Application of GSK-872 in the preparation of drugs for treating traumatic optic neuropathy

Abstract

The present invention discloses the application of GSK-872 in the preparation of a drug for treating traumatic optic neuropathy, and relates to the field of biomedical technology. The present invention provides the application of GSK-872 in the preparation of a drug for treating traumatic optic neuropathy. The present invention has found that GSK-872 has the effects of inhibiting autophagy, anti-inflammatory and neuroprotection. GSK-872 is used to treat traumatic optic neuropathy. Early intravitreal injection of GSK-872 can significantly inhibit the autophagic death caused by excessive activation of autophagy in retinal microglia, reduce local retinal neuroinflammation, improve the inflammatory microenvironment, thereby delaying the process of retinal ganglion cell death, effectively preventing the loss of neurons, and having a significant effect on the protection of visual function, thereby achieving the purpose of treating traumatic optic nerve injury. The present invention provides a new strategy for treating traumatic optic neuropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of GSK-872 in the preparation of a medicine for treating traumatic optic neuropathy. Background Art

[0002] Traumatic Optic Neuropathy (ONT) is a type of intractable eye disease characterized by degeneration and death of retinal ganglion cells (RGCs) as the main pathological changes. The rapid death of degenerated retinal ganglion cells and the limited regeneration ability of the optic nerve after damage ultimately lead to irreversible loss of visual function, seriously affecting the quality of human life.

[0003] The death of retinal ganglion cells is an important pathological basis in optic neuropathy. In this context, there has been controversy over whether autophagy plays a role in "aggravating cell death" or "preventing cell death". Many studies have shown that autophagy mainly plays a neuroprotective role, mainly in the middle and late stages of optic nerve damage.

[0004] Neuroinflammation is an important pathological basis for retinal neurodegenerative diseases. In retinal neurodegenerative diseases such as traumatic optic neuropathy and age-related macular degeneration, there is a common abnormally active retinal inflammatory response: dysfunction of immune cells triggers abnormal immune response and massive release of inflammatory factors, which promotes neuronal damage and degeneration, impairing visual function.

[0005] Current treatments for ONT include surgical optic nerve decompression and the application of corticosteroids. Treatment attempts for ONT include methods such as stem cell transplantation and gene therapy, focusing on the regeneration and functional reconstruction of damaged retinal ganglion cells in the middle and late stages. Although great progress has been made in neuroprotection and neuroregeneration, the effectiveness of these treatments is limited by the rapid death of RGCs after injury and their limited regenerative capacity. Therefore, rescuing retinal ganglion cells in the early stages of injury may provide a new and promising strategy.

[0006] After optic nerve injury, a cascade of changes in cell death markers occurs, and the autophagy signaling pathway is one of the early and important signaling pathways. Autophagy is an intracellular metabolic process in which protein aggregates and damaged organelles are sequestered in autophagosomes and subsequently degraded in lysosomes. The basal level of autophagy under physiological conditions serves as a protective mechanism that contributes to tissue homeostasis, cell differentiation and development, and adaptation to environmental stress. However, under pathological conditions, especially in acute disease states, excessive and uncontrolled autophagy activation may induce "autophagic cell death", which is different from physiological autophagy, in which a large amount of cellular contents are degraded, leading to cell death, inflammation, and imbalance in the body, thereby promoting the development of the disease.

[0007] Microglia play a crucial role as mediators of neuroinflammation in the retina. They regulate inflammatory responses through activation, polarization, phagocytosis, intercellular signaling, and cell death mechanisms such as programmed necrosis. Under pathological conditions, microglia-mediated neuroinflammation significantly affects the fate of other neural cells. Autophagy, as a key mechanism of homeostasis, can affect microglial activation, survival, and inflammatory responses. Excessive autophagy activation, leading to microglial cell death, may be another important form of microglial activation.

[0008] GSK-872 is a cell-permeable quinolyl-aminobenzothiazole compound with the molecular formula C 19 H 17 N3O2S2, molecular weight is 383.49, CAS number is 1346546-69-7, and the structural formula is as follows:

[0009] .

[0010] GSK-872 mainly binds to the RIP3 kinase region with high affinity, thereby specifically blocking RIP3 protein kinase, IC 50 The value was 1.8 nM, and the IC value for inhibiting enzyme activity was 50 The concentration of GSK-872 in the retinal membrane is 1.3 nM. GSK-872 has been shown to have a good therapeutic or alleviating effect in animal models of cardiovascular and cerebrovascular diseases. It improves brain edema and neurological deficits in early brain injury; it can effectively improve brain edema and neurological function after subarachnoid hemorrhage; GSK-872 reduces programmed necrosis of RGCs in glaucoma and improves glutamate-induced retinal damage and neuroinflammation by inhibiting the RIP1 / RIP3 / MLKL pathway. Intervention of autophagy has shown strong therapeutic potential in animal models of tumors, metabolic and neurodegenerative diseases, and cardiovascular diseases, and has become a boon for patients with cardiovascular diseases. There are no reports that GSK-872 plays a positive role in the treatment of traumatic optic neuropathy. Summary of the invention

[0011] The purpose of the present invention is to provide the use of GSK-872 in the preparation of a drug for treating traumatic optic neuropathy to solve the problems existing in the above-mentioned prior art. The present invention has found that GSK-872 has the effects of inhibiting autophagy, anti-inflammatory and neuroprotection. GSK-872 is used to treat traumatic optic neuropathy. Early intravitreal injection of GSK-872 can significantly inhibit the autophagic death caused by excessive activation of autophagy in retinal microglia, reduce local retinal neuroinflammation, improve the inflammatory microenvironment, thereby delaying the process of retinal ganglion cell death, effectively preventing the loss of neurons, and having a significant effect on the protection of visual function, thereby achieving the purpose of treating traumatic optic nerve injury.

[0012] To achieve the above object, the present invention provides the following solutions:

[0013] The present invention provides use of the compound GSK-872 in preparing a medicine for treating traumatic optic neuropathy.

[0014] Furthermore, the dosage form of the drug is an injection.

[0015] The present invention discloses the following technical effects:

[0016] The present invention provides the use of a RIP3 inhibitor, GSK-872, in the preparation of a drug for treating traumatic optic neuropathy. The present invention has found that GSK-872 has the effects of inhibiting autophagy, anti-inflammatory and neuroprotection. GSK-872 is used to treat traumatic optic neuropathy. Early intravitreal injection of GSK-872 can significantly inhibit the autophagic death caused by excessive activation of autophagy in retinal microglia, reduce local retinal neuroinflammation, improve the inflammatory microenvironment, thereby delaying the process of retinal ganglion cell death, effectively preventing the loss of neurons, and having a significant effect on the protection of visual function, thereby achieving the purpose of treating traumatic optic nerve injury. The present invention provides a new strategy for treating traumatic optic neuropathy, and GSK-872 will have broad application prospects in the preparation of pharmaceutical preparations for treating traumatic optic neuropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Western Blot diagram reflecting the expression of programmed necrosis proteins RIP3 and MLKL in the retinal tissue of wild-type C57 mice at different time points after ONC; β-actin is the internal reference protein; Sham is the sham operation group; ONC is the optic nerve injury group; ONC1d, ONC3d, and ONC7d represent different time points after ONC;

[0019] Figure 2 Statistical graph of the expression of programmed necrosis protein RIP3 in the retinal tissue of wild-type C57 mice at different time points after ONC; ns, not significant, * P<0.05, ** P < 0.01;

[0020] Figure 3Statistical graph of the expression of programmed necrosis protein MLKL in the retinal tissue of wild-type C57 mice at different time points after ONC; ns, not significant, * P<0.05, ** P < 0.01;

[0021] Figure 4 The retinal flat mount immunofluorescence staining image reflects the expression of RIP3 protein in microglia in mouse retinal tissue; Sham is the sham operation group; ONC is the optic nerve clamp injury group;

[0022] Figure 5 Western Blot diagram showing the expression of autophagy proteins Beclin-1, LC3-I and LC3-II in wild-type C57 mice and RIP3 knockout mice after ONC; β-actin is the internal reference protein; WT is wild-type C57 mice; RIP3 - / - RIP3 knockout mice;

[0023] Figure 6 The statistical graph of Beclin-1 expression in wild-type C57 mice and RIP3 knockout mice after ONC; * P < 0.05;

[0024] Figure 7 The statistical graphs of LC3-II / LC3-I expression in wild-type C57 mice and RIP3 knockout mice after ONC; ** P < 0.01;

[0025] Figure 8 Immunofluorescence images of frozen sections of mouse retina; DAPI is used to mark cell nuclei, TUNEL is used to mark dead cells, Beclin-1 is used to mark autophagy, and Iba1 is used to mark microglia; WT-ONC is a wild-type C57 mouse after ONC, RIP3 - / - ONC is the RIP3 knockout mouse after ONC;

[0026] Fig. 9 This is a statistical diagram of the number of TUNEL-positive cells in frozen sections of mouse retina; *** P < 0.001;

[0027] Fig.10 Iba1 in mouse retinal cryosections + / Beclin-1 + / TUNEL + Cell number statistics chart; *** P < 0.001;

[0028] Fig.11Immunofluorescence images of frozen sections of mouse retina; DAPI is used to mark cell nuclei, TUNEL is used to mark dead cells, Beclin-1 is used to mark autophagy, and Tuj1 is used to mark RGC; WT-ONC is a wild-type C57 mouse after ONC; RIP3 - / - ONC is the RIP3 knockout mouse after ONC;

[0029] Fig.12 Statistical diagram of the number of TUNEL-positive cells in frozen sections of mouse retina; ns, not significant;

[0030] Fig.13 Tuj1 in mouse retinal cryosections + / Beclin-1 + / TUNEL + Cell number statistics; ns, not significant;

[0031] Fig.14 Immunofluorescence staining of retinal sections after ONC in WT mice and RIP3 knockout mice; WT represents wild-type mice; RIP3 - / - represents RIP3 knockout mice; ONC represents optic nerve clamp injury; Iba1 marks microglia, CD86 marks the M1 polarization state of microglia, IL-6 marks the cell inflammatory factor IL-6, arrows indicate cells showing protein co-localization, scale bar: 50μm;

[0032] Fig.15 The β-tubulin immunofluorescence staining image of the mouse retinal flat mount; β-tubulin was used to mark RGC, Sham+VEH was the blank control group, Sham+GSK872 was the solvent control group, ONC+VEH was the optic nerve injury group, ONC+GSK872 was the GSK872 treatment group, and ONC+3-MA was the 3-MA treatment group;

[0033] Fig.16 The figure is a statistical diagram of RGC cell density in mouse retinal flat mount; ns, not significant, * P<0.05, ** P<0.01, *** P < 0.001;

[0034] Fig.17 Figure 1 shows the pattern visual evoked potential and pattern retinogram after ONC induction for 1 day; PVEP stands for pattern visual evoked potential; PERG stands for pattern retinogram; Sham+GSK872 is the solvent control group; ONC+VEH is the optic nerve injury group; ONC+GSK872 is the GSK872 treatment group; ONC+3-MA is the 3-MA treatment group;

[0035] Fig.18 PERG statistical graph after ONC induction for 1 day; ns, not significant, * P<0.05, ** P<0.01, *** P < 0.001;

[0036] Fig.19 PVEP statistical graph after ONC induction for 1 day; ns, not significant, * P<0.05, *** P < 0.001;

[0037] Fig. 20 The images are of pupil light reflex measurements one day after ONC induction; pre represents before light stimulation; post represents after light stimulation; Sham+GSK872 is the solvent control group; ONC+VEH is the optic nerve injury group; ONC+GSK872 is the GSK872 treatment group; ONC+3-MA is the 3-MA treatment group;

[0038] Fig.21 Statistical graph of pupil contraction of pupil light reflex after ONC induction for 1 day; ns, not significant, ** P<0.01, *** P < 0.001;

[0039] Fig. 22 Statistical graph of contraction latency of pupil light reflex 1 day after ONC induction; ns, not significant; *** P < 0.001;

[0040] Fig.23 Schematic diagram of the open field experiment in mice one day after ONC induction;

[0041] Fig.24 The statistical diagram of the open field test of mice 1 day after ONC induction; where pre means before optic nerve injury; P1d, P3d and P7d respectively mean 1 day, 3 days and 7 days after optic nerve injury; ONC+VEH is the optic nerve injury group; ONC+GSK872 is the GSK872 treatment group; ONC+3-MA is the 3-MA treatment group; * P<0.05. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] Example 1

[0048] 1. Experimental preparation

[0049] 1.1 Experimental animals

[0050] Wild-type C57BL / 6 mice were purchased from Beijing Weitonglihua Company. RIP3 knockout mice were purchased from Shanghai Model Organisms Technology Co., Ltd. Animal ethics and all experimental operations were in accordance with the Regulations on Laboratory Animal Management of the State Science and Technology Commission.

[0051] 1.2 Experimental reagents and antibodies

[0052] GSK-872 (abcam); 3-MA (Sigma); anti-IBA-1 antibody (Wako); anti-RIP3 antibody (abcam); anti-MLKL antibody (Cell Signaling Technology); anti-Beclin-1 antibody (abcam); anti-βIII-tubulin antibody (Biolegend); anti-TNF-α antibody (abcam); anti-GFAP antibody (abcam); anti-LC3B antibody (Cell Signaling Technology); anti-CD86 antibody (abcam); anti-TNF-α antibody (abcam); optimal cutting temperature compound (OCT) embedding solution; anti-β-actin antibody (abcam); donkey anti-rabbit IgG secondary antibody (488); donkey anti-mouse IgG secondary antibody (555); donkey anti-rat IgG secondary antibody (647); DAPI (Life Technologies); TUNEL kit (Roche).

[0053] 1.3 Buffer

[0054] 1) RIPA buffer protein lysis solution: 0.5 M NaCl 1 mL, 2 mM ethylenediaminetetraacetic acid (EDTA) 0.04 mL, phenylmethylsulfonyl fluoride (PMSF) 100 μL, 50 mM tris (hydroxymethylaminomethane) (Tris), pH 7.4 0.5 mL, 1% surfactant NP-40 1 mL, 0.1% sodium dodecyl sulfate (SDS) 0.2 mL, add ultrapure water to make up to 10 mL.

[0055] 2) 10×TAE: 17.4 M glacial acetic acid (11.4 mL), EDTA (3.7 g), Tris (48.4 g), and double distilled water (diluted water) to a total volume of 1.0 L.

[0056] 3) Blocking solution: 50 μL Triton-X-100, 0.5 g fetal bovine serum albumin (BSA), added to 10.0 mL phosphate buffered saline (PBST) or Tris-Borate-Sodium Tween-20 buffer (TBST) to prepare.

[0057] 4) 2× SDS loading buffer: Tris-HCl pH 6.8, β-mercaptoethanol (200 mM), glycerol (20%), SDS (4%), bromophenol blue (0.2%).

[0058] 5) PBS / PBST buffer: NaCl 16.0 g, NaH2PO4 0.46 g, KCl 0.40 g, Na2HPO4 0.24 g, add double distilled water to make the total volume 2.0 L, add Tween-20 at 1‰ to prepare PBST solution.

[0059] 6) Electrophoresis buffer: 94 g 1.25 M glycine, 15.1 g 0.125 M Tris, 5 g 0.5% (W / V) SDS, add double distilled water to make up to 1 L.

[0060] 7) Electroporation buffer: 0.125 M Tris 15.14 g, 1.25 M Glycine 72.07 g, double distilled water 400 mL, add water to 500 mL.

[0061] 1.4 Experimental instruments and equipment

[0062] RETI scan system; Leica cryostat (Leica, CM1950, Germany); laser confocal microscope (Carl Zeiss, LSM710, Germany); fluorescence microscope (A1, AxioVision, Leica, DM4000, Germany); ophthalmic microsurgery instruments; 33G microinjector (Hamilton).

[0063] 1.5 Statistical and analytical software

[0064] GraphPad Prism 6 and Photoshop software (Adobe CC, 2015) were used for experimental data organization and image drawing. SPSS software (version 18.0) was used for statistical analysis. ImageJ software was used for quantitative analysis of morphological results. Student t-test and one way ANOVA were used to analyze the differences among the groups. The results were considered statistically significant when P < 0.05.

[0065] 2. Experimental methods and results

[0066] 2.1 Establishment and treatment of mouse optic nerve crush (ONC) model

[0067] 1) Anesthetize 8-week-old RIP3, MLKL knockout mice and wild-type C57 mice by intramuscular injection of a mixture of 70 mg / mL Zoletil and 20 mg / mL Xylazine hydrochloride (dose 1.0 mL / kg);

[0068] 2) Make a horizontal incision in the conjunctiva of the left fornix, expose the optic nerve by separating the extraocular muscles, and use reverse forceps to compress the optic nerve 1.5 mm behind the eyeball for 5 seconds under an operating microscope;

[0069] 3) Confirmation of effective optic nerve injury by the presence of a clear gap across the optic nerve at the site of compression;

[0070] 4) Wild-type C57 mice were injected intravitreally immediately after ONC.

[0071] 2.2 Intravitreal injection of GSK-872 in mice:

[0072] 1) GSK-872 was dissolved in phosphate buffered saline (PBS) to a concentration of 1 mmol / L.

[0073] 2) Under the surgical microscope, adjust the mouse head position. Use sterile gauze or other sterile materials to pad under the mouse head to keep the eyeball at the level of the corneal limbus.

[0074] 3) Cut the conjunctiva above the eyeball slightly to expose the sclera, pierce the anterior chamber with a microshovel needle to release a little aqueous humor, and insert the needle tip vertically 1-2 mm behind the corneal limbus. Then switch to a 33G microsyringe to slowly push the GSK-872 solution, with an injection volume of 1.0 μL. If the drug is released into the vitreous cavity under the surgical microscope, the intravitreal injection is successful. The control group was injected with PBS solution without drug.

[0075] 4) Experimental animals with complications such as retinal hemorrhage and retinal detachment will be eliminated.

[0076] 2.3 Western blot detection of programmed necrosis protein and inflammatory factor protein expression in mouse retinal tissue:

[0077] 1) Extraction of mouse retinal proteins:

[0078] C57 mice were killed 24 hours after ONC, and the retinas were immediately separated on ice. 100 μL of cell lysis buffer containing protease inhibitors was added to each group of 4 retinas, and ultrasonic disruption was performed on ice (70 Hz, 60 s). The retinas were then centrifuged at 4°C (132,000 rpm, 10 min) to extract the supernatant.

[0079] 2) Determination of protein concentration using BCA kit:

[0080] According to the instructions, add 1 volume of reagent B to 50 volumes of reagent A to prepare a fresh working solution, and prepare 200 μL per well, and mix thoroughly. Dissolve and mix completely with ultrapure water to prepare 0.5 mg / mL BCA protein standard. Add 0, 2.5, 5, 10, 15, 20 and 25 μL of the standard to the standard wells of the 96-well plate in sequence, and add ultrapure water to a final volume of 25 μL. Add 25 μL of protein solution sample to the sample well of the 96-well plate at a 10-fold dilution ratio (2.5 μL sample: 22.5 μL ultrapure water). Add 200 μL of BCA working solution to all standard wells and sample wells, mix well, place in a 37°C incubator for incubation for 30 minutes, and use an enzyme reader to measure the absorbance at a wavelength of 562 nm, and then calculate the sample protein concentration based on the standard curve.

[0081] 3) Preparation of SDS-PAGE polyacrylamide gel

[0082] a. Prepare 10% and 12% separation gel (15 mL). The composition ratios are as shown in Table 1:

[0083] Table 1 Component ratios for preparing 10% and 12% separation gels

[0084]

[0085] b. Install two Bio-Rad glass plates, add the above components in sequence and mix quickly, quickly pour the prepared separation gel solution into the glass plate, leave a certain volume of concentrated gel, gently add a layer of anhydrous ethanol along the glass wall to cover the separation gel, flatten and prevent the gel from drying and concentrating. Wait for about 30 minutes, make sure the separation gel is solidified, carefully pour out the ethanol, and pour in 5% concentrated gel.

[0086] The ratio of each component required to prepare 5% concentrated gel is shown in Table 2 below:

[0087] Table 2 Component ratio for preparing 5% concentrated gel

[0088] Composition and concentration Volume of each component required to prepare separation gel of different concentrations (ml) Double distilled water 5.60 30% Acrylamide 1.32 1.5 M Tris-HCl, pH 6.8 1.00 10% SDS 0.08 10% ammonium persulfate APS 0.08 TEMED 0.008

[0089] After the concentrated gel is poured, immediately insert a 1.5 mm sample comb, be careful to pat out the interlayer gas, and wait for 30 minutes for the concentrated gel to solidify. You can prepare for sample loading or store the gel in a humidified box at 4°C.

[0090] 4) Perform protein electrophoresis

[0091] a. Protein sample preparation:

[0092] Add 5×SDS loading buffer to the sample at a ratio of 4:1, mix well, heat in a boiling water bath for 10 minutes, take out and place on ice, and centrifuge briefly for storage.

[0093] b. Protein electrophoresis:

[0094] Connect the electrophoresis device, add protein pre-stained Marker (Bio-rad, 170 kd) as a protein molecular weight standard in the first lane, and add equal amounts of sample protein solution in the preset order. Adjust the voltage to 80 V. When the sample enters the separation gel from the stacking gel, adjust it to 120 V and continue electrophoresis. When the dye indicator reaches the bottom of the separation gel, take out the gel for transfer.

[0095] c. Electroporation:

[0096] Precool the electrotransfer buffer. Prepare 2 filter papers and 1 PVDF membrane of appropriate size, soak them in electrotransfer buffer, and make a sandwich transfer: place the black side of the electrode plate down, and place 1 soaked filter paper, SDS-PAGE gel, and PVDF membrane in order from bottom to top. Be careful to remove bubbles to avoid damaging the gel, and clamp the electrode plate after alignment. Set the current intensity to 250mA, and set the transfer time to 60-100 minutes according to the molecular weight of the target protein.

[0097] d. Closure:

[0098] After the transfer, remove the filter paper, wash the PVDF membrane quickly with TBST, and pre-stain with Ponceau dye as needed to observe the protein morphology on the membrane. Then put it in the blocking solution (TBST solution containing 5% fetal bovine serum albumin), place it on a slowly shaking shaker platform, and incubate it at room temperature for 1-2 hours.

[0099] e. Antibody incubation:

[0100] Take out the PVDF membrane and place it in the primary antibody diluent at a dilution concentration of 1:1000, and incubate at 4°C overnight. The primary antibodies used include: anti-RIP1 antibody, anti-RIP3 antibody, anti-MLKL antibody, anti-TNF-α antibody, anti-CCL2 antibody, and anti-IL-17 antibody. Take it out and rewarm it for 30 minutes on the second day, and wash the PVDF membrane with PBST three times, 10 minutes each time. Transfer the PVDF membrane to the secondary antibody diluent at a dilution of 1:3000 and incubate at room temperature for 1-1.5 hours. Wash the membrane with PBST solution three times, 10 minutes each time.

[0101] f. Exposure:

[0102] Operate in a dark room according to the instructions of the ECL luminescence kit, prepare fresh luminescence solution (mix equal volumes of solution A and solution B), add it to the PVDF membrane to ensure full contact, incubate at room temperature for 2 minutes, wrap the PVDF membrane with a clean, transparent plastic film, cover it with a piece of film, press it in a dark box, and expose it moderately for about 20 seconds to 1 minute.

[0103] The experimental results are as follows Figure 1-Figure 3 and Figure 5-Figure 7 shown. Figure 2 The results showed that RIP3 increased significantly 1 day after ONC induction. Figure 3 MLKL was shown to be significantly increased two days after ONC induction; Figure 5-Figure 7 Display through RIP3 - / - After the ONC model was established in mice, the expression levels of autophagy-related proteins Beclin-1 and LC3 II were significantly downregulated.

[0104] 2.4 Immunofluorescence staining of retinal frozen sections:

[0105] 24 hours after ONC, RIP3 knockout mice, MLKL knockout mice, and C57 mice were anesthetized and killed by spinal dislocation, and the eyeballs were carefully removed;

[0106] The eyeballs were fixed in 20 times more volume of 4% paraformaldehyde (PFA) for 30-60 min;

[0107] The eyeball was fixed with toothed forceps under a dissecting microscope, the cornea was cut open and the lens was removed, dehydrated with a sucrose solution gradient, embedded in an embedding medium, and stored at -20°C overnight to prepare 8 μm frozen sections;

[0108] The sections were perforated in 0.5% Triton X-100 / PBS for 5 min and washed twice with PBS;

[0109] Block with 5% BSA for 1 hour at room temperature or overnight at 4°C;

[0110] Freshly prepared primary antibodies for immunofluorescence (Beclin-1, iba-1, TNF-α, IL-6, RIP3): diluted in PBS at a ratio of 1:100, incubated at 4°C overnight, and washed with PBS five times, 3 minutes each time;

[0111] The sections were removed and incubated with secondary antibodies. The corresponding secondary antibodies and 4',6-diamidino-2-phenylindole (DAPI) were diluted at 1:1000 for 1-2 hours at room temperature, and protected from light; washed with PBS 5 times, 3 minutes each time;

[0112] Add a little anti-fluorescence quenching sealing agent to seal the slide, and observe and photograph under a fluorescence microscope or confocal microscope.

[0113] The experimental results are as attached Figure 8-Figure 13 shown. Figure 8-Figure 10 showed that after ONC injury, RIP3 was expressed in mice compared with WT mice. - / - TUNEL in the inner retina of mice + Cell reduction, and IBA1 + / Beclin-1 + / TUNEL + The decrease in cell number indicates that the number of microglia undergoing autophagic cell death has decreased, however, silencing RIP3 does not affect the extent of autophagic cell death in RGCs. Fig.14 It was shown that after silencing RIP3, the expression of M1 microglia marked by CD86 and the proinflammatory cytokine IL-6 after ONC could be significantly downregulated.

[0114] 2.5 Immunofluorescence staining of retinal flat mounts:

[0115] C57 mice 1 day after ONC were anesthetized and killed by spinal dislocation, and the eyeballs were carefully removed;

[0116] The eyeballs were fixed in 20 times more volume of 4% PFA for 30-60 min;

[0117] Fix the eyeball with toothed forceps under a dissecting microscope, remove the optic nerve first, clamp the sclera at the optic nerve hole and gently tear it open to expose the retinal tissue, tear it to the corneal limbus in sequence, remove the cornea, iris, lens and other tissues to obtain a complete retina;

[0118] The retina was placed in 0.5% Triton X-100 / PBS for 5 min and then washed twice with PBS;

[0119] Block with 5% BSA for 1 hour at room temperature or overnight at 4°C;

[0120] Freshly prepare primary antibodies for immunofluorescence (iba-1, TNF-α, RIP3, β-tubulin): dilute in PBST or TBST at a ratio of 1:100, place the retina in the antibody, and incubate overnight at 4°C. During this period, gently shake the EP tube every few hours to fully contact the antibody;

[0121] The retina was removed and incubated with secondary antibody at room temperature for 1-2 hours, protected from light; washed with PBST 6 times, 10 minutes each time;

[0122] Transfer the retina to a glass slide, remove impurities with toothless forceps under a dissecting stereo microscope, and slowly peel off the vitreous fibers. Make four radial cuts with the optic disc as the center, with the nerve layer facing upwards, drop a little anti-fluorescence quenching sealing agent to seal the slide, and observe and photograph under a fluorescence microscope or confocal microscope;

[0123] The experimental results are as follows Figure 4 , Figure 15-16 shown. Figure 4 The results showed that RIP3 protein increased in the mouse retina 24 hours after ONC and was mainly located in microglia (marked by iba-1), as shown in Figure 15-16 As shown, treatment with GSK872 or 3-MA can reduce RGC death after optic nerve injury.

[0124] 2.6 Graphic electroretinogram (PERG) evaluation of retinal function in mice after GSK-872 and 3-MA treatment:

[0125] 1) Preparation: PERG was performed on C57 mice 24 hours after ONC using the RETIMAP small animal retinal imaging system (Roland Consult, Germany), and the amplitude of N1-P1 and the peak of P1 were measured using RETI-port recording software (Roland-Consult, Germany). Mice were dark-adapted for 12 hours before examination. Anesthesia was performed by intramuscular injection (a mixture of 70 mg / mL Zoletil and 20 mg / mL Xylazine, dose 1.0 mL / kg), and mydriasis was dilated with compound tropicamide eye drops.

[0126] 2) PERG detection. The whole experiment was carried out under weak red light illumination. The mouse was fixed on the experimental board and the electrodes were installed. A small amount of 3% hydroxypropyl methylcellulose lubricating gel was applied to the cornea to keep the cornea moist, and the recording electrode was gently attached to the cornea. The reference electrode (needle electrode) was placed under the skin of the ipsilateral cheek of the mouse, and the ground electrode was placed under the skin of the mouse tail. Relevant examinations were started after about 5 minutes of dark adaptation again. Dark-adapted ERG (Scot ERG) detection and recording should be performed according to the international clinical visual electrophysiology standardization protocol. The spatial frequency was maintained at 0.02 cycles / degree, the temporal frequency was maintained at 1 Hz, and the number of superpositions was maintained at 200 times. The PERG recording time was 200 ms, and 20.0 cd s / m was recorded. 2 PERG waveform of mice under different light intensity. During recording, the contralateral eye was covered with a black cloth, and the spatial frequency, temporal frequency and number of superpositions of the vertical grating were kept consistent. The waveform was analyzed and counted by application software.

[0127] 3) The experimental results are as attached Figure 17-Figure 18 As shown in Figure 2, it can be observed that the C57 mice in the ONC group showed decreased waveform amplitude and prolonged peak duration; after treatment with GSK-872 and 3-MA, the waveform amplitude of the ONC group decreased and the peak duration of the ONC group increased. 2 Improvements were found in the waveform amplitude and peak time of PERG under different light intensities, indicating that retinal nerve function was partially improved.

[0128] 2.7 Pattern visual evoked potential (PVEP) evaluation of optic nerve conduction function in mice after GSK-872 treatment

[0129] 1) Preparation. PVEP was detected in C57 mice 24 hours after ONC using the RETIMAP small animal retinal imaging system (Roland Consult, Germany), and the amplitude of N1-P1 and the peak of P1 were measured using RETI-port recording software (Roland-Consult, Germany). Mice were dark-adapted for 12 hours before examination. Anesthesia was performed by intramuscular injection (a mixture of 70 mg / mL Zoletil and 20 mg / mL Xylazine, dose 1.0 mL / kg), and mydriasis was dilated with compound tropicamide eye drops.

[0130] 2) PVEP detection. The whole experiment was carried out under weak red light illumination. The mouse was fixed on the experimental board and the electrodes were installed. A small amount of 3% hydroxypropyl methylcellulose lubricating gel was applied to the cornea to keep the cornea moist, and the recording electrode was gently attached to the cornea. The reference electrode (needle electrode) was placed under the skin of the ipsilateral cheek of the mouse, and the ground electrode was placed under the skin of the mouse tail. Relevant examinations were started after about 5 minutes of dark adaptation again. Dark-adapted VEP (Scot ERG) detection and recording should be performed according to the international clinical visual electrophysiology standardization program. The spatial frequency was maintained at 0.02 cycles / degree, the temporal frequency was maintained at 1 Hz, and the number of superpositions was maintained at 240 times. The PVEP recording time was 500 ms, and 20.0 cd s / m was recorded. 2 PVEP waveform of mice under different light intensity. During recording, the contralateral eye was covered with a black cloth, and the spatial frequency, temporal frequency and number of superpositions of the vertical grating were kept consistent. The waveform was analyzed and counted by application software.

[0131] 3) Experimental results are as follows Fig.17 and Fig.19 As shown in Figure 2, it can be observed that the C57 mice in the ONC group showed decreased waveform amplitude and prolonged peak duration; after treatment with GSK-872 and 3-MA, the waveform amplitude of the ONC group decreased and the peak duration of the ONC group increased. 2 The waveform amplitude and peak time of PERG were found to be restored under different light intensities, indicating that the conduction function of the optic nerve was partially improved.

[0132] 2.8 Pupillary light reflex assessment of visual function in mice after GSK-872 treatment

[0133] 1) Place the mice in a dark room for 30 minutes to dark adapt and then anesthetize them;

[0134] 2) Using the 8x objective lens of the surgical microscope to focus and vertically align the pupil midpoint, stimulation was performed using the halogen light source of the surgical microscope (100 W, 12 V) for 20 seconds, followed by 60 seconds of darkness to restore the pupil diameter to the baseline level.

[0135] 3) The pupil state was recorded using the WinFast PVR camera system, and the constricted pupil area was measured using ImageJ software. Twelve eyes of 12 mice in each group were used for statistical analysis.

[0136] The results of pupillary light reflex measurements after 1 day of ONC induction are shown in Figure 20-22 shown. Fig.21 This is a statistical diagram of the pupil contraction amplitude of pupil light reflex one day after ONC induction. It can be seen that the pupil contraction amplitude of the control group is reduced compared with the optic nerve injury group, and the pupil contraction amplitude of the treatment group is increased compared with the optic nerve injury group; Fig. 22 This is a statistical diagram of the contraction time of pupil light reflex one day after ONC induction. It can be seen that the pupil contraction time of the control group was prolonged compared with the optic nerve injury group, and the pupil contraction time of the treatment group was significantly shortened compared with the optic nerve injury group.

[0137] 2.9 Open field experiment

[0138] 1) The experimental apparatus consisted of a cage (45 × 30 × 30 cm) divided into a white open area (300 lux light intensity, 30 × 30 × 30 cm) and a dark area (no lighting, 15 × 30 × 30 cm), with a door (5 × 5 cm) between them;

[0139] 2) Place the mice in the dark chamber for 2 minutes to adapt. After that, open the door and allow the mice to move freely between the two chambers for 5 minutes. The time spent in the dark chamber and the white open space will be recorded. 12 mice in each group were used for statistical analysis.

[0140] The schematic diagram and statistical graph of the open field test of mice after 1 day of ONC induction are shown in Fig.23 and Fig.24 The results showed that in the first three days after optic nerve injury, there was no significant change in the time that mice in the treatment group stayed in the dark room compared with the optic nerve injury group. On the seventh day after optic nerve injury, the time that mice in the treatment group stayed in the dark room was significantly increased compared with the optic nerve injury group.

[0141] 2.8 Experimental Results Summary

[0142] The present invention mainly obtains the following conclusions:

[0143] 1) Immunofluorescence staining and Western Blot experiments confirmed that silencing RIP3 can significantly inhibit the autophagic death of retinal microglia in mice with traumatic optic neuropathy model, reduce their secretion of proinflammatory factors, and alleviate neuroinflammation; intravitreal injection of GSK-872 can significantly reduce the loss of retinal ganglion cells in mice with traumatic optic neuropathy and protect visual function.

[0144] 2) Visual induced potential, retinal potential, pupillary light reflex and open field tests confirmed that intravitreal injection of GSK-872 can significantly improve optic nerve conduction, retinal function and light sensitivity in mice with traumatic optic neuropathy.

[0145] The results of the present invention provide experimental evidence and theoretical basis for the potential clinical application of GSK-872 in treating optic nerve damage and anti-neuroinflammation. GSK-872 will have broad application prospects in the preparation of pharmaceutical preparations for treating neurodegenerative diseases.

[0146] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of the compound GSK-872 in the preparation of a drug for treating traumatic optic neuropathy.

2. The use according to claim 1, characterized in that: The dosage form of the drug is injection.

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