A biological preparation overexpressing seipin and its application in improving pd conditions

Abstract

The present application belongs to the technical field of PD disease improvement treatment, and particularly relates to a biological preparation overexpressing Seipin and application thereof in improving PD disease. The biological preparation for improving Parkinson's disease and overexpressing Seipin is a composition of myristic acid or myristic acid and a Seipin expression promoter. In the composition of myristic acid and the Seipin expression promoter, the mass ratio of myristic acid to the Seipin expression promoter is 1:1. The nucleotide sequence of the Seipin is shown in SEQ ID NO 1. The Seipin expression promoter is a biological preparation containing the nucleotide sequence of SEQ ID NO 1. The present application first finds that myristic acid can significantly up-regulate the expression of Seipin, and myristic acid interacts with the Seipin overexpression vector. Therefore, myristic acid and Seipin can be used as potential therapeutic targets for improving neuroinflammation, regulating lipid metabolism and promoting myelin regeneration, thereby reducing the incidence of PD.

Description

Technical Field

[0001] This invention belongs to the field of PD disease improvement and treatment technology, specifically relating to a biological agent that overexpresses Seipin and its application in improving PD symptoms. Background Technology

[0002] Parkinson's disease, abbreviated as PD, is the second most common neurodegenerative disease worldwide. PD is caused by excessive degeneration and death of dopamine neurons in the substantia nigra pars compacta of the brain, leading to motor dysfunction. PD is accompanied by chronic neuroinflammation, oxidative stress, and the formation of α-synuclein aggregates. Neuroinflammation is a persistent driving factor in the onset and progression of PD. Studies have confirmed that activation of the NLRP3 inflammasome disrupts the function of oligodendrocytes, leading to demyelination. Demyelination forms myelin fragments, which further activate microglia, triggering neuroinflammation. Therefore, demyelination and neuroinflammation form a vicious cycle, ultimately leading to neuronal degeneration and death, accelerating the progression of PD.

[0003] Currently, the core objectives of clinical treatment for PD are to "relieve symptoms, slow progression, and improve quality of life," but there is no cure. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a biological agent that overexpresses Seipin and its application in improving PD symptoms.

[0005] To facilitate understanding of this invention, the materials used in this invention and their abbreviations are listed below: Differentially expressed genes are abbreviated as DEGs. Biological processes are abbreviated as BP. Molecular functions are abbreviated as MF. Cellular components are abbreviated as CC. Dimethyl sulfoxide is abbreviated as DMSO. Polyethylene glycol 300 is abbreviated as PEG300. Double-distilled water is abbreviated as ddH2O. Rabbit anti-NOD-like receptor family protein 3 containing pyrin domain is abbreviated as NLRP3. Mouse anti-interleukin-18 is abbreviated as IL-18. Mouse anti-cysteine ​​aspartate protease-1 is abbreviated as Caspase1. Interleukin-1β is abbreviated as IL-1β. Rabbit anti-apoptosis-associated speckle-like protein is abbreviated as ASC. Mouse anti-tyrosine hydroxylase is abbreviated as TH. Mouse anti-dead bone fragment 1 protein is abbreviated as SQSTM1. Rabbit anti-microtubule-associated protein 1 light chain 3β is abbreviated as LC3B. Mouse anti-Bcl-2 interacting coil-coil protein 1 is abbreviated as Beclin1. Rabbit anti-peroxisome proliferator-activated receptor γ (PPARγ). Rabbit anti-triglyceride lipase (ATGL). Rabbit anti-carnitine palmitoyltransferase 1A (CPT1A). Mouse anti-phosphatidylinositol-3-kinase type I (p110α) catalytic subunit complex (PI3K p100α). Rabbit anti-phosphatidylinositol-3-kinase type I (p110δ) catalytic subunit complex (PI3K p100δ). Rabbit anti-phosphorylated phosphatidylinositol-3-kinase type I (p110α) catalytic subunit complex (p-PI3K p100α). Rabbit anti-protein kinase B (AKT). Mouse anti-phosphorylated protein kinase B (p-AKT). Rabbit antigen myosin-associated kinase B (Trkb). Mouse anti-glial cell-derived neurotrophic factor (GDNF). Rabbit anti-myelin basic protein (MBP). Rabbit anti-brain-derived neurotrophic factor (BDNF). Mouse anti-glial fibrillary acidic protein (GFAP). Rabbit anti-oligodendrocyte transcription factor 2 (Oligo2). Horseradish peroxidase-labeled IgG antibody (IgG-HRP). Rabbit anti-α-synuclein. Mouse anti-neuron-specific class III microtubule protein (Tuj-1). Mouse anti-dead bone fragment 1 protein (SQSMT1). Rabbit anti-NOD-like receptor family protein 3 containing pyrin domain (NLRP3). Mouse anti-caspase-1 (Casp-1). Mouse anti-apoptosis-associated speckle-like protein (ASC). Mouse anti-peroxisome proliferator-activated receptor γ (PPARγ). Mouse anti-CD68 molecule (CD68). Rabbit anti-arginase 1 (ARG1). Rabbit anti-CC chemokine receptor 7 (CCR7). Mouse anti-phosphorylated protein kinase B (p-AKT). Mouse anti-glial fibrillary acidic protein (GFAP). Rabbit anti-oligoglia transcription factor 2 (Oligo2). Rabbit anti-myelin basic protein (MBP).Alexa Fluor 488 is a fluorescent dye, abbreviated as Alexa Fluor 488. Alexa FluorCy3 is a fluorescent dye, abbreviated as Alexa Fluor Cy3. Alexa Fluor 647 is a fluorescent dye, abbreviated as Alexa Fluor 647.

[0006] The first objective of this invention is to provide a biological agent for improving Parkinson's disease and overexpressing Seipin, wherein the biological agent overexpressing Seipin is myristic acid or a combination of myristic acid and a Seipin expression promoter.

[0007] In the composition of myristic acid and seipin expression promoter, the mass ratio of myristic acid to seipin expression promoter is 1:1.

[0008] The nucleotide sequence of the seipin is shown in SEQ ID NO1.

[0009] The Seipin expression promoter is a biological product containing the nucleotide sequence SEQ ID NO1.

[0010] The Seipin expression promoter improves motor dysfunction, autophagy dysfunction, neuroinflammation, and lipid metabolism abnormalities caused by Parkinson's disease by increasing Parkin expression.

[0011]

[0012] Preferably, the Seipin expression promoter is an overexpression vector containing the nucleotide shown in SEQ ID NO.1 or a lentivirus containing the overexpression vector.

[0013] Preferably, the overexpression vector is an m-Seipin-G4S-EGFP vector containing the nucleotides shown in SEQ ID NO.1.

[0014] A second objective of this invention is the use of a biological agent that overexpresses Seipin and is used to improve Parkinson's disease in the preparation of a medicament for improving Parkinson's disease.

[0015] Preferably, the drug uses a biological agent that overexpresses Seipin as its sole active ingredient.

[0016] Preferably, the drug is made from a biological agent that overexpresses Seipin and pharmacologically acceptable excipients.

[0017] Preferably, when the drug is myristic acid, the excipients are dimethyl sulfoxide, polyethylene glycol 300, and Tween 80.

[0018] Preferably, the injection is a solution of myristic acid.

[0019] Preferably, the myristic acid solution is prepared by the following method: 2.5 mg of myristic acid is added to 150 µL of DMSO, then 1200 µL of PEG300 is added, mixed well, then 150 µL of Tween 80 is added, and finally 1500 µL of ddH2O is added and mixed well to obtain the myristic acid solution.

[0020] Preferably, the dosage of the injection is 5 mg / kg, calculated as myristic acid.

[0021] Preferably, when the drug is a seipin expression promoter, the excipient is a sterile buffer solution.

[0022] Preferably, the dosage form of the drug is a solution, injection, or syrup.

[0023] Preferably, the drug has at least one of the following uses: (1) Treatment of nerve inflammation; (2) Treatment of lipid metabolism disorders; (2) Promotes myelin regeneration; (3) Treatment of motor dysfunction.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The biological agent for overexpressing Seipin of the present invention is myristic acid or a Seipin overexpression preparation. The nucleotide sequence of the Seipin is shown in SEQ ID NO1. The biological agent for overexpressing Seipin improves motor dysfunction, autophagy dysfunction, neuroinflammation, and lipid metabolism abnormalities caused by Parkinson's disease by increasing Parkin expression. The myristic acid and Seipin overexpression preparations of the present invention can significantly improve NLRP3 inflammasome activation, lipid metabolism disorders, and myelin regeneration, thereby improving motor dysfunction in mice. In Parkin-deficient PD mice, Seipin expression is significantly decreased, and myristic acid can significantly upregulate Seipin expression, and myristic acid interacts with the Seipin protein. Therefore, myristic acid and Seipin overexpression preparations can be used to improve neuroinflammation in PD, regulate lipid metabolism, and promote myelin regeneration, thereby alleviating PD symptoms. Attached Figure Description

[0025] Figure 1 This is a gene expression differential analysis and enrichment plot for the present invention. A is a volcano plot of differentially expressed genes between the normal and PD groups in the human dataset. B is a volcano plot of differentially expressed genes between the normal and PD groups in the mouse dataset.

[0026] Figure 2 This diagram illustrates the protein-protein interaction between Parkin and Seipin in this invention. A shows the results of Parkin's immunoprecipitation. B shows the immunofluorescence results and co-localization analysis of Parkin and Seipin in PC12 cells. C shows the immunofluorescence co-localization results of Parkin and Seipin in the SNc region of the mouse brain.

[0027] Figure 3 The overexpression of Seipin in this invention can improve Parkin + / - Figure 1 shows the motor dysfunction in mice. A represents the assessment of maximum and fixed rotation speeds in the fatigue rotarod test. B represents the assessment of Rotation Time and Total Time in the Pole Test. C represents the total distance traveled in the open field test in mice. D represents the movement trajectory in the open field test in mice. E represents TH staining of the substantia nigra of the mouse brain. F represents α-Syn immunofluorescence staining of mouse brain tissue sections.

[0028] Figure 4 This is a Western blotting result showing the effect of Seipin upregulation on inhibiting Parkin deficiency-induced inflammatory response in PC12 cells according to the present invention. In the figure, A represents the Western blotting result of PC12 cell protein neuroinflammatory markers, and B represents their grayscale quantitative result.

[0029] Figure 5The present invention describes the immunofluorescence staining of PC12 cells NLRP3, ASC, Caspase-1, etc., induced by Seipin upregulation to inhibit Parkin loss.

[0030] Figure 6 This image shows the Western Blot results of how overexpression of Seipin improves neuroinflammation in Parkin-deficient mice, as described in this invention. In the image, A represents the Western Blot results of the neuroinflammation markers in mouse brain tissue, and B represents their quantitative grayscale values.

[0031] Figure 7 The present invention describes the use of overexpression of Seipin to improve the immunofluorescence staining of NLRP3, ASC, and Caspase-1 in the SNc region of Parkin-deficient mouse brain tissue.

[0032] Figure 8 This invention demonstrates that Seipin overexpression can inhibit microglia activation induced by Parkin deficiency. In this diagram, A represents the Western blotting results of a mouse brain tissue protein marker for microglia, and B represents its grayscale quantitative results.

[0033] Figure 9 This invention demonstrates how Seipin overexpression can inhibit microglia differentiation into M1 cells and increase differentiation into M2 cells induced by Parkin deficiency. A shows immunofluorescence staining of a section of the SNc region of mouse brain tissue.

[0034] Figure 10 The image shows a Western blotting plot illustrating how seipin upregulation can rescue autophagy dysfunction caused by Parkin deficiency, as presented in this invention. In the plot, A represents the Western blotting results of autophagy-related markers of cellular proteins, B represents the quantitative results of these markers, C represents the Western blotting results of autophagy-related markers of tissue proteins, and D represents the quantitative grayscale values ​​of these markers.

[0035] Figure 11 Immunofluorescence and projection scanning electron microscopy images of seipin upregulation to rescue autophagy dysfunction caused by Parkin deficiency, as presented in this invention. A shows the immunofluorescence staining results of LC3B in PC12 cells. B shows the immunofluorescence staining results of P62 in the SNc region of the substantia nigra in brain tissue. C shows the projection scanning electron microscopy results of the cells.

[0036] Figure 12 This is a simulation diagram of the MA molecular docking of the present invention. In the diagram, A represents the three-dimensional structure of the Seipin-MA complex obtained from the molecular docking simulation. B represents the key amino acid residues involved in protein-ligand interactions.

[0037] Figure 13 This is a cell-level concentration diagram of myristic acid in PC12 cells according to the present invention. In the diagram, A represents the Western Blot results after 24 hours of treatment with LV-Parkin-infected PC12 cells at concentrations of 50, 100, and 150 μM myristic acid; B represents the quantification by grayscale value. C represents the Western Blot results after 48 hours of treatment with LV-Parkin-infected PC12 cells at concentrations of 50, 100, and 150 μM myristic acid; and D represents the quantification by grayscale value.

[0038] Figure 14 The Myristic acid of this invention can improve Parkin + / - Figure 1 shows the motor dysfunction in mice. A represents the assessment of maximum and fixed rotation speeds in the fatigue rotarod test. B represents the assessment of Rotation Time and Total Time in the Pole Test. C represents the total distance traveled in the open field test. D represents the mouse's movement trajectory in the open field test. E represents TH staining of the substantia nigra of the mouse brain. F represents α-Syn immunofluorescence staining of mouse brain tissue sections.

[0039] Figure 15 This is a Western blotting image showing the improvement of autophagy dysfunction caused by Parkin deficiency by myristic acid, as described in this invention. In the image, A represents the Western blotting results of cellular protein autophagy-related markers, and B represents their quantitative grayscale values. C represents the Western blotting results of tissue protein autophagy-related markers, and D represents their quantitative grayscale values.

[0040] Figure 16 Immunofluorescence and projection scanning electron microscopy images of myristic acid improving autophagy dysfunction caused by Parkin deficiency, as presented in this invention. A shows the immunofluorescence staining results of LC3B in PC12 cells. B shows the immunofluorescence staining results of P62 in the SNc region of the substantia nigra of brain tissue. C shows the projection scanning electron microscopy results of the cells.

[0041] Figure 17 This is a diagram illustrating the inhibition of NLRP3 inflammasome activation induced by myristic acid in Parkin-deficient mice according to the present invention. In the diagram, A represents the Western blotting results of neuroinflammatory markers in mouse brain tissue proteins, and B represents their quantitative grayscale values.

[0042] Figure 18 This diagram illustrates the inhibition of NLRP3 inflammasome activation induced by myristic acid in Parkin-deficient PC12 cells, as described in this invention. A represents the Western blotting results of PC12 cell protein neuroinflammatory markers, and B represents their grayscale quantitative analysis.

[0043] Figure 19This diagram illustrates the inhibition of Parkin deficiency-induced neuroinflammation by myristic acid, as described in this invention. A shows immunofluorescence staining of NLRP3, ASC, and Caspase-1 in the SNc region of mouse brain tissue. B shows immunofluorescence staining of PC12 cells for NLRP3, ASC, and Caspase-1.

[0044] Figure 20 This invention demonstrates how myristic acid can inhibit microglia activation induced by Parkin deficiency. In the diagram, A represents the Western blotting results of microglia markers from mouse brain tissue, and B represents their quantitative grayscale values.

[0045] Figure 21 The myristic acid of this invention can inhibit the differentiation of microglia into M1 cells and increase the differentiation into M2 cells induced by Parkin deficiency. A is an immunofluorescence staining of a section of the SNc region of mouse brain tissue.

[0046] Figure 22 This is a bubble diagram of KEGG pathway enrichment according to the present invention. A is a bubble diagram of KEGG pathway enrichment.

[0047] Figure 23 The myristic acid injection and overexpression of seipin in this invention can improve lipid metabolism abnormalities. In the figure, A represents the Western blotting results of markers related to protein-lipid metabolism and signaling pathways in mouse brain tissue, and B represents their grayscale quantitative values.

[0048] Figure 24 The myristic acid injection and overexpression of seipin in this invention can improve abnormal lipid metabolism. A is a Bodipy staining of a section of the SNc region of mouse brain tissue. B is an immunofluorescence staining of a section of the SNc region of mouse brain tissue. C is an immunofluorescence staining of a section of the SNc region of mouse brain tissue.

[0049] Figure 25 This is a Western blotting image of myristic acid and upregulation of seipin promoting myelin regeneration, as presented in this invention. In the image, A represents the Western blotting results of neurotrophic factors and myelin-related markers in mouse brain tissue, and B represents their quantitative grayscale values.

[0050] Figure 26 The images show fluorescence images of myristic acid and seipin upregulation promoting myelin regeneration, as presented in this invention. A is a section of mouse brain tissue SNc region stained with MBP. B is a section of mouse brain tissue SNc region stained with immunofluorescence. C is a section of mouse brain tissue SNc region stained with immunofluorescence. Detailed Implementation

[0051] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0052] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0053] The main materials used in this invention include DMEM, fetal bovine serum, 0.25% trypsin, penicillin-streptomycin antibiotics, paraformaldehyde, OCT, donkey serum, BSA, Triton-100, DAPI, solid mounting medium, RIPA lysis buffer, protease inhibitor PMSF, phosphatase inhibitor, PVDF membrane, chemiluminescent solution, isoflurane, immunohistochemistry pen, Cover slip, PAGE gel rapid preparation kit, Bodipy kit, Fetal Bovine Serum, myristic acid, LV3-Parkin-shRNA-mCherry, AAV-Seipin-Myc-EGFP, and LV3-Seipin-Myc-EGFP.

[0054] The following products were purchased: DMEM (model C11965500BT, product name: DMEM basic / High glucose) from Thermo Fisher Scientific (China) Co., Ltd.; Fetal bovine serum (FBS) (model 10099-141, product name: FBS); 0.25% trypsin (model 25200072, product name: Trypsin-EDTA (0.25%), containing phenol red) from Thermo Fisher Scientific (China) Co., Ltd.; Penicillin-streptomycin antibiotic (model 15240062, product name: Fungal-Antibiotic (100X)) from Thermo Fisher Scientific (China) Co., Ltd.; Paraformaldehyde (model 013439851, product name: Paraformaldehyde) from Shanghai Titan Technology Co., Ltd.; and OCT (model 4583, product name: OCT embedding agent) from Shanghai Titan Technology Co., Ltd. Donkey serum, purchased from Shanghai Titan Technology Co., Ltd., model number 017-000-121, product name: Normal Donkey Serum. BSA, purchased from Shanghai Titan Technology Co., Ltd., model number A8010, product name: Bovine Serum Albumin Complete Components. Triton-100, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., model number 9036-19-5, product name: Triton™ X-100. DAPI, purchased from Shanghai Beyotime Biotechnology Co., Ltd., model number C1002, product name: DAPI. Solid mounting medium, purchased from Thermo Fisher Scientific (China) Co., Ltd., model number P36930, product name: ProLong™ Gold Anti-Quenching Mounting Medium. RIPA Lysis Buffer, purchased from Shanghai Beyotime Biotechnology Co., Ltd., model number P0013B, product name: RIPA Lysis Buffer (Strong). The following products were purchased: PMSF (protease inhibitor), ST507-10ml, PMSF Solution (100mM), from Shanghai Beyotime Biotechnology Co., Ltd.; Phosphatase inhibitor, C51003, Phosphatase Inhibitor Cocktail (2 Tubes, 100×), from Beijing Bio-Sens Biotechnology Co., Ltd.; PVDF membrane, IPVH00010, Immobilon® -P PVDF Membrane, from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Chemiluminescent solution, WBKLS0500, Immobilon Western chemiluminescence HRP substrate, from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; and Isoflurane, R510-22-10, isoflurane anesthetic, from Shenzhen Ruiward Life Science Co., Ltd.Immunohistochemistry pen, model AR1181, purchased from Wuhan Boster Biological Engineering Co., Ltd. Cover slip, model 72196-12, purchased from Electron Microscopy Science, Inc. PAGE gel rapid preparation kit, model PG112, purchased from Shanghai Yamei Biomedical Technology Co., Ltd. Bodipy kit, model C2050S, purchased from Shanghai Beyotime Biotechnology Co., Ltd. Fetal bovine serum, model 35-081-CV, purchased from Shanghai Titan Technology Co., Ltd. Myristic acid, model s5617, purchased from Shanghai Titan Technology Co., Ltd. LV3-Parkin-shRNA-mCherry, Parkin-inhibiting lentivirus expression, purchased from General Biotechnology (Anhui) Co., Ltd. AAV-Seipin-Myc-EGFP was purchased from General Biotechnology (Anhui) Co., Ltd., and its product name is Seipin-overexpressing adeno-associated virus. LV3-Seipin-Myc-EGFP was also purchased from General Biotechnology (Anhui) Co., Ltd., and its product name is Seipin-overexpressing lentivirus.

[0055] Healthy C57BL / 6J mice used in this invention were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. Healthy Parkinson's mice... + / - The mice were purchased from Suzhou Saiyuan Biotechnology Model Organism Research Center Co., Ltd.

[0056] The experimental methods used in this invention are as follows: 1. Laboratory animals Animals were housed in clean-grade animal facilities with an ambient temperature controlled at 23±1℃ and a relative humidity of 60%, with a 12-hour light-dark cycle. Experimental procedures were conducted in accordance with the requirements of my country's "Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T 35892-2018)," ensuring the proper implementation of laboratory animal welfare ethics. All experiments were designed and reported in accordance with the "Animal Research: In Vivo Reporting (ARRIVE)" guidelines. The use of laboratory animals and all experimental procedures were approved by the Ethics Committee of Bengbu Medical University (IACUC), approval number 2024-565.

[0057] 2. Data Acquisition and Processing Human and mouse gene expression profiles were obtained from the GSE20295 and GSE31458 datasets in the GEO database, respectively. The raw data were .CEL files generated by the Affymetrix platform. All preprocessing analyses were performed in the R language environment. The oligo v1.66.0 package was used to read the raw .CEL files. Subsequently, the RMA algorithm was used to perform background correction and quantile normalization on the data, and probe signals were summarized to the gene level to obtain the final gene expression matrix at the log2 scale for subsequent analysis.

[0058] 3. Expression visualization and gene set enrichment analysis DEG expression heatmaps were generated using pheatmap v1.0.12 and hierarchical clustering was performed. Box plots of key genes were plotted using ggplot2 to verify intergroup expression differences. ROC curves of core genes were plotted using pROC v1.18.0, and the area under the curve was calculated to evaluate diagnostic efficacy. Simultaneously, GSEA was performed on the whole genome data using the gseKEGG function of cluster Profiler, and pathway enrichment ridge maps were generated.

[0059] 4. Multi-database integration and PPI network construction To screen potential targets closely related to Parkinson's disease, relevant gene sets were first obtained from multiple public databases. Then, the intersection of these gene sets with the differentially expressed genes identified in this invention was calculated using the online tool Venny, and a Venn diagram was generated to identify common targets. All drug prediction results were screened using the pharmMapper algorithm. All prediction results were standardized using UniProt data, converted to official HoganNC gene symbols, and duplicate and non-human source targets were removed to ensure data accuracy and authenticity. Simultaneously, protein-protein interaction relationships corresponding to DEGs were obtained using STRING v11.5 with a confidence level >0.7. PPI network visualization was performed using Cytoscape v3.10.3, and the degree values ​​of nodes in the network were analyzed using NetworkAnalyzer. Core targets were determined based on the degree value ranking.

[0060] 5. Molecular docking simulation The core protein crystal structure (PDB ID: pdb_00006ds5) was downloaded from the RCSB PDB database. Water molecules, pristine ligands, and irrelevant ions were removed from the structure using PyMOL v3.1, and the protein was then hydrogenated. The candidate ligand 3D structure (CID: 11005) was downloaded from the PubChem database. Gasteiger charges were assigned to the ligand using AutoDock Tools v1.5.7, and rotatable bonds were established. Semi-flexible docking was performed using AutoDock Vina v1.2.3. The active pocket spacing angstrom was set to 1 Å = 10⁻¹⁰ m = 0.1 nm. Finally, the protein-ligand complex structure and key interactions were visualized using PyMOL.

[0061] 6. Differentially expressed genes and functional enrichment analysis Based on the expression matrix obtained from preprocessing, differential analysis was performed on the standardized matrix using limma v3.52.4. The screening criteria were: |log2 fold change (log2FC)|>1 and after adjustment P <0.05, the results were plotted as a volcano plot using ggplot2 v3.4.0. DEGs were enriched using clusterProfiler v4.4.4 for GO, Kyoto genes, and KEGG (BP, MF, CC), with the following selection criteria: P <0.05 and after correction Q The result was less than 0.05, and a bubble chart was plotted using ggplot2 to display the result.

[0062] 7. Cell Culture PC12 cells were cultured in complete medium at 37°C in a 5% CO2 incubator. The complete medium was high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The nucleotide sequence of LV3-Parkin-shRNA-mCherry is shown in SEQ ID NO.2. The nucleotide sequence of SEQ ID NO.2 is GCTTTGAACCTGATCACCAGC. The nucleotide sequence of LV3-Seipin-Myc-EGFP was obtained from NM_001136064.3 in the NCBI database. One day before lentiviral infection of PC12 cells, the cells were seeded in 96-well plates, and the cell count was performed using a cell counting chamber, with 1 × 10⁶ cells per well. 4Add 100 μL of complete culture medium to each well and incubate overnight at 37°C with 5% CO2. The next day, add 5 μg / mL of Polybrene and serially diluted virus, remove the cell culture medium, add complete culture medium containing virus, and incubate at 37°C with 5% CO2. After 48 h of infection, discard the original culture medium, add fresh complete culture medium, and add puromycin to remove uninfected cells. After 48 h, replace with fresh complete culture medium and continue culturing. Detect the optimal MOI (Minimum Intake) for virus infection using fluorescence microscopy. Repeat the above steps based on the optimal MOI and expand the culture of infected cells.

[0063] 8. Intraperitoneal injection of myristic acid and bilateral lateral ventricle injection of AAV-Seipin-Myc-EGFP Wild and Parkin animals aged 8 weeks and weighing 22g-25g were selected. + / - Male mice were divided into: wild-type, Parkin knockout, and Parkin gene knockout groups. + / - +PBS group, Parkin + / - +Myristic acid group. The wild-type group is designated as the WT group. The Parkin gene knockout group is designated as the Parkin group. + / - Group. WT Group, Parkin + / - Group, Parkin + / - +Vehicle group, Parkin + / - +AAV-Seipin group, 16 mice in each group. Parkin + / - Mice in the +Myristic acid group were intraperitoneally injected with 5 mg / kg myristic acid once daily for one week. Parkin + / - In the +AAV-Seipin group, based on brain atlases, mice were fixed in place using a stereotaxic instrument, and 2 µl of AAV-Seipin-Myc-EGFP was microinjected at 0.2 µl / min into two sites in the bilateral lateral ventricles: AP(Y)-0.5 mm, ML(X)±1.10, and DV(Z)-2.5 mm. Empty viral vectors were also injected stereotaxically as Parkin. + / - +Vehicle group. After surgery, the mice were placed in a temperature- and humidity-controlled indoor environment. All animals were coded. One month later, six mice from each group were sacrificed for protein extraction and immunofluorescence staining of brain sections. The remaining experimental animals in each group underwent behavioral testing by personnel who were completely unaware of the experiment.

[0064] 9. Mouse behavioral testing Open field test: The open field test chamber measures 50cm × 50cm × 40cm. 24 hours before the formal test, mice were placed in the test room to acclimatize and reduce stress. Each mouse was gently placed in the center of the test chamber and allowed to explore freely for 10 minutes. Each mouse was placed in the center of the test chamber from the same location and direction. After each test, the experimental equipment was sprayed with 75% alcohol to thoroughly remove excrement and odor. Video analysis was performed using the ANY-maze Video Tracking System 7.1.6.

[0065] Pole Test: The pole-climbing device was placed in a square box lined with bedding. The pole was fixed in place, and each group of mice was placed on top of the pole with their noses facing upwards. They were then allowed to turn around and climb to the bottom. This training was conducted for 2 days to allow the mice to adapt to the pole-climbing device. The formal test was conducted on the 3rd day. The test was conducted for two consecutive days to avoid errors caused by the mice. After each test, the scent of each mouse was cleaned with 75% alcohol. Turning Time was recorded as the time it took for the animal to turn its head downwards, and Down Time was recorded as the time it took for the animal to climb to the bottom of the pole.

[0066] Fatigue rotundus test: The rotundus apparatus was set to 20 r / min for 10 min, and mice in each group were trained for 2 consecutive days to adapt to fatigue rotundus. The fatigue rotundus apparatus was cleaned with 75% alcohol. Formal testing was conducted on the third day, and the test was repeated for two consecutive days. After each mouse's test, a test was performed using the maximum rotation speed, with uniform acceleration from 300 s to 40 r / min for 10 min, and the time spent on the rotundus was recorded. A time spent on the rotundus was then performed using uniform acceleration from 20 s to 20 r / min for 20 min, and the time spent on the rotundus was recorded.

[0067] 10. Western Blot Detection Mouse SNc tissue or cell proteins were placed in lysis buffer containing RIPA, PMSF, and a phosphatase inhibitor, and sonicated on ice for 30 min. The lysate was then centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was collected to obtain the complete lysate. The protein lysis buffer was prepared by mixing 1 mL of RIPA, 10 μL of PMSF, and 20 μL of a phosphatase inhibitor. Total protein samples were subjected to constant-voltage SDS-PAGE electrophoresis for 1 hour, followed by constant-current transfer of proteins to PVDF membranes. The membranes were then incubated overnight with antibodies purchased from Abcam, including mouse anti-Parkinson's disease, rabbit anti-congenital lipodystrophy type 2 protein, NLRP3, IL-18, Caspase1, IL-1β, ASC, TH, SQSTM1, LC3B, Beclin1, PPARγ, ATGL, CPT1A, PI3K p100α, PI3K p100δ, p-PI3K p100α, AKT, p-AKT, Trkb, GDNF, MBP, BDNF, GFAP, and Oligo2. The following day, the membranes were incubated with IgG-HRP at room temperature for 1 hour. Chemiluminescence immunoassay was used to detect antigen-antibody binding bands, and images were acquired using a gel imaging system. Protein expression levels were analyzed using ImageLab software.

[0068] 11. Co-IP testing SNc tissue protein from wild-type C57BL / 6J mice was added to protein lysis buffer and sonicated on ice for 30 min. The lysate was then centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was collected to obtain complete lysate. The supernatant was incubated with Parkin antibody at room temperature for 2 h or overnight at 4 °C. The antigen or antibody complex was then bound to protein A / G magnetic beads at room temperature for 1 h. The magnetic beads were washed twice with immunoprecipitation lysis or rinsing buffer, followed by one wash with pure water. The antigen or antibody complex was eluted, and then Western blotting was performed to detect seipin and Parkin.

[0069] 12. Preparation of brain tissue sections and immunofluorescence staining of PC12 cells Mice in different groups were anesthetized with isoflurane gas and then perfused with 1xPBS (pH 7.4) and 4% paraformaldehyde in a pre-cooled solution. The mouse brain tissue was then removed and fixed by immersion in 4% paraformaldehyde. Gradual dehydration was performed using PBS and sucrose solutions with mass fractions of 15% and 30%. After dehydration, the tissue was frozen at -80°C overnight. The next day, the tissue was embedded using OCT and then cut into 13 μm thick coronal frozen sections, which were then mounted on gelatin-coated slides. Cryosectioned brain tissue sections were blocked for 1 hour with a mixture of PBS, donkey serum, 2% BSA, and 10% Triton-100, then washed with PBS. The sections were incubated overnight at 4°C with rabbit anti-congenital lipodystrophy type 2 protein, α-syn, Tuj-1, SQSMT1, TH, NLRP3, Casp-1, ASC, PPARγ, CD68, ARG1, CCR7, rabbit anti-Parkinson's, p-AKT, GFAP, Oligo2, and MBP (purchased from Abcam). The samples were then incubated for 1 hour with the corresponding secondary antibodies Alexa Fluor 488, Alexa FluorCy3, and Alexa Fluor 647 (purchased from Jackson Labs), followed by DAPI counterstaining. Observation and recording were performed using a confocal microscope.

[0070] PC12 cells were cultured on Cover slip sheets, fixed with 4% PFA for 18 min, washed with PBS, and blocked with 0.2% Triton X-100, 1% BSA, and 10% NDS diluted with PBS for 1 h at room temperature, followed by washing with PBS. Cells were then incubated overnight at 4°C with rabbit anti-selenoprotein, NLRP3, mouse anti-caspase-1 (Casp-1), mouse anti-apoptosis-associated speckle-like protein (ASC), LC3B, IL-1β, IL-18, and rabbit anti-Parkinson's disease, respectively. Cells were then incubated with secondary antibodies Alexa Fluor 488, Alexa FluorCy3, and Alexa Fluor 647 for 1 h in the dark at room temperature, followed by DAPI counterstaining for 15 min. Results were observed under a fluorescence microscope.

[0071] 13. Statistical Analysis All data were analyzed using three independent experiments. SPSS 26.0 software was used for analysis, and GraphPad Prism 8.0 software was used for visualization. Differences between two groups were analyzed using an independent samples t-test. For comparisons of differences across multiple samples, if the data conformed to a normal distribution and homogeneity of variance, one-way ANOVA was used, with pairwise comparisons performed using LSD. If not, the Kruskal-Wallis test, a nonparametric test for multiple independent samples, was used, with P < 0.05 considered statistically significant.

[0072] Example 1 Obtaining genes that improve Parkinson's disease involves the following steps: 1. Differentially expressed gene screening and GSEA pathway analysis Analysis of the human Parkinson's disease dataset GSE20295 and the MPTP-induced Parkinson's disease mouse dataset GSE31458 revealed 20 significantly differentially expressed genes in humans and 20 significantly differentially expressed genes in mice. The gene expression differential analysis and enrichment results of this invention are as follows: Figure 1 As shown in the figure. The results showed that Bscl2 expression was significantly decreased in both humans and mice. Bscl2 is also known as Seipin.

[0073] 2. Protein-protein interactions between Parkin and Seipin proteins The protein interaction results between Parkin and Seipin in this invention are as follows: Figure 2 As shown in the figure. The results indicate a significant protein-protein interaction between Parkin and Seipin proteins. Immunofluorescence results showed that Parkin and Seipin proteins were significantly co-expressed in PC12 cells. Immunofluorescence results of the substantia nigra region of mouse brain tissue showed that Parkin and Seipin proteins had significant co-localization in the substantia nigra SNc region. These results indicate that there is a significant interaction between Parkin and Seipin proteins in the SNc region of the mouse substantia nigra and in PC12 cells, and the two proteins are positively correlated. Therefore, overexpression of Seipin can increase the expression of Parkin protein and could be used to treat Parkinson's disease.

[0074] Example 2 Overexpression of Seipin improves Parkin + / - Its application in mouse motor dysfunction includes the following steps: The vector plasmid m-Seipin-G4S-EGFP carrying the target gene Seipin was extracted with high purity and free of endotoxin, and then co-transfected with m-Seipin-G4S-EGFP using High Fectin transfection reagent into 293T cells. Cell pellet was collected 72 h post-transfection. A high-titer adeno-associated virus (AAV) preservation solution was obtained by density gradient centrifugation.

[0075] The Seipin overexpression of Parkin of the present invention + / - The results of the mouse's motor function are as follows Figure 3 As shown in the figure. * indicates... P <0.05, ** indicates P <0.01, *** indicates P<0.001, the same below. One month after stereotactic injection of Seipin-overexpressing virus into the brain, Parkin was detected by fatigue rotarod test. + / - Four-month-old mice had a significantly shorter rod time than four-month-old WT mice, Parkin + / - +AAV-Seipin mice and Parkin + / - Compared to mice, the maximum rotational speed and the time spent on the rod at a fixed rotational speed were significantly prolonged. PoleTest results showed that Parkin... + / - Four-month-old mice were used for a longer period than four-month-old WT mice, while Parkin... + / - +AAV-Seipin mice required significantly less time than Parkin mice. + / - Mice. In the open field test, Parkin + / - Compared with 4-month-old WT mice, 4-month-old mice had a significantly reduced total distance traveled, and compared with Parkin mice... + / - Compared to mice, Parkin + / - +AAV-Seipin mice showed a significant increase in total movement distance. Immunofluorescence results of frozen sections of the substantia nigra in mouse brain tissue showed that Parkin... + / - Compared with WT mice, TH expression was significantly reduced in 4-month-old mice, Parkin + / - +AAV-Seipin mice TH expression and Parkin + / - The improvement was significant compared to that in mice. Parkin + / - The expression level of α-Syn in 4-month-old mice increased sharply, while Parkin... + / - +AAV-Seipin mice compared to Parkin + / - The expression level of α-Syn in mice was significantly reduced. Figure 6 A in Figure 8 The Western blotting results of A in the data also confirmed this conclusion.

[0076] The results showed that Parkin knockout mice exhibited increased α-Syn aggregation and significant damage to dopaminergic neurons, leading to motor dysfunction. Overexpression of Seipin, however, could improve neuronal damage and motor dysfunction caused by Parkin deficiency.

[0077] Example 3 The application of seipin overexpression in improving neuroinflammation caused by Parkin deficiency includes the following steps: Western blotting experiments were performed on PC12 cell proteins to detect the expression levels of inflammation-related markers. The grayscale quantitative results of Western blotting of PC12 cells with upregulated seipin expression in this invention are shown below. Figure 4As shown. The immunofluorescence staining results of NLRP3, ASC, and Caspase-1 in PC12 cells expressing Seipin in this invention are as follows. Figure 5 As shown in the figure, the results indicated that upregulation of Seipin expression in Parkin deficiency led to a significant downregulation of inflammatory markers. Cellular immunofluorescence results also showed that increased Seipin expression significantly improved NLRP3 inflammasome activation and the release of pro-inflammatory factors IL-1β and IL-18.

[0078] Frozen sections of mouse brain tissue and protein extraction from the substantia nigra were performed, and Western blot and immunofluorescence detection were performed. The Western blot results of neuroinflammation in Seipin-overexpressing mice of this invention are as follows: Figure 6 As shown. The immunofluorescence staining results of NLRP3, ASC, and Caspase-1 in the SNc region of brain tissue from Seipin-overexpressing mice of this invention are as follows. Figure 7 As shown in the figure, the results indicate that upregulating Seipin expression significantly improves NLRP3 inflammasome activation. These results suggest that Parkin deficiency induces NLRP3 inflammasome activation and the release of pro-inflammatory factors, and that upregulating Seipin expression can rescue neuroinflammation caused by Parkin deficiency.

[0079] Example 4 The application of seipin overexpression in improving microglial activation induced by Parkin deficiency includes the following steps: Western blot analysis was performed using CD11β and IBA1, common markers of microglia, and CD68, a common marker of microglia activation. The Western blot results of the Seipin-overexpressing mouse brain tissue protein microglia markers of this invention are as follows: Figure 8 As shown. The immunofluorescence staining results of the SNc region sections of mouse brain tissue overexpressing Seipin according to the present invention are as follows. Figure 9 As shown. The results show that Parkin + / - +AAV-Seipin mice and Parkin + / - Compared to mice, the expression levels of CD11β and IBA1 in the substantia nigra of the brain were significantly reduced, and the expression level of CD68 was also significantly decreased. To further elucidate the effects of Seipin overexpression on Parkin... + / - The effects of M1 and M2 microglia subtypes on mouse brain were investigated using immunofluorescence staining with CD68, a common marker of microglia activation, and specific markers for M1 and M2. The fluorescence results showed that Parkin... + / - +AAV-Seipin mice and Parkin + / -Compared with mice, the expression levels of CD68 and CCR7 in the brain were significantly decreased, while the expression level of Arg1 was significantly increased.

[0080] The results showed that upregulation of Seipin expression could inhibit the activation of microglia in the substantia nigra of mouse brain tissue and their transformation into pro-inflammatory M1 cells induced by Parkin deficiency, and promote the differentiation of microglia into anti-inflammatory M2 cells.

[0081] Example 5 The application of seipin overexpression in improving autophagy dysfunction caused by Parkin deficiency includes the following steps: Western blotting analysis of seipin and autophagy-related markers at the tissue and cellular levels in the substantia nigra showed that seipin expression was significantly decreased in the absence of Parkin, further confirming the positive correlation between Parkin and seipin. The Western blotting results of the autophagy-related markers that upregulated seipin expression in cellular and tissue proteins according to this invention are as follows: Figure 10 As shown. The immunofluorescence and projection scanning electron microscopy results of autophagy-disordered mice and PC12 cells of the present invention are as follows. Figure 11 As shown in the figure. The results showed that after upregulating Seipin expression, the expression levels of LC3B and Beclin-1 increased significantly, while the expression level of P62 was significantly downregulated. The immunofluorescence results were consistent with the Western Blot results. These results indicate that Parkin deficiency leads to significant autophagy dysfunction, and upregulating Seipin expression can rescue this phenomenon. Transmission electron microscopy examination of mitochondrial morphology and function further confirmed that upregulating Seipin expression improved mitochondrial morphology and swelling. The green arrows indicate that mitochondrial size tends to be normal, and the cristae structure of mitochondria is clearly restored and their number is significantly restored.

[0082] Example 6 The application of myristic acid in upregulating Parkin expression includes the following steps: 1. Construction of myristic acid and protein-protein interaction network and molecular docking simulation To investigate whether myristic acid can serve as an effective treatment for Parkinson's disease and whether it targets seipin to exert a therapeutic effect, we obtained disease-related genes from the GeneCards and OMIM databases, respectively. We also obtained known drug targets from the TTD and DrugBank databases. A PPI network of genes intersecting with Parkinson's disease was constructed, and molecular docking simulations were performed. The molecular docking simulation results for myristic acid in this invention are as follows: Figure 12As shown in the figure. The results showed that disease-gene association evidence was obtained from the DisGeNET database, with a total of 10,894 disease genes and 337 myristic acid drug targets plotted in a Venn diagram. The 337 myristic acid drug targets were then screened for potential targets using Pharm Mapper prediction software. Data processing was performed on 103 potential targets and disease genes, yielding 91 intersection genes for subsequent analysis. Based on statistical analysis, approximately 88.35% of the myristic acid targets are associated with disease targets, indicating that myristic acid could be an effective drug for treating Parkinson's disease.

[0083] A protein-protein interaction network was constructed using genes intersecting with the drug target and Parkinson's disease. After four rounds of filtering, the core genes of the network were obtained, with Seipin, PPARG, STAT1, and USP90AB1 being the most prominent. Then, Vina software was used to perform molecular docking of Seipin and myristic acid to identify the docking sites and binding free energies between the small molecule ligands and protein receptors. The sites with the lowest free energies were selected for molecular docking, yielding the docking results. The docking results, as shown in the figure, not only exhibited strong binding sites on the surface but also demonstrated the lowest binding free energy.

[0084] 2. Myristic acid upregulates Parkin expression at the PC12 cell level. To investigate the effects of myristic acid on PC12 cells, after treatment with myristic acid for 24 h and 48 h, Western blotting was used to examine its influence on the expression levels of NLRP3, TH, and Seipin in PC12 cells infected with LV-Parkin virus, which inhibits the expression of these proteins. Furthermore, immunofluorescence staining was used to detect the inflammatory release factors IL-1β and IL-18. The Western blotting results of myristic acid treatment of LV-Parkin infected PC12 cells for 24 h and 48 h are as follows: Figure 13 As shown in the figure. The results indicate that treatment of PC12 cells with 150 µM myristic acid for 48 h can significantly inhibit the expression of NLRP3 caused by Parkin deficiency, restore the expression level of TH, significantly upregulate the expression level of Seipin, and significantly inhibit the release of inflammatory factors.

[0085] Example 7 Myristic acid improves Parkin + / - The application in the treatment of motor dysfunction in mice includes the following steps: Mice were subjected to a fatigue rotarod test seven days after intraperitoneal injection of myristic acid. This invention utilizes the Parkin... + / - Mouse motor function results as follows Figure 14 As shown. The results show that Parkin + / -+Myristic acid mice and Parkin + / - Compared to mice, Parkin exhibited significantly prolonged maximum rotational speed and time spent on the rod at a fixed rotational speed in the Pole Test. + / - The time required for +Myristicacid mice was significantly shorter than that for Parkin mice. + / - Mice. In open field tests, compared with Parkin + / - Compared to mice, Parkin + / - +Myristic acid significantly increased the total distance traveled in mice. Immunofluorescence results of frozen sections of the substantia nigra in mouse brain tissue showed that Parkinson's disease... + / - TH expression in +Myristic acid mice and Parkin + / - The improvement was significant compared to that in mice.

[0086] The results showed that intraperitoneal injection of myristic acid could also improve excessive loss of dopaminergic neurons and motor dysfunction caused by Parkin deficiency.

[0087] Example 8 The application of myristic acid in improving autophagy dysfunction caused by Parkin deficiency includes the following steps: Western blotting was used to detect seipin and autophagy-related markers at the tissue and cellular levels in the substantia nigra region. The Western blotting results of the cellular and tissue protein autophagy-related markers of this invention are as follows: Figure 15 As shown. The immunofluorescence and projection scanning electron microscopy results of autophagy dysfunction in this invention are as follows. Figure 16 As shown in the figure. The results showed that after intraperitoneal injection of myristic acid, Seipin expression was upregulated, LC3B and Beclin-1 expression levels were significantly increased, and P62 expression level was significantly downregulated. The immunofluorescence results were consistent with the Western blot results. These results indicate that myristic acid can upregulate Seipin expression and rescue autophagy dysfunction. Transmission electron microscopy examination of mitochondrial morphology and function further confirmed that myristic acid treatment improved mitochondrial morphology and swelling, normalized mitochondrial size, and restored clear cristae structure and significantly increased mitochondrial number.

[0088] Example 9 The application of myristic acid in improving NLRP3 inflammasome activation caused by Parkin deficiency includes the following steps: Western blot experiments were performed on proteins extracted from the substantia nigra and PC12 cells of mouse brain tissue to detect changes in the expression levels of neuroinflammatory markers. The Western blot results of the mouse brain tissue protein neuroinflammatory markers of this invention are as follows: Figure 17As shown. The Western blot results of the PC12 cell protein neuroinflammatory markers of this invention are as follows. Figure 18 As shown. Immunofluorescence staining of the SNc region and PC12 cells in mouse brain tissue of the present invention is as follows. Figure 19 As shown in the figure. The results showed that one week after injection of myristic acid, neuroinflammatory markers in the substantia nigra were significantly downregulated. Immunofluorescence detection of the substantia nigra and cellular levels in mouse brain tissue revealed that the NLRP3 inflammasome was significantly activated after Parkin deficiency, and myristic acid could significantly improve the activation of the NLRP3 inflammasome. These results indicate that myristic acid can rescue neuroinflammation caused by Parkin deficiency by upregulating seipin expression levels.

[0089] Example 10 The application of myristic acid in improving microglial activation induced by Parkin deficiency includes the following steps: To investigate the effect of intraperitoneal injection of myristic acid on microglia activation, Western blot analysis was performed using common microglia markers CD11β and IBA1, and the common microglia activation marker CD68. The Western blot results for microglia in this invention are as follows: Figure 20 As shown. The immunofluorescence staining results of the SNc region sections of mouse brain tissue in this invention are as follows. Figure 21 As shown. The results show Parkin + / - +Myristic acid mice and Parkin + / - Compared to mice, the expression levels of CD11β and IBA1 in the substantia nigra of the brain were significantly reduced, and the expression level of CD68 was also significantly decreased. To further elucidate the effects of myristic acid on Parkinson's disease... + / - The effects of M1 and M2 microglia subtypes on mouse brain were investigated using immunofluorescence staining with CD68, a common marker of microglia activation, and specific markers for M1 and M2. The fluorescence results showed that Parkin... + / - +Myristic acid mice and Parkin + / - Compared with mice, the expression levels of CD68 and CCR7 in the brain were significantly decreased, while the expression level of Arg1 was significantly increased. These results indicate that myristic acid can inhibit the activation of microglia and their transformation into pro-inflammatory M1 cells in the substantia nigra of mouse brain tissue induced by Parkin deficiency by upregulating seipin expression, and promote the differentiation of microglia into anti-inflammatory M2 cells.

[0090] Example 11 The application of myristic acid and seipin overexpression in improving lipid metabolism includes the following steps: 1. Functional enrichment analysis of core target genes Functional enrichment and signaling pathway enrichment analyses were performed on the previously obtained core target genes using R language and the Bioconductor package. The KEGG pathway enrichment results of this invention are as follows: Figure 22 As shown in the figure. The results show that among the three components of the GO enrichment analysis, the core target genes are closely related to lipid metabolism, and the PI3K / AKT signaling pathway, which is most prominent in KEGG enrichment, is also inseparable from lipid metabolism.

[0091] 2. Myristic acid and Seipin overexpression and lipid metabolism To investigate whether myristic acid and seipin overexpression could improve lipid metabolism and related signaling pathway abnormalities induced by Parkin deficiency in mice, Western blotting was performed using the adipogenesis marker PPARγ, the lipolysis marker ATGL, the fatty acid β-oxidation marker CPT1A, and markers related to the PI3K / AKT signaling pathway. The Western blotting results of lipid metabolism following myristic acid injection and seipin overexpression are as follows: Figure 23 As shown. The lipid metabolism fluorescence results of myristic acid injection and seipin overexpression in this invention are as follows. Figure 24 As shown in the results, the expression levels of PPARγ, ATGL, and CPT1A were significantly decreased in Parkin deficiency, and the expression and phosphorylation of p100α, p100δ, and AKT, which are related to the PI3K / AKT signaling pathway, were also significantly reduced. Treatment with myristic acid and overexpression of Seipin significantly upregulated the expression levels of PPARγ, ATGL, and CPT1A, and increased the phosphorylation and expression of p100α, p100δ, and AKT. The immunofluorescence staining results were consistent with the Western blot results. Furthermore, Bodipy staining of the SNc region of mouse brain tissue revealed that the number of lipid droplets was significantly reduced in the case of decreased expression levels of the lipid droplet synthesis protein Seipin due to Parkin deficiency. Treatment with both methods significantly increased lipid droplet synthesis.

[0092] The results showed that Parkin deficiency in mice resulted in reduced fat uptake, decreased lipid droplet synthesis (i.e., fat storage capacity), impaired lipolysis and β-oxidation, and inhibition of the PI3K / AKT signaling pathway. Treatment with myristic acid and overexpression of Seipin activated the PI3K / AKT signaling pathway, thereby improving fat uptake and storage capacity in Parkin-deficient mice, increasing lipolysis and β-oxidation, and alleviating lipid metabolism abnormalities in Parkin-deficient mice.

[0093] Example 12 The application of myristic acid and seipin overexpression in promoting myelin regeneration includes the following steps: To investigate whether myristic acid and seipin overexpression could improve demyelination caused by Parkin deficiency, we performed Western blotting using the oligodendrocyte marker Oligoglia 2 and the myelin-associated marker MBP. The Western blotting results of myristic acid and seipin upregulation promoting myelin regeneration in this invention are as follows: Figure 25 As shown. The fluorescence results of myristic acid and upregulation of seipin promoting myelin regeneration in this invention are as follows. Figure 26 As shown in the results, in the absence of Parkin, the expression levels of oligodendrocyte marker Olig2 and myelin-associated marker MBP were significantly decreased. Treatment with myristic acid and seipin overexpression significantly restored their expression levels. Further analysis of the expression levels of astrocyte markers GFAP, astrogenic neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, and their receptor Trkb revealed that in Parkin deficiency, the expression levels of GFAP, BDNF, and Trkb were all reduced, while GDNF expression increased sharply. After treatment, the expression levels of GFAP, BDNF, and Trkb recovered somewhat, while GDNF showed no significant change after myristic acid treatment. Seipin overexpression reduced GDNF expression. The immunofluorescence results were consistent with the Western blot results.

[0094] The above results indicate that Parkin gene deletion significantly reduces the number of oligodendrocytes and astrocytes in the substantia nigra of mouse brain tissue, decreases the release of brain-derived neurotrophic factor (BDNF), reduces the number of its receptors, and leads to increased myelin loss. Myristic acid and Seipin overexpression treatments significantly improved the reduction in oligodendrocyte and astrocyte numbers caused by Parkin deletion, increased BDNF release, and significantly upregulated its receptor expression, thereby rescuing myelin loss. However, in Parkin deletion, the expression level of BDNF was significantly increased, and myristic acid treatment did not significantly affect its expression level. This may be because in Parkin deletion, neurons are significantly damaged, and astrocytes compensatorily upregulate their ability to release GDNF to cope with neuronal damage, but this does not rescue dopaminergic neuron loss or myelin loss. After myristic acid treatment, the number of astrocytes increased, and the expression of GDNF and BDNF was also significantly increased, thus rapidly and effectively reducing dopaminergic neuron loss and promoting myelin regeneration. While overexpression of Seipin in neurons significantly increased the number of astrocytes, it did not significantly regulate the release of GDNF, but it did significantly increase the secretion of BDNF, thereby rescuing dopaminergic neuronal damage and myelin loss.

[0095] Conclusion 1. Overexpression of myristic acid and seipin can improve autophagy function and motor dysfunction. In the pathogenesis of neurodegenerative diseases, autophagy dysfunction is a crucial factor that cannot be ignored, with mitochondria playing a vital role. In the development and progression of Parkinson's disease, the Parkin gene has been shown to be closely related to the disease. As an important protein regulating autophagy, it is typically recruited to the mitochondrial membrane surface to exert its function. Studies have demonstrated that the loss of Parkin expression prevents the timely and effective clearance of damaged mitochondria, leading to autophagy dysfunction, damage to dopaminergic neurons, and ultimately, motor dysfunction.

[0096] Myristic acid, a 14-carbon long-chain fatty acid obtainable from food, has been previously studied to improve autophagy by inhibiting the cGAS-STING signaling cascade. Seipin, encoded by the BSCL2 gene, is a key protein located on the endoplasmic reticulum regulating lipid droplet synthesis. Loss of seipin expression leads to impaired lipid droplet synthesis, resulting in systemic lipodystrophy, which in turn causes mitochondrial dysfunction and autophagy impairment. Seipin knockout mice also exhibit age-related motor dysfunction and dopaminergic neuron loss. A study on cerebral ischemia-reperfusion injury found that overexpression of seipin improved neuronal apoptosis and autophagy impairment induced by cerebral ischemia-reperfusion. However, studies have not yet confirmed how seipin expression levels change in human PD patients and mouse PD models, whether there is an interaction between Parkin and seipin, or whether there is a molecular interaction between myristic acid and seipin. Whether myristic acid can mediate seipin's improvement of neuronal autophagy and thus rescue motor dysfunction in the absence of Parkin remains unknown.

[0097] By analyzing the transcriptomes of human PD patients and mouse PD models, we discovered for the first time that the expression level of seipin is significantly downregulated in Parkinson's disease. Immunoprecipitation and immunofluorescence co-localization analyses revealed a significant protein-protein interaction between Parkin and seipin, positively correlated with Parkin expression. Further drug target screening and molecular docking assays showed that seipin is one of the core targets of myristic acid and Parkinson's disease, and that a strong molecular interaction exists between myristic acid and seipin. To further clarify the effects of myristic acid and seipin overexpression on autophagy and motor function in Parkin gene knockout PD mouse models, we investigated the effects of myristic acid and seipin overexpression on autophagy and motor function in Parkin gene knockout PD mice. + / -Mice were injected intraperitoneally with myristic acid and stereotactically injected into the brain via bilateral lateral ventricles with adeno-associated virus overexpressing Seipin. The results showed that Seipin expression was upregulated and P62 expression was significantly reduced after myristic acid treatment. LC3B and Beclin1 expression levels were increased. Furthermore, myristic acid treatment could upregulate the reduced Seipin expression caused by Parkin deficiency, ultimately improving autophagy function and rescuing motor dysfunction.

[0098] 2. Overexpression of myristic acid and seipin can improve neuroinflammation and lipid metabolism abnormalities and promote myelin regeneration. In current research on neurodegenerative diseases, lipid metabolism and myelin are increasingly playing a crucial role in maintaining the normal physiological functions of the nervous system. Abnormal lipid metabolism is closely related to the pathogenesis of various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Previous studies have shown that lipid accumulation and reduced fat degradation in AD model mice lead to lipid metabolism disorders, which further exacerbate neuroinflammatory responses, ultimately resulting in increased myelin fragmentation (PMID: 41162676). In the pathogenesis of Parkinson's disease, chronic neuroinflammation has received increasing attention as a persistent driving factor, with the role of the NLRP3 inflammasome being extensively studied. Our previous research found that Parkin deficiency induces rapid assembly and activation of the NLRP3 inflammasome, which then cleaves IL-1β and IL-18 precursors, converting them into active forms and exerting pro-inflammatory effects. Microglia, as the main immune cells in the central nervous system, are activated when myelin fragmentation increases, leading to the phagocytosis of myelin fragments. However, excessive fragmentation can also increase microglia activation, exacerbating neuroinflammatory responses. In existing studies, myristic acid has been shown to exert anti-inflammatory effects and participate in lipid metabolism. Seipin knockout mouse models have shown that the absence of seipin expression induces neuroinflammatory responses, lipid metabolism disorders, and myelin loss, thereby impairing cognitive and memory function in mice. However, no studies have yet demonstrated whether overexpression of myristic acid and seipin in Parkin deficiency can improve neuroinflammatory and lipid metabolism abnormalities, thereby reducing myelin fragmentation and myelin regeneration.

[0099] We used Western blotting and immunofluorescence to detect the expression levels of neuroinflammatory markers such as NLRP3, ASC, and Caspase-1 at the cellular and tissue levels after treatment with myristic acid and seipin overexpression. We found that both myristic acid and seipin inhibited neuroinflammation caused by Parkin deficiency and suppressed microglial activation, promoting the transformation of microglia from M1 to M2. Further analysis of lipid metabolism markers PPARγ, ATGL, and CPT1A, myelin-related marker MBP, and changes in related pathways revealed that both treatments improved lipid metabolism disorders caused by Parkin deficiency, activated the PI3K / AKT lipid pathway, and further promoted oligodendrocyte and myelin regeneration, providing a potential drug target for the treatment of Parkin-related neuropathic disorders (PD). However, whether myristic acid directly regulates seipin expression and exerts a series of effects, and whether there are other interactions between Parkin and seipin, require further investigation.

[0100] In summary, the experimental results indicate that the expression level of seipin plays a crucial role in the pathogenesis of Parkinson's disease (PD). Intraperitoneal injection of myristic acid and overexpression of seipin significantly alleviated autophagy dysfunction, neuroinflammation, lipid metabolism abnormalities, and myelin loss induced by Parkin deficiency, thereby rescuing dopaminergic neuron damage and improving motor dysfunction in PD mice. We are the first to discover a significant association between Parkin and seipin in the substantia nigra (SNc) tissue of the mouse brain, and that seipin expression significantly decreased in SNc tissue after Parkin deficiency. More importantly, we also found that myristic acid and seipin have a molecular interaction and can mediate the therapeutic effect of seipin.

[0101] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0102] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention is also intended to include such modifications and variations.

Claims

1. A biological agent for improving Parkinson's disease and overexpressing Seipin, characterized in that, The biological agent for overexpressing Seipin is myristic acid or a combination of myristic acid and a Seipin expression promoter. In the composition of myristic acid and seipin expression promoter, the mass ratio of myristic acid to seipin expression promoter is 1:1; The nucleotide sequence of the seipin is shown in SEQ ID NO1; The Seipin expression promoter is a biological product containing the nucleotide sequence SEQ ID NO1.

2. The biological agent for improving Parkinson's disease and overexpressing Seipin according to claim 1, characterized in that, The seipin expression promoter is an overexpression vector containing the nucleotides shown in SEQ ID NO.1 or a lentivirus containing the overexpression vector.

3. The use of the biological agent for improving Parkinson's disease and overexpressing Seipin as described in claim 1 in the preparation of a drug for improving Parkinson's disease.

4. The use of the biological agent overexpressing Seipin for improving Parkinson's disease according to claim 3 in the preparation of a drug for improving Parkinson's disease, characterized in that, The drug uses a biological agent that overexpresses Seipin as its sole active ingredient.

5. The use of the biological agent overexpressing Seipin for improving Parkinson's disease according to claim 3 in the preparation of a drug for improving Parkinson's disease, characterized in that, The drug is made from a biological agent that overexpresses Seipin and pharmacologically acceptable excipients.

6. The use of the biological agent overexpressing Seipin for improving Parkinson's disease according to claim 5 in the preparation of a drug for improving Parkinson's disease, characterized in that, When the drug is myristic acid, the excipients are dimethyl sulfoxide, polyethylene glycol 300, and Tween 80.

7. The use of a biological agent overexpressing Seipin according to claim 5 in the preparation of a drug for improving Parkinson's disease, characterized in that, When the drug is a Seipin expression promoter, the excipient is a sterile buffer solution.

8. The application of the biological agent overexpressing Seipin according to claim 3 in the preparation of a drug for improving Parkinson's disease, characterized in that, The dosage form of the drug is a solution, injection, or syrup.

9. The application of a biological agent overexpressing Seipin according to claim 3 in the preparation of a drug for improving Parkinson's disease, characterized in that, The drug has at least one of the following uses: (1) Treatment of nerve inflammation; (2) Treatment of lipid metabolism disorders; (2) Promotes myelin regeneration; (3) Treatment of motor dysfunction.

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