Application of BCL3 inhibitor in preparation of medicine for treating glaucoma

Abstract

The invention discloses an application of a BCL3 inhibitor in preparation of a medicine for treating glaucoma. According to the invention, the effect of a BCL3 / NF-kB / NLRP3 signaling pathway in glaucoma is found, and a BCL3 inhibitor (such as JS-6) is never reported about reduction of apoptosis and pyroptosis of retinal ganglion cells, and is used for treating glaucoma-related diseases.

Description

Technical Field

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

[0002] Glaucoma is the leading irreversible blinding eye disease in the world. It is mainly caused by pathologically high intraocular pressure (ph-IOH), which leads to 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 whole 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-related risk factors include high intraocular pressure, age, family inheritance and many other factors, among which high intraocular pressure is the most critical. Therefore, the existing clinical treatments for glaucoma, whether drugs, lasers or surgery, are mainly to reduce and control intraocular pressure. However, in actual clinical work, many glaucoma patients do not have abnormal increases in intraocular pressure during the onset of the disease, such as patients with normal intraocular pressure glaucoma, and patients with ocular hypertension whose intraocular pressure is higher than normal but who do not suffer from glaucoma. In addition, the drug treatment of glaucoma has problems such as poor patient compliance, poor efficacy in some patients, drug resistance in patients, and unavoidable side effects of the drugs themselves. These patients then choose surgical treatment, but surgical treatment has the problem of poor postoperative intraocular pressure control, increased intraocular pressure again due to scarring of the filtering bleb, certain risks and unavoidable complications of the surgery itself, and contraindications for patients with other eye diseases and systemic underlying diseases and cannot receive surgical treatment. Moreover, even if the patient is treated smoothly and the intraocular pressure is well controlled, the vision loss that has occurred cannot be reversed, and some patients still experience worsening and progression of glaucoma. It can be seen that abnormal increase in intraocular pressure is an important factor in the development of glaucoma but not the only factor. There are many unknown causes of the death of RGCs in the development 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 mechanism of retinal nerve damage caused by glaucoma has been widely studied. The current theories about the factors of optic nerve damage in glaucomatous eyes include mechanical damage caused by high intraocular pressure, vascular dysfunction, oxidative stress, neuroinflammatory response and excitotoxicity. Among them, oxidative stress and inflammation play a crucial role in the occurrence and progression of RGC damage. Acute glaucoma can trigger a self-reinforcing destructive cascade involving neuronal depolarization, calcium influx and blood-retinal barrier rupture, and also lead to extreme release of free radicals and excessive inflammatory response. It overwhelms the normal cellular antioxidant defense, ultimately leading to the death of retinal ganglion cells (RGCs), retinal morphological degeneration and impaired retinal function. Therefore, fighting inflammation may be a promising strategy to save glaucoma-damaged RGCs.

[0005] NF-κB signaling is a key pathway that influences 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, TNF-α, and inflammasomes such as NLRP3, one of the most important inflammasomes regulating neuroinflammation and pyroptosis. The most abundant form of NF-κB activated via the classical 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 the transcription of target genes. BCL3 is an atypical member of the IκB protein family and plays a crucial role in regulating the activity of the nuclear factor NF-κB. Depending on the cell type and the nature of the stimulus, BCL3 can promote or inhibit the NF-κB signaling pathway, thereby regulating downstream gene transcription, ultimately playing a dual role in inflammation, exerting either pro-inflammatory 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 regard.

[0006] JS-6 is a novel small molecule with strong intracellular BCL3 activity inhibition. JS-6 is designed to inhibit the protein interaction between the ankyrin repeat domain of BCL3 and its regulatory protein 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 tumors, including retinal diseases.

[0007] Therefore, further clarifying the therapeutic effect of BCL3-related signal transduction 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 provide an application 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 the 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 reduced 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 treatment strategy for the treatment of glaucoma.

[0010] The present invention provides an application of a BCL3 inhibitor in preparing 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 finds 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 a glaucoma disease caused by acute high intraocular pressure injury.

[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 in which the number of intermediate RBPMS-positive cells in retinal flat mounts is reduced.

[0017] In the present invention, the glaucoma may be a glaucoma disease in which the number of peripheral RBPMS-positive cells in the retinal flat mount is reduced.

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

[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 with 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 being in accordance with the common sense in the art, 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 and progressive effects of the present invention are:

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

[0026] The present invention discloses the application of BCL3 inhibitor JS-6 in the treatment, prevention and alleviation of glaucomatous optic nerve damage. Experimental data show that 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 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 alleviating or saving 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) in retinal flat mounts were labeled by immunofluorescence using RBPMS antibody. From left to right are the whole retinal flat mount results (upper) and the local magnified images of the selected areas (lower) of the normal control group, the group 12 hours after AOH injury, the group 24 hours after AOH injury, and the group 48 hours after AOH injury, scale bar = 200 μm.

[0029] Figure 2b The results of quantitative analysis of the density of RBPMS-positive cells in retinal flat mounts at different time points (normal control, 12h, 24h, 48h) after pathological high intraocular pressure-induced injury. One-way ANOVA was used for comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group.

[0030] Figure 2c The HE-stained retinal tissue sections show the changes in retinal structure at different time points (normal control, 12h, 24h, 48h) after pathological high intraocular pressure was induced. Scale bar = 20 μm; representative images of the central, middle and peripheral areas of the retina were selected.

[0031] Figure 2d Quantitative analysis of cell counts in the ganglion cell layer (GCL) in HE-stained retinal tissue sections at different time points (normal control, 12h, 24h, 48h) after pathological high intraocular pressure-induced injury. One-way ANOVA was used for comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group.

[0032] Figure 3a To label retinal ganglion cells (RGCs) in retinal flat mounts by immunofluorescence using RBPMS antibody. From left to right are the whole retinal flat mount results (top) and the local magnified images of the selected areas (bottom) of the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group, scale bar = 200μm.

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

[0034] Figure 3cThe results of quantitative analysis of the RBPMS-positive cell density in the central part of the retinal flat mounts of the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group were compared by 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 The results of quantitative analysis of the RBPMS-positive cell density in the middle part of the retinal flat mounts of the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group were compared by 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 The results of quantitative analysis of the RBPMS-positive cell density in the peripheral part of the retinal flat mounts of the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group were compared by 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 The retinal tissue sections after HE staining show the changes in the structure of the eyeball (upper image × 4 times) and selected retinas (lower image × 63 times) 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. Scale bar = 200μm (upper), 20μm (lower); representative images of the middle area of ​​the retina were selected.

[0038] Figure 3g The results of quantitative analysis of cell counts in the ganglion cell layer (GCL) of HE-stained retinal tissue sections in the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group were shown. One-way ANOVA was used for comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group.

[0039] Figure 3h The waveforms of flash visual evoked potential (F-VEP) in 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 The results of quantitative analysis of the N1-P1 amplitude and P1 latency of flash visual evoked potential (F-VEP) in the uninjured control group, AOH-injured group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group were shown. One-way ANOVA was used for inter-group comparison. *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 The results of quantitative analysis of the amplitude of the a-wave and b-wave of the flash electroretinogram (F-ERG) in the normal control group, AOH injury group, AOH+1mM JS-6 group, AOH+2.5mM JS-6 group, and AOH+5mMJS-6 group were shown. One-way ANOVA was used for comparison among the groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group.

[0043] Figure 4a The morphology, number and distribution of microglia in the retinal flat mounts of the normal control group, 5mM JS-6 group, AOH injury group and AOH+5mM JS-6 group were taken under a 40× objective lens after co-staining with Iba1 (green) and MHCII (red); i is the local fluorescence image of the retinal flat mount stained with Iba1; ii is the black and white image obtained after color conversion of the above image; iii is the morphology of microglia extracted from the above image. Scale bar = 20μm.

[0044] Figure 4b The morphology, number and distribution of microglia in the retinal flat mounts of the normal control group, 5mM JS-6 group, AOH injury group and AOH+5mM JS-6 group were taken under a 20× objective lens after co-staining with Iba1 (green) and MHCII (red); i is the local fluorescence image of the retinal flat mount stained with Iba1; ii is the local fluorescence image of the retinal flat mount stained with MHCII; iii is the fluorescence co-staining merged image of the above two images. Scale bar = 20μm.

[0045] Figure 5aThe results of protein immunoblot (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, 5mM JS-6 group, AOH injury group, and AOH+5mM JS-6 group. One-way ANOVA was used for inter-group comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group.

[0047] Figure 5c Quantitative analysis of the relative expression levels of IL-1β to Actin in the normal control group, 5mM JS-6 group, AOH injury group, and AOH+5mM JS-6 group. One-way ANOVA was used for comparison among the groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con 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 relative expression level of Actin in the normal control group, 5mM JS-6 group, AOH injury group, and AOH+5mM JS-6 group. One-way ANOVA was used for comparison among the groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group.

[0049] Figure 5e Quantitative analysis of the ratio of Bax to Bcl-2 (Bax / Bcl-2) relative to the relative expression level of Actin in the normal control group, 5mM JS-6 group, AOH injury group, and AOH+5mM JS-6 group. One-way ANOVA was used for comparison among the groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the Con group. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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. After activation, it can cleave GSDMD and cause cell pyroptosis.

[0055] Cleaved-Caspase-1: The cleavage product of Caspase-1, which is a sign 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] Male C57BL6 mice aged 8 weeks were obtained from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China) and maintained under SPF conditions provided by the Laboratory Animal Center of Xiangya Hospital, Central South University with a 12-h light-dark cycle and adequate food and water. All procedures used in the animal experiments were approved by the Laboratory Animal Welfare Ethics Committee of Central South University and were 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 intraocular 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 The appropriate volumes of JS6 were freshly diluted to concentrations of 1 mM, 2.5 mM, and 5 mM with phosphate-buffered saline (PBS; 0.01 M; pH 7.4) for each use.

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

[0065] On the second day, the animals were anesthetized and AOH injury was induced by inserting a micro glass needle connected to a saline reservoir into the anterior chamber of the eye to maintain 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 without increasing intraocular pressure. After surgery, 0.3% tobramycin dexamethasone ointment (saAlcon-Couvreur nv) was applied to the eyes. A heating pad was used to keep the animal body temperature at 37 ° C.

[0066] 3. Retinal flat mounts for quantitative analysis of retinal ganglion cell (RGC) density

[0067] The eyes were immediately removed after the mice were killed 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 drop, 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. After washing with PBS five times the next day, the retinal tissue was incubated with fluorescent secondary antibodies (Alexa Fluor). 488, CST4412, USA) were incubated at room temperature for 1 hour. Images were acquired using a fluorescent microscope (Nicon, N2-DM4B). The number of RBPMS-positive RGCs was counted in three non-overlapping areas along the midline of each quadrant, from the optic disc to the border, at intervals of 400 μm, for a total of 12 areas. The results are shown as the percentage loss of RGCs compared to the control group.

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

[0069] Retinal cross-sections were subjected to histological evaluation. After acute ocular hypertension injury, the retinas were stained with H&E to observe and count cells in the ganglion cell layer (GCL). The eyes were removed immediately 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 an optical 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 for 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] The recording and reference electrodes were inserted subcutaneously in the mouse, contacting the surface of the occipital and frontal bones, respectively; the ground electrode was inserted subcutaneously near the mouse's tail. During the recording process, the unstimulated eye was occluded. F-VEP was recorded continuously at different light intensities, and 100 consecutive flash stimuli were recorded and averaged to obtain a waveform for each intensity. The first positive peak in the F-VEP waveform was designated as P1, and the first negative peak was designated as N1. The N1-P1 amplitude was measured and analyzed.

[0073] Before F-ERG recording, the animal preparation steps were the same as for F-VEP, while mice were dark-adapted for 12 h before examination and red light illumination was used to maintain a dark testing environment. Gold wire loop electrodes were placed on the corneal surface, and carboxymethylcellulose eye drops were applied to increase current conduction and corneal moisturizing. The placement of the ground electrode was the same as that of the above F-VEP recording, and the reference electrode was inserted subcutaneously on both sides of the nose. Stimulation and detection were based on the ISCEV standard. After the detection was completed, the amplitudes of the a-wave and b-wave of each group were analyzed.

[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) at a ratio of 100:1 with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail, and then centrifuged (12000 g, 10 minutes, 4 ° C). The supernatant was extracted and measured by a protein assay kit (Thermo). 20 μg of protein samples from each animal were 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 for 1 hour at room temperature 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 (1:10000, Proteintech, 60004-1) or β-actin (1:10000, Proteintech, 811115) were incubated overnight at 4°C and then washed five times with PBST for 5 minutes each. Secondary antibodies (1:10000, Proteintech, SA00001-1; SA00001-2) were then added and the PVDF membrane was incubated at room temperature for 1 hour. After incubation with the secondary antibody, the membrane was washed five times with PBST for 5 minutes each. Bands were developed using the ECL (NCM Biotech, Suzhou, China) Western blotting detection kit and quantitative analysis was performed using Image J software. After normalizing the samples for 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 555 conjugate (CST4409, USA) was used for incubation. 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 repetitions of independent experiments. Statistical analysis was performed using GraphPad Prism software. To evaluate the differences between different groups, this study used one-way analysis of variance (ANOVA) combined with Tukey's HSD (Honestly Significant Difference) multiple comparison test to calculate P values. For the comparison of two independent groups of samples, this study used Student's t-test 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 using mice. Retinal injury 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 a decrease in RGCs density over time was observed 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 a 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 BCL3 / NF / κB p50 signaling pathway in AOH injury and the therapeutic effect of inhibiting this pathway in glaucoma, the present invention compared and verified the BCL3 inhibitor JS-6 with simple AOH injury.

[0086] Compared with the AOH group, the number of RBPMS-positive cells in the retinal flat mounts of the JS-6 group increased in a dose-dependent manner ( 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 seemed to increase with increasing JS-6 concentration.

[0087] The retinal morphology shown by HE staining also confirmed the same trend. As the dose of JS-6 increased, the cell count in the 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. And the degree of protection increased with the increase of JS-6 concentration. According to the above results, 5mM was selected as the optimal concentration of JS-6 in the next experiment. In addition, the present invention proposes that the use of JS-6 to inhibit the Bcl3 signaling pathway can reduce retinal ganglion cell death and restore retinal function, and at the same time shows the importance of the Bcl3 / NF-κB pathway in the survival of retinal ganglion cells.

[0089] 3. JS-6 alleviates AOH-induced retinal neuroinflammation

[0090] Given that the NF-κB signaling pathway is an inflammatory regulatory pathway, the present invention explores the activation changes of microglia after JS-6 induction. Therefore, the present invention also 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. The number of microglia in the retina increased after AOH injury, 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 massive 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 protein expression levels of pyroptosis, including NLRP3 / IL-1β / cleaved-caspase 1 (p20), and apoptosis markers, including Bax and BCL2. We found that AOH injury caused an increase in NLRP3 / IL-1β / cleaved-caspase1 (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 protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. Use of a BCL3 inhibitor in the preparation of a drug for treating glaucoma.

2. The use according to claim 1, characterized in that The BCL3 inhibitor is JS-6, and the chemical formula of JS-6 is 3. The use according to claim 1, characterized in that The glaucoma is a glaucomatous disease in which the number of central RBPMS-positive cells in retinal flat mounts is reduced.

4. The use according to claim 1, characterized in that The glaucoma is a glaucomatous disease in which the number of intermediate RBPMS-positive cells in retinal flat mounts is reduced.

5. The use according to claim 1, characterized in that The glaucoma is a glaucoma disease in which the number of peripheral RBPMS-positive cells in retinal flat mounts is reduced.

6. The use according to claim 1, characterized in that The glaucoma is a glaucomatous disease in which the amplitude of the P1 wave in the flash visual evoked potential is reduced and the latency is prolonged.

7. The use according to claim 1, characterized in that The glaucoma is a glaucoma disease in which the a wave and the b wave of the flash electroretinogram are decreased.

8. The use according to claim 1, characterized in that The glaucoma is a glaucoma disease characterized by elevated levels of NLRP3, IL-1β, and cleaved-caspase1 proteins.

9. The use according to claim 1, characterized in that The glaucoma is a glaucomatous disease in which the Bax / Bcl2 expression ratio is increased.

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