Application of external vesicles to treatment of neuronal injury

Abstract

The invention particularly discloses application of an outer vesicle to treatment of neuron injury, and relates to the technical field of biological medicines. The extracellular vesicle is secreted after exercise, a mouse SCI model is used for proving that the plasma extracellular vesicle of an athlete can remarkably inhibit neuronal ferroptosis, so that the recovery of the exercise function after injury is promoted, and further mechanism research shows that the extracellular vesicle can be used for inhibiting neuronal ferroptosis of the athlete. The plasma extracellular vesicles of athletes play a key role in ferroptosis inhibition and functional recovery after SCI through RNF216. Generally speaking, the invention discloses an important role of the plasma extracellular vesicles of athletes in neuronal protection and motor function recovery after SCI, provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of an athlete's plasma extracellular vesicle in the preparation of a medicine for treating spinal cord injury. Background Art

[0002] Spinal cord injury (SCI) is a disease with a very poor prognosis, which can lead to transient or permanent loss of function. It has a high morbidity, high disability and mortality rates, high treatment costs, and extremely serious social harm. Studies have shown that the total number of SCI patients in China is 759,302, with 66,374 new cases each year. SCI can be divided into traumatic SCI and non-traumatic SCI according to different etiologies. According to different pathophysiological developments, SCI can be divided into primary and secondary injuries. Primary injury is related to direct destruction of neurons, while secondary injury is the main factor aggravating tissue damage and nerve cell loss. Therefore, targeted neuroprotective treatment is particularly important. However, the effects of surgery, drugs or other treatments are extremely limited, and there are few clear clinical treatments that can effectively promote functional recovery in SCI patients. Therefore, there is an urgent need to develop a safe and effective neuroprotective method. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a use of an extracellular vesicle in preparing a medicament for treating neuronal injury or damage and / or diseases and / or symptoms associated with neuronal injury or damage.

[0004] In one embodiment, the extracellular vesicles are athlete-derived extracellular vesicles.

[0005] In one embodiment, the extracellular vesicles overexpress RNF216 protein.

[0006] In one embodiment, the extracellular vesicle is selected from exosomes, microvesicles, apoptotic bodies, oncosomes, multivesicular bodies, migratoria, exosomes or a combination thereof.

[0007] In one embodiment, the extracellular vesicles are derived from blood, urine, cerebrospinal fluid, the extracellular space of various tissues, saliva, milk, semen, ascites, or a combination thereof.

[0008] In one embodiment, the extracellular vesicles are derived from blood.

[0009] In one embodiment, the extracellular vesicles are derived from plasma.

[0010] In one embodiment, the neuronal injury or damage and / or the disease and / or symptom associated with neuronal injury or damage is spinal cord injury.

[0011] On the other hand, the present invention also provides a method for preparing the above-mentioned extracellular vesicles, which specifically comprises the following steps: after collecting whole blood, centrifuge at 4°C and 2000g for 10 minutes to separate plasma, and then centrifuge at 3000g and 10000g for 20 minutes to remove cell debris and macromolecules in the plasma to obtain plasma. The plasma is centrifuged at 140,000g for 70 minutes at 4°C using an ultracentrifuge, the supernatant is removed, the precipitate is resuspended in PBS, and then centrifuged again at 140,000g for 70 minutes to obtain EVs.

[0012] In another aspect, the present invention further provides a pharmaceutical composition for use in treating neuronal injury or damage and / or diseases and / or symptoms associated with neuronal injury or damage, the pharmaceutical composition comprising:

[0013] (1) a therapeutically effective amount of the above-mentioned extracellular vesicles;

[0014] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0015] In one embodiment, the pharmaceutical composition is formulated for administration via an administration route selected from the group consisting of intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral, and intracerebral administration routes, and combinations thereof.

[0016] In one embodiment, the pharmaceutical composition is formulated for administration via an intravenous route of administration.

[0017] In another aspect, the present invention further provides a use of a pharmaceutical preparation for treating neuronal injury or damage and / or diseases and / or symptoms associated with neuronal injury or damage, wherein the pharmaceutical preparation comprises the above-mentioned pharmaceutical composition.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. AEVs promote motor function recovery after SCI. During the 4-week recovery period after SCI, mice treated with AEVs showed significantly better functional recovery in walking, hind paw placement, and hind limb movement in the BMS score compared with mice treated with EVs; the rotarod test also showed that mice treated with AEVs had better functional recovery of hind limb and tail balance; footprint analysis results also showed that mice treated with AEVs showed significantly faster gait recovery and improved motor coordination; MRI images showed that the spinal cords of mice treated with AEVs were more intact, the cysts were smaller, and the treatment effect was better.

[0020] 2. AEVs play an important role in the regulation of ferroptosis after SCI, which will provide new treatment methods for SCI. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 for AEVs identification and uptake; Figure 1 A. Observation of the morphology of EVs and AEVs under TEM; Figure 1 B. NTA detection of EVs and AEVs particle size distribution; Figure 1 C. The particle size of the two groups of extracellular vesicles; Figure 1 D. The number of particles per milliliter of the two groups of extracellular vesicles; Figure 1 EF. Surface Zeta potential of EVs and AEVs; Figure 1 G. Western blot was used to detect the expression of extracellular vesicle markers.

[0023] Figure 2 AEVs promote motor function recovery after SCI. Figure 2 A. Flow chart of animal experiments; Figure 2 B. BMS scores of mice during the recovery period 28 days after injury; Figure 2 C. Statistical analysis of the results of the mouse rotarod test; Figure 2 D. Statistical analysis of the results of the von Frey filament test in mice; Figure 2 EF. Mouse footprint analysis; Figure 2 GH. Statistical analysis of the gross morphology of the spinal cord and the injury area; *P<0.05; **P<0.01; ***P<0.001.

[0024] Figure 3 AEVs treatment alleviates ferroptosis caused by oxidative stress after SCI. Figure 3 AB. Representative Western blots showing the expression of ferroptosis-related proteins in the injured spinal cord and statistical analysis on day 7 after injury; *P<0.05; **P<0.01; ***P<0.001.

[0025] Figure 4 AEVs inhibit oxidative stress-induced neuronal cell ferroptosis in vitro. Figure 4 AB. Representative Western blots showing the expression of ferroptosis-related proteins in neuronal cells; *P<0.05; **P<0.01; ***P<0.001.

[0026] Figure 5AEVs promote motor function recovery and inhibit ferroptosis after SCI by transferring RNF216 in vivo. Figure 5 A. The protein expression level of RNF216 in EVs and AEVs was detected by Western blot; Figure 5 B. Detect the protein expression level of RNF216 in neuronal cells by Western blot and perform statistical analysis; Figure 5 C. Overexpression of RNF216 in neural stem cells; Figure 5 D. The expression level of RNF216 protein in Ad-Vec-NEVs and Ad-RNF216-NEVs was detected by Western blot; Figure 5 E. The protein expression level of RNF216 in neuronal cells was detected by Western blot and statistical analysis was performed; Figure 5 F. Grouping information for this part of the experiment; Figure 5 G. BMS scores of mice treated with Ad-Vec-NEVs and Ad-RNF216-NEVs during the recovery period 28 days after injury; Figure 5 H. Statistical analysis of the results of the rotarod test in two groups of mice; Figure 5 IJ. Footprint analysis of two groups of mice; Figure 5 K. Statistical analysis of the results of the von Frey filament test in two groups of mice; Figure 5 NO. Representative Western blot showing the expression of ferroptosis-related proteins in the injured spinal cord and statistical analysis; *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0029] definition

[0030] Extracellular vesicles

[0031] As used herein, the terms "extracellular vesicle" and "EV", "EVs" are used interchangeably herein and refer to a vesicle derived from a cell, which comprises a membrane surrounding an internal space. Typically, the diameter of an extracellular vesicle ranges from 30nm to 1000nm, and may include various macromolecular cargoes within the internal space, displayed on the outer surface of the extracellular vesicle, and / or include various macromolecular cargoes across the membrane. The cargo may include nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. The term extracellular vesicle includes the terms "exosomes", "microvesicles", "apoptotic bodies", "tumor bodies", "multivesicular bodies", "migrating bodies", "exosomes".

[0032] As used in this article, the term "exosomes" are produced through the endosomal pathway, released outside the cell after the fusion of multivesicular bodies in the cell with the cell membrane, with a diameter usually between 30-150nm, and contain biologically active substances such as proteins, nucleic acids (such as mRNA, miRNA, etc.), lipids, and specific protein markers such as CD9, CD63, and CD81 on the membrane surface.

[0033] As used in this article, the term "microvesicle" is formed by direct outward budding and shedding of the cell membrane. Its diameter is generally between 100-1000nm. Its protein composition is similar to that of the cell membrane, containing some membrane receptors, cytoskeletal proteins, etc. It can also carry substances such as nucleic acids and lipids. Its surface markers include CD40, CD62E, etc.

[0034] As used in this article, the term "apoptotic body" refers to the process of cell apoptosis in which the cell shrinks, chromatin condenses, and nuclear fragments, and the cell membrane invaginates to form apoptotic bodies and are released outside the cell. The diameter ranges from about 50nm to 5μm, and it contains various components in the cell, such as broken chromatin, organelle fragments, proteins, etc. There may also be some apoptosis-related signaling molecules, such as phosphatidylserine, which are everted on the surface of the apoptotic body and can be recognized by phagocytes.

[0035] As used in this article, the term "tumor body" is an extracellular vesicle unique to tumor cells, secreted by tumor cells, with a diameter generally around 100-1000nm, and contains tumor cell-specific proteins, nucleic acids and other biologically active substances, such as tumor-associated antigens, oncogenes, etc., which can promote tumor growth, invasion and metastasis.

[0036] As used in this article, the term "multivesicular bodies" refers to intracellular multivesicular bodies that can be directly released outside the cell to form extracellular vesicles under certain circumstances. They are about 200-500nm in diameter and contain multiple small vesicles inside. Their composition is similar to that of exosomes, but may differ in the types and contents of proteins and nucleic acids. They also have certain biological functions.

[0037] As used in this article, the term "migrasome" refers to contractile filaments generated at the posterior end of the cell during cell migration. The contractile filaments break to form migrasomes that are released outside the cell. They are about 100-300nm in diameter and contain cytoskeletal proteins, signal transduction molecules, etc. They are closely related to processes such as cell migration, tissue repair and tumor metastasis, and can regulate cell-to-cell interactions and remodeling of the extracellular matrix.

[0038] As used in this article, the term "exosome" is secreted by cells through a special mechanism, is related to cell metabolism and stress response, has a diameter of about 50-200nm, is rich in proteins such as heat shock proteins, and may also carry some small molecule metabolites and signaling molecules, and participate in processes such as intercellular signal transmission and immune regulation.

[0039] The size of the EV may be greater than 2 nm. The size of the EV may be greater than 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm. The size of the EV may be greater than 100 nm, such as greater than 150 nm. The size of the EV may be approximately 200 nm or greater.

[0040] The size of an EV may be within a range, for example, between 2nm and 20nm, 2nm and 50nm, 2nm and 100nm, 2nm and 150nm, or 2nm and 200nm. The size of an EV may be between 20nm and 50nm, 20nm and 100nm, 20nm and 150nm, or 20nm and 200nm. The size of an EV may be between 50nm and 100nm, 50nm and 150nm, or 50nm and 200nm. The size of an EV may be between 100nm and 150nm, or 100nm and 200nm. The size of an EV may be between 150nm and 200nm. The size of an EV may be between 100nm and 600nm, 150nm and 500nm, or 200nm and 400nm.

[0041] The size can be determined in various ways. In principle, the size can be determined by size fractionation and filtration through a membrane with a relevant size cutoff. The particle size can then be determined by following the separation of the component proteins using SDS-PAGE or by biological assays.

[0042] The size may include a hydrodynamic radius. The hydrodynamic radius of the EV may be less than 100 nm. It may be between about 30 nm and about 70 nm. The hydrodynamic radius may be between about 40 nm and about 60 nm, such as between about 45 nm and about 55 nm. The hydrodynamic radius may be about 50 nm. The hydrodynamic radius of the EV may be determined by any suitable means, for example, laser diffraction or dynamic light scattering.

[0043] As described above, the extracellular vesicle is derived from a cell. The terms "derived from" and "originated from" are used interchangeably herein and refer to a vesicle produced in, by, or from a specific cell, cell type, or cell population. As used herein, the terms "parent cell," "producer cell," and "original cell" include any cell from which an extracellular vesicle is derived and isolated. The term also encompasses cells that share the protein, lipid, sugar, or nucleic acid components of an extracellular vesicle. For example, a "parent cell" or "producer cell" includes a cell used as a source of an extracellular vesicle. For example, the cell is a eukaryotic cell.

[0044] athlete

[0045] As used herein, the term "athlete" refers to a person who trains and competes in sports and has a certain level of athletic ability and skill. The difference between an "athlete" and an "ordinary person" is that:

[0046] In terms of sports skills and professional level: athletes have undergone long-term, systematic and professional training, and have superb sports skills and a high level of professionalism in specific sports. They are proficient in various technical movements, tactical strategies of the project, and can flexibly apply them in the game to achieve the best competitive performance. For example, professional table tennis players can not only skillfully hit various spin balls and fast attack balls, but also formulate corresponding tactics according to the characteristics of their opponents, and show superb skills on the court. Ordinary people's sports skills are relatively basic and amateur. They have not undergone systematic professional training. They participate in sports mostly for entertainment, fitness or leisure. Their sports level is limited, and there is a big gap between them and athletes in terms of technical standardization, proficiency and tactical application. For example, ordinary people playing table tennis may simply hit the ball back, and their ability to control the rotation, speed and landing point of the ball is weak.

[0047] In terms of training intensity and commitment: In order to improve their competitive level, athletes need to conduct high-intensity and high-volume training. Their training plans are usually carefully formulated by professional coaches based on their physical condition, sports characteristics and competition goals, including daily physical training, technical training, psychological training, etc. The training time is long and regular, and the degree of commitment to training is extremely high. Almost most of their time and energy are devoted to sports training and competition preparation. For example, track and field athletes preparing for competitions may have to conduct high-intensity training for several hours every day, including short-distance sprints, long-distance endurance training, strength training, and various special technical training. Ordinary people have a low training intensity and relatively irregular exercise time. They often choose the appropriate exercise time and method according to their personal work, study and life arrangements. The purpose of their participation in sports is mainly to maintain physical health, relieve stress or increase the fun of life. They will not devote themselves to sports training like athletes. For example, ordinary people may go to the park for jogging or playing badminton on weekends or in their spare time. Each exercise time is generally short, and the exercise intensity is also appropriate for themselves to bear.

[0048] In terms of physical fitness and health status: athletes usually have better physical fitness than ordinary people in terms of strength, speed, endurance, agility, flexibility, etc. due to their long-term high-intensity professional training. After long-term training and adaptation, their physical functions can withstand greater exercise loads and pressures, and have better fatigue resistance and recovery capabilities. Ordinary people have relatively ordinary physical fitness, and various physical function indicators are within the normal range, but compared with athletes, they are obviously weaker in strength, speed, etc.

[0049] RNF216 protein

[0050] As used herein, “RNF216” and “RNF216 protein (polypeptide)” are used interchangeably and refer to RNF216 protein.

[0051] Pharmaceutical composition

[0052] As used herein, the term "pharmaceutical composition" refers to a composition comprising extracellular vesicles formulated together with one or more pharmaceutically acceptable carriers.

[0053] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated using methods known in the art to provide rapid, continuous or delayed release of the active ingredient after administration to a mammal. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonades, perfumed waters, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic agents, capsules, creams, lozenges, tinctures, pastes, pills and soft or hard gelatin capsules.

[0054] Pharmaceutically acceptable carrier

[0055] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch or talc, or liquid carriers, such as water, fatty oils or liquid paraffin. Other examples of the carrier include culture media, such as DMEM or RPMI; and low temperature storage media, comprising components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates and ion concentrations, which can balance the intracellular state at low temperatures; and mixtures of organic solvents and water.

[0056] The pharmaceutical composition is formulated for administration by an administration route selected from intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral and intracerebral administration routes. According to one embodiment, the pharmaceutical composition is formulated for intranasal administration. According to some embodiments, this pharmaceutical composition is in the form of a liquid solution, nasal drops, spray, or metered spray. According to other embodiments, the pharmaceutical composition is formulated into a preparation for injection, such as intralesional, intrathecal or intravenous injection. According to this embodiment, the pharmaceutical composition is in the form of a sterile injection solution.

[0057] Any known method can be used to administer the pharmaceutical composition of the present invention. One of the various methods known to those skilled in the art can be used to implement the term "giving" or "administering" a substance, compound or medicament to a subject. For example, a compound or medicament can be administered intranasally (e.g., by inhalation), intrathecal (entering the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., by a skin catheter). Compounds or agents can also be appropriately introduced by rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or preparations), which provide extended, slowed, or controlled release for compounds and agents. It can also be administered, for example, once, multiple times, and / or over one or more extended periods.

[0058] Therapeutically effective amount

[0059] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level includes the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors well known in the medical field.

[0060] Spinal cord injury

[0061] As used herein, the terms "spinal cord injury" and "SCI" ​​are used interchangeably herein and refer to damage to the spinal cord. According to one embodiment, the injury is the result of trauma. According to another embodiment, the injury or damage is the result of degeneration or disease. Depending on the location of the spinal cord and nerve roots damaged, symptoms may vary widely, for example, from pain to paralysis to incontinence. Spinal cord injuries are described as "incomplete" to varying degrees, from no effect on the patient to "complete" injuries that mean complete loss of function. Spinal cord injuries have many causes, but are generally associated with major trauma caused by motor vehicle accidents, falls, sports injuries, and violence. Therefore, according to one embodiment, SCI is selected from complete and incomplete SCI. According to some embodiments, the spinal cord injury is selected from acute or chronic SCI. The spinal cord injury may be susceptible to secondary tissue damage, including but not limited to: glial scar formation, myelin inhibition, demyelination, cell death, lack of neurotrophic support, local ischemia, free radical formation, and excitotoxicity.

[0062] Such spinal cord diseases include, but are not limited to, autoimmune diseases (eg, multiple sclerosis), inflammatory diseases (eg, arachnoiditis), neurodegenerative diseases, poliomyelitis, spina bifida, and spinal cord tumors.

[0063] Subjects that can be treated according to the teachings of the present invention include mammalian subjects, such as humans, mice, rats, monkeys, dogs, and cats.

[0064] treat

[0065] The term "treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially ameliorating or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition, or disorder, and avoiding harmful or annoying symptoms. Treatment also refers to accomplishing one or more of the following: (a) lessening the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and / or (e) limiting the recurrence of symptoms in patients who were previously free of symptoms of the condition.

[0066] Materials and methods

[0067] Spinal cord neuron cell culture

[0068] The mice were killed by cervical dislocation and the spinal cord was isolated under sterile conditions using a stereomicroscope. After removing the dorsal root ganglia, the spinal cord was cut into pieces with ophthalmic scissors with a volume of about 1 mm. 3 Small pieces. Add 0.25% trypsin (Thermo Fisher Scientific) and digest for 20 minutes in a cell culture incubator at 37°C and 5% CO2, then add 2 ml of horse serum to stop digestion. Use a 70 μm cell filter to filter tissue fragments to obtain a single cell suspension, centrifuge at 300g for 5 minutes, remove the supernatant, add an appropriate amount of seed plate solution containing 10% horse serum, mix well by blowing, count and plate, incubate for 4 hours, and replace with neuronal culture medium after the cells are fully attached to the wall. Change the medium by 1 / 2 every 2 days.

[0069] Extraction and identification of plasma EVs

[0070] Corresponding peripheral blood samples were obtained from healthy people aged 20-35 years and healthy athletes aged 20-35 years. Blood collection was approved by the hospital ethics committee, and each participant provided informed consent. After whole blood was collected, plasma was separated by centrifugation at 2000g for 10 min at 4°C, followed by centrifugation at 3 000g and 10 000g for 20 min to remove cell debris and macromolecules in the plasma. Plasma was centrifuged at 140,000g for 70 min at 4°C using an ultracentrifuge. After removing the supernatant, the pellet was resuspended in PBS and then centrifuged again at 140,000g for 70 min to obtain EVs.

[0071] EVs isolated from normal human plasma are labeled "GEVs", and EVs isolated from athlete plasma are labeled "AEVs"

[0072] The morphology of the obtained EVs was observed using transmission electron microscopy (TEM).

[0073] Nanoparticle tracking analysis (NTA) was used to measure the size and distribution characteristics of the obtained EVs.

[0074] The EV protein concentration was determined using a BCA kit, and Western blot was used to detect EV protein markers: CD9, CD63, CD81, ALIX, etc.

[0075] Plasmid and cell transfection

[0076] Constructs expressing Flag-tagged RNF216, Flag-tagged RNF216C688A, and Myc-tagged NOX1 were generated by cloning their N-terminal Flag or Myc sequences (Beijing Baijing Biotechnology Co., Ltd.). Plasmids encoding HA-tagged ubiquitin K63 and ubiquitin K48 were from Beijing Baijing Biotechnology Co., Ltd. RNF216 knockout and overexpression (shRNF216 and Ad-RNF216) adenovirus constructs were constructed by Hanheng Biotechnology (Shanghai) Co., Ltd. Random adenovirus constructs (Ad-Vec) were used as negative controls. Transfection was performed using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's instructions. To achieve overexpression of NOX1, adeno-associated virus (AAV)-Syn-MCS-NOX1-3xFlag (AAV-NOX1) and adeno-associated virus AAV-Syn-MCS-3xFlag (AAV-Vec) as a control were constructed by Hanheng Biotechnology (Shanghai) Co., Ltd. Immediately after SCI, the mice were fixed in a stereotaxic apparatus, and a total of 1 × 10 9 vg viral vector.

[0077] Western blot

[0078] For cell proteins, the culture dish was placed on ice, and the cells were washed with PBS and then an appropriate amount of lysis buffer was added. For tissue proteins, 1 mL of lysis buffer was added for every 100 mg of tissue, and the mixture was homogenized using a homogenizer. The samples were lysed on ice for 30 min and then transferred to a centrifuge tube and centrifuged at 4°C 12000g for 15 min. The supernatant after centrifugation was transferred to a centrifuge tube and the precipitate was discarded. The protein concentration was determined by the BCA method. 25 μg of protein per sample was loaded onto 10% SDS-PAGE (Bio-Rad), and then electrotransferred to a polyvinylidene fluoride membrane with a pore size of 0.2 μm (Millipore). After blocking with 5% skim milk powder for 2 h, the membrane was incubated with the primary antibody (Table S1) and the secondary antibody (Jackson ImmunoResearch, 1:10000) in sequence, and the exposure solution was evenly applied to the target protein position. The results were observed using an imaging analysis system, and the grayscale value was quantified using Image J.

[0079] Table S1. Primary antibodies used for immunoblotting.

[0080]

[0081]

[0082] Spinal cord injury model

[0083] Healthy 6-8 week old C57BL / 6J mice were purchased from the University Animal Center. After isoflurane-induced anesthesia, sodium pentobarbital was injected intraperitoneally for anesthesia, the mice were fixed in a prone position, the T10 spinous process was located, the skin was prepared, the back surgical area was disinfected with 75% alcohol, and a sheet was laid. The epidermis was incised and the muscles were separated to clarify the T10 spinous process, the T10 spinous process and lamina were bitten off, and the spinal dura mater sac was exposed. A 5 g rod (1 mm in diameter, Reward, Shenzhen, China) was used to drop from a height of 6 cm to cause spinal cord impact contusion. In the Sham group, only the spinal cord was exposed, and no impact was performed. After surgery, the mice received artificial bladder drainage every day until signs of normal bladder function were restored.

[0084] Functional Behavior Analysis

[0085] BMS score: The movement and hindlimb reflex of mice after SCI were scored. The mice were placed in the room for a period of time. After they adapted to the surrounding environment, the motor function of the mice was recorded and scored by three trained experimenters according to the severity of the motor dysfunction of the mice (9 points: normal motor function; 0 points: completely limited motor function). The assessment was performed at different time points after SCI.

[0086] Footprint analysis: Footprint analysis was performed on mice 28 days after SCI. The front and hind paws of the mice were painted with different colored inks, and then the mice were placed in a runway with absorbent paper on the bottom and encouraged to walk. Footprint images were collected and motor function recovery was evaluated.

[0087] Rotarod test: The motor coordination ability of mice was tested using a rotarod fatigue instrument 28 days after SCI. The mice were placed on a rotating rod with adjustable speed (0-40 rpm), and the speed and duration of the mice when they fell were recorded. The mice were trained once, followed by two tests, and the results were averaged.

[0088] Von Frey Filament Test: The tactile sensation of mouse skin is assessed using the Von Frey mechanical prick device. The thickness of the nylon filament and its degree of stretch determine the intensity of the stimulus provided. The mouse is placed on a metal grid to touch the plantar surface of the paw. The force of the needle prick is adjusted from light to strong. Hindlimb withdrawal is considered a positive response and the threshold is determined accordingly.

[0089] Statistical analysis

[0090] GraphPad Prism (8.0.2) software was used for statistical analysis and graphical data interpretation. All data are presented as mean ± standard deviation (SD). Data were analyzed using Student's t test or one-way analysis of variance (ANOVA) for two groups, followed by Tu key post hoc test for three or more groups. In all analyses, P < 0.05 was considered statistically significant.

[0091] Example 1

[0092] Identification and uptake of AEVs:

[0093] Plasma collected from volunteers was cleared of cell debris and EVs were obtained by ultracentrifugation. Ultracentrifugated EVs were characterized according to the MISEV guidelines. TEM showed typical round nanoparticles with diameters ranging from 50 to 150 nm ( Figure 1 A), NTA showed similar size distribution in both groups ( Figure 1 B). No significant differences were observed between the two groups of EVs in terms of size, concentration, and zeta potential ( Figure 1 CF). Western blot analysis showed that CD9, CD63, CD81, ALIX and TSG101 were enriched in EVs and AEVs particles ( Figure 1 G).

[0094] Example 2

[0095] AEVs promote motor function recovery after SCI:

[0096] Different treatments were used for spinal cord injury model mice. 1×1010 AEVs, EVs or an equal volume of PBS buffer were injected into the tail vein of the mice, and functional behavior was evaluated at designated time points ( Figure 2A). During the 4-week recovery period after SCI, mice treated with AEVs showed significantly better functional recovery in walking, hindpaw placement, and hindlimb movement compared with mice treated with EVs, as quantified by BMS scores ( Figure 2 B). The rotarod test also showed that the AEVs group had better functional recovery of hind limb and tail balance ( Figure 2 C), which was further demonstrated in the von Frey filament experiment ( Figure 2 D). Footprint analysis results also showed that mice treated with AEVs showed significantly faster gait recovery and improved motor coordination compared with the EVs group ( Figure 2 EF). We then evaluated tissue damage in the thoracic SCI mouse model. Figure 2 G shows that the gross morphology of the injured spinal cord clearly shows the injured part of the spinal cord. The lesion area after AEVs treatment was significantly smaller than that in the PBS and EVs groups ( Figure 2 H).

[0097] Example 3

[0098] AEVs treatment alleviates ferroptosis caused by oxidative stress after SCI:

[0099] Previous studies have shown that plasma-derived extracellular vesicles released after endurance exercise have antioxidant effects. Next, we investigated whether AEVs could antagonize ferroptosis caused by oxidative stress in the injured spinal cord. Western blot results showed that compared with the normal spinal cord, the expression of positive regulatory markers of ferroptosis (ACSL4, 4HNE, and PTGS2) was increased and the level of negative regulatory marker (GPX4) was decreased after SCI. Compared with the PBS group, the administration of EVs and AEVs could significantly reduce the expression of positive regulatory markers of ferroptosis (ACSL4, 4HNE, and PTG S2) and increase the level of negative regulatory marker (GPX4) ( Figure 3 AB). However, the therapeutic effect of AEVs was more significant compared with EVs alone ( Figure 3 AB).

[0100] Example 4

[0101] AEVs inhibit oxidative stress-induced ferroptosis in neuronal cells in vitro:

[0102] To further determine the role of AEVs in inhibiting ferroptosis, we treated neuronal cells with Erastin (10uM, 6h) and then co-cultured with EVs or AEVs. Western blot analysis confirmed that EVs or AEVs treatment could reverse the increase of ACSL4, 4HNE and PT GS2 and the decrease of GPX4 in ferroptosis ( Figure 4 AB).

[0103] Example 5

[0104] AEVs promote motor function recovery and inhibit ferroptosis after SCI by transferring RNF216 in vivo

[0105] The results of in vivo and in vitro experiments showed that AEVs inhibited ferroptosis caused by oxidative stress and promoted motor function recovery after SCI to a greater extent than EVs alone. Previous studies have shown that endurance exercise can modulate the content of circulating EVs, leading to an increase in antioxidant proteins with damage-protective functions. Therefore, we hypothesized that the protein content of AEVs may be different from that of EVs. Based on proteomic analysis of EVs and AEVs, we found that RNF216 was the most significantly upregulated protein in AEVs compared with EVs. To confirm the RNF216 protein profile data, we measured the expression level of RNF216 in vitro and found that the protein expression level of RNF216 was significantly increased in AEVs and in neuronal cells ( Figure 5 AB).

[0106] To further investigate the role of plasma extracellular vesicles containing RNF216, we selected extracellular vesicles (NEVs) secreted by neural stem cells with neuroprotective effects in vitro to load RNF216, and then further investigated its effects on functional recovery and ferroptosis. We first extracted neural stem cells and then used a lentiviral-based approach to overexpress RNF216 in neural stem cells ( Figure 5 C). NEVs were then isolated and co-cultured with neuronal cells. The expression of RNF216 was significantly increased in Ad-RNF216-NEVs compared with Ad-Vec-NEVs ( Figure 5 D). Compared with neurons treated with Ad-Vec-NEVs, the expression level of RNF216 protein in neurons treated with Ad-RNF216-NEVs was significantly increased ( Figure 5 E) These results suggest that RNF216 is upregulated in EVs and translocated to neuronal cells.

[0107] To further investigate the role of RNF216 in AEVs-mediated ferroptosis suppression and functional recovery in vivo, we administered Ad-Vec-NEVs and Ad-RN F216-NEVs to mice after SCI. Treatment with Ad-RNF216-NEVs resulted in better functional recovery than treatment with Ad-Vec-NEVs, as quantified by BMS scores, rotarod test, von Frey filament test, and footprint analysis ( Figure 5 FK).

[0108] Further investigation of the functional role of RNF216 in regulating ferroptosis in neuronal cells by Western blot analysis showed that Ad-RNF216-NEVs treatment further reduced the expression of positive ferroptosis regulatory markers (ACSL4, 4HNE, and PTGS2) and increased the level of negative ferroptosis regulatory marker (GPX4) ( Figure 5 NO). Collectively, these data suggest that the ability of AEVs to promote functional recovery and ferroptosis inhibition depends in part on RNF216.

[0109] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an extracellular vesicle in the preparation of a medicament for treating neuronal injury or damage and / or diseases and / or symptoms associated with neuronal injury or damage; the extracellular vesicle is an athlete-derived extracellular vesicle.

2. The use according to claim 1, characterized in that The extracellular vesicles overexpress RNF216 protein.

3. The use according to claim 2, characterized in that: The extracellular vesicles are selected from exosomes, microvesicles, apoptotic bodies, oncosomes, multivesicular bodies, migratoria, exosomes or a combination thereof.

4. The use according to claim 3, characterized in that The extracellular vesicles are derived from blood, urine, cerebrospinal fluid, extracellular spaces of various tissues, saliva, milk, semen, ascites, or a combination thereof.

5. The use according to claim 4, characterized in that The extracellular vesicles are derived from blood; preferably, the extracellular vesicles are derived from plasma.

6. The use according to claim 1, characterized in that: The neuronal injury or damage and / or the disease and / or symptom associated with neuronal injury or damage is spinal cord injury.

7. Use of a pharmaceutical composition for treating neuronal injury or damage and / or diseases and / or symptoms associated with neuronal injury or damage, characterized in that: The pharmaceutical composition comprises: (1) a therapeutically effective amount of the extracellular vesicles according to any one of claims 1 to 5; (2) A pharmaceutically or immunologically acceptable carrier or excipient.

8. The use according to claim 7, characterized in that The pharmaceutical composition is formulated for administration via an administration route selected from the group consisting of intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral and intracerebral administration routes and combinations thereof.

9. The use according to claim 8, characterized in that The pharmaceutical composition is formulated for administration via the intravenous route of administration.

10. Use of a pharmaceutical preparation for treating neuronal damage or injury and / or diseases and / or symptoms associated with neuronal damage or injury, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 7 to 9.

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