Application of Astrocyte Exosomes and Melatonin Pretreatment in the Treatment of Optic Nerve Injury

Abstract

The present invention belongs to the technical fields of biomedicine and molecular biology, and specifically relates to the application of astrocyte exosomes and melatonin pretreatment in the treatment of optic nerve injury. The present invention explores the effects on retinal and visual behavioral changes in mice after ONC by establishing an ONC model and treating with exosomes derived from astrocytes (EVs) and exosomes derived from astrocytes pretreated with melatonin (MT-EVs). The results show that exosome treatment can improve the survival rate of RGCs, and at the same time, visual behavioral tests verify its certain effect on the long-term vision improvement of mice. Moreover, compared with EVs, MT-EVs have better effects, have important clinical value, and also provide new ideas for exosome transplantation therapy, so it has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of astrocyte exosomes and melatonin pretreatment in the treatment of optic nerve injury. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Central nervous system (CNS) neurons usually cannot regenerate after injury, resulting in the loss of nerve function. The difficulty of regeneration after CNS injury in adult mammals remains one of the difficult problems in the medical field and has also been a hot topic of research for a long time. The optic nerve is a part of the CNS, composed of the axons of retinal ganglion cells (RGCs), and can transmit visual information collected by the retina to the cerebral cortex. Glaucoma, eye trauma, neoplastic lesions, and drug poisoning can all cause damage to the optic nerve. Once the optic nerve is damaged, it often causes visual decline, color vision impairment, and even vision loss. Repairing ONC can not only improve the patient's vision, improve visual function, and help the patient regain light, but also has important significance for exploring the repair mechanism of central nerve injury and promoting central nerve regeneration. The recovery of optic nerve function is closely related to the survival number of RGCs and the regeneration of their axons. How to reduce RGC apoptosis and promote the regeneration of their axons is the key issue in the treatment of ONC.

[0004] Astrocytes (ASTs) account for up to 40% of the human brain and are involved in multiple processes of health and disease, with complex mechanisms of action under physiological and pathological conditions. Studies have found that ASTs can participate in cell communication by secreting EVs and maintain the normal function and injury repair of the CNS. Exosomes are a lipid bilayer structure secreted by cells, containing many molecular regulatory substances such as lipids, proteins, and nucleic acids. The information including signal proteins, coding, and regulatory RNAs can be absorbed by target cells, thus promoting the transmission of multi-level information. Research has shown that exosomes released by ASTs help neurons resist neurotransmitter toxicity and promote neurite growth. As a nano-level transport carrier, exosomes are smaller in volume and lower in immunogenicity compared to cells, and are considered to be a potentially ideal way for future alternative cell therapy.

[0005] Melatonin is a neuroendocrine hormone secreted by the pineal gland with a wide range of physiological and pharmacological effects. It can inhibit apoptosis by scavenging free radicals and inhibiting nitric oxide synthase. Melatonin has a neuroprotective effect on CNS diseases, especially neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The neuroprotective effect of melatonin is mainly attributed to its antioxidant, anti-inflammatory and anti-apoptotic properties. Studies have shown that melatonin-treated extracellular vesicles can achieve diabetic wound healing by inhibiting inflammation. At present, melatonin-stimulated cell-derived extracellular vesicles have become a safe and effective new tool for the treatment of inflammatory diseases in regenerative medicine. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides the application of astrocyte extracellular vesicles and melatonin pretreatment in the treatment of optic nerve injury. The present invention established an ONC model and explored the effects of extracellular vesicles derived from astrocytes (EVs) and melatonin-pretreated astrocytes (MT-EVs) on retinal and visual behavioral changes in mice after ONC. The results showed that exosome treatment could improve the survival rate of RGCs, and at the same time, visual behavioral tests verified its role in improving the long-term vision of mice, and MT-EVs had better effects than EVs. Based on the above research results, the present invention was completed.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided the application of extracellular vesicles in the preparation of a product for the treatment of optic nerve injury.

[0009] Specifically, the treatment of optic nerve injury is specifically manifested as:

[0010] (a) Promote the survival of retinal ganglion cells after minor optic nerve injury;

[0011] (b) Improve the recovery of visual function in mice after optic nerve injury;

[0012] Among them, in (b), the recovery of visual function includes, but is not limited to, the function of retinal ganglion cells, retinal function and visual acuity recovery.

[0013] The extracellular vesicles are astrocyte-derived extracellular vesicles; the diameter of the extracellular vesicles is 100-200 nm.

[0014] More specifically, the astrocytes can be co-incubated with melatonin, and the obtained exosomes can be named MT-EVs, while the exosomes obtained without co-incubation with melatonin are named EVs. Through research, the present invention has found that the above-mentioned exosomes can all play a therapeutic effect on optic nerve injury. However, compared with EVs, MT-EVs have a better effect. Therefore, the exosomes are preferably MT-EVs.

[0015] In the second aspect of the present invention, a product is provided, and the product contains the above-mentioned exosomes. More specifically, the exosomes are astrocyte-derived exosomes; further preferably, the exosomes are astrocyte-derived exosomes (MT-EVs) co-incubated with melatonin.

[0016] The product has the following effects:

[0017] (a) Promote the survival of retinal ganglion cells after optic nerve injury;

[0018] (b) Improve the recovery of visual function after optic nerve injury in mice;

[0019] Among them, in (b), the recovery of visual function includes but is not limited to the function of retinal ganglion cells, retinal function, and vision recovery.

[0020] In the third aspect of the present invention, a treatment method for optic nerve injury is provided, and the method includes: administering the above-mentioned exosomes or the above-mentioned product to a subject.

[0021] The beneficial effects of the above one or more technical solutions:

[0022] The above technical solutions use astrocyte exosomes (EVs group) and astrocyte exosomes treated with melatonin (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 after treatment with EVs or MT-EVs, the survival rate of RGCs after ONC increases significantly. At the same time, visual behavioral experiments prove that both EVs and MT-EVs have a certain effect on the long-term recovery of visual function in mice. Our research suggests that astrocyte exosomes are expected to become an effective therapeutic drug for optic nerve injury and other central nervous system injuries, and melatonin pretreatment can improve the therapeutic effect of exosomes, which has important clinical value and also provides new ideas for exosome transplantation therapy. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0024] Figure 1 Isolation and identification of exosomes derived from ASTs in the embodiments of the present invention.

[0025] A and B: Under transmission electron microscopy, the sizes of MT-EVs and EVs exosomes are approximately 100 nm - 150 nm, and there is no significant difference between the two. Scale bar: 200 nm; C: Western blot was used to detect the protein levels of exosome-specific biomarkers CD63 / TSG101, and there is no significant difference between the two.

[0026] Figure 2 Effect of MT-EVs on the survival of RGCs after ONC in mice in the embodiments of the present invention.

[0027] At 14 days after optic nerve crush injury in the Control group, ONC group, EVs group, and MT-EVs group: A: RBPMS was used to label RGCs to detect the surviving RGCs in each group, scale bar 100 μm; B: The proportion of surviving RGCs was statistically analyzed through the RGCs counting results (n = 5). The data results are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0028] Figure 3 MT-EVs improve visual function abnormalities in mice after ONC in the embodiments of the present invention.

[0029] At 60 days after optic nerve crush injury in the Control group, ONC group, EVs group, and MT-EVs group, it shows: A: Schematic diagram of representative FERG. B: Schematic diagram of representative FVEP. C: Bar graph showing FVEP-P2 amplitude (n = 6). D: Bar graph showing FVEP-P2 latency (n = 5). E: Bar graph showing FERG-b2 amplitude (n = 6). F: Quantification of GCC thickness (n = 6). G: Cross-section of the in vivo RGCs complex of a representative optical coherence tomography OCT image. Scale bar = 200 μm. GCC: Ganglion cell complex, including retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) layer; indicated by a one-way arrow. H: Bar graph showing the visual cliff of the visual behavior test, the percentage of time of different age and different animal groups on the cliff side (n = 6). I: Bar graph showing the black and white box of the visual behavior test, the percentage of time of different age and different animal groups in the light chamber (n = 6). The data results are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns is meaningless. Detailed implementation manners

[0030] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the following will be described in detail in conjunction with specific embodiments.

[0032] As mentioned above, CNS neurons usually cannot regenerate after injury, resulting in the loss of nerve function. The difficulty of regeneration after CNS injury in adult mammals remains one of the difficult problems in the medical field and has also been a hot research topic for a long time.

[0033] The inventors' research found that astrocyte exosomes are expected to become an effective therapeutic drug for optic nerve injury and other central nervous system injuries, and melatonin pretreatment can enhance the therapeutic effect of exosomes.

[0034] In view of this, in a typical specific embodiment of the present invention, there is provided the use of exosomes in the preparation of a therapeutic product for optic nerve injury.

[0035] Specifically, the treatment of optic nerve injury is specifically manifested as:

[0036] (a) Promote the survival of retinal ganglion cells after minor optic nerve injury;

[0037] (b) Improve the recovery of visual function after optic nerve injury in mice;

[0038] Among them, in (b), the recovery of visual function includes but is not limited to the function of retinal ganglion cells, retinal function, and visual acuity recovery.

[0039] The exosomes are astrocyte-derived exosomes; the diameter of the exosomes is 100 - 200 nm.

[0040] In another specific embodiment of the present invention, the astrocytes can be co-incubated with melatonin (treatment concentration is 0.5 - 5 μM, preferably 1 μM), and the obtained exosomes can be named MT-EVs, while the exosomes obtained without co-incubation with melatonin are named EVs. The present invention found through research that the above-mentioned exosomes can all play a therapeutic effect on optic nerve injury, but MT-EVs have a better effect than EVs. Therefore, the exosomes are preferably MT-EVs.

[0041] In yet another specific embodiment of the present invention, a product is provided, and the product contains the above-mentioned exosomes. More specifically, the exosomes are astrocyte-derived exosomes; further preferably, the exosomes are astrocyte-derived exosomes co-incubated with melatonin (MT-EVs).

[0042] The product has the following effects:

[0043] (a) Promote the survival of retinal ganglion cells after optic nerve injury;

[0044] (b) Improve the recovery of visual function after optic nerve injury in mice;

[0045] Among them, in (b), the recovery of visual function includes but is not limited to the function of retinal ganglion cells, retinal function, and vision recovery.

[0046] It should be noted that the product can be a drug or a test reagent, and the test reagent is for basic research use, so it can be used to construct relevant cell or animal models.

[0047] When the product is a drug, the drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a conditioner, a surfactant, a dispersant or an antifoaming agent.

[0048] The drug may further 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 carrier of the pharmaceutically acceptable carrier can be a gel-like material, a liposome, a biocompatible polymer (including natural polymers and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial virus envelope, an inorganic (including metal) particle, and a bacterium or a virus (such as a baculovirus, an adenovirus and a retrovirus), a phage, a cosmid or a plasmid vector.

[0049] The drug may also be used in combination with other drugs for preventing and / or treating optic nerve injury and other central nervous system injuries. Other preventive and / or therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition.

[0050] The drug can also be administered to other preventive and / or therapeutic compounds in a separate composition or in a dosage form different from that of the main active ingredient. A partial dose of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dose of the drug of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response to the treatment regimen.

[0051] The drugs of the present invention can be administered into the body by known methods. For example, they can be systemically delivered via the vein or locally injected into the tissue of interest. Optionally, administration can be carried out via intravenous, intraocular, dermal, nasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0052] In another specific embodiment of the present invention, a method for treating optic nerve injury is provided, and the method includes: administering the above-mentioned exosomes or the above-mentioned product to a subject.

[0053] The subject in the present invention refers to an animal that has been the subject of treatment, observation or experiment, preferably a mammal, such as a mouse, a rat, a guinea pig, a chimpanzee, a monkey, etc., and most preferably a human.

[0054] The present invention is further explained and illustrated below by examples, but does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. All raw materials used in the examples are commercially available unless otherwise specified.

[0055] Examples

[0056] I. Materials and Methods

[0057] 1. Preparation of animal model: Animal experiments were carried out in accordance with the International Guiding Principles for Animal Research provided by the Council for International Organizations of Medical Sciences (CIOMS), and the procedures have been approved by the Animal Ethics and Welfare Committee of Shandong University. Adult Kunming mice were selected as experimental subjects, and adult female Kunming mice were randomly divided into four groups, with 6 mice in each group.

[0058] ① Control group (Control group) (only expose the optic nerve without clamping)

[0059] ② Negative control group (ONC group) (inject 2 μl of normal saline into the vitreous body after injury)

[0060] ③ Astrocyte exosome group (EVs group) (inject 2 μl of astrocyte exosomes into the vitreous body after injury)

[0061] ④ Melatonin-treated astrocyte exosome group (MT-EVs group) (inject 2 μl of melatonin-treated astrocyte exosomes into the vitreous body after injury)

[0062] 1.1 Optic nerve injury:

[0063] After the animals in the ONC group were anesthetized intraperitoneally with 1% sodium pentobarbital, topical anesthesia of the eyeball surface was performed using oxybuprocaine hydrochloride eye drops. Under the field of view of a surgical dissecting microscope, a pair of spring scissors was used to cut the conjunctiva of one eye at about the 4 o'clock position on the temporal side of the eyeball. The orbital muscles were gently deflected to expose the white optic nerve. A clamping injury was applied to the optic nerve at a distance of 2 mm from the eyeball for about 5 s. After the crush injury was completed, the incision was sutured.

[0064] 1.2 Inclusion criteria for the postoperative model: The lens of the mouse was not cloudy; there was no retinal hemorrhage or detachment; the blood supply to the fundus was normal; the eyeball muscles recovered normally, retracted normally without protrusion and could rotate freely.

[0065] 2. Extraction and identification of exosomes

[0066] 2.1 Extraction and treatment of primary astrocytes: Newborn Kunming mice within 3 days after birth were taken. After taking the cerebral cortex tissue, it was ground and homogenized, and then seeded into a cell culture flask. The medium was changed every 3 days and cultured for 9 - 10 days. After the cell confluence exceeded 95%, the medium containing melatonin (1 μM) was changed for further culture.

[0067] 2.2 Extraction of exosomes: The cell supernatant was taken. After centrifugation at 200 g for 10 min at 4°C, the supernatant was retained and the cell debris and other precipitates were discarded. The supernatant after the second low-speed centrifugation was centrifuged at 10000 g for 35 min at 4°C, and the supernatant was retained again and the precipitate was discarded. The aforementioned supernatant was added to an ultracentrifugation tube. After strictly balancing the centrifugation tube, it was ultracentrifuged at a centrifugal force of 100000 g for 70 min at 4°C. The supernatant was discarded, and PBS solution was added again to wash the precipitate. The resuspended liquid was ultracentrifuged again at a centrifugal force of 100000 g for 70 min at 4°C, and the supernatant was discarded, thus obtaining exosomes. The extracted exosomes could be resuspended with 100 μl of PBS solution and stored at -80°C in a refrigerator for later use (strict aseptic operation was required throughout the experimental process).

[0068] 2.3 Identification of exosomes by Western blot

[0069] Lyse exosomal proteins. Mix the exosomal protein sample with 5x protein loading buffer at a ratio of 4:1 and heat it at 100 °C in a metal bath for 10 min for denaturation. Install the prepared gel plate on the electrophoresis support. Apply a voltage of 90 V for about 30 - 40 min. After the protein sample runs out of the stacking gel into the separating gel, adjust the voltage to 110 V and terminate the electrophoresis in a timely manner according to the experimental requirements with reference to the protein Marker. After terminating the electrophoresis, transfer the membrane at a voltage of 110 V for 1 h 40 min. Immerse the PVDF membrane in 5% non-fat milk prepared with TBST at room temperature for 2 h or shake slowly overnight at 4 °C. Discard the blocking solution, add the primary antibody and incubate it on a shaker at 4 °C overnight with slow shaking. Recover the primary antibody, wash it 3 times with TBST, 12 min each time, add the secondary antibody and incubate it on a shaker at room temperature for 2 h with slow shaking. Recover the secondary antibody, wash it 3 times with TBST, 12 min each time, and perform chemiluminescent imaging.

[0070] 2.4 Identification of exosome morphology by transmission electron microscopy: Take 10 μl of exosome suspension and drip it onto a carbon film copper grid for sample loading at room temperature. Let it stand for 5 min and then blot the liquid from the side with filter paper. Add 7.5 μl of uranium dye, flip and rinse 2 times, then add another 7.5 μl of dye, let it stand for 30 s and then blot the liquid from the side with filter paper. After blotting the residual liquid with filter paper, dry the copper grid at room temperature. After drying on the filter paper, observe it using a transmission electron microscope.

[0071] 3. Intravitreal injection in mice

[0072] After anesthetizing the mice, place them under a surgical microscope, perform surface anesthesia using Oxybuprocaine Hydrochloride Eye Drops, and dilate the pupils of the mice using Compound Tropicamide Eye Drops. Use a 33G needle Hamilton syringe to inject the exosome suspension into the vitreous cavity. Leave the needle in place for 30 s and then slowly withdraw the needle. The intravitreal injection substances required in this experiment are: PBS for the vehicle control group, EVs and MT-EVs for the administration group. All eyes with cataracts and intraocular hemorrhage were excluded from the experimental analysis.

[0073] 4. Visual behavioral experiments in mice

[0074] 4.1 Black / white transition box

[0075] Use a black / white box. As described previously, this box consists of a dark chamber (16 - 16 - 25 cm) and a light chamber of the same size (illuminated with bright white light). There is a 10 - 12 cm hole in the wall separating the black and white chambers, allowing the mouse to move freely from one chamber to the other. At the start of the experiment, place a mouse in the middle of the light chamber in the laboratory. After 5 min, take it out of the box and measure the time the mouse stays in the light chamber.

[0076] 4.2 Visual cliff

[0077] Before the cliff test, the mice were placed on a small glass box with their heads facing away from the cliff side. A camera was suspended and fixed above the glass box, and video recordings were made through the camera for 5 minutes for each animal to observe the entire exploration process of the mice. After the experiment, the animal behavior automatic tracking software ToxTrac was used to analyze the video of the mice's movement behavior, and then the experimental data was further processed to calculate the proportion of the time each mouse spent on the cliff side (also known as the deep side) in the total time of the test process.

[0078] 4.3 Flash visual evoked potential (FVEP)

[0079] After weighing and anesthetizing the mice, FVEP examinations were performed. The mice were placed in a dark environment in advance for dark adaptation. After 12 hours of dark adaptation, they were anesthetized. The mice were placed on the fixed platform of the electrophysiological instrument. During the examination, one eye was covered with a black eye mask, and a special needle electrode for FVEP examination was installed. Electrode positions: the positive electrode was located at the visual center of the calcarine fissure in the occipital lobe of the animal's brain (about 5 mm from the line connecting the two ears), the negative electrode was located on the ipsilateral cheek of the animal, and the grounding wire was located subcutaneously at the animal's tail. The operation was carried out using the operating system of the British OPTOPROBE electrophysiological instrument. A flickering light was used as the stimulating light, the stimulation frequency was 1 Hz, the frequency bandwidth was 0.5 - 85.0 Hz, the analysis time was 250 ms, and it was superimposed 60 times. It was continuously measured at least 3 times, and the amplitude and latency of the P2 wave were recorded.

[0080] 4.4 Flicker electroretinogram (FERG)

[0081] After 12 hours of dark adaptation of the mice, they were anesthetized and mydriatic, and the electrodes were connected: the recording electrode, the reference electrode, and the ground electrode were connected respectively. First, the ground electrode was connected to the root of the mouse's tail, and then the reference electrode was connected to the bilateral cheeks of the mouse. The mouse's head was placed directly facing the stimulating light source and put into the Ganz fild (the above operations were all carried out under dim red light). The program was opened to perform the FERG recording program, the dark-adapted rod-cone mixed response (dMax-ERG) was analyzed, the amplitude of the FERG-b wave measured was marked, and the differences in the b-wave amplitudes among the four groups were compared.

[0082] 4.5 OCT (Optical Coherence Tomography, OCT)

[0083] After anesthesia and mydriasis, a transparent eye gel was applied to both corneas for imaging and analysis, during which the corneas were kept moist at all times. Mouse retinal images around the optic nerve head were captured and measured using an Optoprobe from the UK. The thickness of the measured retinal ganglion cell complex (GCC), including the RNFL, ganglion cell layer (GCL), and inner plexiform layer (IPL), was used as the measurement result. Built-in software was used to segment the GCC and quantify its thickness.

[0084] 5. Immunofluorescence staining

[0085] 5.1 Retinal flat mount: After enucleation, the eyeballs were rinsed with PBS and then placed in a 4% paraformaldehyde solution for fixation overnight at 4°C. The fixed mouse eyeballs were used to remove the anterior cornea, lens, and vitreous in PBS to make an "eyecup". Using a curved corneal scissors, 4 cuts were made along the course of the blood vessels on the retina to divide the retina into 4 equal parts on average. The iris retractor was used to carefully separate the retina from the underlying choroid and sclera of the "four-leaf clover" - shaped eyecup to obtain a "four-leaf clover" - shaped retina, which was placed in methanol pre-cooled to -20°C for preparing retinal flat mount immunofluorescence staining.

[0086] 5.2 Retinal immunofluorescence staining: After blocking and permeabilizing for 4 hours, the primary antibody was added and incubated in a refrigerator at 4°C for 24 h (the dilution concentration of the primary antibody RBPMS was 1:200); the slides were washed 3 times, 10 min each time; the fluorescent secondary antibody was incubated for 4 hours (the dilution concentration of the secondary antibody Dylight488 was 1:1000); after washing, an anti-fluorescence quencher was added for mounting the slides. Observation and photography were carried out under a fluorescence microscope. 3 photos were selected from each retina at a distance from the optic disc. The StarDist2D plugin in ImageJ (v153) software was used to count the number of RGCs in each photo, and the average density of RGCs was calculated.

[0087] 6. Statistical analysis

[0088] Each experiment in this chapter was repeated 3 times or more. Statistical analysis was performed using GraphPad Prism 90 software, and the data were expressed in the form of mean ± standard error. One-way ANOVA was used for comparison between more than two groups of data, and the differences between groups were detected by Bonferroni post hoc test and non-parametric Kruskal-Wallis test with Dunn multiple comparison test. A p < 0.05 was considered to indicate a statistically significant difference.

[0089] II. Experimental results

[0090] 1. Extraction and identification of exosomes

[0091] After the isolated ASTs were co-incubated with melatonin for 48 hours, exosomes were isolated from the supernatants of melatonin-treated and untreated ASTs by ultracentrifugation. The exosomes were verified by transmission electron microscopy and Western blotting. The morphology of EVs and MT-EVs was observed using transmission electron microscopy. We observed that, as Figure 1 shown, both types of exosomes were oval bilayer lipid membrane vesicles with a diameter of approximately 100 - 200 nm, and there was no significant difference between them. CD63, Tsg101, GAPDH, and Calnexin of EVs and MT-EVs were detected by Western blotting, and there was no obvious difference between the two.

[0092] 2. Melatonin-treated astrocyte exosomes promote the survival of retinal ganglion cells after optic nerve injury in mice

[0093] To detect the protective effects of EVs and MT-EVs on RGCs after ONC, it was found by immunofluorescence that, as Figure 2 shown in A, the number of RBPMS-positive RGCs (green) in the EVs group was significantly higher than that in the injury group. At the same time, the number of RBPMS-positive RGCs in the MT-EVs group was higher than that in the EVs group, indicating that EVs could rescue the death of RGCs, and the therapeutic effect of MT-EVs was better.

[0094] 3. Melatonin-treated astrocyte exosomes improve the recovery of visual function after optic nerve injury in mice

[0095] To evaluate whether EVs and MT-EVs rescued the visual function of mice, we used FVEP to detect the function of RGCs in the treated animals 60 days later; FERG to detect the overall function of the retina; black and white box, visual cliff to detect the visual acuity recovery of mice. As Figure 3 shown, the FVEP results showed that the amplitude of the P2 wave decreased in the ONC group; the latency of the P2 wave was prolonged; the amplitude of the b wave of FERG decreased; the residence time of mice in the light chamber and on the cliff side was prolonged. After treatment with EVS, FVEP showed that the amplitude of the P2 wave in mice recovered, the latency of the P2 wave was shorter than that in the ONC group, the amplitude of the b wave of FERG recovered, and the residence time of mice in the light chamber was shortened, but the results of the visual cliff showed that there was no statistical significance between the EVs group and the ONC group. The MT-EVs group showed greater advantages in RGCs function, retinal function, and visual acuity recovery. In addition, we also detected the thickness of the GCC of the retina by OCT and found that the GCC layer became thinner after ONC, while the thickness of the GCC layer in the EVs group increased compared with the ONC group, and the GCC layer in the MT-EVs group was thicker.

[0096] The results indicate that EVs and MT-EVs can significantly promote the recovery of visual function in mice, and the therapeutic effect of MT-EVs is more obvious.

[0097] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of exosomes in the preparation of a therapeutic product for optic nerve injury; The specific manifestations of the treatment of optic nerve injury are as follows: (a) Promote the survival of retinal ganglion cells after optic nerve injury in mice; (b) Improve the recovery of visual function after optic nerve injury in mice; The exosomes are exosomes derived from astrocytes; The exosomes are obtained by co-incubating astrocytes with melatonin at a concentration of 0.5 - 5 μM.

2. The application according to claim 1, characterized in that In (b), the recovery of visual function includes the function of retinal ganglion cells, retinal function, and visual acuity recovery.

3. The application according to claim 1, characterized in that The product is a drug or a test reagent.

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