads to the irreversible death of selective retinal ganglion cells (RGCs), resulting in progressive and centripetal visual field loss in patients. Glaucoma patients require lifelong treatment, which will bring great living burden and economic losses to patients and the entire society. At present, the specific pathogenesis of glaucoma is still unclear, and there is still a lack of effective treatment methods for glaucomatous optic nerve damage in clinical practice.

[0003] Glaucoma risk factors include elevated intraocular pressure (IOP), age, and family heredity, among others. Elevated IOP is the most critical. Therefore, existing clinical treatments for glaucoma, whether medication, laser therapy, or surgery, primarily focus on lowering and controlling IOP. However, in actual clinical practice, many glaucoma patients do not experience abnormally elevated IOP during their disease course, such as those with normal-tension glaucoma and those with ocular hypertension who have elevated IOP but do not have glaucoma. Furthermore, glaucoma medications are associated with poor patient compliance, poor efficacy in some patients, drug resistance, and unavoidable side effects. These patients then opt for surgical treatment, which carries risks such as poor postoperative IOP control, increased IOP due to scarring of the filtration bleb, inherent risks and unavoidable complications, and contraindications for patients with other eye and systemic conditions. Furthermore, even with successful treatment and good IOP control, existing vision loss cannot be reversed, and some patients may experience worsening glaucoma. It can be seen that abnormal increase in intraocular pressure is an important factor in the pathogenesis of glaucoma but not the only factor. There are many unknown causes of the death of RGCs in the pathogenesis of glaucoma, which makes protecting the optic nerve and reducing the death and loss of RGCs the focus and difficulty in the treatment of glaucoma.

[0004] The mechanisms of glaucoma-induced retinal nerve damage have been extensively studied. Current theories regarding the factors contributing to glaucomatous optic nerve damage include mechanical injury caused by elevated intraocular pressure, vascular dysfunction, oxidative stress, neuroinflammatory responses, and excitotoxicity. Oxidative stress and inflammation play crucial roles in the development and progression of RGC damage. Acute glaucoma triggers a self-reinforcing, destructive cascade involving neuronal depolarization, calcium influx, and blood-retinal barrier disruption, leading to extreme release of free radicals and an excessive inflammatory response. This overwhelms normal cellular antioxidant defenses, ultimately leading to retinal ganglion cell (RGC) death, retinal morphological degeneration, and impaired retinal function. Therefore, combating inflammation may be a promising strategy to rescue glaucoma-damaged RGCs.

[0005] NF-κB signaling is a key pathway influencing various cellular functions, such as proliferation and differentiation, induction of apoptosis, and immune responses. One of its best-known roles is as a key mediator of inflammatory responses, leading to the expression of inflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as inflammasomes such as NLRP3, one of the most important inflammasomes regulating neuroinflammation and pyroptosis. The most abundant form of NF-κB activated through the canonical pathway is the p50 / RelA heterodimer. Classical NF-κB activation leads to the release of p50 / RelA, which acts as a transcription factor to activate target gene transcription. BCL3, an atypical member of the IκB protein family, plays a crucial role in regulating the activity of nuclear factor NF-κB. Depending on the cell type and the nature of the stimulus, BCL3 can promote or inhibit NF-κB signaling, thereby regulating downstream gene transcription. Ultimately, it plays a dual role in inflammation, exerting either pro- or anti-inflammatory effects in different contexts. However, its role in retinal diseases has not been explored, and no studies have investigated the BCL3 / NF-κB / NLRP3 pathway and its regulation in this context.

[0006] JS-6 is a novel small molecule with potent intracellular BCL3 activity inhibition. JS-6 is designed to inhibit the protein interaction between the ankyrin repeat domain of BCL3 and its regulatory partner, p50. Although Zhao et al. questioned its anti-tumor efficacy because JS-6 does not inhibit all BCL3 functions in tumor cells, JS-6 has been shown to prevent tumor growth and metastasis. Currently, JS-6 has not been studied in diseases other than cancer, including retinal diseases.

[0007] Therefore, further clarifying the therapeutic effects of BCL3-related signaling pathways on retinal diseases such as glaucoma and providing new strategies for their treatment are technical issues that need to be urgently addressed in this field. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the defect in the prior art that the therapeutic effect of the BCL3-related signal transduction pathway on retinal diseases such as glaucoma is unclear, and to provide a use of a BCL3 inhibitor in the preparation of a drug for treating glaucoma.

[0009] The present invention creatively discovered that BCL3 inhibitors (such as JS-6) have the effect of protecting ganglion cells in glaucoma models. Specifically: In an in vivo experiment, in order to simulate the intraocular environment of glaucoma disease, mice were subjected to acute ocular hypertension (AOH) injury of glaucoma, and the BCL3 inhibitor JS-6 was administered by intravitreal drug injection before the injury. By comparing the electrophysiology, morphology, neuroinflammation and molecular proteins of mice after acute high intraocular pressure injury of glaucoma, it was found that the BCL3 inhibitor JS-6 alleviated oxidative stress damage by inhibiting the BCL3 / NF-κB / NLRP3 signaling pathway, thereby reducing retinal ganglion cell apoptosis and pyroptosis, and played a role in reducing or rescuing glaucomatous optic nerve damage, providing a new therapeutic strategy for the treatment of glaucoma.

[0010] The present invention provides an application of a BCL3 inhibitor in the preparation of a medicine for treating glaucoma.

[0011] In the present invention, the BCL3 refers to Recombinant B-Cell CLL / Lymphoma 3 (Bcl3), which is an atypical member of the IκB protein family and plays a vital role in regulating the activity of nuclear factor NF-κB.

[0012] In a preferred embodiment of the present invention, the BCL3 inhibitor is JS-6, and the chemical formula of JS-6 is

[0013] The present invention discovered that JS-6, as an inhibitor of BCL3-related signal transduction, can treat retinal diseases such as glaucoma, and provides a new therapeutic strategy for the treatment thereof.

[0014] In the present invention, the glaucoma may be a conventional glaucoma disease in the art, such as glaucoma disease caused by acute high intraocular pressure damage.

[0015] In the present invention, the glaucoma may be a glaucoma disease in which the number of central RBPMS-positive cells in retinal flat mounts is reduced.

[0016] In the present invention, the glaucoma may be a glaucoma disease characterized by a decrease in the number of intermediate RBPMS-positive cells in retinal flat mounts.

[0017] In the present invention, the glaucoma may be a glaucoma disease characterized by a decrease in the number of peripheral RBPMS-positive cells in retinal flat mounts.

[0018] In the present invention, the glaucoma may be a glaucomatous disease characterized by decreased amplitude and prolonged latency of the P1 wave in flash visual evoked potential (F-VEP).

[0019] In the present invention, the glaucoma may be a glaucoma disease in which a wave and b wave in a flash electroretinogram (F-ERG) decrease.

[0020] In the present invention, the glaucoma may be a glaucoma disease characterized by elevated levels of NLRP3, IL-1β, and cleaved-caspase1 (p20) proteins.

[0021] In the present invention, the glaucoma may be a glaucoma disease in which the Bax / Bcl2 expression ratio is increased.

[0022] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0023] The reagents and raw materials used in the present invention are commercially available.

[0024] The positive progress effect of the present invention is:

[0025] The present invention discovered the role of the BCL3 / NF-κB / NLRP3 signaling pathway in glaucoma, and that BCL3 inhibitors (such as JS-6) have never been reported to reduce retinal ganglion cell apoptosis and pyroptosis, and are used to treat glaucoma-related diseases. Specifically:

[0026] The present invention discloses the pharmaceutical application of the BCL3 inhibitor JS-6 in the treatment, prevention, and mitigation of glaucomatous optic nerve damage. Experimental data show that the BCL3 inhibitor JS-6 has a definite improvement and therapeutic effect on glaucomatous optic nerve damage and can significantly improve ganglion cell death caused by oxidative stress. Animal experiments show that the BCL3 inhibitor JS-6 can reduce oxidative stress damage by inhibiting the BCL3 / NF-κB / NLRP3 signaling pathway, thereby reducing retinal ganglion cell apoptosis and pyroptosis, and play a role in reducing or rescuing glaucomatous optic nerve damage. Through experimental verification, the present invention provides a new treatment strategy for the field of ophthalmic medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the structural formula of JS-6.

[0028] Figure 2aRetinal ganglion cells (RGCs) were immunofluorescently labeled using RBPMS antibodies in retinal flat mounts. From left to right, whole retinal flat mounts (top) and magnified images of selected regions (bottom) from the normal control group, the AOH group 12 hours after injury, the AOH group 24 hours after injury, and the AOH group 48 hours after injury. Scale bar = 200 μm.

[0029] Figure 2b Quantitative analysis of the density of RBPMS-positive cells in retinal flat mounts at different time points (normal control, 12 hours, 24 hours, and 48 hours) after pathologically elevated intraocular pressure-induced injury. Comparisons were made using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Con group.

[0030] Figure 2c HE-stained retinal tissue sections show changes in retinal structure at different time points (normal control, 12 hours, 24 hours, and 48 hours) after induction of pathologically elevated intraocular pressure. Scale bar = 20 μm; Representative images of the central, intermediate, and peripheral regions of the retina were selected.

[0031] Figure 2d Quantitative analysis of cell counts in the ganglion cell layer (GCL) of retinal tissue sections stained with hematoxylin and eosin at different time points (normal control, 12 hours, 24 hours, and 48 hours) after pathologically elevated intraocular pressure (IOP)-induced injury. Comparisons were made using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group.

[0032] Figure 3a Immunofluorescence labeling of retinal ganglion cells (RGCs) in retinal flat mounts using the RBPMS antibody. From left to right, whole retinal flat mounts (top) and magnified images of selected regions (bottom) are shown for the normal control group, AOH-injured group, AOH + 1 mM JS-6 group, AOH + 2.5 mM JS-6 group, and AOH + 5 mM JS-6 group. Scale bar = 200 μm.

[0033] Figure 3b Schematic diagram of the selected area for counting RBPMS-positive cells in retinal flat mounts.

[0034] Figure 3cQuantitative analysis of the density of RBPMS-positive cells in the central portion of retinal flat mounts in the normal control group, AOH injury group, AOH + 1 mM JS-6 group, AOH + 2.5 mM JS-6 group, and AOH + 5 mM JS-6 group. Comparisons were made using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Con group.

[0035] Figure 3d Quantitative analysis of the density of RBPMS-positive cells in the middle portion of retinal flat mounts in the normal control group, AOH injury group, AOH + 1mM JS-6 group, AOH + 2.5mM JS-6 group, and AOH + 5mM JS-6 group. Comparisons were made using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Con group.

[0036] Figure 3e Quantitative analysis of the density of RBPMS-positive cells in the peripheral retinal flat mounts of the normal control group, AOH injury group, AOH + 1mM JS-6 group, AOH + 2.5mM JS-6 group, and AOH + 5mM JS-6 group. Comparisons were made using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Con group.

[0037] Figure 3f HE-stained retinal sections show structural changes in the eyeballs (upper image, ×4x) and selected retinas (lower image, ×63x) in the normal control group, the AOH-injured group, the AOH + 1mM JS-6 group, the AOH + 2.5mM JS-6 group, and the AOH + 5mM JS-6 group. Scale bars = 200 μm (upper), 20 μm (lower); Representative images of the central retinal region.

[0038] Figure 3g Figure 3. Quantitative analysis of cell counts in the ganglion cell layer (GCL) of HE-stained retinal tissue sections from the normal control group, AOH-injured group, AOH + 1 mM JS-6 group, AOH + 2.5 mM JS-6 group, and AOH + 5 mM JS-6 group. Comparisons were made using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group.

[0039] Figure 3h Flash visual evoked potential (F-VEP) waveforms of the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group;

[0040] Figure 3i Figure 3. Quantitative analysis of the flash visual evoked potential (F-VEP) N1-P1 amplitude and P1 latency in the uninjured control group, AOH-injured group, AOH + 1 mM JS-6 group, AOH + 2.5 mM JS-6 group, and AOH + 5 mM JS-6 group. Group comparisons were performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Con group.

[0041] Figure 3j Flash electroretinogram (F-ERG) waveforms of the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group;

[0042] Figure 3k Figure 3. Quantitative analysis of the flash electroretinogram (F-ERG) a-wave and b-wave amplitudes in the normal control group, AOH injury group, AOH + 1 mM JS-6 group, AOH + 2.5 mM JS-6 group, and AOH + 5 mM JS-6 group. Group comparisons were performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group.

[0043] Figure 4a Figure 4. Retinal flat mounts from the control group, the 5 mM JS-6 group, the AOH-injured group, and the AOH + 5 mM JS-6 group. Images showing the morphology, number, and distribution of microglia were taken under a 40x objective lens after co-staining with Iba1 (green) and MHCII (red). (i) A focal fluorescence image of the Iba1-stained flat mount; (ii) a black-and-white image obtained by color conversion of the above image; and (iii) an extracted image of microglia from the above image. Scale bar = 20 μm.

[0044] Figure 4b Figure 2: Images of the morphology, number, and distribution of microglia in retinal flat mounts from the normal control group, 5 mM JS-6 group, AOH-injured group, and AOH + 5 mM JS-6 group, obtained under a 20× objective lens and co-stained with Iba1 (green) and MHCII (red). (i) A focal fluorescence image of the Iba1-stained flat mount; (ii) A focal fluorescence image of the MHCII-stained flat mount; and (iii) A merged image of the two co-stained images. Scale bar = 20 μm.

[0045] Figure 5aThe results of Western Blot (WB) showed the relative expression levels of NLRP3, IL-1β, Caspase-1 and its cleavage product Caspase-1 (Cleaved-Caspase-1), Bax and Bcl-2 relative to Actin in the normal control group, 5mM JS-6 group, AOH injury group, and AOH+5mM JS-6 group.

[0046] Figure 5b Quantitative analysis of the relative expression levels of NLRP3 relative to Actin in the normal control group, 5 mM JS-6 group, AOH injury group, and AOH + 5 mM JS-6 group. Group comparisons were performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Control group.

[0047] Figure 5c Quantitative analysis of the relative expression levels of IL-1β to Actin in the normal control group, 5 mM JS-6 group, AOH injury group, and AOH + 5 mM JS-6 group. Group comparisons were performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Control group.

[0048] Figure 5d Quantitative analysis of the ratio of caspase-1 cleavage product to total caspase-1 (Cleaved-Caspase-1 / Caspase-1) relative to the expression level of actin in the normal control group, 5 mM JS-6 group, AOH injury group, and AOH + 5 mM JS-6 group. One-way analysis of variance was used for inter-group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group.

[0049] Figure 5e Quantitative analysis of the ratio of Bax to Bcl-2 (Bax / Bcl-2) relative to Actin expression in the normal control group, 5 mM JS-6 group, AOH injury group, and AOH + 5 mM JS-6 group. Group comparisons were performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the Control group. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] In the present invention, the terms are explained as follows:

[0052] NLRP3: NACHT, LRR and PYD domain protein 3, is a key component of the inflammasome and can promote the activation of Caspase-1 after activation.

[0053] IL-1β: Interleukin-1β is an important proinflammatory cytokine whose maturation and secretion depend on the NLRP3 inflammasome.

[0054] Caspase-1: Caspase-1 is an effector protein of the NLRP3 inflammasome. Upon activation, it can cleave GSDMD, leading to cell pyroptosis.

[0055] Cleaved-Caspase-1: The cleavage product of Caspase-1, a marker of its activation.

[0056] Bax: Bcl-2 homologous antagonist, a pro-apoptotic protein. An increase in the ratio of Bax to Bcl-2 can promote cell apoptosis.

[0057] Bcl-2: B cell lymphoma-2, is an anti-apoptotic protein, and its increased expression can inhibit cell apoptosis.

[0058] Part I: Experimental Methods

[0059] 1. Animals

[0060] Eight-week-old male C57BL6 mice were obtained from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China) and maintained under SPF conditions with a 12-h light-dark cycle and adequate food and water at the Laboratory Animal Center of Xiangya Hospital, Central South University. All procedures used in the animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Central South University and performed in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).

[0061] 2. Animal Model of Acute Hypertension Injury in Retinal Glaucoma and Drug Management

[0062] To verify the effect of inhibiting BCL3 on retinal nerve damage, the animals were randomly divided into four groups: control group, control treatment group (JS-6), AOH (acute ocular hypertension) injury group and AOH+JS6 group.

[0063] JS-6 powder (BCL3 inhibitor JS6, catalog PC-38199, ProbeChem, Shanghai, China; its structural formula is as follows Figure 1 Before each use, an appropriate volume of JS6 was freshly diluted with phosphate-buffered saline (PBS; 0.01 M; pH 7.4) to a concentration of 1 mM, 2.5 mM, and 5 mM, respectively.

[0064] Animals were anesthetized with 100 mg / kg of 1% sodium pentobarbital solution. Mydriasis and local corneal anesthesia were achieved using 0.5% tropicamide-phenylephrine eye drops (Sanen Pharmaceutical Co., Ltd., Shiga Plant) and 0.4% oxybuprocaine hydrochloride eye drops (Benoxil; Santen Pharmaceutical Co., Ltd.). Intravitreal administration was performed by tunnel injection using a 5-μL Hamilton syringe with a 32G needle, with a volume of 2 μL. Another group of mice received an equal volume of 0.9% saline injected into the vitreous cavity as a control.

[0065] On the second day, after anesthesia, the animals were induced to AOH by inserting a micro glass needle connected to a saline reservoir into the anterior chamber of the eye and maintaining the intraocular pressure at 120 mmHg for 60 minutes. The successful establishment of the model was verified by observing the whitening of the anterior segment of the eye and the fading of conjunctival blood vessels under an operating microscope (BELONA, China). The control group underwent a sham operation and did not increase the intraocular pressure. After the operation, 0.3% tobramycin dexamethasone ointment (saAlcon-Couvreur nv) was applied to the eyes. A heating pad was used to maintain the animal body temperature at 37 ° C.

[0066] 3. Retinal Flat-Plate for Quantitative Analysis of Retinal Ganglion Cell (RGC) Density

[0067] Immediately after the mice were sacrificed, the eyes were removed and fixed with 4% paraformaldehyde solution for 2 hours. Retinal flat mounts were prepared under a surgical microscope and incubated in 0.3% TritonX-100 for 30 minutes. After removing the TritonX-100 droplet, 5% bovine serum albumin (BSA) was added to block the antigen for 1 hour at room temperature. After aspirating the blocking solution, RGCs were labeled with antibodies against multiple spliced ​​RNA binding proteins (RBPMS) at 4°C overnight. The next day, after washing with PBS five times, the retinal tissue was incubated with fluorescent secondary antibodies (Alexa Fluor 500). 488, CST4412, USA) were incubated at room temperature for 1 hour. Images were acquired using a fluorescence microscope (Nicon, N2-DM4B). The number of RBPMS-positive RGCs was counted in three non-overlapping fields along the midline of each quadrant, from the optic disc to the border, at 400 μm intervals, for a total of 12 fields. Results are presented as the percentage of RGC loss compared to the control group.

[0068] 4. Hematoxylin and Eosin (H&E) Staining

[0069] Retinal cross-sections were subjected to histological evaluation. After acute intraocular hypertension injury, the retinas were stained with H&E to observe and count cells in the ganglion cell layer (GCL). The eyes were immediately removed after the mice were sacrificed and fixed with FAS eye fixative (Servicebio, China) for 24 hours at 4°C. The eyes were embedded in paraffin and cut into 5 μm thick sections through the optic disc and parallel to the optic nerve, prepared in a standard manner, and then stained with hematoxylin and eosin. Retinal morphology was observed using a light microscope, and scanning micrographs were taken. Sections containing the optic nerve stump were selected to maintain consistency between groups, and at least three discontinuous sections from each animal were analyzed using CaseViewer software.

[0070] 5. Flash visual evoked potential (F-VEP) and flash electroretinogram (F-ERG)

[0071] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital and their body temperature was maintained at 37°C. Pupils were dilated with 0.5% tropicamine eye drops.

[0072] Recording and reference electrodes were inserted subcutaneously in mice, contacting the occipital and frontal bones, respectively; a ground electrode was inserted subcutaneously near the tail. During recording, the unstimulated eye was occluded. F-VEPs were recorded continuously at various light intensities. 100 consecutive flashes were recorded and averaged to produce a single waveform for each intensity. The first positive peak in the F-VEP waveform was designated P1, and the first negative peak was designated N1. The N1-P1 amplitude was measured and analyzed.

[0073] For F-ERG recording, animal preparation procedures were identical to those for F-VEP. However, mice were dark-adapted for 12 hours before examination, and red light was used to maintain darkness in the testing environment. A gold wire-loop electrode was placed on the corneal surface, and carboxymethylcellulose eye drops were applied to enhance current conduction and corneal moisturization. The ground electrode was placed as described for F-VEP recording, and a reference electrode was inserted subcutaneously on either side of the nose. Stimulation and detection procedures followed the ISCEV standard. After completion of the test, the amplitudes of the a- and b-waves were analyzed for each group.

[0074] 6. Western Blot (WB)

[0075] Western blotting was used to detect the levels of NF-κB, BCL3, NLRP3, IL-1β, CASP1, Bax, and BCL2 in the retina. Mouse retinas were homogenized in RIPA lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with a 100:1 ratio of 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail, followed by centrifugation (12,000 g, 10 minutes, 4°C). The supernatant was extracted and measured using a protein assay kit (Thermo). 20 μg of protein sample from each animal was subjected to SDS-polyacrylamide gel electrophoresis at a constant voltage of 120 V for 60 minutes and then transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated with 5% skim milk at room temperature for 1 hour and washed with PBST. Western blots were performed with antibodies against NF-κB p105 / p50 (1:1000, Abcam ab32360), Bcl3 (1:500, Santa Cruz sc-32741), NLRP3 (1:1000, Cell Signaling Technology 15101), IL-1β (1:1000, Abcam Ab254360), CASP1 (1:2000, Proteintech, 22915), BAX (1:2000, Abclonal, A19684), BCL2 (1:2000, HUABIO, ET1702-53), and GAPDH (1:10,000, Proteintech, 60004-1) or β-actin (1:10,000, Proteintech, 811115) antibodies were incubated overnight at 4°C and then washed five times with PBST for 5 minutes each. Secondary antibodies (1:10,000, Proteintech, SA00001-1; SA00001-2) were then added and the PVDF membrane was incubated at room temperature for 1 hour. After secondary antibody incubation, the membrane was washed five times with PBST for 5 minutes each. Bands were visualized using an ECL Western blotting detection kit (NCM Biotech, Suzhou, China), and quantitative analysis was performed using Image J software. After normalizing the samples to GAPDH or β-actin levels, the expression ratio was determined.

[0076] 7. Immunohistochemical Staining

[0077] Retinal flat-mount samples were prepared according to the above method and incubated with primary antibodies Anti-Iba1 (1:200, WAKO 019-19741, USA) and Anti-MHCII (1:200, Abcam, ab233990) at 4°C overnight. The next day, after three washes with PBS, the samples were incubated with secondary antibodies Alexa 488 conjugate (CST4412, USA) and Alexa The cells were incubated with a 555 conjugate (CST4409, USA). Nuclear staining was performed with DAPI (EK-5103, Ecoto, China). Images were acquired using a confocal microscope (Zeiss, AirScan) or a fluorescence microscope (Nicon, N2-DM4B). The staining intensity of the target protein was quantitatively analyzed using Image J software.

[0078] 8. Statistical Analysis

[0079] Data are presented as mean ± standard deviation (SD), where n represents the number of replicates for independent experiments. Statistical analysis was performed using GraphPad Prism software. To assess differences between groups, one-way analysis of variance (ANOVA) combined with Tukey's HSD (Honestly Significant Difference) multiple comparison test was used to calculate P values. For comparisons between two independent groups, Student's t-test was used for statistical analysis. P values ​​less than 0.05, 0.01, and 0.001 were considered statistically significant, highly significant, and extremely significant, respectively.

[0080] Part II: Experimental Results

[0081] 1. AOH injury causes RGC death

[0082] A retinal AOH injury model was established in mice, which was caused by transient increase in intraocular pressure (ph-IOP). At 12 hours, 24 hours, and 48 hours after AOH injury, the whole retina was collected for flat mount and HE staining. Figure 2a ) showed that the number of RGCs positively stained with RBPMS decreased over time. 24 hours after AOH injury, the number of RGCs had decreased by more than 50%, and decreased slightly after 48 hours ( Figure 2b HE staining also confirmed this trend, and RGCs density was observed to decrease over time in the AOH group ( Figure 2cThe ganglion cell layer (GCL) showed discontinuity, with some vacuoles present. Other retinal structures, including the inner nuclear layer (INL) and outer nuclear layer (ONL), also became loose and disorganized over time, especially in the 24-hour and 48-hour groups. Retinal damage occurred mainly in the peripheral areas of the 24-hour and 48-hour groups. The 48-hour group also showed more severe loss of RGCs in the intermediate and central positions and retinal thinning in the peripheral areas ( Figure 2d ).

[0083] Based on these results, significant RGCs loss and morphological changes were shown 24 hours after high intraocular pressure, so 24 hours was selected as the observation time point for subsequent experiments.

[0084] 2. Intravitreal injection of JS-6 rescues RGC death and retinal function

[0085] In order to study the role of the BCL3 / NF / κB p50 signaling pathway in AOH damage and the therapeutic effect of inhibiting this pathway in glaucoma, the present invention compared and verified the BCL3 inhibitor JS-6 with simple AOH damage.

[0086] Compared with the AOH group, the number of RBPMS-positive cells in the retinal flat mounts increased in a dose-dependent manner in the JS-6 group ( Figure 3a 、 Figure 3b 、 Figure 3c 、 Figure 3d 、 Figure 3e After AOH injury, RBPMS-positive cells were significantly reduced. However, JS-6 was able to reverse this effect, and the protective effect appeared to increase with increasing JS-6 concentration.

[0087] The retinal morphology shown by HE staining also confirmed the same trend. With the increase of JS-6 dose, the cell count in GCL gradually increased ( Figure 3f 、 Figure 3g The above results indicate that JS-6 can significantly rescue RGC death and alleviate retinal morphological changes with increasing doses.

[0088] The present invention also confirmed the protective effect of JS-6 through FVEP and FERG. The amplitude of the P1 wave in FVEP caused by AOH injury was reduced and the latency was prolonged ( Figure 3h 、 Figure 3i ), and the decline of a- and b-waves in FERG ( Figure 3j 、 Figure 3k) were rescued by JS-6. The degree of protection increased with increasing JS-6 concentration. Based on these results, 5 mM was selected as the optimal JS-6 concentration in subsequent experiments. Furthermore, the present invention proposes that inhibiting the Bcl3 signaling pathway with JS-6 can mitigate retinal ganglion cell death and restore retinal function, demonstrating the importance of the Bcl3 / NF-κB pathway in retinal ganglion cell survival.

[0089] JS-6 alleviates AOH-induced retinal neuroinflammation

[0090] Given that the NF-κB signaling pathway is an inflammatory regulatory pathway, the present invention explored the changes in microglial activation after JS-6 induction. Therefore, the present invention further illustrates the ability of JS-6 to combat retinal neuroinflammation caused by ph-IOP. Iba1 (green) and MHCII (red) were co-stained on retinal flat mounts, and the morphology, number, and distribution of microglia were observed. After AOH injury, the number of microglia in the retina increased, the branches decreased, and the location was superficial ( Figure 4a ), where a large number of Iba1-positive cells were seen fused with MHCII perinuclear-positive cells ( Figure 4b ), indicating a substantial activation of microglia in the ganglion cell layer after AOH injury. In contrast, JS-6 treatment inhibited microglial activation and redistribution; therefore, it can be concluded that JS-6 exhibits anti-inflammatory properties and provides support for JS-6 as a protective factor against retinal AOH injury.

[0091] 4.JS-6 attenuates AOH-induced RGC pyroptosis and apoptosis

[0092] To evaluate whether JS-6 inhibits the BCL3 pathway to regulate pyroptosis and apoptosis of RGCs in glaucoma, we compared the expression levels of pyroptosis proteins, including NLRP3 / IL-1β / cleaved-caspase 1 (p20), and apoptosis markers, including Bax and BCL2. We found that AOH injury induced an increase in NLRP3 / IL-1β / cleaved-caspase 1 (p20) protein levels, while JS-6 treatment significantly downregulated them ( Figure 5a 、 Figure 5b 、 Figure 5c 、 Figure 5d The increase in Bax / Bcl2 expression ratio was also restored by JS-6 ( Figure 5e These data indicate that blocking the BCL3 signaling pathway by JS-6 can significantly inhibit the pyroptosis and apoptosis of RGCs in glaucomatous eyes.

[0093] Experimental conclusion: BCL3 inhibitor JS-6 can effectively rescue the death and loss of retinal ganglion cells in glaucoma.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. Use of a BCL3 inhibitor in the preparation of a drug for treating glaucoma; The BCL3 inhibitor is JS-6, and the chemical formula of JS-6 is

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