Application of JAK1 inhibitor in preparation of medicine for treating optic neurodegenerative diseases

Abstract

The invention relates to application of a JAK1 inhibitor in preparation of a medicine for treating optic neurodegenerative diseases. According to the application disclosed by the invention, the activation of JAK1 in optic ganglion cells (RGC) in the optic nerve degenerative change process is defined, and a series of in-vivo experiments show that the JAK1 inhibitor can be used as a drug target for improving the optic nerve degenerative change, so that the survival rate of the RGC is remarkably increased, the thickness of the GCC is increased, the optic nerve axons are protected, and the application of the JAK1 inhibitor to the optic nerve degenerative change is realized. And independent of the reduction of intraocular pressure, the composition is used for treating optic neurodegenerative diseases. In general, the invention provides experimental basis and theoretical basis for research and development and clinical application of drugs for treating optic neurodegenerative diseases by using the JAK1 inhibitor, opens up a new convertible way for optic neurodegenerative diseases, and has important clinical application prospects and social value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of a JAK1 inhibitor in the preparation of a medicine for treating optic nerve degenerative diseases. Background Art

[0002] Optic nerve degeneration is a type of intractable blinding eye disease caused by the degeneration and eventual death of retinal ganglion cells (RGCs), including retinal vascular occlusion, diabetic retinopathy, glaucoma, etc., manifested as decreased vision and visual field defects, posing a serious threat to visual health. The RGCs in the inner retina are the only neurons that directly transmit visual information to the brain. RGCs are most susceptible to the effects of insufficient blood perfusion in the retina's optic nerve. Insufficient blood perfusion in the retina's optic nerve often leads to local retinal occult ischemia and hypoxia, which triggers a series of inflammatory reactions and oxidative stress, leading to irreversible degenerative changes in the optic nerve. Therefore, ischemia is one of the main pathological factors hypothesized for this type of disease. In optic nerve degeneration, such as optic nerve degeneration caused by high intraocular pressure, retinal insufficiency and ischemia can occur independently of elevated intraocular pressure. Targeting retinal ischemia is expected to develop neuroprotective or restorative therapies independent of lowering intraocular pressure. It is a new direction to improve the treatment of optic nerve degeneration and also has broad significance and value in the treatment of other types of optic nerve degeneration.

[0003] The JAK / STAT pathway is an evolutionarily conserved transmembrane signal transduction variation mechanism that enables cells to communicate with the external environment. JAK / STAT mediates key signal transduction mechanisms such as a variety of interferons and cytokines. These factors bind to specific receptors on the cell surface, causing conformational changes in the receptors, thereby activating Janus kinases (JAKs) bound to the intracellular part of the receptors, further phosphorylating tyrosine residues on the receptors to form phosphorylation sites. After receptor phosphorylation, signal transducers and activators of transcription (STATs) are recruited, and JAKs further phosphorylate STAT proteins. Phosphorylated STATs dimerize and then translocate into the cell nucleus, bind to specific DNA sequences, and regulate the expression of specific genes. The JAK family usually includes JAK1, JAK2, JAK3, and TYK2, and STAT usually includes members such as STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6. In the interferon receptor pathway, the JAK family mediates the phosphorylation of STAT1 and STAT2; phosphorylated STAT1 and STAT2, together with interferon regulatory factor 9 (IRF9), form the ISGF3 transcription factor complex, enter the cell nucleus, and stimulate the expression of a series of IFN-stimulated genes (ISGs).

[0004] Dysregulated JAK-STAT signaling and related gene mutations are closely related to immune activation and a variety of diseases. The JAK / STAT signaling pathway has been found to play an important role in brain neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and ischemic stroke. As a member of the JAK family, JAK1 inhibition can protect and significantly reduce neurodegeneration and neuroinflammatory responses in Parkinson's mouse models, and improve brain damage and neuroinflammation induced by cerebral hemorrhage. In recent years, the successful application of JAK1 inhibitors in autoimmune diseases has attracted widespread attention. Drugs such as filgotinib and upadacitinib have shown good efficacy and safety in the clinical treatment of rheumatoid arthritis and ulcerative colitis. Although the JAK1 signaling pathway may play an important role in brain neurodegenerative diseases, its specific role and mechanism in optic nerve degeneration have not been deeply studied. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art of optic nerve degeneration. Based on the analysis of retinal single cell atlas data of the high intraocular pressure model of cynomolgus monkeys, it is clarified that the JAK1 signal is significantly enriched in RGC and is upregulated in the early stage of optic nerve degeneration. And through the optic nerve degeneration mouse model, the conservation of JAK1 activation in RGC is clarified, and it is explained that JAK1 inhibitors treat optic nerve degeneration by targeted inhibition of JAK1 pathway activation in RGC. Provide experimental basis and theoretical basis for the drug development and clinical application of optic nerve degeneration.

[0006] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0007] The first aspect of the present invention provides the use of a JAK1 inhibitor in the preparation of a drug for treating diseases related to optic nerve degeneration and / or improving physiological indicators of diseases related to optic nerve degeneration.

[0008] Preferably, the optic nerve degeneration-related diseases include, but are not limited to, one or more of ischemic lesions caused by acute retinal blood flow interruption and optic nerve degeneration caused by chronic high intraocular pressure.

[0009] Preferably, the JAK1 inhibitor is selected from one or more of Filgotinib and Upadacitinib.

[0010] Preferably, the physiological index is selected from one or more of retinal ganglion cell survival rate, retinal ganglion cell function, retinal ganglion cell complex thickness, optic nerve axon degeneration, and optic nerve axon gliosis.

[0011] The second aspect of the present invention provides the use of a reagent for detecting the expression level of JAK1 in the preparation of a product for auxiliary diagnosis of diseases related to optic nerve degeneration.

[0012] Preferably, the reagent for detecting the JAK1 expression level includes a reagent for detecting the JAK1 gene level and / or a reagent for detecting the JAK1 protein level.

[0013] Preferably, the reagent for detecting JAK1 protein level is selected from one or more of Jak1 Rabbit mAb (Cell Signaling, #3344) and Phospho-Jak1 Rabbit mAb (Cell Signaling, #74129).

[0014] Preferably, the optic nerve degeneration-related diseases include, but are not limited to, one or more of ischemic lesions caused by acute retinal blood flow interruption and optic nerve degeneration caused by chronic high intraocular pressure.

[0015] The third aspect of the present invention provides a pharmaceutical composition for treating diseases related to optic nerve degeneration and / or improving physiological indicators related to optic nerve degeneration, comprising a JAK1 inhibitor and a pharmaceutically acceptable carrier.

[0016] Preferably, the optic nerve degeneration-related diseases include, but are not limited to, one or more of ischemic lesions caused by acute retinal blood flow interruption and optic nerve degeneration caused by chronic high intraocular pressure.

[0017] Preferably, the JAK1 inhibitor is selected from one or more of Filgotinib and Upadacitinib.

[0018] Preferably, the physiological index is selected from one or more of retinal ganglion cell survival rate, retinal ganglion cell function, retinal ganglion cell complex thickness, optic nerve axon degeneration, and optic nerve axon gliosis.

[0019] Preferably, the pharmaceutically acceptable carrier includes one or more of a solvent, a solubilizer, a surfactant, an antibacterial agent, an antioxidant, a chelating agent, a filler, a binder, a disintegrant, a lubricant, a flavoring agent, and a colorant. The administration method may include but is not limited to oral administration, intravitreal injection, eye drops, and the like.

[0020] It should be noted that, unless otherwise specified, the "JAK1 inhibitor" or similar expressions in the context of the present invention refer to preparations that can specifically antagonize or inhibit JAK1, including but not limited to small molecule inhibitors, shRNA, CRISPR-Cas9 complexes, etc., which can be obtained commercially or prepared by themselves. In order to fully display the relevant experiments and technical solutions, and to facilitate the understanding of those skilled in the art, the commercially available Filgotinib (Selleckchem, S7605) and Upadacitinib (Selleckchem, S8162) are used in the context of the present invention, but the inhibitors used are not used to limit the technical solutions and protection scope of the present invention, and all preparations that can specifically antagonize or inhibit JAK1 should be understood as the scope of the "inhibitor" mentioned in the present invention. In addition to the antibodies listed in the present invention, those skilled in the art are fully capable of designing corresponding primers, primer pairs, and antibodies based on the JAK1 gene sequence using conventional methods in the field including but not limited to molecular biology, and screening the designed primers, primer pairs, and antibodies by conventional experimental means, or obtaining them commercially, as long as they can achieve specific detection of JAK1 expression and phosphorylation levels.

[0021] The present invention found that in the key target cells RGC of optic nerve degeneration, JAK1 is activated by autophosphorylation as Janus kinase and activates the downstream ISGF3 transcription factor complex. In the animal model of optic nerve degeneration, JAK1 inhibitors can inhibit its signal transduction, independently of the intraocular pressure regulation, and reduce the degeneration and functional loss of RGC in the optic nerve degeneration model, and can be used as a target for the treatment of optic nerve degeneration.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses optic nerve degeneration models such as mouse retinal ischemia reperfusion and magnetic bead chronic high intraocular pressure, combined with cynomolgus monkey high intraocular pressure retinal single cell atlas analysis, and finds that in the key target cells RGC of optic nerve degeneration, JAK1 is activated by autophosphorylation as Janus kinase, and activates the downstream ISGF3 transcription factor complex; and deeply studies the key effects of JAK1 inhibitors on optic nerve degeneration and its related physiological indicators, and clarifies that inhibiting JAK1 helps to significantly improve RGC survival rate, increase GCC thickness, protect optic nerve axons, and improve visual electrophysiological function, and can be used as a key target for the treatment of optic nerve degeneration. In general, the present invention proposes that JAK1 inhibitors can target RGC to treat optic nerve degeneration, and provides an experimental theoretical basis for optic nerve degeneration as a clinical JAK1 inhibitor drug treatment indication, which has important clinical application prospects and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the results of Western blot validation of JAK1 phosphorylation activation in the early stages of ischemic optic neuropathy in a retinal ischemia-reperfusion mouse model.

[0025] Figure 2 Schematic diagram of the results of Western blot validation of ISGF3 complex activation in the early stages of ischemic optic neuropathy in a retinal ischemia-reperfusion mouse model.

[0026] Figure 3 Schematic diagram of Western blot verification of JAK1 inhibitor inhibiting ISGF3 complex activation in retinal ischemia-reperfusion mouse model.

[0027] Figure 4 Schematic diagram of the results of JAK1 inhibitor protecting RGC function in the retinal ischemia-reperfusion mouse model.

[0028] Figure 5 Schematic diagram of the results of JAK1 inhibitor Filgotinib increasing RGC survival in the retinal ischemia-reperfusion mouse model.

[0029] Figure 6 Schematic diagram of the results of JAK1 inhibitor Filgotinib increasing GCC thickness in the retinal ischemia-reperfusion mouse model.

[0030] Figure 7 Schematic diagram of the results showing that the JAK1 inhibitor Upadacitinib increased RGC survival in the retinal ischemia-reperfusion mouse model.

[0031] Figure 8 Single-cell mapping of the retina in the cynomolgus monkey model of intraocular hypertension revealed that JAK1 was significantly enriched in RGCs.

[0032] Fig. 9 Schematic diagram of the retinal single-cell atlas of the crab-eating macaque model of intraocular hypertension, which found that JAK1 expression was significantly elevated in RGCs in the early stages of optic nerve degeneration.

[0033] Fig.10 Schematic diagram of the results of Western blot verification of ISGF3 complex activation in the early stage of optic nerve degeneration in the magnetic bead chronic ocular hypertension mouse model.

[0034] Fig.11 Schematic diagram of the results of Western blot verification of JAK1 inhibitors inhibiting ISGF3 complex activation in the magnetic bead chronic ocular hypertension mouse model.

[0035] Fig.12 Schematic diagram of the inhibitory results of JAK1 inhibitors in inhibiting ISGF3 complex activation in RGCs in the magnetic bead chronic ocular hypertension mouse model verified by immunofluorescence.

[0036] Fig.13 This is a schematic diagram showing the results that JAK1 inhibitors have no regulatory effect on intraocular pressure in the magnetic bead chronic ocular hypertension mouse model.

[0037] Fig.14 Schematic diagram of the results of JAK1 inhibitor increasing RGC survival rate and GCC thickness in the magnetic bead chronic ocular hypertension mouse model.

[0038] Fig.15 Schematic diagram of the results of JAK1 inhibitor protecting optic nerve axons in the magnetic bead chronic ocular hypertension mouse model. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Unless otherwise specified, the reagents used in the present invention are all commercially available. The experimental methods used in the present invention, such as molecular experiments, cell experiments, animal experiments, etc., are all conventional methods and techniques in the art. For the conduct of animal experiments, the experimental protocols were approved by the Animal Experiment Ethics Committee of Zhongshan Eye Center of Sun Yat-sen University and followed its guidelines.

[0041] Representative results selected from the biological experiment repetitions are presented in the context figures, and the data are displayed as mean±SD or mean±SEM as specified in the figure. All experiments were repeated at least three times. The data were analyzed using GraphPad Prism9.0 or SPSS22.0 software. Conventional medical statistical methods such as t-test, chi-square test, analysis of variance, rank sum test, etc. were used to compare the mean differences between two or more groups. p<0.05 was considered a significant difference. For the intracellular / in vivo JAK1 expression level, conventional methods in the art (such as PCR, Western blot, etc.) can be used for detection; and the present invention verifies the detection specificity of the Jak1 Rabbit mAb (Cell Signaling, #3344) and Phospho-Jak1 Rabbit mAb (Cell Signaling, #74129) antibodies set forth in the present invention for the expression level and phosphorylation level of JAK1 through specific experiments. For the inhibition of JAK1, in addition to the listed Filgotinib, the present invention is also verified by Upadacitinib, which can specifically inhibit the expression level of JAK1 and produce a therapeutic effect similar to that of Filgotinib. In view of the fact that the detection and inhibition of the expression level of specific genes / proteins belong to conventional means in the art, and are not the main improvement direction of the present invention, the relevant test results are not specifically presented in the present invention, and those skilled in the art can perform detection and verification according to the experimental methods described in the present invention or other conventional methods in the prior art as needed. In addition, in addition to the relevant antibodies listed in the present invention, those skilled in the art can also design relevant primers / primer pairs / antibodies, inhibitors according to the JAK1 gene sequence and / or protein structure, or obtain commercial reagents from the market to complete the detection or inhibition of the expression level of JAK1, so the specific information such as primer pairs, antibodies, inhibitors, etc. listed in the context of the present invention does not constitute a limitation on the actual protection scope of the present invention.

[0042] Example 1 Study on the activation of the JAK1-ISGF3 complex pathway in the mouse retinal ischemia-reperfusion ischemic optic neuropathy model

[0043] First, establish a mouse retinal ischemia-reperfusion (IR) model. The specific steps are as follows:

[0044] (1) The perfusion device is a 250 mL saline infusion bag and an infusion tube. The needle on the infusion tube is replaced with a 32G insulin needle and hung at a position 147 cm above the eye level of the mouse.

[0045] (2) After inducing general anesthesia in mice as described above and performing eyelid disinfection, corneal anesthesia and mydriasis, a 32G insulin needle was inserted into the anterior chamber, the infusion switch was turned on, the needle position was fixed, and the hydrostatic pressure of the liquid in the infusion bag at this height was connected to the eye to induce intraocular pressure to rise to about 120 mmHg, compressing the intraocular blood vessels to reduce blood perfusion and cause retinal ischemia, which lasted for 1 hour, after which the infusion switch was turned off, the needle was removed from the anterior chamber, and the intraocular pressure returned to normal and the intraocular blood vessels resumed perfusion. After surgery, topical antibiotic eye ointment was applied to the cornea and the mouse was rewarmed on a heating pad to wake up.

[0046] Then, the phosphorylation level of JAK1 and the level of downstream ISGF3 complex protein in the retina after modeling were detected by Western-blot. The specific steps are as follows:

[0047] (1) Mouse eyeballs were collected at different time points. After the retina was separated on ice, RIPA lysis buffer (containing 1% PMSF and 1% phosphatase inhibitor) was added to fully lyse for half an hour, and the supernatant was collected by centrifugation.

[0048] (2) Determine the protein concentration using a BCA kit, add SDS loading buffer and denature at 95°C for 10 min to prepare the protein sample.

[0049] (3) After electrophoresis, transfer to the membrane, block the PVDF membrane in order, apply primary antibodies to Jak1, p-Jak1, p-Stat1, Stat1, Irf9, β-Actin, or Gapdh, and secondary antibodies to HRP, prepare fresh luminescent solution, and develop using an exposure device.

[0050] (4) Quantitative analysis of the target protein was performed using ImageJ, and the relative expression level was obtained by normalization based on the internal reference of each sample, followed by statistical analysis.

[0051] The experimental results are as follows Figure 1-2 The results showed that in the mouse retinal ischemia-reperfusion model, JAK1 was phosphorylated and activated in the early stage of optic nerve degeneration, and the level of p-JAK1 was significantly increased (see Figure 1 ), while ISGF3 complex is activated (represented by p-Stat1, Irf9 and Isg15) (see Figure 2 ).

[0052] Example 2 Effect of JAK1 inhibition on ISGF3 complex activation in retinal ischemia-reperfusion model

[0053] First, the mouse retinal ischemia-reperfusion model was established as described previously.

[0054] Subsequently, the Jak1 inhibitor Filgotinib (Selleckchem, S7605) was administered to the model mice by oral gavage. The specific steps are as follows:

[0055] (1) The drug powder was fully dissolved in 6% DMSO to prepare a stock solution, which was then dissolved in corn oil and diluted to a working concentration.

[0056] (2) The dosage was 10 mg / kg body weight, administered orally twice a day, with a volume of 200 μL each time.

[0057] (3) Drug administration began 1 hour before IR modeling, and the control group was gavaged with corn oil at the same frequency and volume.

[0058] Finally, the levels of ISGF3 complex protein in the retina after modeling were detected by Western-blot as described above.

[0059] The experimental results are as follows Figure 3 The results showed that administration of Filgotinib before IR significantly reduced the activation of pStat1, Isg15, and Irf9 24 hours after modeling, verifying the key role of JAK1 in the activation of the IFN signaling pathway.

[0060] Example 3 Effect of JAK1 inhibition on optic nerve degeneration in retinal ischemia-reperfusion model

[0061] The mouse retinal ischemia-reperfusion model was established as described above, and JAK1 inhibitor was administered 1 hour before IR modeling for 3 days. The control group was gavaged with corn oil at the same frequency and volume. Electroretinogram was performed 7 days later. The specific steps were as follows:

[0062] (1) The day before the examination, mice were dark-adapted overnight.

[0063] (2) Mice were anesthetized as described above and placed on a heating pad. After dilation of the pupil and topical anesthesia of the cornea, a reference electrode was inserted subcutaneously in the forehead, a ground electrode was clamped on the tail, and a recording electrode (lubricated with sodium hydroxymethylcellulose) was placed in contact with the cornea.

[0064] (3) After the mouse is securely placed, electrophysiological stimulation is performed, and the STR detection mode of ERG is selected, with a stimulation intensity of 10 - 4 cd·s·m -2 The flash stimulation was performed at intervals of 12 seconds, and the waveforms superimposed after 5 flash stimulations were recorded and analyzed. The amplitude from the baseline level to the first positive wave peak was recorded as the detection value of STR.

[0065] The experimental results are as follows Figure 4The results showed that the amplitude of pSTR in IR eyes was significantly reduced compared with the sham-operated group, while Filgotinib treatment significantly increased the amplitude of pSTR, which can reflect the function of ganglion cells in rodents, indicating that JAK1 inhibitors effectively rescued the function of RGCs.

[0066] Subsequently, retinal ischemic optic neuropathy testing was performed, and the specific steps were as follows:

[0067] (1) Mice were killed after deep anesthesia, and their eyeballs were removed and fixed in 4% paraformaldehyde at room temperature for 1 hour.

[0068] (2) After dehydration through 10%, 20% and 30% sucrose gradient, the cells were embedded in OCT and frozen at -80°C overnight.

[0069] (3) Use a microtome to slice the eyeball at 10 μm. Only the eyeball slices containing the optic disc structure are selected and mounted on a glass slide. After drying, fluorescent staining sections are prepared.

[0070] (4) The optic nerve about 1 mm behind the eyeball was isolated and fixed in glutaraldehyde, dehydrated in ethanol, and then embedded in resin for semi-thin sections with a thickness of 1.5 μm. The sections were stained with toluidine blue and tested for optic neuropathy.

[0071] The indicators for optic neuropathy detection are as follows:

[0072] (1) RGC survival analysis: RGCs were quantified by counting RGCs. RGCs were labeled with the primary antibody Rbpms. Three non-adjacent sections or 12 random areas of retinal flat mounts were selected for each eye. RBPMS was analyzed using ImageJ. + RGCs were counted blindly. RGC survival rate was obtained by dividing the counts of the modeling group by the baseline counts of the control group.

[0073] (2) Analysis of retinal ganglion cell complex (GCC) thickness: The thickness of the retinal ganglion cell complex (GCC) was defined as the distance from the inner limiting membrane of the retina to the edge of the inner nuclear layer, including the RGC nerve fiber layer, ganglion cell layer, and inner plexiform layer of RGC dendrites. Thickness was measured in three different retinal regions: central (500 μm from the midpoint of the optic disc), middle (midpoint between the midpoint of the optic disc and the edge of the retina), and peripheral (150 μm from the edge of the retina). Three representative sections were randomly selected from each retina, and the average of the measurements of each three cross-sections was calculated using ImageJ software to ensure data reliability.

[0074] The experimental results are as follows Figure 5-7 The results showed that the JAK1 inhibitor Filgotinib-treated mice had a higher RGC survival rate 1 week after IR modeling compared with the control group gavaged with corn oil (see Figure 5 ), GCC is thicker (see Figure 6 ), indicating that ischemic optic neuropathy was significantly improved. Similarly, the RGC survival rate of mice treated with the JAK1 inhibitor Upadacitinib was significantly improved 1 week after IR modeling (see Figure 7 ).

[0075] In summary, treatment with the JAK1 inhibitor Filgotinib effectively improved ischemic optic neuropathy in the retinal ischemia-reperfusion model.

[0076] Example 5: Spatiotemporal activation characteristics of JAK1 revealed by single cell atlas analysis of retina in cynomolgus monkey model of intraocular hypertension

[0077] (1) Using Rstudio and Seurat software packages to analyze the retinal single-cell atlas data of the cynomolgus monkey model of intraocular hypertension and analyze the expression of JAK family members in different retinal populations

[0078] (2) Using bioinformatics algorithms to find differentially expressed genes, we analyzed the differences in gene expression at different stages of optic nerve degeneration.

[0079] The experimental results are as follows Figure 8-9 The results showed that JAK1 was significantly expressed in RGCs in the retinal cells of the cynomolgus monkey model of intraocular hypertension (see Figure 8 In the early stages of optic nerve degeneration, JAK1 expression is significantly increased in RGCs (see Fig. 9 ), suggesting that JAK1 activation also occurs during high-pressure optic neuropathy.

[0080] Example 6 Study on the activation of ISGF3 complex pathway by JAK1 inhibitor in mouse magnetic bead chronic intraocular hypertension (COHT) model

[0081] First, establish the mouse magnetic bead chronic ocular hypertension (COHT) model. The specific steps are as follows:

[0082] (1) After the mice were anesthetized and the eyelids were disinfected, 0.5% proparacaine hydrochloride eye drops were applied topically for corneal anesthesia. After mydriasis was dilated with 1% tropicamide eye drops, magnetic beads were injected into the anterior chamber using a glass needle with a diameter of 100 μm. The magnetic beads were sucked into the anterior chamber at the contralateral corneal limbus using a magnet and evenly distributed in the chamber angle. After the operation, antibiotic eye ointment was applied topically to the cornea and the mice were rewarmed on a heating pad.

[0083] (2) After modeling, the intraocular pressure increase was monitored and recorded weekly. After anesthetizing the mice, they were immobilized with their heads horizontal. The intraocular pressure at the central position of the cornea was measured with a handheld tonometer. The average of the three measured values ​​was recorded as the intraocular pressure at that time, and the measurement time period was kept consistent.

[0084] As described above, the protein level of ISGF3 complex in the retina after modeling was detected by Western-blot. The experimental results are as follows Fig.10 The results showed that in the mouse COHT model, ISGF3 complex activation (p-Stat1, Irf9) was activated at an early stage.

[0085] Subsequently, the model mice were given the Jak1 inhibitor Filgotinib (Selleckchem, S7605) by oral gavage as described above. The administration began 1 week after magnetic bead modeling, and the control group was gavaged with corn oil at the same frequency and volume. Three weeks after COHT induction, the peak time point of ISGF3 complex protein activation, the samples were collected, and the regulation of JAK1 inhibitor on ISGF3 complex activation was examined by WB and immunofluorescence experiments as described above.

[0086] The experimental results are as follows Figure 11-12 The results showed that Filgotinib was given 1 week after model establishment, and the activation of pSTAT1 and IRF9 was significantly reduced at 3 weeks after model establishment (see Fig.11 ); Immunofluorescence validation showed that Filgotinib significantly reduced the activation of IRF9 in RGCs (see Fig.12 ), together verifying the key role of JAK1 in activating the ISGF3 complex in RGCs.

[0087] Example 7 Study on the Effect of JAK1 Inhibitor on Optic Neuropathy in Mouse Magnetic Bead Chronic Intraocular Hypertension Model

[0088] The mouse COHT model was established as described above. The Jak1 inhibitor Filgotinib was administered to the model mice by oral gavage. The administration began 1 week after the magnetic bead model was established. The control group was gavaged with corn oil at the same frequency and volume. During this period, intraocular pressure was measured and analyzed as described above.

[0089] The experimental results are as follows Fig.13 The results showed that there was no significant difference in intraocular pressure between the two groups, suggesting that JAK1 inhibitors had no significant regulatory effect on intraocular pressure.

[0090] Subsequently, samples were collected at 6 weeks of modeling and optic neuropathy was detected as described above. The detection indicators were RGC survival analysis and GCC thickness analysis.

[0091] The experimental results are as follows Fig.14 The results showed that compared with the control group gavaged with corn oil, the mice in the JAK1 inhibitor Filgotinib treatment group had a higher RGC survival rate and thicker GCC thickness 6 weeks after magnetic bead modeling.

[0092] Furthermore, the optic nerve axon was quantitatively analyzed: five different 50×50 μm square areas were sampled from the peripheral area of ​​each nerve section and analyzed using ImageJ software. The average number of axons in each optic nerve was calculated and compared with the number per 250 square microns.

[0093] The experimental results are as follows Fig.15 The results showed that optic nerve axon degeneration and gliosis occurred in the COHT model, and this lesion could be significantly improved by the Jak1 inhibitor Filgotinib, and the retention of optic nerve axons was significantly increased, indicating that the optic neuropathy caused by chronic high intraocular pressure was significantly improved.

[0094] It can be seen that the JAK1 inhibitor Filgotinib can effectively inhibit the activation of ISGF3 complex in RGCs and improve the occurrence of optic neuropathy in the magnetic bead chronic high intraocular pressure model. This therapeutic effect is independent of the reduction of intraocular pressure.

[0095] In summary, the present invention found that in the key target cells RGC of optic nerve degeneration, JAK1 is activated by autophosphorylation as Janus kinase, and activates the downstream ISGF3 transcription factor complex. In the animal model of optic nerve degeneration, JAK1 inhibitors can inhibit its signal transduction, and independently of the regulatory effect on intraocular pressure, reduce RGC degeneration and functional loss in the ischemic retinal disease model, and can be used as a target for the treatment of optic nerve degeneration. In general, the present invention proposes that JAK1 inhibitors can target RGC to treat optic nerve degeneration, and provides an experimental theoretical basis for optic nerve degeneration as a clinical JAK1 inhibitor drug treatment indication, which has important clinical application prospects and social value.

[0096] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of a JAK1 inhibitor in the preparation of a drug for treating diseases related to optic nerve degeneration and / or improving physiological indicators of diseases related to optic nerve degeneration.

2. The use according to claim 1, characterized in that: The diseases related to optic nerve degeneration include, but are not limited to, one or more of ischemic lesions caused by acute retinal blood flow interruption and optic neuropathy caused by chronic high intraocular pressure.

3. The use according to claim 1, characterized in that: The physiological index is selected from one or more of retinal ganglion cell survival rate, retinal ganglion cell function, retinal ganglion cell complex thickness, optic nerve axon degeneration, and optic nerve axon gliosis.

4. The use according to claim 1, characterized in that: The JAK1 inhibitor is selected from one or more of Filgotinib and Upadacitinib.

5. Use of reagents for detecting JAK1 expression levels in the preparation of products for auxiliary diagnosis of diseases related to optic nerve degeneration.

6. The use according to claim 5, characterized in that: The reagents for detecting the JAK1 expression level include reagents for detecting the JAK1 gene level and / or reagents for detecting the JAK1 protein level.

7. The use according to claim 5, characterized in that: The diseases related to optic nerve degeneration include, but are not limited to, one or more of ischemic lesions caused by acute retinal blood flow interruption and optic neuropathy caused by chronic high intraocular pressure.

8. A pharmaceutical composition for treating diseases related to optic nerve degeneration and / or improving physiological indicators of diseases related to optic nerve degeneration, characterized in that: It comprises a JAK1 inhibitor and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The diseases related to optic nerve degeneration include, but are not limited to, one or more of ischemic lesions caused by acute retinal blood flow interruption and optic nerve degeneration caused by chronic high intraocular pressure.

10. The pharmaceutical composition according to claim 8, characterized in that The JAK1 inhibitor is selected from one or more of Filgotinib and Upadacitinib.

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