Star-shaped glial cell-derived exosome and preparation method and application thereof

Abstract

This invention relates to astrocyte-derived exosomes, their preparation method, and applications. Specifically, it involves using photostimulation with PBM to induce the generation of astrocyte-derived exosomes. The generated exosomes can protect neurons and improve cognitive impairment and mood disorders. The exosomes obtained by this invention can be used for the treatment and / or prevention of cognitive impairment and / or mood disorders, demonstrating excellent application prospects and research value.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to an astrocyte-derived exosome, its preparation method, and its application. Background Technology

[0002] With the accelerated pace of modern life and intensified social competition, most people are under varying degrees of stress. Prolonged excessive stress has been identified as a significant contributing factor to many major fatal diseases in humans, and approximately 70% of human diseases are closely related to stress-induced damage. The brain is the regulatory center for sensing and responding to stress, and it is also a target organ susceptible to stress damage, especially cognitive functions involved in higher brain activities, which are particularly vulnerable to stress. Epidemiological surveys show that the prevalence of mild cognitive impairment and dementia in people under chronic stress is more than twice that of non-stressed individuals of the same age. Furthermore, chronic stress has been found to be a key risk factor for mood disorders such as depression and anxiety. The hippocampus is a key brain region regulating cognitive function and emotion. Stress can cause changes in hippocampal structure and function, including a reduction in hippocampal volume, a decrease in the number of dendritic spines in pyramidal cells, abnormal synaptic plasticity, and obstruction of dentate gyrus nerve development, thereby leading to cognitive impairment and mood abnormalities. However, since the molecular mechanisms by which stress induces cognitive impairment and emotional abnormalities remain unclear, there are currently no effective drugs for the prevention and treatment of stress-induced brain dysfunction in clinical practice.

[0003] Photobiomodulation (PBM) refers to the technology that utilizes the non-thermal effects induced in biological tissues by red to near-infrared light (wavelengths of 600-1100 nm) emitted from lasers or light-emitting diodes (LEDs) to stimulate or modulate various biological processes, alleviate pain and inflammation, and repair bodily damage. In recent years, PBM has been discovered as an innovative approach to stimulate neural activity and improve brain function. Currently, a growing body of basic research confirms the protective effects of PBM on various neurological diseases, such as Alzheimer's disease, Parkinson's disease, ischemic stroke, and age-related cognitive decline. However, whether PBM has a protective effect against stress-induced cognitive impairment and mood disorders, and the molecular mechanisms underlying its regulation, remain unclear.

[0004] Astrocytes are the most numerous type of glial cell in the central nervous system, playing a crucial role in maintaining neuronal function by providing nutritional support to neurons, participating in signal transduction, and regulating synaptic plasticity. Astrocytes can also communicate with other nerve cells by releasing exosomes, maintaining homeostasis in the central nervous system. Exosomes are important carriers for communication between astrocytes and neurons, microglia, and other cells. Exosomes are vesicles with a diameter of approximately 40–160 nm released from cells and containing bioactive molecules such as proteins, nucleic acids, and lipids. They can further regulate gene expression and biological functions in target cells by transferring various proteins, mRNAs, and non-coding RNAs they carry to target cells. Because exosome preparations lack cellular structure, they offer greater safety and operability, are easier to store, and can be used for multi-drug delivery for combination therapy, targeted modification, immune escape modification, and controlled drug release modification, showing great promise for future applications.

[0005] Therefore, it is necessary to further study the function of astrocytes and their associated exosomes in order to develop drugs for treating functional impairment and mood disorders. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides exosomes derived from astrocytes, their preparation method, and applications, particularly for the treatment and / or prevention of cognitive impairment and / or mood disorders. Specifically,

[0007] In a first aspect, the present invention provides a method for preparing exosomes derived from astrocytes, the method comprising inducing astrocytes using photobiomodulation (PBM).

[0008] Preferably, the PBM employs near-infrared light.

[0009] Specifically, the preparation method includes the following steps:

[0010] (1) Culture astrocytes;

[0011] (2) Photostimulation of astrocytes induces exosome production;

[0012] (3) Collect the supernatant and extract the exosomes.

[0013] Preferably, in step (2), the stimulation parameters include light wavelengths of 700nm-2526nm (e.g., 700, 750, 780, 790, 800, 810, 850, 900, 1000, 1500, 2000, 2500, 2526nm), and more preferably 700-850nm;

[0014] The stimulation time is 300-400s (e.g., 300, 310, 320, 330, 340, 350, 370, 380, 390, 400s);

[0015] Power density is 40-60 mW / cm³ 2 (For example, 40, 42, 44, 45, 47, 49, 50, 52, 55, 57, 60 mW / cm) 2 );

[0016] Output power is 3-4W (e.g., 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4W);

[0017] Energy density is 15-25 J / cm³ 2 (For example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 J / cm) 2 );

[0018] Total energy is 1000-1500J (e.g., 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500J); continuous wave.

[0019] More preferably, in step (2), the stimulation parameters are a wavelength of 810 nm, a stimulation time of 360 s, and a power density of 50 mW / cm². 2 Output power 3.75W, energy density 18J / cm³ 2 Total energy 1350J, continuous wave.

[0020] Preferably, step (3) can be performed using any existing method for exosome extraction, including but not limited to ultracentrifugation, density gradient centrifugation, ultrafiltration, magnetic bead immunoassay, or size exclusion chromatography.

[0021] Preferably, the exosomes overexpress the transcription factor Yy-1 (YIN-YANG 1).

[0022] Preferably, the exosomes are saucer-shaped or hemispherical.

[0023] Preferably, the average particle size of the exosomes is 100-200 nm (e.g., 100, 110, 120, 130, 140, 150, 160, 165, 166.3, 170, 180, 190, 200 nm), particularly 120-180 nm.

[0024] In one specific embodiment, the average particle size of the exosomes is 166.3 nm.

[0025] Preferably, the exosomes express tumor susceptibility gene 101 (TSG101), transmembrane 4L six family 1 (TM4SF1 or CD81), and leukocyte differentiation antigen 9 (CD9 molecule, CD9).

[0026] Preferably, the astrocytes express calnexin.

[0027] Preferably, the exosomes have a neuroprotective effect; more preferably, the protective effect includes enhancing neuronal cell viability and inhibiting apoptosis; and even more preferably, the cells are derived from hippocampal neurons.

[0028] Preferably, the exosomes reduce the expression of cleaved-Caspase 3, reduce the expression of Bcl-2 Associated X Protein 2 (BAX-2), promote the expression of B-cell lymphoma-2 (BCL-2), and inhibit apoptosis.

[0029] Preferably, the exosomes promote the expression of brain-derived neurotrophic factor (BDNF) by overexpressing transcription factor Yy-1.

[0030] In a second aspect, the present invention provides an exosome obtained by the above preparation method.

[0031] A third aspect of the present invention provides a drug comprising exosomes obtained by the above preparation method.

[0032] Preferably, the drug can be administered by any suitable method, including but not limited to oral administration, injection (such as intravenous injection, intramuscular injection, subcutaneous injection, etc.), inhalation administration, local administration, nasal administration, intracerebral administration, etc.

[0033] Preferably, the drug can be formulated into any suitable dosage form, such as, but not limited to, cream, foam, ointment, cream, emulsion, liquid solution, eye drops, injection, powder for injection, gel, spray, suspension, microemulsion, etc.

[0034] In a fourth aspect, the present invention provides an application of the above-described exosomes or the above-described drug.

[0035] Preferably, the application includes:

[0036] 1) Its application in promoting BDNF expression;

[0037] 2) Application in the preparation of drugs for the prevention and / or treatment of diseases associated with neurological dysfunction.

[0038] Preferably, the neurological impairment includes hippocampal neuronal damage.

[0039] Preferably, the neurological function impairment includes cognitive function impairment and / or mood disorders; more preferably, the cognitive function impairment includes impairment of learning and memory abilities; and the mood disorders include decreased mood regulation ability, depression, anxiety, etc.

[0040] Preferably, the diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, brain injury syndrome, traumatic brain injury, spinal cord injury, ischemic brain injury, epilepsy, depression, bipolar disorder, anxiety disorder, vascular dementia, diabetic neuropathic pain, intracranial infection, stress disorder, etc. More preferably, the stress disorder includes acute stress disorder, chronic stress disorder, post-traumatic stress disorder, adaptation stress disorder, etc.

[0041] Preferably, the exosomes or drugs have neuroprotective effects; more preferably, the protective effects include improving neuronal cell viability and inhibiting neuronal apoptosis.

[0042] More preferably, the treatment includes improvement of symptoms such as cognitive impairment and / or mood disorders accompanying the above-mentioned diseases, and the improvement includes partial improvement and complete improvement.

[0043] In one specific embodiment, the disease includes stress disorder, which is accompanied by cognitive impairment and / or mood disorders. More preferably, the cognitive impairment includes impairment of learning and memory abilities; the mood disorders include decreased mood regulation ability, depression, anxiety, etc.

[0044] The technical advantages of this invention are as follows:

[0045] This invention discovers that near-infrared light stimulation can induce astrocytes to produce a large number of exosomes, which can improve neurological function impairment. These exosomes can enhance neuronal viability and inhibit stress-induced neuronal apoptosis. Furthermore, near-infrared light induces an increase in Yy-1 content in astrocyte exosomes; after entering neurons, Yy-1 binds to the BDNF promoter, promoting BDNF expression and thus achieving a protective effect on neurons. The exosomes obtained by this invention can be used for the treatment or prevention of diseases accompanied by cognitive impairment and / or mood disorders, showing great application prospects and research value.

[0046] The above only summarizes some aspects of the present invention and is not, and should not be considered as limiting the present invention in any way.

[0047] All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications may be made to this invention without departing from its spirit or scope. The following embodiments further illustrate the invention in detail and should not be construed as limiting the scope of the invention or the specific methods described herein. Attached Figure Description

[0048] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0049] Figure 1 Electron micrograph of astrocyte exosomes.

[0050] Figure 2 Image showing the NTA detection results of astrocyte exosomes.

[0051] Figure 3 Analysis of astrocyte exosome concentration, ****P<0.001.

[0052] Figure 4 Standard curve for determining exosomal protein concentration using the BCA method.

[0053] Figure 5 Western blot detection of exosome markers.

[0054] Figure 6 Morris water maze test for spatial learning and memory function in mice: A, escape latency during training period, *P<0.05.

[0055] **P<0.01 vs CTRL-PBS, #P<0.05 vs CUMS-Ast-Ctrl-exo; B, escape latency during the test period; C, number of times the platform was crossed during the test period; D, proportion of time spent in the target quadrant during the test period; *P<0.05, **P<0.01.

[0056] Figure 7 The novel object recognition experiment was used to test the long-term memory function of mice. ***P<0.001.

[0057] Figure 8 The sucrose preference test was used to detect pleasure in mice. **P<0.01.

[0058] Figure 9 Tail suspension test was used to detect despair in mice. **P<0.01.

[0059] Figure 10 Effects of astrocyte exosomes on the viability of HT22 cells.

[0060] Figure 11 Effects of astrocyte exosomes on GC-induced apoptosis in HT22 cells. The left figure shows the results of flow cytometry staining, and the right figure shows the percentage of apoptosis.

[0061] Figure 12 Effects of astrocyte exosomes on cleaved-caspase 3 expression.

[0062] Figure 13 The effect of astrocyte exosomes on the expression of key molecules regulating apoptosis.

[0063] Figure 14 Near-infrared light induced differential expression of mRNA, lncRNA, and circRNA in astrocyte exosomes compared to control exosomes.

[0064] Figure 15 Volcano plot of differentially expressed mRNA between near-infrared light-induced astrocyte exosomes and control exosomes.

[0065] Figure 16 Yy1 mRNA levels in exosome sequencing, *P<0.05.

[0066] Figure 17 Effects of astrocyte exosomes on Yy-1 levels in HT22 cells.

[0067] Figure 18 ChIP assay was used to detect the binding of Yy1 to the BDNF promoter in mouse hippocampus.

[0068] Figure 19 EMSA experiments were used to detect the binding of Yy1 to the BDNF promoter. Detailed Implementation

[0069] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0070] Example 1: Near-infrared light-induced astrocyte exosomes and their preparation and identification

[0071] 1. Astrocyte culture and near-infrared light stimulation

[0072] Culturing C8-D1A astrocytes or primary astrocytes, and after passage, seeding the cells into 10cm culture dishes, marking the cell attachment time as 0h, and applying near-infrared light stimulation at 0h, 2h, 18h, and 48h, with stimulation parameters of 810nm wavelength, 360s stimulation time, and 50mW / cm² power density. 2 Output power 3.75W, energy density 18J / cm³ 2 Total energy 1350J, continuous wave. Supernatant was collected immediately after the last near-infrared light stimulation, and exosomes were extracted (Ast-PBM-exo). Control group cells were not given near-infrared light stimulation, and supernatant was collected for exosome extraction (Ast-sham-exo).

[0073] 2. Exosome extraction

[0074] Exosomes can be extracted using commercially available exosome extraction kits, ultracentrifugation, magnetic bead extraction, and other effective methods. The magnetic bead extraction method will be used as an example below.

[0075] 1) Sample preparation. Centrifuge 10,000g of the supernatant at 4°C for 10 min and transfer it to a new centrifuge tube.

[0076] 2) Magnetic bead preparation. Vortex the bottled magnetic beads for 30 seconds, add 350 μL to a centrifuge tube, centrifuge at 3,000 g and 4 °C for 2 min, and discard the supernatant.

[0077] 3) Wash the magnetic beads. Add 10 mL of PBS pre-cooled at 4℃ and filtered through a 0.22 μm filter, vortex to mix for 30 sec, centrifuge at 3,000 g at 4℃ for 5 min, and discard the supernatant.

[0078] 4) Exosome adsorption. Add 4 mL of Buffer EXA, 1 mL of Buffer EXB and 14.65 mL of supernatant to a rotary mixer and mix at 4°C for 40 min.

[0079] 5) Magnetic bead collection. Remove the centrifuge tube, place it on a magnetic rack, and let it stand at 4°C for 10 minutes until the magnetic beads gather. Discard the supernatant.

[0080] 6) Centrifuge at 3,000g for 1 min at 4℃ and discard the residual liquid.

[0081] 7) Elute exosomes. Add 0.5 mL of Buffer EXE and mix well. Centrifuge at 7,000 g, 4 °C for 2 min. Transfer the supernatant to an EP tube.

[0082] 8) Filtration to remove impurities. After rinsing syringe filters A and B with EXE, filter the exosome solution separately to obtain a purified exosome solution.

[0083] 9) The purified exosomes should be used immediately for testing or stored in a -80°C freezer.

[0084] 3. Exosome identification

[0085] The extracted exosomes were detected using transmission electron microscopy, NTA nanoparticle detection technology, protein concentration determination, and immunoblotting to obtain the morphology, particle size, number, and concentration of the extracted exosomes, and to verify that the samples conformed to the characteristics of exosomes.

[0086] 3.1 Transmission Electron Microscopy

[0087] 1) Drop 10 μL of exosome solution onto a copper grid, incubate at room temperature for 10 min, wash with sterile distilled water, and blot off excess liquid with absorbent paper.

[0088] 2) Take 10 μL of 2% uranium acetate and drop it onto a copper grid for negative staining for 1 min. Absorb the floating liquid with filter paper and dry for 2 min.

[0089] 3) Place the copper mesh under a transmission electron microscope for observation and image it at 80kV.

[0090] Transmission electron microscopy results showed that the exosomes extracted from the sample, when photographed at 100 nm, 200 nm, and 500 nm, exhibited a saucer-like or hemispherical morphology. Figure 1 ).

[0091] 3.2 NTA Nanoparticle Detection

[0092] A laser light source was used to irradiate the nanoparticle suspension, and the scattered light from the nanoparticles was detected. The concentration of the nanoparticles was calculated by counting the number of scattered particles. Specifically, the concentration of the separated exosomes was determined using a ZetaView PMX 110 particle matrix under 405nm emission light, and the exosomes were diluted with PBS to a concentration of 1*10. 7 Particles / mL ~ 1*10 9The number of exosomes was measured per mL, and their size and mass were determined. Simultaneously, the particle trajectories of exosomes were analyzed.

[0093] The results showed that the average particle size of exosomes in the Ast-sham-exo group was 173.8 nm, the main peak was 126.8 nm, the percentage of the main peak was 98.5%, and the final concentration was 1.0 × 10⁻⁶. 11 Particles / mL; the average particle size of the Ast-PBM-exo sample was 166.3 nm, the main peak was 127.5 nm, the percentage of the main peak was 97.9%, and the final concentration was 1.3 × 10⁻⁶. 11 Particles / mL ( Figure 2 and Figure 3 ).

[0094] 3.3 Protein Concentration Determination

[0095] Exosomal proteins were extracted, and the protein concentrations of the two groups of exosomal proteins were determined using the BCA method, based on a standard curve. Figure 4 The protein concentrations of exosomes were calculated, and the results showed that the protein concentrations of the extracted exosomes were all within a reasonable range (Table 1).

[0096] Table 1. Detection and Calculation of Exosomal Protein Concentration

[0097]

[0098] 3.4 Detection of exosome markers

[0099] The expression of exosome markers (TSG101, CD81, CD9) and non-exosome protein (Calnexin) was detected by Western blot. The results showed that TSG101, CD81, and CD9 proteins were expressed in exosomes, while Calnexin was expressed in cells. Figure 5 ).

[0100] Example 2: Near-infrared light-induced astrocyte exosomes improve stress-induced cognitive impairment in mice.

[0101] 1. Establish an animal disease model of stress-induced stress disorder accompanied by cognitive impairment.

[0102] C57BL / 6 mice were subjected to chronic unpredictable mild stress (CUMS) for 6 weeks to induce cognitive impairment. Subsequently, Ast-PBM-exo and Ast-Ctrl-exo (2 μL / mouse, exosome concentration 1 μg / μL, once daily) were injected into the hippocampus of the mice via stereotactic injection for 7 consecutive days. Behavioral experiments were then conducted to examine the effects of astrocyte exosomes on the cognitive function of the stressed mice.

[0103] 2. Near-infrared light-induced astrocyte exosomes improve cognitive function in stressed mice.

[0104] The effects of astrocyte exosomes on cognitive function in stressed mice were investigated using the Morris water maze test and novel object recognition test.

[0105] The Morris water maze experiment setup consisted of a circular pool, 100 cm in diameter and 60 cm deep, painted white to allow the software to more accurately identify and record the behavioral trajectories of black mice. Furthermore, to provide spatial cues, the pool walls and surrounding obstructions were marked with different shapes and colors. The experiment was divided into a training phase and a testing phase. In the training phase, mice underwent five platform exploration sessions, each half-day apart. During training, the underwater platform was fixed in one quadrant, with its top approximately 1 cm below the water surface. The experimenter gently placed the mouse in the opposite quadrant of the platform and guided it to find it. If the mouse failed to find the platform within 3 minutes, it was guided to the platform and held there for 15 seconds to reinforce its learning. During this process, the experimental software recorded the time it took for the mouse to find the platform (i.e., the escape latency) and its movement trajectory. In the testing phase, the platform was removed, and the mouse was placed into the pool from a random location for 3 minutes. At this point, the experimental software will record the time required for the mouse to first find the original platform location (i.e., the escape latency), the number of times it crosses the original platform location, and the percentage of time spent in the quadrant where the platform is located. These experimental indicators can effectively reflect the mouse's spatial learning and memory abilities.

[0106] Morris water maze test results showed that, compared with Ast-Ctrl-exo injection, stress-induced mice injected with Ast-PBM-exo experienced a significant reduction in maze platform finding time and distance, an increase in the number of platform crossings, and a longer dwell time in the target quadrant. This indicates that near-infrared light-induced astrocyte exosomes can significantly improve the spatial learning and memory abilities of stress-induced mice. Figure 6 ).

[0107] The novel object recognition experimental setup consisted of a black, square box with an open top and a base side length of 50 cm. Before the experiment, each mouse underwent 5 minutes of acclimatization training with the same objects. During this time, the mouse was placed inside the box and exposed to two identical objects to familiarize itself with the experimental environment. To ensure accuracy, the inside of the box was wiped clean with 75% ethanol before each mouse's experiment to minimize the impact of odor differences on mouse behavior. Furthermore, each mouse started from the opposite side of the object, in roughly the same position, to ensure consistency of experimental conditions. After the acclimatization training, at intervals of 3-4 hours, one object in the box was replaced with a new object of approximately the same size. Subsequently, camera tracking software recorded the time spent by the mice exploring the two objects. By calculating the proportion of time spent exploring the new object to the total time spent exploring both objects, we obtained the novel object recognition index, thereby assessing the mouse's ability to recognize or learn new objects.

[0108] The results of the novel object recognition experiment showed that, compared with the mice injected with Ast-Ctrl-exo, the novel object recognition index was significantly increased in the stress group mice after injection of Ast-PBM-exo. Figure 7 The above results indicate that near-infrared light-induced astrocyte exosomes can significantly improve stress-induced cognitive impairment.

[0109] Example 3: Near-infrared light-induced astrocyte exosomes improve stress-induced mood disorders in mice.

[0110] 1. Establish an animal disease model of stress-induced stress disorder accompanied by mood disorders.

[0111] A mouse model of stress disorder accompanied by mood disturbances was established by subjecting C57BL / 6 mice to chronic unpredictable mild stress (CUMS) for 6 weeks. Subsequently, Ast-PBM-exo and Ast-Ctrl-exo (2 μL / mouse, exosome concentration 1 μg / μL, once daily) were injected into the hippocampus of the mice via stereotactic injection for 7 consecutive days. Behavioral experiments were then conducted to examine the effects of astrocyte exosomes on the mood of the stressed mice.

[0112] 2. Near-infrared light-induced astrocyte exosomes improve depressive mood in stressed mice.

[0113] The regulatory effect of astrocyte exosomes on the mood of stressed mice was investigated using a sucrose preference test and tail suspension test.

[0114] A sucrose preference test was used to detect depressive-like behavior. Before the experiment, mice were housed alone in cages for 48 hours to acclimatize to the experimental environment. Two water dispensers were placed in the cages, one containing plain drinking water and the other containing a 1% sucrose solution, along with normal food. The purpose of this phase was to familiarize the mice with the two water dispensers and the presence of the sucrose solution. To avoid preference bias due to memory of the water dispenser locations, the locations of the water dispensers were changed every 8 hours. After the acclimatization period, the mice were fasted and deprived of water for 24 hours. Subsequently, the two water dispensers were reintroduced (one containing plain drinking water and the other containing a 1% sucrose solution), and the initial weights of the water dispensers were recorded. During the experiment, the locations of the water dispensers were changed every 12 hours to further eliminate the influence of location preference on the results. After the 24-hour test, the weights of the water dispensers were weighed again. By calculating the weight difference between the water dispensers, the amount of plain water and sucrose solution consumed by the mice within 24 hours could be accurately determined. The proportion of sucrose water consumed to the total water intake was calculated as the sucrose preference index.

[0115] The results of the saccharide preference experiment showed that, compared with the mice injected with Ast-Ctrl-exo, the saccharide preference index of mice in the stress group increased after injection of Ast-PBM-exo. Figure 8 ).

[0116] The tail suspension test can reflect the despair state of mice. The test chamber is 55 cm high. The tip of the mouse's tail is suspended from the apparatus, with its head facing downwards and the tail tip approximately 30 cm from the ground. A video recording system is used to record for 6 minutes. The duration of stillness for the last 4 minutes is analyzed; a longer period of stillness generally indicates a more severe degree of depression in the mouse.

[0117] The results of tail suspension test showed that, compared with the injection of Ast-Ctrl-exo, the tail suspension immobility time of the stressed mice was significantly reduced after injection of Ast-PBM-exo. Figure 9 The above results indicate that near-infrared light-induced astrocyte exosomes can significantly improve stress-induced depressive mood.

[0118] Example 4: Near-infrared light-induced astrocyte exosomes inhibit stress-induced hippocampal neuronal damage.

[0119] Stress induces abnormal activation of the hypothalamic-pituitary-adrenal axis (HPA axis), leading to a significant increase in plasma glucocorticoid (GC) levels and hippocampal GC levels in mice. GC is a key stress hormone, and high-concentration GC-induced neuronal damage has been found to be an important pathological mechanism in the development of cognitive dysfunction. High-concentration GC can induce decreased viability and increased apoptosis in hippocampal HT22 neurons. Therefore, a stress-induced neuronal damage model was established using high-concentration GC stimulation to investigate the protective effect of near-infrared light-induced astrocyte exosomes on neurons.

[0120] 1. Near-infrared light-induced astrocyte exosomes enhance hippocampal neuronal cell viability.

[0121] Mouse hippocampal HT22 cells were cultured and seeded into 96-well plates. A stress-induced cell injury model was established by stimulation with GC (50 μmol / mL). Near-infrared light-induced astrocyte exosomes (1 mg / mL) and control exosomes (1 mg / mL) were added to the culture medium. The effects on hippocampal neuron viability were assessed at 48 and 72 hours using a CCK8 cell proliferation-toxicity assay kit (Dojin Chemical, Japan). Results showed that high concentrations of GC induced a decrease in hippocampal neuron viability, while near-infrared light-induced astrocyte exosomes significantly enhanced cell viability, antagonizing the GC-induced decrease in cell viability. Figure 10 ).

[0122] 2. Near-infrared light-induced astrocyte exosomes inhibit hippocampal neuronal apoptosis.

[0123] 2.1 Flow cytometry detection of apoptosis

[0124] HT22 cells were seeded into 6-well plates and induced with GC (50 μmol / mL). Near-infrared light-induced astrocyte exosomes (1 mg / mL) and control exosomes (1 mg / mL) were added to the culture medium. After 24 h, cells were collected and stained with the Annex V-APC / PI apoptosis detection kit (Wuhan Yilairuit Biotechnology Co., Ltd.). The effect of exosomes on GC-induced hippocampal neuronal apoptosis was detected by flow cytometry. The results showed that high concentrations of GC induced increased hippocampal neuronal apoptosis, while near-infrared light-induced astrocyte exosomes significantly inhibited GC-induced apoptosis. Figure 11 ).

[0125] 2.2 Detection of apoptosis-related protein levels using immunoblotting technology

[0126] HT22 cells were seeded into 6-well plates and induced with GC (50 μmol / mL). Near-infrared light-induced astrocyte exosomes (1 mg / mL) and control exosomes (1 mg / mL) were added to the culture medium. Cells were collected after 24 h, and cellular proteins were extracted. Western blot analysis was performed to detect the expression levels of key apoptosis proteins. The results showed that high concentrations of GC induced an increase in cleaved-Caspase 3 levels in hippocampal neurons, while near-infrared light-induced astrocyte exosomes significantly reduced GC-induced cleaved-Caspase 3 expression. Figure 12 ).

[0127] 2.3 Real-time quantitative PCR (qRT-PCR) detection of expression levels of key apoptosis regulatory proteins

[0128] HT22 cells were seeded into 6-well plates and induced with GC (50 μmol / mL). Near-infrared light-induced astrocyte exosomes (1 mg / mL) and control exosomes (1 mg / mL) were added to the culture medium. After 24 h, cells were collected, cellular proteins were extracted, and qRT-PCR was performed to detect the mRNA levels of BAX-2 and BCL-2. The results showed that high concentrations of GC induced increased BAX-2 mRNA expression and decreased BCL-2 mRNA expression in hippocampal neurons; while near-infrared light-induced astrocyte exosomes antagonized the GC-induced changes in apoptosis protein expression. Figure 13 ).

[0129] Example 5: Near-infrared light induces an increase in the content of Yy-1 transcripts in astrocyte exosomes.

[0130] 1. Exosome whole transcriptome sequencing

[0131] Near-infrared light-induced astrocyte exosomes and control exosomes were collected. A biotechnology company was commissioned to perform whole-transcriptome sequencing of the exosomes to obtain the levels of mRNA, lncRNA, and circRNA within the exosomes, and the effects of near-infrared light stimulation on the contents of astrocyte exosomes were analyzed. Results showed that, using a fold change ≥ 2.0 and a p-value < 0.05 as the differential screening thresholds, compared with control exosomes, 179 differentially expressed mRNAs, 81 differentially expressed lncRNAs, and 6 differentially expressed circRNAs were detected in near-infrared light-induced astrocyte exosomes. Figure 14 ).

[0132] 2. Near-infrared light significantly increased the content of Yy-1 mRNA in astrocyte exosomes.

[0133] Analysis of differentially expressed mRNAs revealed a significant increase in the mRNA level of transcription factor Yy-1 in near-infrared light-induced astrocyte exosomes. Figure 15 & Figure 16 ).

[0134] Further, exosomes were added to a GC-induced neuronal injury model, and changes in Yy-1 levels in HT22 cells were detected. qRT-PCR results showed that, compared with control exosomes, near-infrared light induction significantly increased Yy-1 levels in HT22 cells. Figure 17 ).

[0135] 3. Transcription factor Yy-1 binds to the BDNF promoter to promote BDNF expression.

[0136] Brain-derived neurotrophic factor (BDNF) is one of the most important neurotrophic factors in the brain. BDNF participates in regulating neural development and neuronal survival. A decrease in BDNF can lead to neurological disorders such as abnormal neural development, mood disorders, and cognitive impairment. Chromatin immunoprecipitation (ChIP) was used to detect the binding of transcription factor Yy-1 to the BDNF promoter in the hippocampus of CUMS mice. The results showed that, compared with control mice, the binding of Yy-1 to the BDNF promoter was reduced in stressed mice. Figure 18 This indicates that chronic stress induces a weakened DNA-binding capacity of the transcription factor Yy-1, which is an important regulatory molecule for stress injury.

[0137] The binding efficiency of Yy-1 to specific sites on the BDNF promoter was further verified using an electrophoresis gel migration assay (EMSA). The BDNF promoter DNA double-stranded sequence was synthesized in vitro, and an EMSA reaction system was prepared. Different concentrations of recombinant Yy-1 protein were added to the system, and the reaction products were subjected to agarose gel electrophoresis. The results showed that with increasing concentration of Yy-1 protein, the binding of Yy-1 to the BDNF promoter was enhanced, while the free DNA gradually decreased. Figure 19 This indicates that the Yy-1 protein can bind to the BDNF promoter, and the binding ability increases with increasing Yy-1 levels.

[0138] In summary, near-infrared light stimulation can induce astrocytes to produce a large number of exosomes, which can improve the negative effects of chronic stress on cognitive function and mood. These exosomes can enhance neuronal vitality and inhibit stress-induced neuronal apoptosis. Furthermore, near-infrared light can increase the content of Yy-1 mRNA in astrocyte exosomes. After Yy-1 enters neurons, it binds to the BDNF promoter to promote BDNF expression, thereby achieving a protective effect on neurons.

[0139] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0140] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0141] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for preparing exosomes derived from astrocytes, characterized in that, The preparation method includes inducing astrocytes using photobiomodulation (PBM) technology, and the preparation method includes the following steps: (1) Culture astrocytes; (2) Photostimulation of astrocytes induces exosome production; (3) Collect the supernatant and extract exosomes; In step (2), the stimulation parameters include a light wavelength of 700-850 nm and a power density of 40-60 mW / cm². 2 Energy density is 15-25 J / cm³ 2 Continuous wave, stimulation time is 300-400s.

2. The preparation method according to claim 1, characterized in that, In step (2), the stimulation parameters also include an output power of 3-4W and a total energy of 1000-1500J.

3. The preparation method according to claim 1, characterized in that, The exosomes overexpress transcription factor Yy-1.

4. The preparation method according to any one of claims 1-3, characterized in that, The average particle size of the exosomes is 100-200 nm.

5. An exosome obtained by the preparation method according to any one of claims 1-4.

6. A drug comprising the exosomes of claim 5.

7. The use of the exosomes of claim 5 in the preparation of a medicament for treating stress-induced cognitive impairment.

8. The use of the exosomes of claim 5 in the preparation of a medicament for treating stress-related mood disorders.

9. The use of the exosomes of claim 5 in the preparation of a medicament for treating stress-induced hippocampal neuronal damage.

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