CN115531416A

CN115531416A - Application of astrocyte exosome and melatonin pretreatment in optic nerve injury treatment

Info

Publication number: CN115531416A
Application number: CN202211336855.2A
Authority: CN
Inventors: 郝爱军; 杜静怡; 韩蕾; 周文娟
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2022-12-30

Abstract

The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to an application of an astrocyte exosome and melatonin pretreatment in optic nerve injury treatment. The invention researches the influence of astrocyte Exosomes (EVs) and exosomes (MT-EVs) derived after melatonin pretreatment of astrocytes by establishing an ONC model and treating the astrocyte exosomes and the exosomes (MT-EVs) on the change of retina and visual behavior after mouse ONC. The result shows that the survival rate of RGCs can be improved by exosome treatment, and the visual behavior detection verifies that the RGCs have a certain effect on the long-term vision improvement of mice, and compared with the EVs, the MT-EVs has better effect and important clinical value, and also provides a new idea for exosome transplantation treatment, so the RGCs have good practical application value.

Description

Application of astrocyte exosome and melatonin pretreatment in optic nerve injury treatment

Technical Field

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to an application of astrocyte exosome and melatonin pretreatment in optic nerve injury treatment.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Central Nervous System (CNS) neurons often fail to regenerate after injury, resulting in loss of neural function. The difficulty of regeneration after injury of the adult mammalian CNS remains one of the difficulties of medical community and is a hot spot of research for a long time. The optic nerve, which is part of the CNS, is composed of axons of Retinal Ganglion Cells (RGCs) that carry visual information collected by the retina to the cerebral cortex. Glaucoma, ocular trauma, neoplastic disease, drug intoxication can all lead to damage to the optic nerve. Once damaged, the optic nerve often causes visual deterioration, impaired color vision and even loss of vision. The ONC can be repaired, so that the vision of a patient can be improved, the visual function of the patient can be improved, the patient can be helped to regain the eyesight, and the ONC has important significance in researching a central nerve injury repair mechanism and promoting central nerve regeneration. The recovery of optic nerve function is closely related to the survival number of RGCs and the axon regeneration of the RGCs, and how to reduce the apoptosis of the RGCs and promote the axon regeneration is a key problem for treating ONC.

Astrocytes (ASTs) account for up to 40% of the human brain, participate in multiple processes of health and disease, and have complex mechanisms of action under physiological and pathological conditions. Research shows that ASTs can participate in intercellular communication by secreting EVs, and maintain normal functions of CNS and repair injury. Exosomes are lipid bilayer structures secreted by cells, and contain many molecular regulatory substances such as lipids, proteins and nucleic acids, which contain signaling proteins, coding and regulatory RNAs, and other information, that can be taken up by the target cell, thereby facilitating the delivery of multi-level information. Studies have shown that exosomes released by the ASTs help neurons fight neurotransmitter toxicity and promote neurite outgrowth. Exosomes as nano-scale transport vectors are smaller in size and less immunogenic than cells, and are considered to be an ideal mode for carrying out alternative cell therapy in the future.

Melatonin is a neuroendocrine hormone secreted by the pineal body and having a wide range of physiological and pharmacological actions, and can inhibit apoptosis by scavenging free radicals and inhibiting nitric oxide synthase. Melatonin has neuroprotective effects on CNS disorders, especially neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The neuroprotective effects of melatonin are mainly attributed to its antioxidant, anti-inflammatory and anti-apoptotic properties. Studies have shown that melatonin-treated extracellular exosomes can achieve diabetic wound healing by inhibiting inflammation. At present, melatonin-stimulated cell-derived exosomes have become a safe and effective novel tool for regenerative medicine in the treatment of inflammatory diseases.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of astrocyte exosome and melatonin pretreatment in optic nerve injury treatment. The invention researches the influence of astrocyte Exosomes (EVs) and exosomes (MT-EVs) derived after melatonin pretreatment of astrocytes by establishing an ONC model and treating the astrocyte exosomes and the exosomes (MT-EVs) on the change of retina and visual behavior after mouse ONC. The results show that the survival rate of RGCs can be improved by exosome treatment, and the long-term vision improvement of mice is realized by visual behavior detection, and the MT-EVs has better effect than the EVs. The present invention has been completed based on the above results.

Specifically, the technical scheme of the invention is as follows:

in a first aspect of the invention, there is provided the use of an exosome in the preparation of a product for the treatment of optic nerve injury.

Specifically, the optic nerve injury treatment is characterized by comprising the following steps:

(a) Promoting survival of retinal ganglion cells after damage of the small optic nerve;

(b) Improving the visual function recovery of the mouse after the optic nerve injury;

wherein in (b), the recovery of visual function includes, but is not limited to, retinal ganglion cell function, retinal function, and vision recovery.

The exosome is an exosome derived from astrocytes; the diameter of the exosome is 100-200nm.

More specifically, the astrocytes may be treated by co-incubation with melatonin, and the exosomes obtained therefrom may be designated as MT-EVs, while the exosomes obtained without co-incubation with melatonin are designated as EVs. According to the invention, researches show that the exosome can play a role in treating optic nerve injury, but MT-EVs have better effect than EVs, so that the exosome is preferably MT-EVs.

In a second aspect of the invention, there is provided a product comprising an exosome as described above. More specifically, the exosomes are astrocyte-derived exosomes; further preferably, the exosome is astrocyte-derived exosome (MT-EVs) co-incubated with melatonin.

The product has the following effects:

wherein in (b), the visual function recovery includes, but is not limited to, retinal ganglion cell function, retinal function and vision recovery.

In a third aspect of the present invention, there is provided a method of treating optic nerve damage, the method comprising: administering the exosome or the product to a subject.

The beneficial effects of one or more of the above technical solutions are as follows:

the technical scheme adopts the astrocyte exosomes (EVs group) and the melatonin-treated astrocyte exosomes (MT-EVs group) to treat optic nerve injury, thereby reducing the apoptosis of RGCs and promoting the recovery of optic nerve injury. The results show that the survival rate of RGCs after ONC is obviously improved by treating EVs or MT-EVs, and meanwhile, visual behavior experiments prove that the EVs and the MT-EVs have certain effect on the long-term visual function recovery of mice. Our research suggests that astrocyte exosomes are expected to be effective therapeutic drugs for optic nerve injury and other central nervous system injuries, and melatonin pretreatment can improve the therapeutic effect of exosomes, so that the melatonin has important clinical value and also provides a new idea for exosome transplantation therapy, and therefore, the astrocyte exosomes have good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.

FIG. 1 shows the isolation and characterization of exosomes from ASTs in an example of the present invention.

A and B: the sizes of MT-EVs and EVs exosomes under a transmission electron microscope are about 100nm-150nm, the MT-EVs and EVs exosomes have no obvious difference, and the MT-EVs and EVs exosomes are scaled according to the following rules: 200nm; c: the western blot detects the protein level of the exosome-specific biomarker CD63/TSG101, and the two are not obviously different.

FIG. 2 is a graph showing the effect of MT-EVs on the survival of RGCs after ONC in mice according to the present invention.

Control, ONC, EVs and MT-EVs groups were shown 14d after optic nerve crush injury: a: RBPMS labeled RGCs detects the surviving RGCs of each group, and the ruler is 100 mu m; b: the proportion of survival of the RGCs was counted by counting the RGCs (n = 5) and the data results were expressed as mean ± standard deviation, # P <0.05, # P < 0.01, # P < 0.0001.

FIG. 3 is a graph showing the abnormal visual function of mice after improving ONC by MT-EVs in the present invention.

After optic nerve crush injury, 60d in the Control group, ONC group, EVs group, and MT-EVs group, a: representative FERG schematic. B: representative FVEP schematic. C: a bar graph of FVEP-P2 amplitude is shown (n = 6). D: bar graphs of FVEP-P2 latency are shown (n = 5). E: a bar graph of the FERG-b2 amplitude is shown (n = 6). F: quantification of GCC thickness (n = 6). G: representative optical coherence tomography OCT images of RGCs complex cross-sections in vivo. Scale bar =200 μm. GCC: a ganglion cell complex including a Retinal Nerve Fiber Layer (RNFL), a Ganglion Cell Layer (GCL), and an Inner Plexiform Layer (IPL) layer; indicated as a single-directional arrow. H: bar graphs of visual cliffs of visual behavior tests are displayed, percentage of time in cliff side for different age groups of animals (n = 6). I: bar graphs of black and white boxes showing visual behavior tests, percentage of time in the light room for different age groups of animals (n = 6). Data results are expressed as mean ± sd, # P <0.05, # P < 0.01, # P < 0.001, # P < 0.0001, ns is meaningless.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the following detailed description is given with reference to specific embodiments.

As previously mentioned, CNS neurons are often unable to regenerate after injury, resulting in loss of neural function. The difficulty of regeneration after injury of the adult mammalian CNS remains one of the difficulties of medical community and is a hot spot of research for a long time.

The inventor finds that astrocyte exosomes are expected to be effective treatment drugs for optic nerve injury and other central nervous system injuries, and melatonin pretreatment can improve the treatment effect of exosomes.

In view of the above, in an exemplary embodiment of the present invention, there is provided a use of an exosome for preparing a product for treating optic nerve injury.

(a) Promoting retinal ganglion cell survival after the small optic nerve injury;

(b) Improving the recovery of visual function of the mouse after the optic nerve injury;

In still another embodiment of the present invention, the astrocytes may be treated by co-incubation with melatonin (treatment concentration of 0.5-5. Mu.M, preferably 1. Mu.M), and the exosomes obtained therefrom may be named as MT-EVs, while the exosomes obtained without co-incubation with melatonin are named as EVs. According to the invention, researches show that the exosome can play a role in treating optic nerve injury, but MT-EVs have better effect than EVs, so the exosome is preferably MT-EVs.

In a further embodiment of the invention, there is provided a product comprising an exosome as described above. More specifically, the exosome is an astrocyte-derived exosome; further preferably, the exosomes are astrocyte-derived exosomes (MT-EVs) treated with co-incubation with melatonin.

The product has the following effects:

It is noted that the product may be a pharmaceutical or a test agent for use in basic research and thus may be used to construct relevant cellular or animal models.

When the product is a pharmaceutical, the pharmaceutical may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, emulsifier, suspending agent, stabilizer, preservative, excipient, filler, coagulant and blender, surfactant, dispersing agent or antifoaming agent.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a virus, a microcapsule, a liposome, a nanoparticle, or a polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier can be a gel-like material, a liposome, a biocompatible polymer (including natural and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial viral envelope, an inorganic (including metal) particle, and a bacterial or viral (e.g., baculovirus, adenovirus and retrovirus), phage, cosmid, or plasmid vector.

The medicament may also be combined with other medicaments for the prevention and/or treatment of optic nerve damage and other central nervous system injuries, and the other prophylactic and/or therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The medicament may also be administered separately to other prophylactic and/or therapeutic compounds, either as a separate composition or in a different dosage form than the main active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the agents of the invention may be adjusted during the course of treatment depending on the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The medicament of the present invention can be administered into the body by a known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, ocular, dermal, nasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In yet another embodiment of the present invention, there is provided a method for treating optic nerve damage, the method comprising: administering the exosome or the product to a subject.

The subject of the present invention refers to an animal, preferably a mammal, such as a mouse, rat, guinea pig, chimpanzee, monkey, etc., and most preferably a human, who has been the subject of treatment, observation or experiment.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. All the starting materials used in the examples are, unless otherwise specified, commercially available.

Examples

1. Materials and methods

1. Preparing an animal model: animal experiments were conducted according to the International guidelines for animal research, supplied by the International organization for medical science (CIOMS), and procedures were approved by the animal ethics and welfare Committee, university of Shandong. Adult Kunming mice were selected as the subject and were randomly divided into four groups of 6 mice each.

(1) Control group (Control group) (optic nerve exposed without clamping)

(2) Negative control group (ONC group) (vitreous injection 2. Mu.l physiological saline after injury)

(3) Astrocyte exosome group (EVs group) (intravitreal injection 2. Mu.l of astrocyte exosome after injury)

(4) Melatonin-treated astrocyte exosome group (MT-EVs group) (intravitreal injection 2. Mu.l of melatonin-treated astrocyte exosomes after injury)

1.1 optic nerve injury:

in the ONC group, animals were anesthetized with 1% sodium pentobarbital in the abdominal cavity, and then surface anesthesia was performed with oxybutynin hydrochloride eye drops. Under the visual field of a surgical dissection microscope, a spring scissors is used for cutting out the conjunctiva of one eye at about 4 o' clock on the temporal side of the eyeball, the orbital muscles are slightly deflected to expose white optic nerves, a clamp wound is applied to the optic nerves at a position 2mm away from the eyeball for about 5s, and after the extrusion wound is completed, the incision is closed.

1.2 post-operative model inclusion criteria: the lens of the mouse is not turbid; the retina has no bleeding and shedding phenomenon; the blood supply of the eyeground is normal; the muscle of the eyeball recovers to be normal, and the eyeball can rotate freely without protrusion after recovering to be normal.

2. Exosome extraction and identification

2.1 extraction and treatment of primary astrocytes: collecting newborn Kunming suckling mouse within 3d, grinding and homogenizing cerebral cortex tissue, inoculating into cell culture bottle, changing liquid every 3d, and culturing for 9-10d. After the degree of cell fusion exceeded 95%, the medium containing melatonin (1. Mu.M) was replaced for culture.

2.2 exosome extraction: collecting cell supernatant, centrifuging at 4 deg.C for 10min at 200g, collecting supernatant, and discarding cell debris and other precipitate. Centrifuging the supernatant after the secondary low-speed centrifugation for 35min at 10000g at 4 ℃, reserving the supernatant again, and discarding the precipitate. Adding the supernatant into an ultracentrifuge tube, strictly balancing the centrifuge tube, ultracentrifuging at 4 ℃ for 70min by using a centrifugal force of 100000g, discarding the supernatant, adding PBS (phosphate buffer solution) again to wash the precipitate, ultracentrifuging the resuspended liquid at 4 ℃ for 70min by using a centrifugal force of 100000g again, and discarding the supernatant to obtain the exosome. The extracted exosomes can be resuspended in 100. Mu.l PBS solution and then frozen in a-80 ℃ refrigerator for later use (strict sterility is not required during the whole experiment).

2.3 exosome Westernblot identification:

and (3) cracking the exosome protein, uniformly mixing an exosome protein sample and a 5x protein loading buffer solution according to a ratio of 4. And (3) mounting the prepared rubber plate on an electrophoresis bracket, keeping the voltage at 90V for about 30-40 min, adjusting the voltage to 110V after the protein sample runs out of the concentrated gel to the separation gel, and timely terminating electrophoresis according to the experimental requirement by referring to a protein Marker. After the electrophoresis is finished, the membrane is rotated, and the voltage is 110V and lasts for 1h40min. PVDF membrane was immersed in 5% skimmed milk powder in TBST formulation and shaken slowly overnight at room temperature for 2h or 4 ℃. The blocking solution was discarded and primary antibody was added to the solution and incubated on a shaker at 4 ℃ with slow shaking overnight. Recovering primary antibody, rinsing with TBST for 3 times (12 min each time), adding secondary antibody, and shaking slowly at room temperature for 2h. Recovering secondary antibody, rinsing 3 times for 12min each time by TBST, and developing by luminescence.

2.4 transmission electron microscope morphological identification of exosomes: and (3) dripping 10 mu l of exosome suspension on a sample-carrying carbon film copper net at room temperature, standing for 5min, sucking liquid from the side by using filter paper, adding 7.5 mu l of uranium dye, turning and rinsing for 2 times, then adding 7.5 mu l of dye, standing for 30s, and sucking dry by using the side filter paper. After the residual liquid was absorbed by filter paper, the copper mesh was dried at room temperature, placed on the filter paper, air-dried, and observed using a transmission electron microscope.

3. Mouse vitreous injection

After anaesthetizing, mice were placed under an operating microscope, and surface anaesthesia was performed using the olbutine hydrochloride eye drops, and the mouse pupils were dilated using the compound tropicamide eye drops. The exosome suspension was injected into the vitreous gun using a 33G needle hamilton syringe, leaving 30 seconds after injection and slowly withdrawing the needle. The intravitreal injections required in this experiment were PBS for the vehicle control group, EVs and MT-EVs for the administration group. All eyes with cataracts and intra-bulbar hemorrhage were excluded from the experimental analysis.

4. Visual behavior experiment of mouse

4.1 Black and white box (Black/white transition box)

With a black and white box, as previously described this box consists of a dark room (16-16-25 cm) and a light room of the same size (illuminated with bright white light). A 10-12cm hole in the wall separates the black and white compartments, allowing the mouse to move freely from one compartment to the other. At the beginning of the test, one mouse was placed in the middle of the laboratory in the bright room, and after 5min, it was taken out of the box and the residence time of the mouse in the bright room was measured.

4.2 Vision cliff (Visual cliff)

Prior to the cliff test, the mouse was placed on a small glass box with the head facing the non-cliff side. And (3) fixing a camera on the glass box in a hanging manner, recording video through the camera for 5min for each animal, and observing the whole exploration process of the mouse. After the experiment is finished, the animal behavior automatic tracking software toxTrac is applied to analyze the mouse movement behavior video, so that the experimental data is further processed, and the proportion of the time spent by each mouse on the deep side (also called cliff side) to the total time of the testing process is calculated.

4.3 Flash Visual Evoked Potential (FVEP)

Weighing and anaesthetizing the mouse, then carrying out FVEP examination, putting the mouse in a dark environment in advance for dark adaptation, anaesthetizing the mouse after dark adaptation for 12 hours, putting the mouse on a fixed platform of an electrophysiology instrument, covering one side of an eye by using a black eye mask during examination, and installing a needle electrode special for FVEP examination. Electrode position: the positive pole is positioned at the distance schizophrenic center (about 5mm of the connecting line of the two ears) of the occipital lobe of the brain of the animal, the negative pole is positioned at the cheek part on the same side of the animal, the grounding line is positioned under the skin of the tail of the animal, and the operation is carried out by using an operating system of an OPTOPROBE electrophysiology instrument in England. Using scintillation light as stimulating light, stimulating frequency is 1Hz, transmission bandwidth is 0.5-85.0 Hz, analyzing time is 250ms, superposing is carried out for 60 times, at least 3 times of continuous measurement are carried out, and P2 wave amplitude and latency are recorded.

4.4 Flashlight Electroretinogram (FERG)

And (3) after the mouse adapts to the dark for 12h, anesthetizing the mydriasis, connecting electrodes, namely respectively connecting a recording electrode, a reference electrode and a ground electrode, firstly connecting the ground electrode to the tail root of the mouse, then connecting the reference electrode to the two sides of the cheek of the mouse, and putting the head of the mouse, which is just opposite to the stimulating light source, into the Ganz film (the operations are all carried out under dark red light). The program was turned on to perform the FERG recording program, the dark adaptation rod-cone mixed response (dMax-ERG) was analyzed, the measured FERG-b wave amplitudes were labeled, and the differences between the four sets of b wave amplitudes were compared.

4.5OCT(Optical Coherence Tomography,OCT)

After anesthesia of mydriasis, the cornea of both eyes is coated with a clear eye gel and imaged and analyzed while keeping the cornea constantly moist. Images of the mouse retina around the optic nerve head were captured and measured using Optoprobe, uk. The thickness of the obtained complex of RGCs (GCC) was measured as a measurement result, including RNFL, ganglion Cell Layer (GCL), and Inner Plexiform Layer (IPL). Built-in software is used to segment the GCC and quantify its thickness.

5. Immunofluorescent staining

5.1 retinal plating: washing an eyeball by PBS, placing the eyeball into 4% paraformaldehyde solution, fixing at 4 ℃ overnight, removing anterior cornea, crystalline lens and vitreous body of the fixed mouse eyeball in the PBS to prepare an eye cup, and averagely dividing the retina into 4 segments by using a curve-head corneal scissors and a 4-blade traveling scissors along the blood vessel on the retina. The retina of the "clover" eye cup was carefully separated from the underlying choroidal sclera using an iris restorer to obtain a "clover" retina, which was placed in-20 ℃ pre-chilled methanol in preparation for immunofluorescent staining of the retina plate.

5.2 immunofluorescent staining of retina: blocking and permeabilizing for 4 hours, adding primary antibody, and incubating for 24 hours in a refrigerator at 4 ℃ (the dilution concentration of the primary antibody RBPMS is 1; washing for 10min for 3 times; fluorescent secondary antibody was incubated for 4 hours (secondary antibody Dylight488 dilution concentration: 1; after washing, the anti-fluorescence quencher is added dropwise for sealing. The film was photographed by observation under a fluorescence microscope. Each retina selects 3 pictures distant from the papilla. The average density of the RGCs was calculated by counting the number of RGCs in each picture using the StarDist2D plug-in ImageJ (v 153) software.

6. Statistical analysis

Each experiment in this chapter was repeated 3 or more times, and statistical analysis was performed using GraphPadprism90 software, with data expressed as mean. + -. Standard error. Comparison between the two or more groups of data was performed using one-way anova and differences between the groups were detected by bonferroplast hoc test and non-parametric Kruskal-Wallis test with Dunn multiple comparison test, p <0.05 considered statistically significant.

2. Results of the experiment

1. Exosome extraction and identification

After 48 hours of co-incubation of the isolated ASTs with melatonin, exosomes were isolated from supernatants of melatonin-treated and non-melatonin-treated ASTs by ultracentrifugation. The exosomes were verified by transmission electron microscopy and Western blotting. The morphology of Evs and MT-Evs was observed by transmission electron microscopy. We observed that both types of exosomes are ellipsoidal bilayer lipid membrane vesicles, approximately 100-200nm in diameter, with no significant difference between the two, as shown in figure 1. Western blotting detects CD63, tsg101, GAPDH and Calnexin of Evs and MT-Evs, and the two have no obvious difference.

2. Melatonin-treated astrocyte exosomes promote retinal ganglion cell survival in mice after optic nerve injury

In order to detect the protective effect of EVs and MT-EVs on the RGCs after ONC, the number (green) of RBPMS positive RGCs in the EVs group is obviously higher than that in the damaged group through immunofluorescence discovery, as shown in figure 2A, and meanwhile, the number of the RBPMS positive RGCs in the MT-EVs group is higher than that in the EVs group, which shows that the EVs can save the death of the RGCs, and meanwhile, the treatment effect of the MT-EVs is better.

3. Melatonin-treated astrocyte exosomes improve visual function recovery following optic nerve injury in mice

To assess whether EVs and MT-EVs rescued the visual function of mice, we tested mice for RGCs function using FVEP after 60d treatment of animals; FERG detects the integral function of retina; and detecting the vision recovery condition of the mouse by using a black and white box and a visual cliff. As shown in fig. 3, the FVEP results showed a decrease in the ONC group P2 amplitude; the P2 latency is prolonged; a decrease in the amplitude of the FERGb wave; the residence time of the mouse on both the bright room and cliff side was extended. After EVS treatment, FVEP shows that the P2 wave amplitude of the mice is recovered, the P2 latency period is shortened compared with the ONC group, the b wave amplitude of FERG is recovered, the residence time of the mice in the bright room is shortened, but the result of the visual cliff shows that the result has no statistical significance compared with the result of the ONC group. The MT-EVs group showed greater advantages in RGCs function, retinal function, and vision recovery. In addition, the thickness of the GCC of the retina is detected by OCT, and the thickness of the GCC layer after ONC is thin, while the thickness of the GCC layer in EVs group is increased compared with that in ONC group, and the thickness of the GCC layer in MT-EVs group is thicker.

The results show that EVs and MT-EVs can remarkably promote the visual function recovery of mice and the treatment effect of MT-EVs is more obvious.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of the exosome in preparing a product for treating optic nerve injury;

(b) Improve the recovery of the visual function of the mouse after the optic nerve injury.

2. The use of claim 1, wherein in (b), the recovery of visual function comprises retinal ganglion cell function, retinal function, and vision recovery.

3. The use of claim 1, wherein said exosomes are astrocyte-derived exosomes.

4. The use of claim 3, wherein said exosomes are obtained after co-incubation of astrocytes with melatonin; further, the melatonin treatment concentration is 0.5-5 μ M.

5. A product, wherein said product comprises exosomes; the exosome is an astrocyte-derived exosome.

6. The product of claim 5, wherein said exosomes are obtained after co-incubation of astrocytes with melatonin.

7. The product of claim 6, wherein said product has the following effects:

8. A product as in claim 7 wherein in (b), visual function recovery comprises retinal ganglion cell function, retinal function and visual recovery.

9. The product of claim 7, wherein the product is a pharmaceutical or a test agent.

10. A method of treating optic nerve injury, the method comprising: administering to the subject the product of any one of claims 5-7.