Application of GMFB in preparation of medicine for treating traumatic optic neuropathy

Abstract

The invention relates to application of GMFB in preparation of a medicine for treating traumatic optic neuropathy. Research finds that on the third day of a rat ONC model, compared with normal control, GMFB expression is remarkably up-regulated; cholera toxin B subunit (Cholera Toxin Subunit B, CTB) tracing shows that the axons of a rat ONC model with the GMFB knocked out are remarkably lengthened (compared with those of a wild rat ONC model), and LFB (luxoLuxol Fast Blue) myelination staining shows that remyelination of the denatured axons can be effectively improved, axon regeneration is promoted, and the visual function is improved by knockout of the GMFB.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of GMFB in the preparation of a medicament for treating traumatic optic neuropathy. Background Art

[0002] Traumatic optic neuropathy is one of the common and serious complications of craniocerebral injury, accounting for about 2% - 5% of craniocerebral trauma. More than 90% of optic nerve injuries are indirect injuries in the optic canal segment. Once damaged, it will lead to partial or even total vision loss, seriously affecting the quality of life of patients. The death of retinal ganglion cells (RGC) and axonal degeneration are common pathological changes in all types of optic neuropathy. Neuroprotection, axon regeneration, and remyelination are the main treatment strategies for optic neuropathy. Currently, the clinical treatment methods for traumatic optic nerve injury include drug treatment and surgical treatment. Drug treatment includes pulsed glucocorticoid treatment and neurotrophic treatment. For surgical treatment, there is a one-week treatment time window for optic canal decompression surgery. Currently, other treatment methods include mesenchymal stem cell transplantation, which promotes the repair and survival of damaged optic nerves by secreting neurotrophic factors, etc. The overall treatment effect is not good. The very limited regenerative potential of the adult mammalian optic nerve is a major challenge for restoring vision after optic nerve injury.

[0003] Currently, there are three theories used to explain the mechanism of failed optic nerve regeneration: The first theory is the exogenous inhibition theory, that is, there are inhibitory proteins in the environment of optic nerve regeneration that inhibit axon growth and regeneration, including inhibitory proteins produced by myelin and glial scars. Scientists have successively identified the protein components in myelin that inhibit axon growth, including Inhibitor of neurite growth 1 (IN-1), Myelin associated glycoprotein (MAG), and Oligodendrocyte myelin glycoprotein (OMgp). However, knocking out the three major myelin inhibitory proteins simultaneously did not cause significant axon regeneration. The exogenous inhibition theory mainly involves oligodendrocytes and astrocytes.

[0004] The second theory is the endogenous factor theory, and its main view is that the axon regeneration ability of RGC itself is low. The major breakthrough of the endogenous factor theory comes from the research of He Zhigang's laboratory. Conditional knockout of PTEN in the optic nerve injury model can significantly promote RGC axon regeneration. Subsequently, a series of endogenous factors and molecular pathways regulating central axon regeneration have been discovered, including Socs3 / stat3, KLFs, Sox11, DCLKs, lin28, and Myh9 / 10, etc. However, in traumatic optic nerve injury, axonal degeneration is the primary pathological change, while RGC death is the secondary pathological change.

[0005] The main view of the third exogenous promoting factor theory is that the regenerative environment lacks neurotrophic factors. Scholars have tried IGF1 and osteopontin to promote the growth of the optic nerve or corticospinal tract by activating neuronal IGFR and mTOR. After optic nerve injury, researchers have combined various intervention measures to promote axon regeneration effects, including intravitreal injection of zymosan to induce an inflammatory response, intravitreal injection of a cAMP analog to enhance the RGC axon regeneration signal, knockout of the transcription factor PTEN, etc. This combined treatment has achieved the regeneration of RGC axons in mice with optic nerve injury, and partial visual function has been restored.

[0006] However, most of the regenerated RGC axons obtained by current methods for promoting optic nerve regeneration remain unmyelinated and are ineffective in improving visual function. The research of He Zhigang's group found that optic nerve injury induces the expression of GPR17 in oligodendrocyte precursor cells (OPCs), and long-term activation of microglia inhibits the differentiation of OPCs into mature myelinating oligodendrocytes. The synergistic effect of GPR17 and microglia promotes the extensive myelination of regenerated axons and effectively improves visual function.

[0007] Currently, it is believed that axon regeneration after optic nerve injury requires complex processes such as the establishment of axon regeneration channels and regenerative microenvironments, and the remyelination of axons. Among them, the microenvironment of axon repair and regeneration plays an important role.

[0008] The optic nerve crush (ONC) model is widely used as a preclinical model to study neuronal survival and regeneration. This model provides a tool for studying the pathway from axon injury to neuronal death, and also provides a good tool for studying the axon regeneration microenvironment and axon remyelination. In the ONC model, axon injury is the primary injury, while extensive neurodegeneration and ganglion cell death are secondary pathological results, making it an ideal animal model for simulating optic nerve injury. The ONC model is easy to make and has good repeatability.

[0009] In the microenvironment of axonal remyelination, the main cell types are astrocytes (AS), neurons, and oligodendrocytes. Communication between astrocytes and oligodendrocytes is a determinant of damaged axonal remyelination.

[0010] Myelin is an extension of the oligodendrocyte plasma membrane in the central nervous system (CNS). After CNS injury, the failure of myelin regeneration around neuronal axons leads to neurological dysfunction. Due to the high turnover rate of myelin, oligodendrocytes are very sensitive to oxidative stress. Cholesterol is not only a major structural component of myelin but also essential for myelin growth and axonal wrapping. Therefore, maintaining a relatively high cholesterol level is crucial for the growth of the myelin sheath. When the catalytic enzymes in the cholesterol biosynthesis pathway in oligodendrocytes are depleted or inactivated, most axons show abnormally thin myelin sheaths or even no myelin sheaths, but their morphology is normal.

[0011] The brain is the organ richest in cholesterol, accounting for about 25% of the total body cholesterol content, and the cholesterol in myelin accounts for 80% of the total CNS cholesterol. Astrocytes are considered the main net producers of CNS cholesterol, and neurons are considered net consumers. Astrocytes have a higher cholesterol level, and neurons obtain cholesterol from astrocytes. Oligodendrocyte-mediated myelination may require a large amount of cholesterol. In chronic demyelinating diseases, neuronal cholesterol also promotes remyelination by promoting the proliferation of oligodendrocyte precursor cells. However, in 2023, the research group of Williams A used in vivo / in vitro rodent models to study the crosstalk between astrocytes and oligodendrocytes and clarified that the interaction between astrocytes and oligodendrocytes forming mature myelin is a determinant of axonal remyelination.

[0012] Glia maturation factor beta (GMFB) is a 17-kd acidic cytoplasmic protein with 142 amino acid residues, named because it can promote glial cell differentiation. GMFB is highly conserved in evolution and is mainly expressed in CNS astrocytes. It plays an important role in brain tissue growth, differentiation, and regeneration. Its expression is upregulated during development and significantly decreased in adulthood. In the intact sciatic nerve of adult rats, neither myelinating nor non-myelinating Schwann cells showed detectable GMFB immunostaining. As a biomarker of gliosis, GMFB is more sensitive than GFAP. GMFB is closely related to neurodegeneration and neuroinflammation, such as highly expressed in the brains of Alzheimer's disease and Parkinson's disease. There is currently no report on GMFB in optic nerve crush injury (ONC). Summary of the Invention

[0013] The object of the present invention is to provide the application of GMFB in the preparation of a medicament for treating traumatic optic neuropathy so as to overcome the defects existing in the above-mentioned prior art.

[0014] The object of the present invention can be achieved by the following technical solutions:

[0015] First of all, the present invention provides the application of the Gmfb gene in the preparation of a medicament for treating traumatic optic neuropathy.

[0016] Furthermore, the present invention provides the application of the Gmfb gene in the preparation of a medicament for treating optic nerve crush injury.

[0017] Still further, the present invention provides the application of the Gmfb gene in the preparation of a medicament for promoting axonal regeneration after optic nerve injury and remyelination of degenerated axons after optic nerve injury so as to treat optic nerve crush injury.

[0018] In one embodiment of the present invention, the medicament is an inhibitor for inhibiting the functional expression of the Gmfb gene.

[0019] In one embodiment of the present invention, the inhibitor is a reagent for knocking out the Gmfb gene by the CRISPR / Cas9 technology.

[0020] The present invention further provides the application of the Gmfb gene in screening candidate compounds for preventing or treating traumatic optic neuropathy.

[0021] Furthermore, the present invention provides the application of the Gmfb gene in screening candidate compounds for preventing or treating optic nerve crush injury.

[0022] Furthermore, the present invention provides the application of the Gmfb gene in screening candidate compounds for treating and promoting axonal regeneration after optic nerve injury and remyelination of degenerated axons after optic nerve injury.

[0023] The present invention also provides the application of a reagent for detecting the expression level of the Gmfb gene in the preparation of a product for diagnosing traumatic optic neuropathy.

[0024] Furthermore, the present invention also provides the application of a reagent for detecting the expression level of the Gmfb gene in the preparation of a product for diagnosing optic nerve crush injury.

[0025] In one embodiment of the present invention, the product includes: a probe for specifically recognizing the Gmfb gene; or a primer for specifically amplifying the Gmfb gene; or an antibody or ligand for specifically binding to the protein encoded by the Gmfb gene.

[0026] The present invention finds that on the third day of the rat ONC model, compared with the normal control, the expression of GMFB is significantly up-regulated; traced by Cholera Toxin Subunit B (CTB), it shows that the axons of the rat ONC model with GMFB knockout are significantly longer (compared with the wild rat ONC model), and the transverse section of the optic nerve stained with LFB (luxoLuxol Fast Blue) for myelin shows that knocking out GMFB can effectively increase the remyelination of degenerated axons, promote axon regeneration, and improve visual function.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] 1) Knocking out GMFB can effectively promote the regeneration of degenerated axons in the ONC model;

[0029] 2) After knocking out GMFB, the remyelination of degenerated axons in the ONC model can be significantly improved. Brief Description of the Drawings

[0030] Figure 1 : Traced by CTB, it shows that knocking out GMFB can significantly promote the regeneration of damaged axons in ONC;

[0031] Figure 2 : Stained with LFB for myelin, it shows that knocking out GMFB promotes the remyelination of degenerated axons in the ONC model. Detailed Embodiments

[0032] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0033] Example 1

[0034] Traced by CTB, it shows that knocking out GMFB can significantly promote the regeneration of damaged axons in ONC

[0035] 1) Preparation of the ONC model

[0036] In the research of this application example, SD rats were used as the experimental subjects for the optic nerve crush model.

[0037] This experiment strictly complied with the relevant regulations in "The Use and Management of Laboratory Animals" of the School of Medicine of Tongji University and the School of Life Sciences of Tongji University, and met the requirements of the Association for Research in Vision and Ophthalmology (ARVO) for animal experiments in the field of ophthalmic research. This research plan has been officially approved by the Animal Experiment Ethics Committee of Tongji University.

[0038] The wild-type (WT) Sprague-Dawley rats used in the experiment were provided by Slac Laboratory Animal Co., Ltd. They were 6-week-old male rats weighing approximately 180 g and were divided into a normal control group (WT-NC) and an optic nerve crush group (WT-ONC). The Gmfb gene knockout (KO) Sprague-Dawley rats were prepared by Cyagen Biosciences using CRISPR / Cas9 technology, raised and bred in a specific pathogen-free (SPF) environment at the Animal Center of Tongji University. When they reached 6 weeks of age and weighed approximately 180 g, they were used for modeling and were also divided into a normal control group (KO-NC) and an optic nerve crush group (KO-ONC) for further experimental research.

[0039] The rat model of optic nerve injury established in this study followed the established standard operating procedures of the laboratory.

[0040] Before ONC modeling in rats, chloral hydrate (300 mg / kg) was intraperitoneally injected for anesthesia. After the rats entered deep sleep, 1% Hypnorm (0.1 mL / 200 g) was injected into the leg muscles until muscle relaxation and exophthalmos occurred. Tetracaine (10 mg / mL) was dropped onto the surface of the rat's eyes for ocular surface anesthesia, and 0.5% tropicamide eye drops were used to dilate the pupils. Under a dissecting microscope, the fascia on the temporal side of the rat's eye was incised no more than 1 / 3 to cut off part of the connection between the eyeball and the orbital wall. A pair of Dumont #5 forceps (FST) was inserted into the incision, and the optic nerve 2 mm behind the eye was clamped, and the optic nerve was crushed with a constant pressure for 60 s to avoid damaging the ophthalmic artery. The nerve injury was verified by whether there was a gap at the compression site, and at the same time, the integrity of the retinal blood vessels was evaluated by fundus microscopy, and the animals that could not ensure the integrity of the retinal blood vessels were excluded. After the operation, PBS buffer was dropped onto the surface of the rat's eyes to prevent keratitis caused by dryness of the ocular surface until the rats woke up.

[0041] Three days before sample collection, 2 μl of Alexa-cholera toxin subunit β (CTB-488, 2 μg) was injected into the vitreous cavity of the rats to trace the regenerated RGC axons, and the number of regenerated axons in the optic nerve was estimated by calculating the number of CTB-labeled axons at different distances from the compression site. The anesthesia method for vitreous cavity injection was the same as that for ONC modeling. After anesthesia, the eyeballs were exposed under a dissecting microscope, and a 10 μL microinjector needle was inserted obliquely towards the optic nerve 2 mm behind the limbus corneoscleral at the temporal angle of the head, and the CTB drug was injected into the vitreous cavity. During this process, the needle was avoided from contacting the lens to prevent traumatic cataract. Taking the day of modeling as day 0, samples were collected at 3 days, 7 days, 14 days, and 28 days after modeling for the evaluation of axon regeneration.

[0042] 2) Immunofluorescence detection of CTB tracing results

[0043] Optic nerve tissue samples were collected on the 3rd, 7th, 14th, and 28th days after model establishment. Before sample collection, the rats were sacrificed by decapitation. The cervical vertebrae of the rats were cut, the rats were separated, the occipital bone and parietal bone were cut, and the brain and cerebellum were carefully removed to expose the optic chiasm. Before collecting the optic nerve of the ONC model, the eyelids of the rats were first cut along the outer canthus, the tissues around the eyeballs were separated, and then the optic chiasm was cut, and the optic nerve was gently removed together with the eyeballs.

[0044] The fresh optic nerve samples were fixed in 4% paraformaldehyde at 4°C overnight, then successively placed in 10%, 20%, and 30% sucrose solutions for sufficient dehydration, and then placed in tissue embedding agent OCT at 4°C for overnight equilibration. The samples were transferred to an embedding mold and frozen in a -80°C ultra-low temperature freezer.

[0045] Serial sections with a thickness of 14 μm were cut using a cryostat. The sections that could observe the complete optic nerve head were selected, air-dried overnight at room temperature in the dark with a table fan, and the air-dried sections could be stored at -80°C for a long time.

[0046] The CTB optic nerve tissue sections stored at -80°C were transferred to room temperature and air-dried in the dark. A small amount of water was placed at the bottom of a wet box, and the air-dried glass slides were placed on the wet box and infiltrated with PBS buffer for 10 min to wash away the residual OCT on the slides (OCT is an embedding agent for embedding tissues, and its full name is: tissue-Tek O.C.T. Compound).

[0047] Remove the PBS, use 5 - 10 μl of anti-fluorescence quenching mounting medium to mount the slides, and observe under a confocal microscope after air-drying. The mounted samples can be placed in a 4°C refrigerator for one week.

[0048] The optic nerve samples were imaged using a 20x magnification objective lens. When taking pictures, start from the farthest regenerated axons and take pictures backward towards the compression site. Draw lines perpendicular to the long axis of the optic nerve at 0.25, 0.5, 0.75, and 1.0 mm from the compression site, and count the CTB-positive axons between these lines. Use image j to analyze the total number of regenerated fibers in all sections from each animal and the average number of axons at the distances of each measurement position.

[0049] The results are shown in Figure 1 , Figure 1 In (A), at 2 weeks and 4 weeks after ONC model establishment, the CTB green fluorescence labeling detected under the microscope indicates axon regeneration. The ★ sign indicated by the arrow points to the optic nerve crush site. Scale bar: 100 μm. WT: Gmfb+ / + wild-type rats (i.e., the optic nerve crush group WT-ONC); KO: Gmfb KO rats (i.e., the optic nerve crush group with Gmfb gene knockout, KO-ONC).

[0050] Figure 1Among them, at 2 weeks and 4 weeks after ONC modeling, the number of regenerated axons in the optic nerve was counted. SD: wild-type rats. One-way ANOVA was used for statistical analysis, and P<0.05 was considered to have a significant difference. **p<0.01, ****p<0.0001. It shows that knocking out GMFB can effectively promote the regeneration of degenerated axons in the ONC model.

[0051] Example 2: LFB myelin staining showed that knocking out GMFB promoted the remyelination of degenerated axons in the ONC model.

[0052] 1) Preparation of optic nerve transection model

[0053] The optic nerve transection rat model established in this example study followed the established standard operating procedures in the laboratory.

[0054] Wild-type (WT) SD rats used in the experiment were provided by Slack Animal Company, 6 weeks old, male, with a body weight of about 180 grams. SD rats with Gmfb gene knockout (KO) were prepared by Cyagen Biosciences using CRISPR / Cas9 technology, raised and reproduced in a SPF (Specific Pathogen Free) - level environment in the Animal Center of Tongji University, at 6 weeks old, with a body weight of about 180 grams.

[0055] Before optic nerve transection modeling in SD rats, chloral hydrate (300 mg / kg) was intraperitoneally injected for anesthesia. After the rats entered deep sleep, 1% sumianxin (0.1 mL / 200 g) was injected into the leg muscles until muscle relaxation and exophthalmos occurred. Tetracaine (10 mg / mL) was dropped on the surface of the rats' eyes for ocular surface anesthesia, and 0.5% tropicamide eye drops were used for mydriasis. Under a dissecting microscope, the fascia on the temporal side of the rats' eyes not exceeding 1 / 3 was cut to cut off part of the connection between the eyeball and the orbital wall. An ophthalmic scissors was inserted into the incision, and the optic nerve was transected 2 mm behind the eyeball to avoid damaging the ophthalmic artery. After the operation, PBS buffer solution was dropped on the ocular surface of the rats to prevent keratitis caused by dryness of the ocular surface until the rats woke up.

[0056] Two weeks after modeling, the optic nerves of the rats were sampled. After the rats were decapitated, the skull was opened to expose the brain. The brain was gently removed with an ophthalmic scissors to expose the complete optic chiasm. The transected optic nerve was gently clamped with forceps and removed, and fixed with 4% PFA overnight.

[0057] 2) Luxol fast blue (LFB) myelin staining

[0058] (1) Paraffin section: The optic nerve tissue was dehydrated and then infiltrated with wax for embedding. The wax block was cut into 4-μm wax ribbons, pasted on glass slides, and dried in an oven at 40°C.

[0059] (2) Myelin LFB staining:

[0060] ① Immerse in xylene for 15 min each time, repeat once;

[0061] ② Immerse successively in 100%, 95%, 75%, and 50% alcohol;

[0062] ③ Rinse thoroughly with running water and then immerse in distilled water;

[0063] ④ Preheat the 0.1% LFB solution in an oven at 60 °C for 30 min in advance. Place the tissue section into the preheated 0.1% LFB solution and stain it hermetically at 60 °C for 8 - 16 h;

[0064] ⑤ Take out the section after natural cooling at room temperature and wash it with tap water until it becomes colorless;

[0065] ⑥ Quickly immerse the section in the 0.05% lithium carbonate differentiating solution for 5 s, then immediately take out the section and place it in 70% ethanol for 10 s. That is, alternately immerse the section into the 0.05% lithium carbonate differentiating solution and 70% ethanol for differentiation. Examine under a microscope until the myelin sheath shows a blue background and becomes colorless, then wash with water to terminate the differentiation;

[0066] (5) Immerse the section in absolute ethanol for dehydration three times, 5 min each time;

[0067] (6) Immerse in xylene for clearing, 5 min each time;

[0068] (7) Mount with neutral balsam.

[0069] The results are shown in Figure 2 . It can be seen that at 2 weeks after optic nerve transection, the myelin sheath morphology of the optic nerve cross-section observed under a microscope. The optic nerve myelin sheath structure of WT rats was significantly absent and irregular in shape. The optic nerve myelin sheath structure of KO rats was relatively uniform and more regular in shape. Scale bar: 150 μm. WT: Gmfb+ / + wild-type rats; KO: Gmfb KO rats. It shows that knocking out GMFB can significantly improve the remyelination of degenerated axons in the ONC model.

[0070] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Application of Gmfb gene in the preparation of drugs for treating traumatic optic neuropathy.

2. The use according to claim 1, characterized in that: Application of Gmfb gene in the preparation of drugs for treating optic nerve crush injury.

3. The use according to claim 1, characterized in that: The application of Gmfb gene in the preparation of drugs for promoting axon regeneration after optic nerve injury and remyelination of degenerated axons after optic nerve injury to treat optic nerve crush injury.

4. The use according to claim 1, characterized in that: The drug is a reagent or drug for knocking out the Gmfb gene, or an inhibitor for inhibiting the functional expression of the Gmfb gene.

5. The use according to claim 4, characterized in that: The inhibitor is a reagent for knocking out the Gmfb gene through CRISPR / Cas9 technology.

6. Application of Gmfb gene in screening candidate compounds for preventing or treating traumatic optic neuropathy.

7. The use according to claim 6, characterized in that: Application of Gmfb gene in screening candidate compounds for preventing or treating optic nerve crush injury.

8. The use according to claim 6, characterized in that: The application of Gmfb gene in screening candidate compounds for promoting axon regeneration and remyelination of degenerated axons after optic nerve injury.

9. Use of a reagent for detecting the expression level of the Gmfb gene in the preparation of a product for diagnosing traumatic optic neuropathy.

10. The use according to claim 9, characterized in that: The product includes: a probe that specifically recognizes the Gmfb gene; or a primer that specifically amplifies the Gmfb gene; or an antibody or ligand that specifically binds to the protein encoded by the Gmfb gene.

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