Application of mitochondrial transplantation based on skeletal muscle transporter in repairing damaged neurons of cerebral apoplexy

Abstract

The invention discloses application of mitochondrial transplantation based on skeletal muscle migrators in repairing damaged neurons in cerebral apoplexy. Researches prove that drug stimulation, electrical stimulation and movement can induce skeletal muscle to generate the migrators, and the migrators are identified to not only contain mitochondria with a complete structure, but also contain a plurality of mitochondria function supporting substances. Therefore, an important basis is provided for applying the skeletal muscle transporter to mitochondrial transplantation treatment. It is further found that the curative effect of repairing the cerebral apoplexy damaged neurons through mitochondrial transplantation based on the skeletal muscle transporter is remarkably superior to that of pure mitochondrial transplantation treatment. Besides, the skeletal muscle transporter can be administered through intravenous infusion and has the targeting property to neurons on the stroke focus side, so that the clinical transformation possibility and transplantation efficiency of mitochondrial transplantation treatment are greatly improved. Therefore, the key bottlenecks of lack of donor sources, insufficient targeting, low transplantation efficiency and the like in the current mitochondrial therapy are successfully overcome, and a technical foundation is laid for clinical transformation of the mitochondrial therapy.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, and particularly relates to application of mitochondrial transplantation based on skeletal muscle migration bodies in repairing damaged neurons in stroke. BACKGROUND

[0002] Acute Ischemic Stroke (AIS) is one of the main causes of death and long-term disability worldwide.

[0003] Mitochondrial damage after ischemia and reperfusion of neurons is one of the core pathological mechanisms of stroke, and mitochondrial transplantation is considered to be a direct and effective way to suppress mitochondrial damage of neurons. According to existing research, mitochondrial transplantation can play a protective role through multiple mechanisms such as restoring energy metabolism balance of neurons, relieving intracellular oxidative stress, preventing calcium overload, and inhibiting apoptosis, and is expected to become a new strategy for the treatment of nervous system diseases. In recent years, significant progress has been made in the research of mitochondrial transplantation targeting neurons. In the field of stem cells, it has been found that mesenchymal stem cells (MSCs) can deliver mitochondria and metabolic enzymes to damaged neurons through direct cell-to-cell contact and exosome transmission, and enhance the energy metabolism and antioxidant capacity of neurons. In the field of nanotechnology, researchers have developed various nanocarriers to improve the stability and targeting of mitochondria. For example, liposomes and nanoparticles are used to load mitochondria or mitochondria-related substances, and the action time of drugs in the brain is prolonged by reducing the particle size of liposomes or modifying polyethylene glycol (PEG) on the surface. However, although mitochondrial transplantation technology has made important breakthroughs in basic research, it still faces many challenges in clinical translation and cost control, and needs to be further optimized. At present, the main problems of mitochondrial transplantation therapy for damaged neurons are as follows: (1) the most suitable source of mitochondria donor tissue / cells is unknown. (2) The efficiency of mitochondrial transplantation is low. Mitochondria themselves as organelles cannot target damaged neurons, and for nervous system diseases, in situ transplantation has limitations in clinical application, and the efficiency of transvascular transplantation is significantly lower than that of in situ transplantation. How to improve the targeting of donor cell mitochondria to damaged neurons needs to be solved. (3) The source of renewable and sustainable mitochondria needs to be explored. Future research needs to focus on improving the safety, efficiency and accessibility of mitochondrial transplantation to promote its wide application in the treatment of stroke. SUMMARY

[0004] To address the above issues, this invention focuses on identifying the most suitable tissue / cell mitochondrial source for mitochondrial transplantation therapy targeting damaged neurons; finding suitable mitochondrial carriers to improve transplantation efficiency; and inventing engineered mitochondrial formulations with neuroprotective effects, providing an effective therapy for mitochondrial transplantation targeting neurons. These methods improve the safety, efficiency, and accessibility of mitochondrial transplantation therapy for damaged neurons, thereby promoting its widespread application in stroke treatment.

[0005] The first objective of this invention is to provide the use of skeletal muscle migration bodies in the preparation of medicaments for treating ischemic stroke.

[0006] Preferably, the ischemic stroke is an acute ischemic stroke.

[0007] Preferably, the skeletal muscle migration body is capable of repairing neurons damaged in stroke.

[0008] Preferably, the skeletal muscle migratory body contains mitochondria.

[0009] Preferably, the skeletal muscle migration body further comprises metabolites and proteins that support mitochondrial function and protect mitochondria.

[0010] Preferably, the skeletal muscle migration bodies can be generated through drug stimulation, electrical stimulation, or exercise induction.

[0011] Preferably, the drug stimulation includes acetylcholine stimulation.

[0012] Preferably, the method of administration of the drug includes injection.

[0013] A second objective of this invention is to provide a medicament for treating ischemic stroke, wherein the medicament comprises skeletal muscle migratory bodies carrying mitochondria as its active ingredient.

[0014] Preferably, the dosage form of the drug includes an injection.

[0015] This invention confirms the existence of skeletal muscle migration bodies and identifies their structure and composition, providing a basis for their application in mitochondrial transplantation therapy. Previously, it was believed that migration bodies were generated during cell migration (absolute displacement), but this invention demonstrates that drug stimulation, electrical stimulation, and exercise can all induce skeletal muscle contraction (relative displacement) and also generate migration bodies. Furthermore, it was identified that skeletal muscle migration bodies contain structurally intact mitochondria and are rich in various mitochondrial functional support substances.

[0016] This invention innovatively proposes mitochondrial transplantation based on skeletal muscle transfer organisms to repair neurons damaged in stroke, with efficacy superior to mitochondrial transplantation alone. It partially addresses existing problems associated with mitochondrial transplantation for neurological diseases: mitochondrial transplantation alone typically requires orthotopic transplantation, limiting its clinical application. Skeletal muscle transfer organisms, administered intravenously and targeted to neurons on the stroke lesion side, significantly improve the clinical translational potential and transplantation efficiency of mitochondrial transplantation.

[0017] Therefore, this invention develops mitochondrial transplantation based on skeletal muscle migration bodies to repair neurons damaged in stroke, which has the following three main advantages compared with previous mitochondrial transplantation therapy research:

[0018] (1) The skeletal muscle-derived mitochondria carried by the skeletal muscle migration body have high biocompatibility and safety for the brain:

[0019] Previous studies have shown that mitochondrial transplantation from various tissues, including stem cells, liver, and skeletal muscle, for neurological diseases has some efficacy. However, no research has yet explored the most suitable mitochondrial source tissue / cell for brain transplantation. Our team, through bioinformatics comparisons of mitochondrial similarity among various mouse tissues, found that skeletal muscle tissue has the highest similarity to brain mitochondria. This provides a basis for selecting skeletal muscle-derived mitochondria for mitochondrial transplantation in neurological diseases. Figure 5 ).

[0020] (2) Mitochondria themselves do not target recipient cells, but skeletal muscle migration bodies carrying mitochondria can target neurons in stroke lesions after intravenous infusion. Mitochondrial transplantation is more efficient and has greater potential for clinical translation.

[0021] For neurological diseases, mitochondria themselves do not target recipient cells, and previous studies have typically involved orthotopic transplantation, limiting their clinical application. This patent, through bioinformatics analysis, discovered that CEACAM1 expression is upregulated on the cell membranes of neurons on the lesion side after stroke, and that various membrane proteins on skeletal muscle transfer organisms can interact with the neuronal receptor CEACAM1. Further experiments verified that intravenously infused skeletal muscle transfer organisms can bind to the CEACAM1 receptor on the cell membranes of neurons on the stroke lesion side and enter the neurons, simultaneously releasing mitochondria into the neurons. The fact that skeletal muscle transfer organisms can be administered intravenously and that they target neurons on the stroke lesion side after infusion makes them more clinically viable and increases the efficiency of mitochondrial transplantation. Figure 7 ).

[0022] (3) Skeletal muscle transplants not only carry mitochondria but are also rich in various metabolites and proteins that support and protect mitochondrial function, thus improving the efficacy of mitochondrial transplantation.

[0023] This invention utilizes DIA proteomics and non-targeted metabolomics to analyze skeletal muscle migration bodies extracted from resting and exercising mice. Bioinformatics analysis revealed that, in addition to containing mitochondria, skeletal muscle migration bodies are rich in various metabolites and proteins that support and protect mitochondrial function. In in vitro experiments, compared to simple skeletal muscle-derived mitochondrial treatment, treatment with skeletal muscle-derived migration bodies better protected primary neurons damaged by oxidative stress. Figure 8 In animal studies, skeletal muscle-derived mitosome therapy showed better recovery of post-stroke cognitive impairment compared to skeletal muscle-derived mitochondrial therapy alone. Figure 9 ). Attached Figure Description

[0024] Figure 1 : Skeletal muscle contraction produces migration bodies. (A) In vitro cultured muscle tubes stained with the migration body-specific dye wheat germ agglutinin (WGA) were treated with acetylcholine (0.1 mM). Confocal video recordings were used to document muscle tube contraction and migration body production within 4 minutes after the addition of acetylcholine; confocal video recordings were used to document muscle tube relaxation and migration body production within 4 minutes after the removal of acetylcholine. (B) Wild-type mouse tibialis anterior muscle stained with WGA was stimulated with electrodes. Confocal video recordings were used to document tibialis anterior muscle contraction and migration body production within 4 minutes after electrical stimulation. (C) Immunofluorescence was used to detect the expression of the migration body marker TSPAN9 (red) in slow-twitch muscle fibers (MYH7, green) and fast-twitch muscle fibers (MYH1 / 2+MYH4, green) of exercised mice.

[0025] Figure 2 To identify the purity of migration bodies extracted from peripheral blood of healthy young adults before and after exercise. Western blot was used to detect the expression of migration body markers (TSPAN7, TSPAN9) and exosome markers (CD63, HSP70).

[0026] Figure 3The number of extracellular vesicles in peripheral blood significantly increased after aerobic exercise. (AB) Particle size determination and migration proportion statistics of all extracellular vesicles in peripheral blood of resting mice and exercising mice (running at 8 m / min for 1 hour on a small animal treadmill) (A), and nano-flow cytometry counting of migration bodies isolated from mouse plasma (B); N = 3-7, ***P < 0.001, Student's first test. (CD) Particle size determination and migration proportion statistics of all extracellular vesicles in peripheral blood of healthy young adults before and after exercise (running for 1 hour at the age-corresponding optimal aerobic heart rate) (C), and nano-flow cytometry counting of migration bodies isolated from plasma of healthy subjects (D); N = 7, **P < 0.01, Paired t test.

[0027] Figure 4 : Skeletal muscle migratory bodies carrying mitochondria. (A) Transmission electron microscopy of the gastrocnemius muscle of a motorized rhesus monkey. (B) Immunofluorescence detection of the gastrocnemius muscle migratory body (TSPAN9, red) and mitochondria (TOM20, green) of a motorized rhesus monkey. The white arrow points to the migratory body structure containing mitochondria. (C) Transmission electron microscopy of the gastrocnemius muscle of a motorized mouse. (D) Immunofluorescence detection of the gastrocnemius muscle migratory body (TSPAN9, red) and mitochondria (TOM20, green) of a motorized mouse. The white arrow points to the migratory body structure containing mitochondria.

[0028] Figure 5 : The mitochondria of skeletal muscle and the brain are highly similar. (AB) Mitochondrial genome expression in various mouse tissues and similarity scores between the mitochondrial genome expression in various tissues and the brain. (C) Expression of all mitochondrial-related genes (including nuclear genome and mitochondrial genome) in various mouse tissues and the GO (Gene Ontology) pathway with the highest expression similarity between different tissues.

[0029] Figure 6 The skeletal muscle migration bodies produced by skeletal muscle contraction after exercise are rich in mitochondrial functional support substances. (A) Bioinformatics analysis of the upregulated and downregulated proteins in the skeletal muscle migration bodies of exercised mice compared to those of resting mice. (B) Bioinformatics analysis of the upregulated and downregulated metabolites in the skeletal muscle migration bodies of exercised mice compared to those of resting mice.

[0030] Figure 7Neurons may recognize and take up skeletal muscle migration bodies and their mitochondria via CEACAM1. (A) Bioinformatics techniques were used to analyze the receptor-ligand composition of upregulated receptors in neurons of tMCAO mouse models (vs. sham-operated mice) and membrane proteins contained in skeletal muscle migration bodies (SKM-Ex-M) from exercise mice. (B) Skeletal muscle migration bodies (SKM-Ex-M) from exercise mice were isolated and labeled with the dye WGA (green). After intravenous infusion of SKM-Ex-M into tMCAO model mice (10 mg / kg), the expression of CEACAM1 molecules (red) on neurons (TUJ1, white) and their binding to skeletal muscle migration bodies (WGA, green) were detected by immunofluorescence. (C) Skeletal muscle migration bodies (SKM-Ex-M) from exercise mice were isolated and labeled with WGA staining, and mitochondria were labeled with Mitotracker staining. SKM-Ex-M was intravenously infused into tMCAO model mice 3 days after surgery (10 mg / kg). The presence of skeletal muscle migration bodies (WGA) in neurons on the lesion side (TUJ1, white) was detected by immunofluorescence. + Mitotracker + ,yellow).

[0031] Figure 8 Skeletal muscle migration bodies can alleviate oxidative stress damage in primary neurons. Primary neurons from mice subjected to oxygen deprivation (OGD) were treated with either skeletal muscle mitochondria (SKM-mito) or skeletal muscle migration bodies (SKM-M). (AB) Neuronal apoptosis was detected by immunofluorescence. + TUNEL + A), Neuronal DNA damage (NeuN) + 8OHGD + (B) (CD) Mitochondrial autophagy (TOM20) in neurons was detected by immunofluorescence and flow cytometry. + LC3 + (C) Mitochondrial membrane potential (D) Mitochondrial ROS (Reactive Oxygen Species) level (E). (F) Neuronal ATP production. N=3, * P<0.05, ** P<0.01, *** P<0.001, # P<0.05, ## P<0.01, ### P<0.001, one-way ANOVA.

[0032] Figure 9Skeletal muscle transfer molecules improve post-stroke cognitive impairment. Sham-operated mice and tMCAO mice were randomly divided into three groups: a resting group, a skeletal muscle mitochondrial transfer molecule treatment group (SKM-Mito, 10 mg / kg, intravenously administered for 3 consecutive days post-surgery, once daily thereafter, once weekly), and a skeletal muscle transfer molecule treatment group (SKM-Ex-M, 10 mg / kg, intravenously administered for 3 consecutive days post-surgery, once daily thereafter, once weekly). Cognitive function of mice in each group was assessed using a water maze test 31-35 days post-surgery; N=5-10. ** P<0.01, *** P<0.001; ## P<0.01, two-way ANOVA. Detailed Implementation

[0033] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0035] 1. Instruments and equipment

[0036] Small Animal Flat Treadmill (8 Channels): Calvin, Item No. KW-PT-8.

[0037] Nanoparticle size and zeta potential analyzer: Anton Paar, model Litesizer 500.

[0038] Analytical flow cytometer: Agilent, model NoVoCyte Advanteon B4R2

[0039] tMCAO molding line bolt: CINONTECH, part number A5-122050.

[0040] 2. Antibodies

[0041] Table 1 Antibodies

[0042]

[0043] 3. Main experimental reagents

[0044] Table 2 Main Experimental Reagents

[0045]

[0046] Example 1: Drug stimulation, electrical stimulation, and exercise can all induce skeletal muscle contraction and generate migration bodies.

[0047] (1) Acetylcholine stimulation induces myotube contraction and migration body production: In vitro cultured myotubes stained with the migration body-specific dye wheat germ agglutinin (WGA) were treated with acetylcholine (0.1 mM). Confocal video recordings were used to document myotube contraction and migration body production within 4 minutes after the addition of acetylcholine. After the acetylcholine was removed, confocal video recordings were used to document myotube relaxation and migration body production within 4 minutes after the removal of acetylcholine. Figure 1 (A) The results showed that after the addition of acetylcholine, the myotubes began to contract, and the production of migratory bodies also increased significantly. After the removal of acetylcholine, the myotubes gradually relaxed, and the production of migratory bodies decreased accordingly. This indicates that acetylcholine can induce myotube contraction and the production of migratory bodies.

[0048] (2) Electrical stimulation-induced contraction of the tibialis anterior muscle to generate migration bodies: The tibialis anterior muscle of wild-type mice stained with WGA was stimulated with electrodes, and the contraction of the tibialis anterior muscle and the generation of migration bodies within 4 minutes after electrical stimulation were recorded by confocal video recording. Figure 1 (B in the original text). The results showed that after electrical stimulation, the tibialis anterior muscle began to contract, and the production of migration bodies also increased significantly. This indicates that electrical stimulation can induce muscle contraction and the production of migration bodies.

[0049] (3) Compared with resting mice, the number of migration bodies in the gastrocnemius muscle tissue of exercising mice was increased: After 8-week-old healthy wild-type C57 mice ran at a speed of 8 m / min for 1 hour on a small animal treadmill, the gastrocnemius muscle was harvested and frozen sections were prepared. The expression of the migration body marker TSPAN9 (red) in slow-twitch muscle fibers (MYH7, green) and fast-twitch muscle fibers (MYH1 / 2+MYH4, green) of the gastrocnemius muscle of resting and exercising mice was detected by immunofluorescence. Figure 1 (C) The results showed that TSPAN9 expression was low in both fast-twitch and slow-twitch muscle fibers at rest. However, TSPAN9 expression increased significantly in both fast-twitch and slow-twitch muscle fibers during exercise. This indicates that exercise can induce the generation of transference molecules, whether in fast-twitch or slow-twitch muscle fibers.

[0050] The above results indicate that drug (acetylcholine) stimulation, electrical stimulation, and exercise can all induce skeletal muscle contraction to produce migration bodies ( Figure 1 ).

[0051] Example 2: Extraction of all extracellular vesicles and kinematosomes from plasma and skeletal muscle tissue

[0052] 10 mL of peripheral blood was drawn from healthy young adults before and after a 1-hour run (maintaining optimal aerobic heart rate = 60%-70% × maximum heart rate MHR, MHR = 220 - age). The plasma was collected after centrifugation at 1000g for 10 min, followed by centrifugation at 4000g for 20 min to remove cell debris. Half of the supernatant was centrifuged at 100000g for 70 min, the precipitate was collected, and the cells were washed twice with PBS to obtain all extracellular vesicles for subsequent nanoparticle size and zeta potential analysis. The other half of the supernatant was centrifuged at 20000g for 40 min, the precipitate was collected, and the cells were washed twice with PBS to obtain migratory bodies for subsequent nanoparticle size and zeta potential analysis, nanoflow cytometry, Western blot detection of migratory body protein markers, transmission electron microscopy, non-targeted metabolomics and DIA proteomics detection, and tMCAO mouse infusion therapy. The purity identification results of the isolated migratory bodies are shown in [link to relevant documentation]. Figure 2 . Figure 2 Western blot analysis revealed high expression of migration markers (TSPAN7 and TSPAN9) in peripheral blood samples extracted before and after exercise, while exosome markers (CD63 and HSP70) were almost undetectable. This indicates high purity of the extracted migrations and a trend of increased migration expression after exercise. Furthermore, Ponceau S staining showed an increase in total protein content after exercise, further supporting this conclusion.

[0053] Eight-week-old healthy wild-type C57 mice were sacrificed after running on a small animal treadmill at a speed of 8 m / min for 1 hour. Plasma was collected and perfused into the skeletal muscles of the lower limbs (gastrocnemius, tibialis anterior, and quadriceps femoris). The procedure for extracting all extracellular vesicles and kinesiology from the plasma was the same as that for the healthy young adults described above. Mouse hind limb skeletal muscle (gastrocnemius, tibialis anterior, quadriceps femoris) was digested with digestive enzymes (containing 10 mg / mL collagenase II, 10000 PU / mL dispase II, 100 mM HEPES, and 2.5 mM CaCl2) at 37°C for 1 hour. After neutralization with an equal volume of PBS containing 2 mM EDTA, the mixture was filtered through a 0.4 μm filter and centrifuged at 4000 g for 20 min to remove cell debris. The supernatant was then centrifuged at 20000 g for 40 min. The precipitate was collected and washed twice with PBS to obtain the migratory bodies, which were used for subsequent nanoparticle size and zeta potential analysis, nanoflow cytometry counting, Western blot detection of migratory body protein markers, transmission electron microscopy, non-targeted metabolomics and DIA proteomics detection, and tMCAO mouse infusion therapy.

[0054] Example 3: Physicochemical properties, structure and composition analysis of skeletal muscle migration bodies

[0055] 1. Nanoparticle size and zeta potential analysis: All extracellular vesicle precipitates extracted from human and mouse plasma were resuspended in 1 mL PBS and their particle composition was analyzed using a nanoparticle size and zeta potential analyzer (Anton Paar, Litesizer 500). The proportion of various extracellular vesicles (exosomes, microvesicles, and migratory bodies) was also classified and statistically analyzed.

[0056] 2. Western blot detection of migration markers: Migration precipitates extracted from human and mouse plasma were lysed using RIPA (Beyotime) and proteins were extracted. Protein concentrations were determined using a BCA protein assay kit (Beyotime). Proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane; the membrane was then blocked with 5% BSA and incubated overnight at 4°C with primary antibodies against TSPAN4, TSPAN9, and Desmin; after washing the membrane with TBST, it was incubated with enzyme-labeled secondary antibody at room temperature for 1 hour, followed by incubation with an enhanced chemiluminescence reagent, and visualized using a chemiluminescence imaging system.

[0057] 3. Nanoscale flow cytometry: Crude migratory precipitates extracted from human and mouse plasma were fixed with fixation and permeabilization reagents at room temperature in the dark for 1 hour, washed twice with 1× flow cytometry permeabilization buffer, and incubated overnight at 4°C with primary antibody anti-TSPAN4. After washing twice with 1× flow cytometry permeabilization buffer, the precipitates were incubated with secondary antibody at room temperature for 30 minutes, washed twice with PBS, resuspended in 200 μL of PBS, and analyzed using a flow cytometer (Agilent, NoVoCyte Advanteon B4R2) to count the number of migratory precipitates per 100 μL of plasma.

[0058] 4. Transmission electron microscopy (TEM) observation of structures: Migratory precipitates extracted from plasma and skeletal muscle, as well as mouse skeletal muscle tissue (1 mm × 1 mm × 1 mm in size), were each added to 1 mL of TEM fixative and fixed for 2 hours at room temperature in the dark. They were then stored at 4°C for later use. Next, the copper mesh with the supporting membrane was brought into contact with the sample solution surface and allowed to stand for 5 minutes. The copper mesh was then removed and blotted dry with filter paper to remove excess liquid. The copper mesh was then stained with 1% uranium acetate for 2 minutes, followed by blotting dry with filter paper and rinsing twice with distilled water. Finally, it was dried before TEM observation.

[0059] 5. Non-targeted metabolomics: Metabolites from the migratory sample are extracted and then analyzed using a high-resolution Q-Exactive (Thermo) mass spectrometer in both positive and negative ion modes, generating a large amount of mass spectrometry data. This analysis first utilizes...

[0060] Peak extraction was performed using XCMS software. XCMS software identifies specific substances based on their primary mass-to-charge ratio (m / z) and retention time, and extracts the primary chromatographic peak area as quantitative information. Metabolite identification is performed by matching the primary m / z of metabolites and the m / z of secondary fragment ions with standards in the database. The quantitative information of metabolites comes from the primary chromatographic peak area. Intensity information for each substance in each sample was extracted using XCMS software. The extracted data was then quality controlled using MetaX software. First, low-quality peaks were removed (ions missing more than 50% in QC samples or more than 80% in actual samples). Then, missing values ​​were filled using the KNN (K-Nearest Neighbors) method, followed by PQN (Probabilistic Quotient Normalization). The corrected data was then filtered, removing ions with a coefficient of variation (CV) > 50% from all QC samples (ions with CV > 50% fluctuated significantly during the experiment and were not included in differential quantitative analysis). The open-source MetaX metabolomics analysis pipeline was used to perform univariate and multivariate analyses to obtain differentially expressed metabolites between groups. The methods included parametric and nonparametric tests, differential expression fold analysis, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) (for samples >3).

[0061] 6. DIA Proteome: Proteins in the migratory body samples were extracted and enzymatically digested. Peptides were enriched and separated, and then detected using high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-MS / MS), generating a large amount of mass spectrometry data. The DIA-NN software was used to identify proteins in the samples, with the following identification conditions: spectral false positive rate (PSM FDR) < 0.01, protein false positive rate (Protein FDR) < 0.01. Data preprocessing was performed on the intensity of all proteins across all samples, including: 1) Missing value handling: removing less than 20% of existing proteins and completing missing values ​​using the KNN method. 2) Data normalization: evaluating data using median, mean, and summation normalization. Pairwise differentially expressed proteins were screened, primarily through univariate analysis. Differentially expressed proteins were screened using a Fold change > 1.5 (default) and a T-test statistical significance (p-value) less than 0.05.

[0062] 7. Results

[0063] (1) The number of migration cells in the peripheral blood of healthy young adults and mice increased after exercise, and the migration cells expressed skeletal muscle-specific markers.

[0064] Extracellular vesicles were extracted from plasma of healthy young adults and mice via gradient centrifugation for particle size analysis. The changes in the proportion of different types of extracellular vesicles (exosomes, microvesicles, and migratory bodies) after exercise were statistically analyzed. The results showed that the number of migratory bodies increased in the peripheral blood of both healthy young adults and mice after exercise. Figure 3 (A and C in the text).

[0065] Crude migratory bodies were further extracted from plasma of healthy young adults and mice via gradient centrifugation. After labeling with the migratory body marker TSPAN4, nanoflow cytometry was used for detection and counting. The results showed that the migratory bodies expressed skeletal muscle-specific markers (TSPAN4). Figure 3 (B and D in the text).

[0066] (2) Transmission electron microscopy detection and identification of mitochondria in skeletal muscle migration bodies

[0067] Transmission electron microscopy and immunofluorescence were used to examine the gastrocnemius muscle of isolated exercise mice (8-week-old healthy wild-type C57 mice running on a small animal treadmill at a speed of 8 m / min for 1 hour) and exercise rhesus monkeys (running at a speed of 4.0 km / h for 1 hour) to identify the structure of skeletal muscle migration bodies. Figure 4 The results showed that exercise promoted the formation of migratory bodies in the gastrocnemius muscles of rhesus monkeys and mice, and that these migratory bodies contained mitochondria.

[0068] (3) Bioinformatics analysis revealed that skeletal muscle mitochondria and brain mitochondria have a high degree of similarity.

[0069] Bioinformatics analysis of data from public databases compared the expression profiles of mitochondrial-related DNA (including mitochondrial and nuclear genomes) from various mouse tissues (brain, heart, skeletal muscle, liver, lung, kidney, spleen, pancreas, bone, bone marrow, skin, small intestine, brown adipose tissue, gonadal adipose tissue, subcutaneous adipose tissue, mesenteric adipose tissue, and leukocytes). The study found that skeletal muscle showed the highest mitochondrial similarity to the brain. This result suggests a high degree of similarity in composition and metabolic state between skeletal muscle and brain mitochondria, thus providing a basis for using skeletal muscle as a potential donor cell for targeted neuronal mitochondrial transplantation. Figure 5 ).

[0070] (4) DIA proteomics and non-targeted metabolomics detection identified that skeletal muscle migration bodies contain a variety of mitochondrial functional support substances.

[0071] Lower limb skeletal muscles (gastrocnemius, tibialis anterior, and quadriceps femoris) were isolated from resting and exercising mice (8-week-old healthy wild-type C57 mice ran for 1 hour on a small animal treadmill at a speed of 8 m / min). Skeletal muscle migration bodies were extracted after digestion with dispersin II and collagenase II and gradient centrifugation (see Example 2 for specific steps), and further DIA proteomics and non-targeted metabolomics were performed. Bioinformatics was used to identify upregulated substances in skeletal muscle migration bodies after exercise (…). Figure 6 The results showed that the skeletal muscle migration bodies (Ex-SKM-M) of exercising mice exhibited significant differences in both proteomics and metabolomics compared to those of resting mice (Se-SKM-M). Ex-SKM-M was rich in proteins and metabolites that support mitochondrial function.

[0072] Example 4: Interaction analysis between skeletal muscle migration bodies and damaged neurons in stroke lesions

[0073] 1. Bioinformatics Analysis of Receptor-Ligand Interactions: On one hand, upregulated receptors in neurons were obtained by analyzing single-cell sequencing data of the brains of tMCAO mice 3 days post-surgery in a public database using bioinformatics analysis. On the other hand, membrane proteins were screened using DIA proteomics data of migratory bodies extracted from skeletal muscle after mouse exercise, and the intersection of membrane proteins from plasma-derived motor migratory bodies and skeletal muscle-derived migratory bodies was collected. The intersection of upregulated receptors in neurons after stroke and membrane proteins from motor migratory bodies was analyzed by retrieving...

[0074] Receptor-ligand interaction analysis was performed using data from the STING database, and the interaction strength was calculated. The analysis revealed that CEACAM1 is upregulated on neurons after stroke, and multiple membrane proteins on motor lobe can bind to and interact with CEACAM1.

[0075] 2. Animal experiments to verify:

[0076] (1) Transient middle cerebral artery occlusion (tMCAO) model: 8-10 week old male C57 mice were used. After weighing, a matching suture was selected. Before the operation, tissues, cotton swabs, surgical instruments, alcohol, PBS, 1-2 cm long 6-0 sutures, microscope, and anesthesia machine were prepared. After adding isoflurane to the anesthesia machine, the main switch was turned on, the oxygen flow rate was adjusted to the 0.5-1 mark, the isoflurane output was turned up to 5, and the machine mode was switched to anesthetize the mice. After anesthesia, the isoflurane was adjusted to 1.5, and the mask mode was switched to maintain anesthesia. After positioning the mice, disinfection and skin preparation were carried out. The skin was cut along the midline of the neck, and the glands and anterior neck muscles were bluntly separated. The common carotid artery with the vagus nerve was found deep in the sternocleidomastoid muscle, and a slipknot was tied at the proximal end. The medial external carotid artery and the lateral internal carotid artery were separated upwards. A dead knot was tied at the tip of the external carotid artery, and a loose knot was tied at the bifurcation. A tight slipknot was tied at the internal carotid artery to prevent bleeding. Trim the suture 2-3 mm beyond the black dot. Make a small incision between the two knots of the external carotid artery and insert the suture. Tie the bifurcation knot to secure the suture. Loosen the slipknot of the internal carotid artery, cut the external carotid artery, and flip the suture. Insert it into the skull towards the internal carotid artery until the black dot of the suture reaches the bifurcation of the internal and external carotid arteries (blood flow is halved). Anesthetize the mouse during suture removal. Quickly pull out the suture and tie the external carotid artery suture tightly. Loosen the slipknot of the common carotid artery to achieve reperfusion. During the operation, a physiotherapy device should be used to maintain the mouse's body temperature at approximately 37°C.

[0077] (2) Skeletal muscle migration bodies (SKM-Ex-M) from exercise mice were isolated and labeled with WGA (green) dye. SKM-Ex-M (10 mg / kg) was injected into mice 3 days after tMCAO via the inner canthus vein, and mouse brain sections were then stained with TUJ1 (labeled neurons) / CEACAM1 co-stained by immunofluorescence staining, and finally detected by confocal microscopy.

[0078] 3. Results:

[0079] For neurological diseases, mitochondria themselves do not target recipient cells, limiting their clinical application in previous studies, which typically involved orthotopic transplantation. This invention, through bioinformatics analysis, discovered that CEACAM1 expression is upregulated on the cell membranes of neurons on the lesion side after stroke, and that various membrane proteins on skeletal muscle transfer organisms can interact with the neuronal receptor CEACAM1. Further animal experiments verified that intravenously infused skeletal muscle transfer organisms can bind to the CEACAM1 receptor on the cell membranes of neurons on the stroke lesion side and enter the neurons, simultaneously releasing mitochondria into the neurons. The fact that skeletal muscle transfer organisms can be administered intravenously and that they target neurons on the stroke lesion side after infusion makes them more clinically viable and increases the efficiency of mitochondrial transplantation. Figure 7 ).

[0080] Example 5: Comparison of therapeutic effects between skeletal muscle migration bodies and skeletal muscle mitochondria alone

[0081] 1. In vitro experiments:

[0082] (1) Mitochondrial isolation from mouse skeletal muscle: Mice were euthanized by cervical dislocation, and the gastrocnemius muscle was quickly removed with a scalpel and immersed in 5 mL of pre-chilled PBS (containing 10 mM EDTA). The muscle was minced and impurities such as fat and ligaments were removed. After washing 2-3 times with pre-chilled PBS (containing 10 mM EDTA), it was resuspended in 5 mL of PBS containing 10 mM EDTA and 0.05% trypsin and incubated for 30 minutes. After centrifugation at 200g for 5 minutes, the supernatant was discarded, and the precipitate was resuspended in 1 IBmL. The muscle was homogenized 10 times with a Teflon pestle at 1600 rpm in pre-chilled (ice bath for 5 minutes) at 4°C (tissue to buffer ratio 1:5-1:10 w:v). The homogenate was transferred to 50 mL. Centrifuge in Falcon tubes at 700g for 10 minutes at 4°C. Transfer the supernatant to a glass centrifuge tube and centrifuge at 8000g for 10 minutes at 4°C. Discard the supernatant, resuspend the precipitate in 5 mL of ice bath IBm2, centrifuge again at 8000g for 10 minutes at 4°C, and finally resuspend the mitochondrial precipitate in the small amount of buffer remaining after discarding the supernatant (avoid air bubbles). Transfer to 14 mL Falcon tubes and store on ice. (IBm1: 100 mL system contains 6.7 mL 1M sucrose, 5 mL 1M Tris / HCl, 5 mL 1M KCl, 1 mL 1M EDTA, and 2 mL 10% BSA, adjusted to pH 7.4 and then diluted to volume with distilled water; IBm2: 100 mL system contains 25 mL 1M sucrose, 3 mL 0.1M EGTA / Tris, and 1 mL 1M Tris / HCl, adjusted to pH 7.4 and then diluted to volume with distilled water).

[0083] (2) Primary neurons from mice subjected to oxygen deprivation (OGD) were treated with either skeletal muscle mitochondria (SKM-mito) or skeletal muscle migratory bodies (SKM-M) from exercised mice. Neuronal apoptosis was detected by immunofluorescence. + TUNEL + Neuronal DNA damage (NeuN) + 8OHGD + Mitochondrial autophagy (TOM20) in neurons was detected by immunofluorescence and flow cytometry. + LC3 + ), mitochondrial membrane potential, ROS (Reactive Oxygen Species) levels in mitochondria, and ATP production in neurons.

[0084] The results are as follows Figure 8As shown, compared with simple skeletal muscle-derived mitochondrial treatment, skeletal muscle-derived migratory body treatment can better protect primary neurons damaged by oxidative stress.

[0085] 2. Animal experiments:

[0086] The sham-operated group and tMCAO mice were divided into a resting group, a skeletal muscle migration body treatment group (SKM-Ex-M, 10 mg / kg, intravenous infusion for 3 consecutive days after surgery, once daily, then once weekly), and a skeletal muscle mitochondrial body treatment group (SKM-Mito, 10 mg / kg, intravenous infusion for 3 consecutive days after surgery, once daily, then once weekly). Cognitive function of the mice in each group was assessed using the water maze test 31-35 days after surgery.

[0087] The results are as follows Figure 9 As shown, compared to simple skeletal muscle-derived mitochondrial therapy, skeletal muscle-derived migration body therapy can better restore post-stroke cognitive impairment.

[0088] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. Application of skeletal muscle transfer bodies in the preparation of drugs for treating ischemic stroke.

2. The application according to claim 1, characterized in that, The ischemic stroke mentioned refers to acute ischemic stroke.

3. The application according to claim 1, characterized in that, The skeletal muscle transfer organism can repair neurons damaged in stroke.

4. The application according to claim 1, characterized in that, The skeletal muscle migration body contains mitochondria.

5. The application according to claim 4, characterized in that, The skeletal muscle migration body also contains metabolites and proteins that support mitochondrial function and protect mitochondria.

6. The application according to claim 1, characterized in that, The skeletal muscle migration bodies can be generated through drug stimulation, electrical stimulation, or exercise induction.

7. The application according to claim 6, characterized in that, The drug stimulation includes acetylcholine stimulation.

8. The application according to claim 1, characterized in that, The drug can be administered via injection.

9. A drug for treating ischemic stroke, characterized in that, The active ingredient is a skeletal muscle migration body carrying mitochondria.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug includes injections.

Images