Application of mitochondria-activated miRNA and its loaded pueraria exosome in targeting anti-parkinson's disease

Abstract

The application discloses a Parkinson's disease targeted application of a pueraria exosome loaded with a mitochondria-activated miRNA, and belongs to the field of biological applications.The miRNA comprises at least one of miRNAs shown in sequences as shown in SEQ ID No, 1 to SEQ ID No, 23.The pueraria exosome and the miRNA delivered thereby in the application show a good treatment effect on Parkinson's disease, and mainly play a role in improving mitochondrial dysfunction, such as promoting mitochondrial autophagy, improving mitochondrial membrane potential, reducing oxidative stress, maintaining the integrity of the mitochondrial inner membrane respiratory chain, and increasing ATP generation.The application further discloses a delivery scheme of the pueraria-derived exosome as the miRNA carrier by using neuron-targeted modification, which can improve the targeting and stability of the miRNA, and overcome the problems of low miRNA drug delivery efficiency and easy degradation.The application is expected to promote the treatment process of Parkinson's disease through the neuron-targeted pueraria exosome and the miRNA delivered thereby.

Description

Technical Field

[0001] This invention belongs to the field of biological applications, specifically relating to the application of mitochondrial activated miRNA and its loaded kudzu exosomes in targeting Parkinson's disease. Background Technology

[0002] A key pathogenic mechanism of Parkinson's disease involves cellular damage caused by mitochondrial dysfunction. Mitochondrial dysfunction affects cellular function in multiple ways, including energy production, oxidative stress, metabolic regulation, and apoptosis. Mitochondria are primarily responsible for intracellular energy production; impaired mitochondrial function leads to reduced adenosine triphosphate (ATP) production, resulting in impaired energy production. Furthermore, impaired mitochondrial function leads to decreased mitochondrial membrane potential and increased free radical production, which can damage intracellular lipids, proteins, and DNA. Mitochondria participate in various metabolic pathways; metabolic disorders caused by their dysfunction can impair lipid and carbohydrate metabolism. Mitochondrial dysfunction also leads to impaired apoptosis signaling, release of apoptosis factors, and expression of apoptosis-related proteins, promoting apoptosis.

[0003] microRNAs (miRNAs) represent a cutting-edge strategy for treating mitochondrial dysfunction, aiming to restore mitochondrial function by regulating gene expression. miRNAs are small non-coding RNA molecules that regulate various intracellular biological processes by binding to the mRNA of target genes, inhibiting their translation or promoting their degradation. In treating mitochondrial dysfunction, miRNAs can target key genes related to mitochondrial function, thereby regulating mitochondrial biosynthesis, improving oxidative stress levels, or reducing mitochondrial-related apoptosis. Through these mechanisms, miRNAs hold promise for improving the symptoms of Parkinson's disease and slowing its progression.

[0004] Regulating the expression of mitophagy-related genes through miRNAs to promote mitophagy and thus ensure normal mitochondrial function is a potential entry point for the effective treatment of Parkinson's disease. Mitophagy plays a crucial role in improving mitochondrial dysfunction by clearing damaged mitochondria, reducing oxidative stress, and promoting mitochondrial renewal. Mitophagy can remove damaged mitochondria that produce excessive free radicals and cause oxidative stress, thereby mitigating the harmful effects on cells. Furthermore, mitophagy helps restore mitochondrial function by clearing old mitochondria and promoting the generation of new mitochondria, maintaining a healthy balance of mitochondria within the cell. This process can also reduce the release of apoptosis factors from mitochondria, thereby protecting cells from unnecessary programmed cell death.

[0005] However, miRNA drug development faces several challenges. Currently, efficient delivery of miRNA drugs remains difficult; traditional delivery methods may be inefficient or trigger immune responses, necessitating the development of novel delivery systems. Furthermore, miRNAs are easily degraded in vivo, making it crucial to improve their stability and prolong their half-life, requiring optimized chemical modifications or delivery strategies. Finally, the difficulties in developing gene-specific miRNA drugs also limit their clinical application and commercialization.

[0006] Exosomes are extracellular vesicles with a diameter of approximately 30-150 nanometers, capable of carrying various biomolecules, including miRNAs. They have been shown to have potential applications in the treatment of various diseases through the miRNAs they contain, significantly aiding in miRNA drug development. Exosomes possess natural biocompatibility, and as cell-derived nanovesicles, they exhibit highly efficient intracellular internalization properties, ensuring intracellular delivery of miRNAs. Furthermore, the miRNAs within exosomes are relatively stable and not easily degraded by in vivo enzymes, thus extending their half-life. The surface of exosomes can also be loaded with specific ligands or antibodies, enhancing targeting specific cells or tissues. As a natural miRNA reservoir, the application of exosomes holds promise for addressing some challenges in traditional miRNA drug delivery systems and provides a rich library of miRNAs for disease treatment, increasing the likelihood of discovering effective miRNA molecules and promoting miRNA drug development.

[0007] Currently, there are few reports on therapeutic miRNAs for Parkinson's disease. The discovery and targeted validation of such miRNAs are expected to advance the treatment of Parkinson's disease. Summary of the Invention

[0008] To address the problems in existing technologies, this invention proposes the application of mitochondrial activated miRNAs and their loaded kudzu exosomes in targeting and treating Parkinson's disease. This invention also elucidates that improving mitochondrial dysfunction is one of its main mechanisms of action against PD.

[0009] In a first aspect, the present invention provides the use of miRNA in the preparation of drugs for treating Parkinson's disease, wherein the miRNA comprises at least one of the miRNAs with sequences as shown in SEQ ID No. 1 to SEQ ID No. 23.

[0010] Furthermore, the miRNA uses exosomes as a carrier, and the miRNA is encapsulated inside the exosomes.

[0011] Furthermore, the exosomes or exosomes encapsulating the miRNA are extracts derived from kudzu root (Pueraria lobata).

[0012] Preferably, the extract is derived from a pulp prepared from kudzu root, and the pulp is one or more of freshly squeezed liquid, extract, and decoction.

[0013] Furthermore, the extract is derived from one or more of the following: fresh kudzu leaves, kudzu flowers, kudzu roots, kudzu vines, kudzu stems, processed kudzu products, and kudzu decoctions.

[0014] Furthermore, the exosomes or exosomes encapsulating the miRNA are functional exosomes with neuron-targeting ligand modifications on their surface. This enhances the ability of the puerarin exosomes and their delivered miRNA to be specifically taken up by pathological neurons, thereby increasing the therapeutic effect on Parkinson's disease.

[0015] Furthermore, the targeting ligand is at least one of small molecules, peptides, antibodies, and lipids.

[0016] Preferably, the neuron-targeting ligand is immobilized on the surface of puerarin exosomes by chemical coupling or physical adsorption to achieve specific targeting of dopaminergic neurons.

[0017] Preferably, the small molecule is a ligand capable of targeting dopamine receptors, and is a synthetically produced selective D1-like or D2-like receptor agonist or antagonist, or a natural product, such as one or more of dopamine, levodopa, benzyltetrahydroisoquinoline alkaloids, apophene-type alkaloids, kullarine alkaloids, phenanthrene alkaloids, berberine and tetrahydroproberberine, and ergot alkaloids; the polypeptide is one or more of transferrin, low-density lipoprotein, insulin, leptin, RVG29, c(RGDyk), c(RGDfk), Angiopep-2, THR, CRT, g7, TGN, TAT, SynB1, RDP, KC2S, LCDX, DCDX, MiniAp-4, and G23; the protein is one or more of an antibody against dopamine receptors, an antibody against dopamine transporters, a neuroadhesion protein, and neurotropin; and the lipid is one or more of phospholipids, ceramides, sphingolipids, and glycolipids.

[0018] In a second aspect, the present invention provides a medicament for treating Parkinson's disease, characterized in that the medicament contains miRNA, wherein the miRNA contains at least one of the miRNAs with sequences as shown in SEQ ID No. 1 to SEQ ID No. 23.

[0019] Thirdly, the present invention provides a medicament for treating Parkinson's disease, characterized in that the medicament comprises exosomes derived from kudzu root (Pueraria lobata), wherein the exosomes encapsulate miRNAs, and the miRNAs comprise at least one of the miRNAs with sequences as shown in SEQ ID No. 1 to SEQ ID No. 23.

[0020] Furthermore, the exosomes are functional exosomes with neuron-targeting ligand modifications on their surface.

[0021] According to a preferred embodiment of the present invention, the miRNA provided in the above aspects is preferably at least one of the miRNAs with sequences such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 12, SEQ ID No. 16, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, and SEQ ID No. 23.

[0022] The advantages of this invention are that the miRNA and drugs for treating Parkinson's disease described in this invention can improve mitochondrial dysfunction, including promoting mitophagy, increasing mitochondrial membrane potential, reducing oxidative stress, maintaining the integrity of the inner mitochondrial membrane respiratory chain complex, and increasing ATP production, among one or more of the following: further, the mitophagy described is PINK1-PARKIN-mediated mitophagy; the inner mitochondrial membrane respiratory chain complex is one or more of complexes I, II, III, IV, and V.

[0023] The key genes involved in improving mitochondrial dysfunction through the miRNA and drugs for treating Parkinson's disease described in this invention are: the miRNA improves mitochondrial dysfunction by reducing the expression of LRRK2, USP8, and USP15 in cells and increasing the expression of TOMM7, PINK1, SQSTM1, NDUFB4, and ATP5J2.

[0024] The miRNAs and drugs for treating Parkinson's disease described in this invention can improve mitochondrial dysfunction by increasing the content of one or more of the endogenous miRNAs mentioned above in cells. Specifically, this includes promoting mitophagy, increasing mitochondrial membrane potential, reducing oxidative stress, maintaining the integrity of the mitochondrial inner membrane respiratory chain complex, and increasing ATP production.

[0025] The miRNAs and drugs for treating Parkinson's disease described in this invention can reduce the expression of LRRK2, USP8, and USP15 in cells and increase the expression of TOMM7, PINK1, SQSTM1, NDUFB4, and ATP5J2 by ​​increasing the content of one or more of the endogenous miRNAs mentioned above in cells. This promotes PINK1-PARKIN-mediated mitophagy, thereby clearing damaged mitochondria and mitigating the cascade of cell damage patterns they cause, and ensuring the integrity of the mitochondrial inner membrane respiratory chain complexes I and V to improve energy supply.

[0026] The neuron-targeting puerarin exosomes and their delivered miRNAs provided by this invention have great potential in the treatment of Parkinson's disease, as they can specifically target neurons and broadly regulate mitochondrial dysfunction in pathological neurons. Furthermore, the puerarin exosome-based miRNA upregulatory agent of this invention has good safety and virtually no toxic side effects. Attached Figure Description

[0027] Figure 1 The particle size of the kudzu exosomes obtained in Example 1;

[0028] Figure 2 This is a transmission electron microscope image of the kudzu exosomes obtained in Example 1;

[0029] Figure 3 Transmission electron micrographs of kudzu exosomes obtained in Example 1 after storage for different times;

[0030] Figure 4 The particle size of the neuron-targeting puerarin exosomes obtained in Example 2;

[0031] Figure 5 Transmission electron microscopy image of neuron-targeted puerarin exosomes obtained in Example 2;

[0032] Figure 6 This is an evaluation of the uptake effect of kudzu exosomes before and after targeted modification in SH-SY5Y neuronal model cells in Example 3;

[0033] Figure 7 The intersection of miRNA contained in kudzu exosomes in Example 4 and differentially upregulated miRNA in SH-SY5Y cells treated with kudzu exosomes;

[0034] Figure 8 , 9 This is a graph showing the KEGG and GO enrichment analysis of SH-SY5Y pathological Parkinson's model cells before and after treatment with kudzu exosomes in Example 5.

[0035] Figure 10The effect of miRNA delivered by kudzu exosomes in Example 6 on the activity of SH-SY5Y cells in a pathological Parkinson's disease model.

[0036] Figure 11 The effect of miRNA delivered by kudzu exosomes in Example 7 on the mitochondrial membrane potential of SH-SY5Y cells in a pathological Parkinson's disease model.

[0037] Figure 12 The effect of miRNA delivered by kudzu exosomes in Example 8 on ATP content in SH-SY5Y cells, a pathological Parkinson's disease model.

[0038] Figure 13-15 This elucidates the targeting and regulatory effects of the miRNA delivered by the kudzu exosomes in Example 9 on LRRK2, USP8, and USP15.

[0039] Figure 16 In Example 10, kudzu exosomes promoted the improvement of mitophagy and mitochondrial morphology in SH-SY5Y cells, a model of pathological Parkinson's disease.

[0040] Figure 17 This refers to the improvement of the activity of SH-SY5Y mitochondrial respiratory chain complex I and V in pathological Parkinson's disease model cells by kudzu exosomes in Example 11.

[0041] Figure 18 , 19 The expression of LRRK2, TOMM7, PINK1, SQSTM1, NDUFB4, and ATP5J2 in the pathological Parkinson's disease model cells SH-SY5Y after treatment with kudzu root exosomes in Example 12.

[0042] Figure 20 The images show the movement trajectories of Parkinson's disease model mice in an open field before and after targeted modification and treatment with kudzu root exosomes in Example 13.

[0043] Figure 21 The rotarod performance of Parkinson's model mice after targeted modification and treatment with kudzu exosomes in Example 14;

[0044] Figure 22 The tail suspension behavior of Parkinson's model mice after targeted modification and treatment with kudzu root exosomes in Example 15;

[0045] Figure 23 The expression of tyrosine hydroxylase in dopaminergic neurons of the substantia nigra in Parkinson's disease model mice before and after treatment with kudzu root exosomes following targeted modification in Example 16.

[0046] Figure 24The expression of Nissl substance in dopaminergic neurons of the substantia nigra in Parkinson's disease model mice before and after treatment with kudzu root exosomes following targeted modification in Example 17.

[0047] Figure 25 , 26 This serves as in vivo molecular validation of the effect of kudzu exosomes on mitochondrial dysfunction before and after targeted modification in Example 18;

[0048] Figure 27 This study evaluates the biosafety of kudzu exosomes before and after targeted modification in Example 19. Detailed Implementation

[0049] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0050] Example 1: Preparation and Characterization of Pueraria Exosomes

[0051] Take 50g of fresh kudzu root, wash away residual soil, and extract juice using a juicer with 150mL of phosphate buffer. Filter the juice through a screen to remove residue, then centrifuge the collected juice sequentially at 4℃, 1000×g for 10min, 4000×g for 40min, and 10000×g for 60min to remove large particles and cell debris. Centrifuge the supernatant at 4℃, 150000×g for 90min, resuspend the bottom precipitate in an appropriate amount of PBS, and centrifuge using a sucrose density gradient centrifugation method (6%, 30%, 45%, 60%) at 4℃, 150000×g for 90min. Combine the 30% and 45% layers, ultra-high temperature, and wash to obtain kudzu exosomes. Measure the particle size of the kudzu exosomes using a Malvern particle size analyzer, photograph the morphology of the exosomes using a transmission electron microscope, and quantify the protein concentration of the exosomes using a BCA kit. Observe the morphology of the kudzu exosomes after storage for different times. (See attached image) Figure 1 As shown, the obtained kudzu exosomes have a particle size of approximately 125 nm and exhibit good dispersion properties. (See attached image.) Figure 2 As shown, kudzu exosomes exhibit a teacup-shaped structure under transmission electron microscopy, consistent with the classic characteristics of exosomes. The outer white region is the lipid bilayer, providing excellent protection for the encapsulated miRNA. (See attached image) Figure 3 As shown, the morphology of kudzu exosomes did not change significantly after storage for approximately 30, 90, and 360 days, indicating their good stability.

[0052] Example 2: Preparation and Characterization of Neuron-Targeted Pueraria Exosomes

[0053] Pueraria exosomes were co-incubated with DSPE-PEG2000-RVG29 at 37℃ and a mass ratio of 1:1 for 0.5 h. After incubation, the free DSPE-PEG2000-RVG29 was removed by centrifugation at 150,000 × g for 60 min at 4℃. The bottom precipitate was resuspended to obtain neuron-targeting pueraria exosomes. The particle size and potential of the exosomes were measured using a Malvern particle size analyzer, and the morphology of the exosomes was photographed using a transmission electron microscope. (See attached image) Figure 4 , 5 As shown, the morphology of neuron-targeted puerarin exosomes did not change significantly, indicating the feasibility of the modification method. Due to the introduction of functional side chains, the particle size increased slightly to about 133 nm.

[0054] Example 3: Evaluation of the uptake effect of kudzu exosomes before and after targeted modification in SH-SY5Y neuronal model cells.

[0055] Pueraria exosomes and neuronal-targeting pueraria exosomes were labeled with the fluorescent dye DiO, and SH-SY5Y cells were collected after incubation for different time periods. Flow cytometry with CytExpert 2.4 software was used to analyze the uptake efficiency of fluorescently positive SH-SY5Y cells at different time points. (See attached image) Figure 6 As shown, at 2 hours, the number of fluorescently positive cells reached nearly 50%, and at 4 hours, the maximum uptake percentage was almost reached, indicating excellent uptake performance of kudzu exosomes by cells. Furthermore, at different time points, the uptake rate of RVG-modified neurons targeting kudzu exosomes was higher than that of pure kudzu exosomes, indicating optimized uptake performance.

[0056] Example 4: Intersection analysis of pueraria exosomes and differentially upregulated miRNAs in SH-SY5Y cells after treatment with pueraria exosomes

[0057] miRNAs were extracted from kudzu exosomes and from SH-SY5Y cells before and after kudzu exosome treatment using an RNA extraction kit. Detailed expression profiles and relative abundances of the miRNAs were obtained using high-throughput sequencing. Differentially upregulated miRNAs in SH-SY5Y cells after kudzu exosome treatment were also identified, and their intersection with miRNAs in kudzu exosomes was calculated. (See attached image.) Figure 7As shown, there were 23 overlapping miRNAs, indicating that kudzu exosomes delivered 23 miRNAs to SH-SY5Y cells, which are fundamental to its drug efficacy. Analysis revealed that these 23 miRNAs are recorded in the miRBase database, with the names hsa-miR-27a-3p, hsa-miR-148a-3p, hsa-miR-194-5p, hsa-miR-185-5p, hsa-miR-15a-5p, hsa-miR-30a-5p, hsa-let-7i-5p, hsa-let-7e-5p, hsa-miR-20a-5p, hsa-let-7c-5p, and hsa-let-7a. -5p, hsa-miR-30e-5p, hsa-miR-22-3p, hsa-miR-199b-3p, hsa-let-7d-5p, hsa-miR-30b-5p, hsa-miR-450a-5p, hsa-miR-15b-5p, hsa-miR-26b-5p, hsa-let-7g-5p, hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-374b-5p, and their corresponding sequences are shown in SEQ ID No. 1 to SEQ ID No. 23 and in Table 1. The rightmost column of Table 1, miR-NO, is the abbreviation of each sequence in the attached figure.

[0058] Table 1: Names and sequences of miRNAs (SEQ ID No. 1 to SEQ ID No. 23)

[0059]

[0060]

[0061] Based on common knowledge in the art, these miRNA sequences can be extracted from kudzu root as described in the embodiments of this application, or they can be synthesized artificially. It should be understood that miRNAs with the same sequence from different sources should have the same or nearly the same activity when used to prepare the same drug for disease treatment. Therefore, this invention does not limit the source of miRNAs. Without loss of generality, the following embodiments will still use miRNAs delivered by kudzu root-derived exosomes to evaluate miRNA-related properties, but their properties should be understood to be equally applicable to miRNAs with the same sequence from other sources.

[0062] Example 5: KEGG and GO enrichment analysis of differentially expressed genes in pathological SH-SY5Y cells before and after treatment with kudzu exosomes.

[0063] Cluster analysis was performed on differentially expressed genes using KEGG and GO enrichment analyses. KEGG enrichment analysis was used to enrich specific metabolic or signal transduction pathways. GO enrichment analysis was used to enrich one or more biological processes, molecular functions, or cellular components. (See attached...) Figure 8 , 9 As shown, enrichment analysis of KEGG and GO revealed a wide range of events related to autophagy (mitochondrial autophagy) and mitochondrial structure, suggesting a potential regulatory role of miRNAs delivered by kudzu exosomes in mitochondrial dysfunction.

[0064] Example 6: Effect of miRNA delivered by kudzu exosomes on the activity of SH-SY5Y cells in a neuronal pathological Parkinson's disease model.

[0065] 5000 SH-SY5Y cells were seeded in each well of a 96-well plate. After overnight adhesion, the cell culture medium was removed. SH-SY5Y cells were treated with 1 mM MPP+ for 24 hours to establish an in vitro pathological cell model of Parkinson's disease. A control group was set up after modeling, treated with 1 mM MPP+. + Model group, 1mM MPP + / miRNA transfection group. After modeling, the normal culture medium was replaced in the model group. The corresponding miRNA of SH-SY5Y was transfected into the transfection group according to the Thermo Fisher Scientific Lipo3000 transfection reagent instructions, with 5 replicates per group. After 24 hours, the cell culture medium was removed and replaced with serum-free medium containing MTT, and incubation continued for 4 hours. Then, 100 μL of Formazan dissolving solution was added to each well, mixed appropriately, and shaken in the dark for 10 min. After all the purple crystals had dissolved, the absorbance was measured at 570 nm. (See attached image) Figure 10 As shown, based on the model group, different miRNAs exhibited different abilities to improve the survival of PD pathological cell models.

[0066] Example 7: Effects of miRNA delivered by kudzu exosomes on mitochondrial membrane potential in SH-SY5Y neuronal pathological Parkinson's disease model cells.

[0067] 200,000 SH-SY5Y cells were seeded in each well of a 6-well plate. After overnight adhesion, the cell culture medium was removed. A control group was set up with 1 ml MMPs. + Model group, 1mM MPP + / miRNA transfection group. Following the instructions of the mitochondrial membrane potential assay kit (BL711A, Biosharp), 1 mL of JC-1 staining working solution was added to each well, mixed thoroughly, and incubated at 37°C for 20 minutes. After incubation, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer (1×). The fluorescence intensity values ​​at 490 nm and 525 nm were measured using a fluorescence microplate reader, and the ratio of red to green fluorescence intensity was calculated. (See attached image) Figure 11 As shown, based on the model group, different miRNAs exhibited different abilities to improve mitochondrial membrane potential in PD pathological cell models.

[0068] Example 8: Effect of miRNA delivered by kudzu exosomes on ATP content in SH-SY5Y cells of a neuronal pathological Parkinson's disease model.

[0069] 200,000 cells were seeded per well in a 6-well plate. After overnight adhesion, the cell culture medium was removed. A control group was set up with 1 mM MPP. + Model group, 1mM MPP + / miRNA transfection group. Following the ATP assay kit instructions (S0026, Beyotime), 200 μL of lysis buffer was added to lyse cells. The cells were repeatedly pipetted to ensure thorough contact with the lysis buffer and complete cell lysis. After lysis, the cells were centrifuged at 12000g for 5 minutes at 4°C, and the supernatant was collected. The ATP standard solution was diluted with ATP assay lysis buffer to create a concentration gradient of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM to construct an ATP standard concentration versus RLU value curve. 100 μL of ATP assay working solution was added to the wells and incubated at room temperature for 3-5 minutes to allow all background ATP to be consumed. Then, 20 μL of sample or standard was added to the wells, and the RLU value was measured using a chemiluminescence analyzer. (See attached...) Figure 12 As shown, based on the model group, different miRNAs exhibited different abilities to improve ATP production in the PD pathological cell model.

[0070] Example 9: Evaluation of the targeting effect of kudzu exosome-delivered miRNA on key genes of mitophagy.

[0071] TargetScan (https: / / www.targetscan.org / vert_80 / ) was used to predict miRNA target sequence sites and the 3′UTR sequences of LRRK2, USP8, and USP15. Based on the 3′UTR sequences (Wide type, WT) of LRRK2, USP8, and USP15 from NCBI (National Center for Biotechnology Information), and the predicted mutant 3′UTR sequences (Mutant type, MUT), primers were designed (Sangon, China). Full-length sequences and sequences containing SacI and XhoI restriction sites were amplified by PCR. The PCR products were then inserted between the SacI and XhoI restriction sites into the firefly / rabbit luciferase reporter vector PmirGLO (Takara, Japan) (Fenghe Bio, China). Recombinant plasmids were then screened using ampicillin (Aladdin, China) to successfully construct the bacterial strain, and the results were verified by DNA sequencing (Sangon, China). SH-SY5Y cells were seeded at 200,000 cells / well in 12-well plates. Sixteen hours later, SH-SY5Y cells were co-transfected with blank plasmid PmirGLO, reporter genotype Pmi-3'UTR WT or Pmi-3'UTR MUT (1 μg / well), and miRNA (negative control, NC model, or miRNA model) (30 ng / well). Blank PmirGLO and miRNA NC models served as controls. Forty-eight hours after transfection, luciferase activity in fireflies and kidney rabbits was continuously measured using a dual-luciferase reporter gene assay kit (YEASEN, China) to identify the binding of the target gene to the miRNA. For each individual analysis, the Renilla luciferase signal was normalized to the firefly luciferase signal. (See attached image.) Figure 13-15 As shown, after miRNA binds to the target gene, the normalized firefly luciferase signal decreases, indicating the direct regulatory effect of miRNA on key genes LPPK2, USP8, and USP15 that regulate mitochondrial dysfunction.

[0072] Example 10: Effects of Pueraria exosomes on mitophagy and mitochondrial morphology in SH-SY5Y cells, a model of neuronal pathological Parkinson's disease.

[0073] Cells were fixed overnight in 2.5% glutaraldehyde solution at 4°C and washed with PBS. Samples were fixed with 1% osmium tetroxide solution for 1–2 hours and washed three times with PBS. Dehydration was performed using gradient concentrations (30%, 50%, 70%, 80%) of ethanol solutions for 15 min each, followed by transitions to 90% and 95% acetone solutions for 15 min each. Finally, samples were treated twice with pure acetone for 20 min each time, then with a mixture of Spurr embedding medium and acetone (V / V = 1 / 1) for 1 h, then with a mixture of Spurr embedding medium and acetone (V / V = 3 / 1) for 3 h, and finally treated overnight with pure embedding medium. The permeated samples were embedded and heated overnight at 70°C to obtain encapsulated samples. Samples were sectioned using a microtome (LEICA EMUC7) to obtain sections with a size of 70–90 nm. Stain with lead citrate solution and uranium peroxide saturated solution in 50% ethanol for 5-10 minutes, then observe under a transmission electron microscope (Hitachi H-7650). (See attached image) Figure 16 As shown, compared with the model group cells, the cells treated with kudzu exosomes showed obvious mitophagy structures and more complete mitochondrial cristae morphology. This indicates that kudzu exosomes can clear damaged mitochondria by promoting mitophagy and maintaining the integrity of the mitochondrial inner membrane respiratory chain complex.

[0074] Example 11: Effects of Pueraria exosomes on the activity of SH-SY5Y mitochondrial respiratory chain complexes I and V in neuronal pathological Parkinson's disease model cells.

[0075] The activities of mitochondrial complexes I and V were detected using the CheKine™ MRC Complex I Activity Assay Kit (KTB1850) and the CheKine™ MRC V Activity Assay Kit (KTB1890). (See attached...) Figure 17 As shown, compared with the model group, the activity of mitochondrial respiratory chain complexes I and V in pathological neurons was improved after treatment with kudzu exosomes.

[0076] Example 12: Molecular validation of the effect of pueraria exosomes on improving SH-SY5Y mitochondrial dysfunction in neuronal pathological Parkinson's disease model cells.

[0077] Total protein was extracted from model group cells and exosome-treated cells using RIPA lysis buffer, and then quantified using a BCA quantitative kit. 8-12% separating gel and 5% stacking gel were prepared, with 60 μg of total protein loaded per well (10-15 μl per well). Electrophoresis was performed at 60V for the stacking gel and 80V for the separating gel for approximately 2 hours. PVDF membranes were soaked in methanol for 20 seconds, then transferred to Tris-Glycine transfer buffer (containing 5% methanol) for at least 5 minutes to equilibrate. SDS-PAGE gels were equilibrated in Tris-Glycine transfer buffer for at least 30 minutes. Transfer was performed under constant voltage (100V) for 2 hours under cooling conditions. Primary antibody was dissolved in T-TBS at a specific ratio and incubated overnight at 4°C; then washed with T-TBS for 5 minutes × 4. Secondary antibody was dissolved in T-TBS at a specific ratio and incubated at room temperature for 1 hour; then washed with T-TBS for 5 minutes × 5. Using West Dura Extended Duration Substrate, approximately 1 ml of ECL working solution was prepared according to the manufacturer's instructions. The transfer membrane was incubated at room temperature for 1 min, then excess ECL reagent was removed. The membrane was sealed with plastic wrap and placed in a dark box with X-ray film for exposure for 5-10 min, followed by development and fixing. Image J 1.8.0 image processing software (USA) was used to analyze the optical density values ​​of the bands. Each band was repeated three times. The relative expression level of the target protein was expressed as {target protein (optical density value) / internal reference (optical density value)} × 10n. Results are expressed as mean ± standard deviation. (See attached image.) Figure 18 , 19 As shown, compared with the model group and the pueraria exosome treatment group, there were significant differences in the grayscale of LRRK2, TOMM7, PINK1, SQSTM1, ATP5J2, and NDUFB4 protein blots, indicating that pueraria exosomes improve mitochondrial dysfunction through the mitophagy pathway and by maintaining the integrity of the mitochondrial inner membrane respiratory chain complex.

[0078] Example 13 Open Field Experiment

[0079] Mice were randomly divided into a blank control group, a Parkinson's disease model group (no drug intervention), a pueraria exosome treatment group, and an RVG-modified neuron-targeting pueraria exosome treatment group. The Parkinson's disease model was established by intraperitoneal injection of MPTP into mice for seven consecutive days. After the treatment course, mice in each group were placed in a 45×45cm... 2 Inside the box, an infrared light wave activity monitor was placed on top to record the mice's movement over 5 minutes. After each test, the test area was cleaned with 75% ethanol to eliminate odor interference. (See attached image) Figure 20As shown, compared with the Parkinson's model group, mice in the pueraria exosome treatment group moved a greater distance in the open field and their trajectories were more complex, indicating that the mice's autonomous movement ability was improved, and the RVG-modified neuron-targeted pueraria exosome treatment had the most significant effect.

[0080] Example 14 Rotating Rod Experiment

[0081] The coordination of mice was measured using an accelerated rotarod apparatus. Mice in the blank control group, Parkinson's model group (no drug intervention), puerarin exosome treatment group, and RVG-modified neuronal-targeted puerarin exosome treatment group were trained before the formal experiment. One day before the experiment, mice were trained on a rotating rod that accelerated from 0 rpm to 40 rpm over 240 seconds. The formal experiment was conducted the next day, measuring the time the mice remained on the rod and their rotational speed upon falling. (See attached diagram) Figure 21 As shown, compared with the Parkinson's model group, mice in the pueraria exosome treatment group exercised for a longer time on the rotarod, indicating that the coordination of the mice's movement was improved, and the RVG-modified neuron-targeted pueraria exosome treatment had the most significant effect.

[0082] Example 15: Tail Suspension Experiment

[0083] Depressive-like behavior was assessed using the tail suspension test. Mice in the control group, Parkinson's model group (no drug intervention), pueraria exosome treatment group, and RVG-modified neuronal-targeted pueraria exosome treatment group were suspended for 10 minutes at a node approximately 1.5 inches from the base of the tail. The immobility time of the mice was assessed within 6 minutes after the test. (See attached...) Figure 22 As shown, compared with the Parkinson's model group, the mice in the pueraria exosome treatment group had a shorter period of rigidity during tail suspension, indicating an improvement in depressive behavior in the mice, and the RVG-modified neuronal-targeted pueraria exosome treatment had the most significant effect.

[0084] Example 16 Immunohistochemical staining of tyrosine hydroxylase

[0085] Brain tissue samples were collected from mice in the blank control group, Parkinson's model group (no drug intervention), kudzu exosome treatment group, and RVG-modified neuronal-targeting kudzu exosome treatment group. The tissues were fixed, dehydrated, sectioned, embedded in paraffin, and blocked with serum at room temperature for 30 min. Primary and secondary antibodies were added sequentially, followed by DAB staining. Cell nuclei were counterstained, dehydrated, mounted, and examined under a microscope, and images were acquired. Hematoxylin stained the cell nuclei blue, while DAB showed positive expression as brownish-yellow. The grayscale values ​​of the positive areas were analyzed. (See attached image.) Figure 23 As shown, compared with the Parkinson's model group, the brownish-yellow color of the DA-positive region in the substantia nigra of mice treated with kudzu exosomes was deeper and the area was larger, indicating a higher level of tyrosine hydroxylase expression. Moreover, the neuronal-targeted kudzu exosome treatment with RVG-modified neurons had the most significant therapeutic effect.

[0086] Example 17 Nissl staining

[0087] Brain tissue samples were collected from mice in the blank control group, Parkinson's model group (no drug intervention), kudzu exosome treatment group, and RVG-modified neuronal-targeted kudzu exosome treatment group. The tissues were fixed, dehydrated, sectioned, embedded in paraffin, dewaxed to water, and washed three times with pure water. Sections were stained with Nissl body staining solution for 5 min. They were then washed three times with pure water. Rapid dehydration was performed with anhydrous ethanol, clearing with xylene, and mounting with neutral resin. Nissl bodies within neurons appeared as dark blue granules, cell nuclei as pale blue, and the background as pale blue. (See attached image) Figure 24 As shown, compared with the Parkinson's model group, the area of ​​Nissl bodies positive regions in mice treated with puerarin exosomes was significantly increased, and the neuronal-targeted puerarin exosomes modified by RVG showed the most significant therapeutic effect.

[0088] Example 18: Validation of the in vivo mechanism by which kudzu exosomes improve mitochondrial dysfunction

[0089] Brain tissue samples were collected from the substantia nigra region of mice in the blank control group, model group, kudzu exosome treatment group, and RVG-modified kudzu exosome treatment group. Tissue homogenization was performed using a tissue homogenizer, and total protein was extracted. The process was the same as in Example 12. (See attached...) Figure 25 , 26 As shown, compared with the model group, there were significant differences in the grayscale of protein blots of LRRK2, TOMM7, PINK1, SQSTM1, ATP5J2, and NDUFB4 in the pueraria exosome treatment group. Furthermore, the expression levels of the corresponding genes in the RVG-modified pueraria exosome treatment group were lower or higher, indicating that pueraria exosomes improve mitochondrial dysfunction through the mitophagy pathway and by maintaining the integrity of the mitochondrial inner membrane respiratory chain complex.

[0090] Example 19 H&E staining

[0091] After dewaxing, wash the sections and then stain with hematoxylin for 5 minutes. Next, briefly expose the sections to hydrochloric acid solution for 2 seconds, followed by differentiation with ammonia solution for 15-30 seconds. Wash the sections to remove any excess staining reagent. Next, dehydrate the sections with 95% alcohol and then stain with eosin solution for 5-8 seconds. After dehydration and sealing, observe the stained sections. (See attached...) Figure 27 As shown, no significant organ damage was produced before and after targeted modification of kudzu exosomes, indicating its good biocompatibility.

[0092] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of kudzu-derived exosomes in the preparation of drugs for treating Parkinson's disease, wherein the exosomes are internally encapsulated with miRNA, and the miRNA has the sequence shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The exosomes derived from kudzu are derived from one or more of the following: fresh kudzu leaves, kudzu flowers, kudzu roots, kudzu vines, and kudzu stems.

3. The application according to claim 1, characterized in that, The exosomes or exosomes encapsulating the miRNA are functional exosomes with neuron-targeting ligand modifications on their surface.

4. The application according to claim 3, characterized in that, The targeting ligand is at least one of small molecules, peptides, antibodies, and lipids.

5. A drug for treating Parkinson's disease, characterized in that, The drug comprises exosomes derived from kudzu root (Pueraria lobata), wherein the exosomes encapsulate miRNAs with sequences as shown in SEQ ID No.

1.

6. The drug according to claim 5, characterized in that, The exosomes are functional exosomes with neuron-targeting ligand modifications on their surface.

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