Application of ACSL1 in alleviating and treating neurodegenerative diseases

Abstract

The present invention provides the use of ACSL1 in alleviating and treating neurodegenerative diseases. By screening differentially expressed genes in microglia from patients with Parkinson's disease (PD) and healthy controls, the authors identified the most critical disease-associated gene, ACSL1. Furthermore, the authors conducted multiple loss-of-function and gain-of-function experiments on ACSL1. The results demonstrated that ACSL1, located in the endoplasmic reticulum, promotes lipid droplet accumulation, thereby exacerbating microglial activation and dopamine neuron death. Modulating ACSL1 to alter lipid droplet biosynthesis may be a potential strategy for treating neuroinflammation in PD patients and inhibiting the pathological progression of PD.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of ACSL1 in alleviating and treating neurodegenerative diseases. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Its primary symptoms include tremor, rigidity, bradykinesia, and gait abnormalities. Its exact pathogenic mechanisms are complex, and currently there is no effective treatment to slow or prevent disease progression. In recent years, microglia have transitioned from a supporting role in the nervous system to a relatively important role in the development and progression of PD. Following the onset of neurodegenerative signals, microglia, which possess immune surveillance capabilities, become activated, migrate, and aggregate in the substantia nigra, transitioning to an abnormally active pathological state. These cells promote the spread of oligomeric α-synuclein (α-Syn), release toxic compounds, and induce abnormal synaptic pruning. These microglia are potential target cells for the early diagnosis and treatment of PD.

[0003] Recent studies based on single-cell sequencing and multi-omics have shown that microglia in the process of neurodegenerative diseases are highly heterogeneous and molecularly diverse, and are closely related to disease progression, such as TREM2 in Alzheimer's disease. high 、CSPG4 high and GALECTIN-3 high Subpopulation, TDP-43 in traumatic brain injury high 、GPR84 high subpopulations, and aging-related p16 high Therefore, interventions targeting microglial subpopulations with specific molecular markers could potentially improve existing treatments and halt the progression of neurodegenerative diseases. However, whether microglial subpopulations characterized by high expression of specific proteins in PD have distinct functional subtypes remains to be fully elucidated. Understanding the functions and formation mechanisms of specific PD-associated microglial subpopulations will provide important theoretical insights into the underlying mechanisms of neuroinflammation in PD.

[0004] Phenotypic shifts in microglial subpopulations during different disease processes are mediated by cellular metabolic reprogramming. Studies have shown that metabolic pathways are significantly altered in activated microglia and are central to controlling microglial inflammatory responses. Lipid metabolism is particularly involved in controlling microglial activation and effector functions, such as migration, phagocytosis, and inflammatory signaling. Pathologically activated microglia exhibit lipid metabolic imbalances. Microglial subpopulations that accumulate lipid droplets (LDs) exhibit impaired phagocytosis, resulting in high levels of reactive oxygen species (ROS) and excessive secretion of proinflammatory cytokines, exacerbating neuroinflammation in aging and neurodegenerative diseases such as Alzheimer's disease. LDs are globular organelles with a core composed of neutral lipids, such as triglycerides and sterol esters. LDs are not simple lipid depots but rather complex organelles with diverse cellular functions. Recent studies have found that microglial subpopulations with LDs accumulate in the substantia nigra of patients with Parkinson's disease (PD), suggesting that LD-accumulating microglia may also contribute to the pathological progression of PD. However, the function and formation mechanism of LDs-accumulating microglia in PD are still unclear. Elucidating the biological processes involved is of great significance for exploring new therapeutic targets. Summary of the Invention

[0005] The present invention aims to alleviate and / or treat neurodegenerative diseases.

[0006] In a first aspect, the present invention provides an inhibitory ACSL1 Use of a substance that inhibits gene expression and / or a substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum in the preparation of a medicament for alleviating and / or treating neurodegenerative diseases, wherein the substance that inhibits ACSL1 gene expression and / or the substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum is selected from at least one of A1) to A10):

[0007] A1) Targeted Inhibition ACSL1 RNA molecules expressed by genes;

[0008] A2) Targeted Inhibition Nrdp1 RNA molecules expressed by genes;

[0009] A3) Targeted Inhibition P65 RNA molecules expressed by genes;

[0010] A4) a DNA molecule encoding any one of the RNA molecules A1) to A3);

[0011] A5) an expression cassette containing the DNA molecule described in A4);

[0012] A6) a recombinant vector containing the DNA molecule described in A4), or a recombinant vector containing the expression cassette described in A5);

[0013] A7) a recombinant microorganism containing the DNA molecule described in A4), or a recombinant microorganism containing the expression cassette described in A5), or a recombinant microorganism containing the recombinant vector described in A6);

[0014] A8) A modified RNA molecule described in A1), or a pharmaceutically acceptable salt thereof;

[0015] A9) Substances that inhibit TBK1 protein activity;

[0016] A10) NF-κB pathway activity inhibitors.

[0017] In the present invention, ACSL1 The gene is a gene encoding long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 Substances that inhibit gene expression may be, for example, ACSL1 Encoding genes or suppressing ACSL1 Encoding gene-related gene expression substances to reduce the cell ACSL1 The substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum refers to a substance that inhibits the localization and accumulation of ACSL1 protein in the endoplasmic reticulum.

[0018] In the present invention, neurodegenerative diseases are characterized by the gradual loss of neurons, resulting in functional impairment. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), multiple sclerosis (MS), spinal muscular atrophy (SMA), and other types of neurodegenerative diseases. In a specific embodiment, the neurodegenerative disease is Parkinson's disease.

[0019] In the present invention, Nrdp1 The gene encoding neuregulin receptor degradation protein 1 (NRP1) inhibits K63 ubiquitination of TANK binding kinase 1 (TBK1). ACSL1Gene expression and endoplasmic reticulum localization. The p65 gene is NF-κB p65, which is an important component of the nuclear factor κB (NF-κB) signaling pathway. NF-κB p65 is a transcription factor. In the NF-κB signaling pathway, p65 is usually bound to the IκB protein in the cytoplasm. When the cell receives an activation signal, the IκB protein is phosphorylated and degraded, releasing p65 and translocating it to the nucleus. p65 can act as a promoter of ACSL1 in PD microglia, thereby activating downstream genes. ACSL1 Gene expression.

[0020] In the present invention, the ACSL1 Gene, Nrdp1 Genes and P65 Genes refer to genes in animals, more specifically genes in mammals. The mammals are selected from bovines, equines, felines, canines, lagomorphs, porcines, camelids, rodents, and primates, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, and gorillas), and humans; preferably, rats, mice, and humans.

[0021] Human ACSL1 The gene ID is 2180 (updated on September 19, 2024), ACSL1 The Gene ID of the gene is 14081 (updated on September 18, 2024), the human Nrdp1 The Gene ID of the gene is 10193 (updated on September 19, 2024), and the Gene ID of the human P65 gene is 5970 (updated on September 19, 2024).

[0022] In a specific embodiment, in A1), the RNA molecule is siRNA or shRNA. ACSL1 Gene, Nrdp1 Genes and P65 The present invention designs siRNA that targets and inhibits the expression of the above genes. ACSL1 The siRNA for gene expression is selected from one of A11)-A12):

[0023] A11) Targeted Inhibition ACSL1 The siRNA for gene expression comprises a first strand and a second strand that are complementary to each other in at least partial regions, wherein the nucleotide sequence of the first strand is SEQ ID NO: 1, and the nucleotide sequence of the second strand is SEQ ID NO: 2;

[0024] A12) Targeted Inhibition ACSL1 The siRNA for gene expression comprises a third strand and a fourth strand that are complementary to each other in at least a portion of their regions, wherein the nucleotide sequence of the third strand is SEQ ID NO: 3, and the nucleotide sequence of the fourth strand is SEQ ID NO: 4;

[0025] SEQ ID NO: 1: 5'-GGAAAGUCCGGCUGAUGAUTT-3';

[0026] SEQ ID NO: 2: 5'-AUCAUCCACGGACUUUCCTT-3';

[0027] SEQ ID NO: 3: 5'-GGAAGGAAGCCGGUCUGAATT-3';

[0028] SEQ ID NO:4: 5'-UUCAGACCGGCUUCCUUCCTT-3'.

[0029] Specifically, targeted inhibition Nrdp1 The gene-expressed siRNA comprises a fifth strand and a sixth strand that are complementary to each other in at least a portion of their regions. The nucleotide sequence of the fifth strand is SEQ ID NO: 5, and the nucleotide sequence of the sixth strand is SEQ ID NO: 6.

[0030] SEQ ID NO:5: 5'-CCUUCAGAACCUGGAGGAGTT-3';

[0031] SEQ ID NO:6: 5'-CUCCUCCAGGUUCUGAAGGTT-3'.

[0032] Specifically, the siRNA targeting and inhibiting the expression of the P65 gene is selected from one of A31)-A32):

[0033] A31) The siRNA targeting and inhibiting P65 gene expression comprises a seventh strand and an eighth strand that are at least partially complementary to each other, wherein the nucleotide sequence of the seventh strand is SEQ ID NO: 7, and the nucleotide sequence of the eighth strand is SEQ ID NO: 8;

[0034] A32) The siRNA targeting and inhibiting the expression of the P65 gene comprises a ninth strand and a tenth strand that are at least partially complementary to each other, wherein the nucleotide sequence of the ninth strand is SEQ ID NO: 9, and the nucleotide sequence of the tenth strand is SEQ ID NO: 10.

[0035] SEQ ID NO:7: 5'-AGAAGACAUUGAGGUGUAUTT-3';

[0036] SEQ ID NO:8: 5'-AUACACCUCAAUGUCUUCUTT-3';

[0037] SEQ ID NO:9: 5'-GAAGAAGAGUCCUUUCAAUTT-3';

[0038] SEQ ID NO: 10: 5'-AUUGAAAGGACUCUUCUUC-3'.

[0039] Understandably, the suppression ACSL1 The substance for gene expression and / or the substance for inhibiting the localization of ACSL1 protein in the endoplasmic reticulum may also include a DNA molecule encoding the above RNA molecule, an expression cassette including the above DNA molecule, a recombinant vector or a recombinant microorganism.

[0040] In one embodiment, the RNA molecule can be obtained by cloning the double-stranded DNA template sequence corresponding to the RNA molecule into the promoter of RNA polymerase III in a vector, thereby expressing the desired RNA molecule in vivo. In addition, it is understood that the RNA molecule can also be obtained by in vitro synthesis.

[0041] In the present invention, the term "modified RNA molecule" refers to a product obtained by modifying the RNA molecule. Various modification methods may be used, including at least one selected from ribose modification, base modification, and phosphate backbone modification. In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that, within the scope of sound medical judgment, is suitable for contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are selected from those generally known in the art.

[0042] In the present invention, inhibition ACSL1 Substances that inhibit gene expression and / or ACSL1 protein localization in the endoplasmic reticulum can also be substances that inhibit TBK1 protein activity. TBK1 is a TANK-binding kinase 1 protein. TBK1 acts as a scaffold protein to facilitate the ACSL1 subcellular localization circuit. Inhibiting TBK1 activity can mitigate PD pathological progression by inhibiting ACSL1 accumulation in the endoplasmic reticulum. In one specific embodiment, the substance that inhibits TBK1 protein activity is GSK8612, whose CAS number is 2361659-62-1.

[0043] In the present invention, inhibition ACSL1The substance that inhibits gene expression and / or the substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum may also be an NF-κB pathway activity inhibitor. The NF-κB pathway activity inhibitor may be JSH-23, and the CAS number of JSH-23 is 749886-87-1.

[0044] In the present invention, inhibition ACSL1 The substance that regulates gene expression and / or inhibits the localization of ACSL1 protein in the endoplasmic reticulum is used to alleviate and / or treat neurodegenerative diseases, specifically in at least one of the aspects B1) to B5):

[0045] B1) Reduce the pro-inflammatory response of microglia;

[0046] B2) inhibit excessive accumulation of lipid droplets in microglia;

[0047] B3) weaken PD neuroinflammation;

[0048] B4) attenuates microglial dopamine neurotoxicity;

[0049] B5) Inhibits dopamine neuron death in PD progression.

[0050] In a second aspect, the present invention provides an inhibitory ACSL1 Use of a substance that regulates gene expression and / or a substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum in any of aspects C1) to C5):

[0051] C1) Use in the preparation of a product for reducing the pro-inflammatory response of microglia;

[0052] C2) application in the preparation of a product for inhibiting excessive accumulation of lipid droplets in microglia;

[0053] C3) Use in the preparation of a product that reduces neuroinflammation in PD;

[0054] C4) Use in the preparation of a product for attenuating dopamine neurotoxicity in microglia;

[0055] C5) Use in the preparation of a product for inhibiting dopamine neuron death in the progression of PD.

[0056] In a third aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned inhibitors ACSL1 A substance that regulates gene expression and / or a substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum and a pharmaceutically acceptable carrier.

[0057] In the present invention, a pharmaceutically acceptable carrier can be a substance used to help the substance effectively enter cells and exert its effect. When the substance is an RNA molecule, it can be a lipid carrier, specifically liposomes, dendrimers, cationic liposomes, etc.; another example can be a polymer carrier, specifically polyamidoamine dendrimers (PAMAM), polylactic-co-glycolic acid (PLGA), polyethyleneimine (PEI), etc.; another example can be inorganic nanoparticles, specifically gold nanoparticles, mesoporous silica nanoparticles (MSNP), etc.; another example can be nanocapsules and polymer micelles. The specific method can be determined based on factors such as the therapeutic purpose, the stability of the RNA molecule, the desired targeting, and the route of administration.

[0058] In the present invention, the pharmaceutical composition can be prepared into various dosage forms, including conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. In one embodiment, the dosage form is an injection.

[0059] In a fourth aspect, the present invention provides a method for constructing a recombinant cell, the method comprising introducing any of the above-mentioned inhibitors into a recipient cell. ACSL1 The recombinant cell is obtained by expressing a substance that inhibits gene expression and / or a substance that inhibits the subcellular localization of ACSL1 protein.

[0060] In a fifth aspect, the present invention provides a method for alleviating and / or treating neurodegenerative diseases, comprising administering to a subject a therapeutically effective amount of any of the above-mentioned inhibitors ACSL1 A substance that inhibits gene expression and / or a substance that inhibits the localization of ACSL1 protein in the endoplasmic reticulum.

[0061] In the present invention, a subject refers to a subject suffering from a neurodegenerative disease, and clinical manifestations include memory impairment, cognitive dysfunction, motor dysfunction, speech disorders, sleep disorders, dysphagia, muscle atrophy and weakness, convulsions and dystonia, autonomic dysfunction, dementia, etc. The effect of the above-mentioned method for alleviating and / or treating a neurodegenerative disease is manifested as any one of B1) to B5):

[0062] B1) Reduce the pro-inflammatory response of microglia;

[0063] B2) inhibit excessive accumulation of lipid droplets in microglia;

[0064] B3) weaken PD neuroinflammation;

[0065] B4) attenuates microglial dopamine neurotoxicity;

[0066] B5) Inhibits dopamine neuron death in PD progression.

[0067] In the present invention, the subject can be a mammal, which can be selected from bovines, equines, felines, canines, lagomorphs, porcines, camelids, rodents and primates, including but not limited to cows, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, orangutans) and humans, preferably rats, mice and humans.

[0068] inhibition ACSL1 The dosage of the substance that inhibits gene expression and / or the substance that inhibits ACSL1 protein subcellular localization varies depending on the mode of administration, route of administration, age and / or weight of the individual, and the condition of the individual being treated, and is ultimately determined by the attending physician.

[0069] The present invention screens the differentially expressed genes in microglia of PD patients and normal healthy individuals and selects the most critical disease-related gene ACSL1. ACSL1 Multiple loss-of-function and gain-of-function experiments were performed on the gene, and the results showed that the ACSL1 protein located in the endoplasmic reticulum promoted the accumulation of lipid droplets, thereby exacerbating microglial activation and dopamine neuron death. Manipulating the biosynthesis of lipid droplets by regulating ACSL1 may be a potential strategy to treat neuroinflammation in PD patients and inhibit the progression of PD pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 snRNA-seq reveals the unique transcriptomic characteristics of ACSL1high glial cells in the substantia nigra of PD patients; among them, a is a schematic diagram showing the dimensionality reduction of cell types in the substantia nigra region of normal people (HC) and PD patients (PD) using UMAP, different colors represent different cell types, and each point represents a cell; b is a volcano plot showing the significantly differentially expressed genes in microglia of PD patients and healthy people, and the dotted line indicates the adjusted p-value < 0.05 as the cutoff value; c is the result of further screening of significantly differentially expressed genes based on WGCNA; d is the feature genes further obtained from the WGCNA brown module using the RFE method; e is the ROC curve obtained after calculation of significantly differentially expressed genes by different machine learning models; f is a bee colony plot showing the ranking of significantly differentially expressed genes obtained from the SV algorithm; g is the expression of ACSL1 in microglia of healthy people (HC) and PD patients (PD), each point represents a cell, and the darker the color, the higher the expression level; h is the cell immunofluorescence image of the substantia nigra region of LPS-PD model mice and control mice induced by LPS, the scale is 20 μm; i is the percentage of ACSL1-positive microglia in the substantia nigra of LPS-PD model mice and control mice induced by LPS, *** indicates pLess than 0.001; j is the division of microglia in the substantia nigra of PD patients into ACSL1-high and ACSL1-low subpopulations based on unsupervised clustering and ACSL1 expression levels; k is the functional analysis result of ACSL1-high microglia using GO-BP (Gene Ontology Biological Process); l is the functional analysis result of ACSL1-high microglia using KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0071] Figure 2The effect of knocking down ACSL1 expression on microglia-mediated neuroinflammation; a is the relative mRNA expression of proinflammatory cytokines IL-6, IL-1β and TNFα after primary microglia transfected with NC or si-ACSL1 were treated with or without LPS; b is the concentration of proinflammatory cytokines IL-6, IL-1β and TNFα in the culture medium after primary microglia transfected with NC or si-ACSL1 were treated with or without LPS; c is the concentration of NO in the culture medium after primary microglia transfected with NC or si-ACSL1 were treated with or without LPS; d is Figure 3 ROS fluorescence intensity of cells after primary microglia transfected with NC or si-ACSL1 were treated with or without LPS; e is the protein expression levels of iNOS and COX2 in cells after primary microglia transfected with NC or si-ACSL1 were treated with or without LPS, with β-actin as the loading control; f is the cell culture medium after primary microglia transfected with NC or si-ACSL1 were treated with or without LPS. After SH-SY5Y cells were cultured for 24 hours, the expression of PARP, Cleaved Protein levels of PARP, caspase3, and cleaved caspase3, with β-actin as a loading control; g is the TUNEL staining result of SH-SY5Y cells after culturing SH-SY5Y cells with or without LPS in the cell culture medium of primary microglia transfected with NC or si-ACSL1 for 24 hours, with a scale bar of 50 μm; h is the quantitative analysis result of TUNEL-positive cells corresponding to g; i is a schematic diagram of the animal experiment process; j is a localization map of microglia in the substantia nigra region of mice, with a scale bar of 20 μm; k is the Western blot result of ACSL1 in the substantia nigra tissue of mouse brain; l is the quantitative result of ACSL1 expression in the substantia nigra tissue of mouse brain, with β-actin as a control; m is a representative image of IBA1+ cells in the substantia nigra region of mice in the NC+PBS, NC+LPS, shACSL1#1+LPS, and shACSL1#2+LPS groups, with a scale bar of 200 μm; n is the representative images of TH+ cells in the substantia nigra of mice in the NC+PBS, NC+LPS, shACSL1#1+LPS, and shACSL1#2+LPS groups, the scale bar is 200 μm, and o is the quantitative analysis result corresponding to n.

[0072] Figure 3ACSL1 knockdown inhibits lipid droplet synthesis; a is Rectome enrichment to visualize the metabolic pathway of microglia with high ACSL1 expression; b is a representative multiplex IHC image of the substantia nigra region of brain tissue of normal mice and LPS-PD model mice, yellow represents ACSL1, green represents lipid droplet-associated protein PLIN2, red represents microglial marker IBA1, and blue represents DAPI; c is BV-2 cells transfected with si-ACSL1#1, si-ACSL1#2 and control small interfering RNA (NC) respectively, and then Representative micrographs of BODIPY-stained BV2 cells after 18 h of treatment with LPS or LPS; d is the statistical result corresponding to c; e is a schematic diagram of LD structural composition, and the colored objects represent LD surface-binding proteins localized to the phospholipid monolayer; f is a representative confocal image of BV-2 cells after si-ACSL1#1, si-ACSL1#2, and control small interfering RNA (NC) were transfected into BV-2 cells, followed by LPS treatment and combined treatment with chloroquine; g is a Western blot evaluation of p62 and LC3B in BV-2 cells in f. hi is the transcriptional level detection results of lipolysis and lipid uptake related indicators in treated BV-2 cells; j is the co-localization result of BODIPY (green) and ACSL1 (red) in BV-2 cells; k is the expression level of ACSL1 in the mitochondria (mt) and cytoplasmic components (cyto) of BV-2 cells evaluated by Western blotting; l is the expression level of ACSL1 in the endoplasmic reticulum (ER) of BV-2 cells evaluated by Western blotting; m is the confocal microscopy analysis of ACSL1 (green) and MitoTracker (red) in BV-2 cells, and the fluorescence confocal microscopy was quantified using ImageJ.

[0073] Figure 4ACSL1 promotes the proinflammatory phenotype of microglia by increasing lipid droplet accumulation; wherein, a is a schematic diagram of the construction of ACSL1-WT and ACSL1-MN; b is the expression of exogenous ACSL1 detected by Western blotting analysis after BV-2 cells were transfected with ACSL1-WT and ACSL1-MN; c is the result of immunofluorescence analysis of BV-2 cells transfected with ACSL1-WT or ACSL1-MN; d is a representative micrograph of BODIPY-stained microglia after BV2 cells transfected with ACSL1-WT or ACSL1-MN were treated with PBS or LPS for 18 h; e is the BODIPY fluorescence quantification result of each cell corresponding to d; f is the relative levels of IL-6, IL-1β and TNFα mRNA in primary microglia transfected with ACSL1-WT or ACSL1-MN after LPS treatment; g is the concentration of IL-6, IL-1β and TNFα corresponding to f; h is the NO of cells corresponding to f. concentration; i is the relative ROS fluorescence of the cells corresponding to f; j is the expression of iNOS and COX2 of the corresponding cells; k is primary microglia transfected with ACSL-WT or ACSL1-MN and exposed to LPS for 24 hours, and then the conditioned medium of different groups was collected to culture SH-SY5Y cells for 24 hours, and the SH-SY5Y cells were subjected to immunoblotting analysis to evaluate the protein levels of PARP, cleaved PARP, caspase3, cleaved caspase3 and β-actin; l is the TUNEL staining results of SH-SY5Y cells after culturing SH-SY5Y cells with the cell culture medium after primary microglia transfected with ACSL-WT or ACSL1-MN were treated with or without LPS for 24 hours, the scale bar is 50 μm; m is the quantitative analysis result of TUNEL-positive cells corresponding to l.

[0074] Figure 5 To inhibit TBK1 activity, we weakened the accumulation of lipid droplets caused by ACSL1. (a) Schematic diagram of the interaction between TBK1 and ACSL1. The upper figure shows resting microglia, and the lower figure shows activated microglia. (b) Western blot analysis of p-TBK1 in BV-2 cells treated with PBS or LPS for 12 hours and then treated with GSK8612 in DMSO or DMSO for 6 hours. S172and TBK1 expression levels; c is the expression level of ACSL1 in the mitochondrial and cytoplasmic parts of the cells after treatment using the method in b, with Tomm20 as a mitochondrial marker; d is a representative micrograph of cells after treatment using the method in b; e is the statistical results of lipid droplets in cells after treatment using the method in b; g is a representative micrograph of BODIPY-stained BV-2 cells transfected with or without GSK8612 and LPS treatment; h is the statistical results of lipid droplets in cells after treatment using the method in g; i is Western blot detection of iNOS, COX2, and p-TBK1 in BV-2 cells S172 、TBK1、p-p65 S536 , and the expression levels of p65; j is the relative mRNA levels of proinflammatory factors IL-6, IL-1β, and TNFα in cells after treatment using the method in g.

[0075] Figure 6 ACSL1 promotes TBK1 activation through Nrdp1-mediated K63 ubiquitination; a is a protein immunoblot analysis of BV-2 cells overexpressing ACSL1 (WT or MN); b is an immunoprecipitation analysis of BV-2 cells overexpressing ACSL1-WT; c is an immunoprecipitation analysis of BV-2 cells with si-ACSL1 knockdown after LPS treatment; d is an immunoprecipitation analysis of BV-2 cells overexpressing ACSL1-WT, ACSL1-MN, TBK1-WT and ubiquitin proteins (WT, K48 and K63); e is an immunoprecipitation analysis of BV-2 cells overexpressing ACSL1-WT, ACSL1-MN, TBK1-WT and ubiquitin protein-K63; f is an immunoprecipitation analysis of ACSL1, Nrdp1, Schematic diagram of the formation of the MIB1 and TBK1 complex; g is immunoblot analysis of BV-2 cells overexpressing ACSL1-WT or silencing Nrdp1; h is immunoprecipitation analysis of BV-2 cells overexpressing ACSL1-WT or silencing Nrdp1; i is the number of lipid droplets and the relative mRNA expression levels of IL-6, IL-1β, and TNFα in microglia stimulated with LPS PBS solution transfected with NC or siNrdp1 or in control cells.

[0076] Figure 7P65 acts as the promoter of ACSL1 to regulate the expression of ACSL1; b is the expression of ACSL1 detected by RT-qPCR after BV-2 cells were cultured with TNFα for different time periods; c is the protein immunoblot analysis after BV-2 cells were treated with TNFα for different time periods; d is the protein immunoblot analysis result of BV-2 cells transfected with si-p65 and treated with TNFα; e is the RT-qPCR analysis result of the cells corresponding to d; f is the immunoblot analysis result of BV-2 cells treated with TNFα alone or in combination with JSH-23; g is the RT-qPCR analysis result of the cells corresponding to f.

[0077] Figure 8 To inhibit TBK1 activity, ACSL1 can be used to suppress PD neuroinflammation and pathological progression; a is a schematic diagram of the animal experiment process; b is a representative image of IBA1+ cells in the substantia nigra region of mice in the PBS+solvent group, LPS+solvent group, PBS+GSK8612 group, and LPS+GSK8612 group; c is a representative image of TH+ cells in the substantia nigra region of mice in the PBS+solvent group, LPS+solvent group, PBS+GSK8612 group, and LPS+GSK8612 group; d is the quantitative statistical result corresponding to c; e is the p-TBK1 in the substantia nigra region of mice in the PBS+solvent group, LPS+solvent group, PBS+GSK8612 group, and LPS+GSK8612 group S172 、TBK1、p-p65 S536 , p65 and PLIN3 expression results; f is the quantitative statistical results corresponding to e.

[0078] The above data are shown as the mean ± SEM of three independent experiments, *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05, and ns indicates no significant difference. DETAILED DESCRIPTION

[0079] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0080] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0081] Male C57BL / 6 mice (6-8 weeks old) used in the following examples were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and housed individually under a 12-hour light-dark cycle at 22 ± 1°C and a relative humidity of 50-60%, with free access to food and water. This study was approved by the Ethics Committee of the Beijing Neurosurgical Institute, Capital Medical University (Protocol No. BNI202308002).

[0082] Example 1 Screening and analysis of genes significantly associated with PD patients

[0083] 1.1. Determine differentially expressed genes based on the transcriptional status of microglia in PD patients and normal subjects.

[0084] The mononuclear cell transcriptome data of the substantia nigra region of 5 PD patient samples and 6 healthy control samples from the public database (GSE157783) were analyzed. The main cell types in the substantia nigra region were determined based on the mononuclear cell transcriptome data, and the data were processed by UMAP for dimensionality reduction. Figure 1 The dimensionality reduction diagram shown in a is based on Figure 1 As shown in Figure a, the cell types in normal people (HC) and PD patients (PD) mainly include oligodendrocytes, astrocytes, microglia, oligodendrocyte precursor cells (OPCs), endothelial cells, pericytes, inhibitory neurons (GABA), ependymal, dopamine neurons (DaNs), excitatory neurons (Excitatory), and other types of cells (Multi-cells), and the abundance of microglia in PD patients is significantly higher than that in the normal group.

[0085] Then, using the Seurat and dplyr packages in R language, we analyzed and visualized the significantly differentially expressed genes in microglia of 5 PD patient samples and 6 healthy control samples. The analysis found that a total of 2235 genes were significantly different between the two population samples ( P <0.05), such as Figure 1As shown in b. Weighted gene correlation network analysis (WGCNA analysis) was used to analyze the top 25% genes with significant differences. The analysis results are shown in Figure 1 As shown in c. Figure 1 As shown in Figure 3, the brown module shows that there are 179 genes that are highly associated with healthy samples and PD patients (r=0.44, P <0.001), these genes are closely related to immune response, phagocytosis and lipid metabolism functions and pathways.

[0086] The RFE algorithm was used to further screen out characteristic genes from the 179 genes shown in the WGCNA brown module. The analysis results are shown in the figure below. Figure 1 As shown in d. Figure 1 As shown in Figure d, 110 characteristic genes were identified.

[0087] The 179 genes selected by the RFE algorithm were learned using Gaussian naive Bayes (GNB), logistic regression (LR), K-nearest neighbor (KNN), support vector machine (SVM), random forest (RF), decision tree (DT), gradient boosting (GBDT), adaptive boosting (AdaBoost), eXtreme Gradient Boosting (XGB), light gradient boosting machine (LGBM), categorical boosting (CatBoost), bootstrap aggregating (bagging), soft voting (SV) and stacked generalization (stacking) algorithms to obtain prediction results. The following is constructed based on the prediction results of the above algorithms and the actual results. Figure 1 The ROC curve shown in e. Figure 1 As can be seen from Figure 5, the SV algorithm has the highest AUC value of 0.8676.

[0088] SHAP (Shapley additive explanations) is used to analyze the corresponding features in the model constructed by the SV algorithm. The analysis results are as follows: Figure 1 As shown in middle f, it can be seen that ACSL1 has the greatest impact on the model output results, indicating that the high expression of ACSL1 is a key gene for the activation of microglia in PD patients.

[0089] The Seurat package was used to detect and visualize the expression levels of the ACSL1 gene in microglia of healthy subjects and PD patients. Figure 1 As shown in middle g, it can be seen that the positive rate and expression level of ACSL1 in microglia of PD patients are significantly increased.

[0090] 1.2、 ACSL1 Gene verification.

[0091] LPS (Lipopolysaccharides) was used to induce mice to construct a PD model. The specific steps included: mixing 0.5 μg of LPS with 1 μl of normal saline to prepare an LPS injection solution, anesthetizing the mice with 0.3% sodium pentobarbital solution by intraperitoneal injection, and then injecting the LPS injection solution into the substantia nigra compacta of the mice to construct LPS-PD model mice. Mice injected with normal saline were used as controls (Control). After culturing the LPS-PD model mice and the control group mice for 7 days, they were killed, brain tissues were collected, and immunofluorescence staining of cells in the substantia nigra region was performed after frozen sections. The staining results are shown in the figure. Figure 1 As shown in h, the statistical results are as follows Figure 1 As shown in Figure 1, it can be seen that the ACSL1 positive rate of microglia in the brain tissue of LPS-PD model mice is higher than that of the control group.

[0092] 1.3, According to unsupervised clustering and ACSL1 expression levels of microglia in the substantia nigra of PD patients, e.g. Figure 1 As shown in middle j, microglia are divided into two subpopulations, ACSL1-high and ACSL1-low. It can be seen that the number of cells in the ACSL1-high subpopulation is significantly higher than that in the ACSL1-low subpopulation.

[0093] 1.4. ACSL1 function prediction.

[0094] GO-BP (Gene Ontology Biological Process) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyze the functions of ACSL1-high subpopulation microglia, and the results were as follows Figure 1 As shown in ki, it can be seen that it plays corresponding functions in cell phagocytosis, inflammatory response and lipid metabolism.

[0095] Example 2 Effect of ACSL1 on microglia-mediated neuroinflammation.

[0096] To investigate the effect of ACSL1 on microglia-mediated neuroinflammation, two siRNAs were designed in this example based on the gene sequence of ACSL1 in the mouse genome to knock down the expression of ACSL1 in cells. The two siRNAs are double-stranded siRNAs with nucleotide sequences shown in SEQ ID NOs: 1-4, respectively, and are named si-ACSL1#1 and si-ACSL1#2. Their nucleotide sequences are as follows:

[0097] si-ACSL1#1:

[0098] SEQ ID NO: 1: 5'-GGAAAGUCCGGCUGAUGAUTT-3';

[0099] SEQ ID NO: 2: 5'-AUCAUCCACGCGGACUUUCCTT-3'.

[0100] si-ACSL1#2:

[0101] SEQ ID NO: 3: 5'-GGAAGGAAGCCGGUCUGAATT-3';

[0102] SEQ ID NO:4: 5'-UUCAGACCGGCUUCCUUCCTT-3'.

[0103] The nucleotide sequence of the negative control NC is as follows:

[0104] NC-1: 5'-UUCUCCGAACGUGUCACGUTT-3';

[0105] NC-2: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0106] 2.1. In vitro validation of the effect of ACSL1 on microglia-mediated neuroinflammation

[0107] 2.1.1. First, si-ACSL1#1, si-ACSL1#2, and NC were synthesized in vitro and transfected into primary microglia extracted from the brains of 0-1 day old C57BL / 6 mice using a transfection kit (purchased from Thermo Fisher Scientific, Cat. No. L3000015). Subsequently, the transfected microglia were injected with LPS injection (a mixed solution of LPS dissolved in PBS, with an LPS concentration of 100 ng / ml) to induce an inflammatory response in microglia. PBS buffer was injected as a control group. 18 hours later, the microglia were collected and the relative expression levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6, as well as the concentrations of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the culture medium were analyzed. The results are shown in Figure 2. Figure 2 As shown in ab.

[0108] The NO concentration in the microglial cell culture medium was tested using a NO concentration detection kit (Cat. No. S0020S, Beyotime Biotechnology Co., Ltd.). The test results are as follows: Figure 2 As shown in c, the ROS fluorescence intensity of the above microglia cells was tested using a reactive oxygen species detection kit (Cat. No. S0033S, Beyotime Biotechnology Co., Ltd.). The test results are shown in Figure 2 As shown in d.

[0109] according to Figure 2 As shown in Figure 5, compared with the PBS group mice, the proinflammatory cytokines TNF-α, IL-1β and IL-6 in the LPS-induced PD model mice were significantly increased, and the levels of NO and ROS were significantly increased. After knocking down the expression of ACSL1, the proinflammatory cytokines TNF-α, IL-1β and IL-6 were significantly decreased, and the levels of NO and ROS were significantly decreased.

[0110] 2.1.2 iNOS and COX2 proteins are two important protein molecules in the NF-κB signaling pathway. They coordinate with each other and are key genes in the inflammatory process. In this example, Western blot analysis was performed on iNOS and COX2 proteins in LPS-induced microglia to determine the expression levels of iNOS and COX2 proteins. The test results are as follows: Figure 2 As shown in e.

[0111] 2.1.3. Studies have shown that DA neuron loss and neuroinflammation play a key role in the pathogenesis of PD. Both DA neuron loss and neuroinflammation are key pathological features and mechanisms of PD. Neuroinflammation can exacerbate DA neuron loss, and abnormal activation of microglia can release excessive proinflammatory cytokines, damaging surrounding DA neurons. Therefore, this example investigated the effects of ACSL1 on DA neurons and neuroinflammation, specifically including the following steps:

[0112] Mouse primary microglia were transfected with si-ACSL1#1, si-ACSL1#2 and NC for 48 hours. The primary microglia cells after 48 hours of transfection were exposed to LPS for 24 hours. The culture medium was collected and cultured with human neuroblastoma cells (SH-SY5Y, purchased from Wuhan Punosai Biotechnology Co., Ltd.) for 24 hours. The SH-SY5Y cells were collected and immunoblotted to detect the protein levels of PARP, Cleaved-PARP, Caspase3 and Cleaved-Caspase3. The results are as follows: Figure 2 At the same time, the collected SH-SY5Y cells were stained with TUNEL and observed and photographed under a microscope. The results are shown in Figure 2 As shown in g, the statistical results of TUNEL positive percentage are as follows Figure 2 As shown in h.

[0113] according to Figure 2 It can be seen from Figure 5 that compared with the LPS control group, inhibiting ACSL1 expression helps to reduce the toxicity of microglial supernatant to DA neurons, the levels of cleaved PARP and cleaved caspase3 proteins are significantly decreased, and the percentage of TUNEL positive cells is significantly decreased.

[0114] 2.2. Verification of the effect of ACSL1 on microglia-mediated neuroinflammation in mice

[0115] Male mice aged 6-8 weeks were randomly divided into six groups, with 10 mice in each group. Three groups of mice were injected with AAV virus containing si-ACSL1#1 sequence (titer: 1.07*10 13 , the solution is the supernatant of the culture medium for producing the virus after ultrafiltration), AAV injection solution including si-ACSL1#2 sequence (titer: 2.98*10 13 ) and AAV injection including NC sequence (titer: 2.13*10 13) were injected into the bilateral substantia nigra region of the mouse brain. After culturing for 3 weeks, LPS solution (LPS concentration was 100 ng / ml, solution was PBS) was injected at the same location to establish a PD model. The other three groups of mice were injected with AAV injection containing the si-ACSL1#1 sequence, AAV injection containing the si-ACSL1#2 sequence, and AAV injection containing the NC sequence, respectively. After culturing for three weeks, PBS solution was injected. After culturing for 1 week, the six groups of mice were sacrificed and brain tissues from the substantia nigra region of the mice were collected for the following tests:

[0116] 2.2.1. Since IBA1 is uniquely expressed in cells of the monocytic lineage, we used antibodies against IBA1 to stain microglia to specifically label microglia, and used EGFP fluorescence signals to observe and analyze the specific expression of the virus in microglia (the virus sequence carries an EGFP tag, and if the virus is successfully expressed, the cells will carry green fluorescence). The results are as follows Figure 2 As shown in jm. Figure 2 As shown in Figure 1, AAV virus is specifically expressed in microglia in the substantia nigra region, and compared with the control group (NC-LPS), the IBA1 fluorescence intensity of the experimental group mice injected with si-ACSL1#1 and si-ACSL1#2 was significantly decreased.

[0117] 2.2.2. Western blot analysis and quantitative analysis were performed on the expression of ACSL1 in the substantia nigra region of mice. The results were as follows: Figure 2 As shown in kl. Figure 2 As can be seen from Figure 1, compared with the control group (NC-PBS), the expression level of ACSL1 in LPS-induced mouse microglia was significantly increased, and the expression level of ACSL1 in the experimental group mice injected with si-ACSL1#1 and si-ACSL1#2 was significantly decreased.

[0118] 2.2.3. TH (Tyrosine Hydroxylase) is an enzyme related to DA synthesis and is often used as a marker for DA neurons. TH-positive cells in microglia were stained using immunofluorescence and the number of TH-positive cells was counted. The results are shown in Figure 2 Indicated by n and o. Figure 2 As shown in n and o, the LPS-induced DA neuron loss in mouse microglia was significantly increased, and the DA neuron loss in the experimental group mice injected with si-ACSL1#1 and si-ACSL1#2 was significantly alleviated.

[0119] Example 3 Analysis of the effect of ACSL1 on microglial lipid droplet metabolism

[0120] 3.1. ACSL1 is a key enzyme involved in lipid synthesis, which may affect the physiological function of PD-related microglia by regulating lipid metabolism. To verify this hypothesis, this example first used Reactome enrichment to visualize the metabolic pathways of microglia with high ACSL1 expression, such as Figure 3 As shown in (a), ACSL1 is involved in the regulation of fatty acid synthesis (Fatty acidmetabolism) and lipid metabolism (Regulation of metabolism by pparalpha).

[0121] 3.2. Multiple immunohistochemical staining was performed on the substantia nigra of the brain tissue of the LPS-induced PD model mice and normal mice to observe the localization of ACSL1 protein and lipid droplet-related protein PLIN2. The staining results are as follows: Figure 3 As shown in middle b, it can be seen that ACSL1 and lipid droplet-associated protein PLIN2 are located in basically the same position in the substantia nigra region of the brain tissue.

[0122] 3.3. BV-2 cells (purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number: CL-0493) were transfected with si-ACSL1#1, si-ACSL1#2, and control small interfering RNA (NC), respectively. 48 hours after transfection, BV-2 cells were treated with PBS solution dissolved with LPS (LPS concentration was 1 μg / ml) for 18 hours. Subsequently, BV-2 cells were stained with BODIPY (purchased from Thermo Fisher Scientific, catalog number: D3922) to detect the distribution of intracellular lipid droplets. The staining results and the statistical results of the number of lipid droplets are shown below. Figure 3 As shown in the CD. Figure 3 It can be seen from the CD4 T cells that knocking down the expression of ACSL1 in cells can significantly reduce the formation of lipid droplets.

[0123] 3.4. BV-2 cells were transfected with si-ACSL1#1, si-ACSL1#2, and control small interfering RNA (NC). 48 hours after transfection, BV-2 cells were treated with LPS-dissolved PBS solution (LPS concentration was 1 μg / ml) for 18 hours and treated with chloroquine (purchased from Sellerk, catalog number: NSC-187208) for 6 hours to inhibit autophagic flux in microglia. BV-2 cells were then stained with BODIPY and immunofluorescence staining for the autophagy marker LC3B protein was performed simultaneously, as shown in Figure 3. Figure 3 As shown in Figure f, knocking down the expression of ACSL1 in cells did not significantly reduce the fluorescence intensity of LC3B. In addition, BV-2 cells were extracted for Western blot analysis. Figure 3As shown in middle g, knockdown of ACSL1 expression in cells did not significantly reduce the expression of LC3B.

[0124] 3.5. BV-2 cells were treated with the method described in 3.3 and RNA was extracted using the precipitation method (purchased from Nanjing Novi Biotech Co., Ltd., Catalog No.: R711-01). After reverse transcription using a reverse transcription kit (purchased from Nanjing Novi Biotech Co., Ltd., Catalog No.: R423-01), real-time fluorescence quantitative PCR analysis (purchased from Nanjing Novi Biotech Co., Ltd., Catalog No.: Q712-02) was performed to detect the transcription levels of lipolysis and lipid uptake related indicators. The test results are as follows. Figure 3 As shown in middle hi, knocking down ACSL1 expression in cells did not significantly change the transcriptional levels of lipolysis and lipid uptake related indicators.

[0125] 3.6. Immunofluorescence staining of ACSL1 and lipid droplets in BV-2 cells treated with PBS solution containing LPS (LPS concentration was 1 μg / ml) for 18 h was performed using immunofluorescence technology. Figure 3 As shown in middle j, ACSL1 protein was not co-labeled with lipid droplets, indicating that ACSL1 does not act as a lipid droplet protein to regulate lipid droplet homeostasis in microglia.

[0126] 3.7. BV-2 cells were treated with PBS solution containing LPS (LPS concentration was 1 μg / ml) for 18 h, and then the expression of ACSL1 in the cytosolic and mitochondrial fractions of the cells was analyzed by immunoblotting. Figure 3 As shown in Figure k, the expression of ACSL1 in the cytoplasmic fraction of LPS-treated microglia was significantly increased. In addition, an endoplasmic reticulum extraction kit (purchased from Beijing Solebow Technology Co., Ltd., catalog number: EX2690) was used and immunoblotting experiments were performed, as shown in Figure 1. Figure 3 As shown in Figure 1, the expression of ACSL1 in the endoplasmic reticulum fraction of LPS-treated microglia was significantly increased. Mitochondria were labeled with Mito-tracker (purchased from Beyotime Biotech, Cat. No. C1049B), and ACSL1 was immunofluorescently stained. Figure 3 As shown in Figure 5(m), the co-localization of ACSL1 with mitochondria was significantly decreased in BV-2 cells treated with LPS-dissolved PBS solution (LPS concentration was 1 μg / ml).

[0127] Example 4 ACSL1 aggravates the pro-inflammatory phenotype of microglia by promoting lipid droplet formation

[0128] like Figure 4As shown in Figure a, by analyzing the amino acid sequence of the ACSL1 protein, the amino acids 1-90 at its N-terminus are a transmembrane domain that is used to transport the ACSL1 protein in the cytoplasm to the mitochondria. To further verify that ACSL1 regulates lipid droplet metabolism, thereby affecting microglial function, this example constructed an ACSL1-WT expression vector and an ACSL1-MN expression vector. The ACSL1-WT expression vector expresses the mouse ACSL1 protein (the amino acid sequence of the mouse ACSL1 protein is shown in SEQ ID NO: 11), and the ACSL1-MN expression vector expresses a protein with 90 amino acids truncated at the N-terminus (i.e., amino acids 91-699 of the amino acid sequence shown in SEQ ID NO: 11).

[0129] SEQ ID NO: 11:

[0130] .

[0131] Specifically, the ACSL1-WT expression vector is obtained by replacing the small fragment between the BamHI restriction site and the HindIII restriction site of the CV702 vector (purchased from GeneChip) with the mouse ACSL1 protein coding sequence of SEQ ID NO: 11; the ACSL1-MN expression vector is obtained by replacing the small fragment between the BamHI restriction site and the HindIII restriction site of the CV702 vector (purchased from GeneChip) with the coding sequence of amino acids 91-699 of the mouse ACSL1 protein of SEQ ID NO: 11.

[0132] BV-2 cells were transfected with ACSL1-WT expression vector and ACSL1-MN expression vector for 48 h, and the expression of exogenous ACSL1 was detected by Western blot analysis. The analysis results are shown in Figure 4 As shown in middle b, it can be confirmed that ACSL1 protein is successfully expressed.

[0133] Immunofluorescence analysis was performed on BV-2 cells transfected with ACSL1-WT expression vector and ACSL1-MN expression vector using anti-Flag and Mito-tracker to detect the distribution of proteins in cells. The analysis results are shown in Figure 2. Figure 4 As shown in middle c, only a small amount of ACSL1-MN co-localized with Mito-tracker, while most ACSL1-WT co-localized with Mito-tracker, indicating that ACSL1-WT can be expressed in mitochondria and endoplasmic reticulum, while ACSL1-MN is not expressed in mitochondria, indicating that the ACSL1-MN vector was successfully constructed.

[0134] BV-2 cells transfected with empty vector CV702 (Vector group), ACSL1-WT expression vector, and ACSL1-MN expression vector were treated with LPS in PBS solution (LPS concentration was 1 g / ml) or PBS solution for 18 hours and named as Vector+PBS, Vector+LPS, ACSL1-WT+LPS, and ACSL1-MN+LPS, respectively. Subsequently, the Vector+PBS group, Vector+LPS group, ACSL1-WT+LPS group, and ACSL1-MN+LPS group were subjected to BODIPY staining to detect lipid droplets composed of intracellular neutral lipids. The detection results are shown in Figure 3. Figure 4 As shown in Figure 5, it can be seen that the content of lipid droplets in the ACSL-MN group + LPS group, which specifically and highly expresses ACSL1 in the endoplasmic reticulum, was significantly higher than that in the Vector + LPS and ACSL1-WT + LPS groups.

[0135] The same method as in Example 2 was used to detect BV-2 cells transfected with Vector vector, ACSL1-WT expression vector and ACSL1-MN expression vector after LPS treatment. The test results are shown in Figure 2. Figure 4 As shown in middle fm, it can be seen that the enrichment of ACSL1 in the endoplasmic reticulum may aggravate the inflammatory manifestations of microglia by regulating lipid droplet accumulation.

[0136] Example 5 Inhibition of TBK1 activity reduces endoplasmic reticulum enrichment of ACSL1, reduces microglial lipid droplet accumulation and pro-inflammatory phenotype

[0137] 5.1、 Figure 5As shown in (a), in resting microglia, ACSL1 and TBK1 protein (TANK-binding kinase 1) bind to each other. In activated microglia, TBK1 is phosphorylated, causing TBK1 activation, resulting in a decrease in the affinity between TBK1 and ACSL1, weakening the ability of TBK1 as a scaffold protein to transport ACSL1 to mitochondria, and causing ACSL1 protein to be enriched in the endoplasmic reticulum, thereby affecting the lipid droplet homeostasis in microglia.

[0138] 5.2. To verify the ability of TBK1 protein to transport ACSL1 to mitochondria as a scaffold protein, this example used the TBK1 protein inhibitor GSK8612 to perform Western blot analysis on BV-2 cells treated with PBS and LPS. Specifically, BV-2 cells were first treated with LPS in PBS for 12 hours. The LPS-treated BV-2 cells were divided into two groups, one of which was treated with the drug solvent dimethyl sulfoxide (DMSO) (purchased from Merck, Catalog No.: D2650) for 6 hours, and the other group was treated with GSK8612 (purchased from Sellerk, Catalog No.: S8872) in DMSO (the concentration of GSK8612 was 5 μM) for 6 hours. At the same time, BV-2 cells were treated with PBS. The cells were treated for 12 hours. The PBS-treated BV-2 cells were divided into two groups, one of which was treated with DMSO for 6 hours, and the other was treated with GSK8612 DMSO solution (GSK8612 concentration was 5 μM) for 6 hours. After the treatment, the cells were collected and lysed, and the proteins were collected and subjected to Western blotting analysis to analyze p-TBK1 S172 (TBK1 protein phosphorylated at Ser172) and the expression level of TBK1 protein. The results are shown in Figure 5 As shown in middle b, GSK8612 can inhibit the phosphorylation of TBK1 at Ser172 induced by LPS treatment.

[0139] Western blot analysis of ACSL1 protein in the mitochondria and endoplasmic reticulum of the above BV-2 cells was performed using TOMM20 as a mitochondrial marker. The analysis results are shown in Figure 2. Figure 5 As shown in middle c, it can be seen that inhibiting the phosphorylation of TBK1 can reduce the enrichment of ACSL1 protein in the endoplasmic reticulum caused by LPS induction.

[0140] Cells treated with PBS / LPS and DMSO / GSK8612 were stained with BODIPY and microscopically observed to observe lipid droplets in the cells. The results are as follows: Figure 5 As shown in Figure 5, it can be seen that inhibiting the phosphorylation of TBK1 can inhibit the excessive accumulation of lipid droplets.

[0141] 5.3. First, the ACSL1-WT expression vector constructed in Example 4 was used to transfect BV-2 cells, and then the BV-2 cells were treated with a PBS solution of LPS for 12 hours. The BV-2 cells after LPS treatment were divided into two groups, one of which was treated with DMSO for 6 hours and named ACSL1-WT+DMSO group, and the other group was treated with a DMSO solution of GSK8612 (the concentration of GSK8612 was 5 μm) for 6 hours and named ACSL1-WT+GSK8612 group; BV-2 cells were transfected with the empty vector CV702, and then the BV-2 cells were treated with a PBS solution of LPS for 12 hours and then treated with DMSO for 6 hours, named Vector+DMSO group; At the same time, BV-2 cells transfected with the ACSL1-WT expression vector and the empty vector CV702 were treated with PBS solution as a control. The cells in each group were stained with BODIPY, and the number of lipid droplets was counted. The results are as follows: Figure 5 As shown in gh.

[0142] Western blot analysis was further performed on the cells in the above groups to evaluate the expression of iNOS, COX2, and p-TBK1 S172 、TBK1、p-p65 S536 The expression levels of IL-6, IL-1β, and TNFα were measured by ELISA. Figure 5 As shown in ij. Figure 5 It can be seen that inhibiting the phosphorylation of TBK1 can reduce the production of pro-inflammatory protein markers and pro-inflammatory cytokines in microglia caused by ACSL1-WT vector transfection, indicating that inhibiting TBK1 activity helps to reduce the endoplasmic reticulum enrichment of ACSL1 and inhibit the accumulation of neutral lipid droplets, thereby alleviating the inflammatory response.

[0143] Example 6 ACSL1 activates TBK1 through ubiquitination pathway

[0144] 6.1. Different concentrations of ACSL1-WT and ACSL1-MN expression vectors were transfected into BV-2 cells (concentrations were 0, 0.5 μg, 1 μg, and 2 μg, respectively). Western blot analysis was performed on BV-2 cells. The results were as follows: Figure 6 As shown in (a), it can be seen that the phosphorylation level of TBK1 increased in a dose-dependent manner, and the phosphorylation level of TBK1 was more obvious in the ACSL1-MN group.

[0145] 6.2. Empty vector CV702 and ACSL1-WT were transfected into BV-2 cells. Then, anti-TBK1 antibody was used to immunoprecipitate TBK1 protein using an immunoprecipitation kit (purchased from Thermo Fisher Scientific, Cat. No. 88804). The ubiquitination level of TBK1 protein was detected by western blotting. The results were as follows: Figure 6 As shown in middle b, it can be seen that transfection of ACSL1-WT increased the ubiquitination level of TBK1.

[0146] After 48 hours of transfection of NC, siACSL1#1, and si-ACSL1#2 in BV-2 cells, the cells were treated with LPS in PBS for 18 hours to detect the ubiquitination level of TBK1 protein. Figure 6 As shown in middle c, knockdown of ACSL1 expression in cells inhibited the ubiquitination level of TBK1 induced by LPS.

[0147] 6.3. A TBK1 expression vector, named HA-TBK1, was constructed. This expression vector was constructed by replacing the coding sequence of TBK1 protein with the small fragment between the BamHI and XhoI restriction sites of the pCDNA3.1 vector (purchased from Thermo Fisher Scientific, Catalog No. V79027), while keeping the rest of the sequence unchanged. The amino acid sequence of TBK1 protein is SEQ ID NO: 12:

[0148] SEQ ID NO: 12:

[0149] .

[0150] A Ubiquitin-WT expression vector was constructed. The Ubiquitin protein coding sequence was substituted for a small fragment between the BamHI and XhoI restriction sites of the pCDNA3.1 vector, while maintaining the remaining sequence unchanged. The vector expresses the Ubiquitin protein, and the amino acid sequence of the Ubiquitin protein is shown in SEQ ID NO: 13.

[0151] SEQ ID NO: 13:

[0152] MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIFAGKQLEDGRRTLSDYNIQKESTLHLVLRLRGG.

[0153] The Ubiquitin-K48 expression vector and the Ubiquitin-K63 expression vector were constructed by replacing the small fragment between the BamHI restriction site and the XhoI restriction site of the pCDNA3.1 vector with the Ubiquitin-K48 protein coding sequence and the Ubiquitin-K63 protein coding sequence, while keeping the other sequences unchanged. The vectors express the Ubiquitin-K48 protein (SEQ ID NO: 13, except for the K at position 48, where all other Ks are mutated to Rs) and the Ubiquitin-K63 protein (SEQ ID NO: 13, except for the K at position 63, where all other Ks are mutated to Rs), respectively.

[0154] The constructed TBK1 expression vector, ACSL1-WT, ACSL1-MN expression vector, Ubiquitin-WT, Ubiquitin-K48, and Ubiquitin-K63 expression vectors were transfected into HEK-293T cells (purchased from Pronocell Life Science Co., Ltd., catalog number CL-0005), respectively. TBK1 protein was immunoprecipitated using anti-TBK1 antibody, and the ubiquitination level of TBK1 protein was detected by western blotting. Figure 6 As shown in de, ACSL1-WT transfection significantly increased the K63 ubiquitination level of TBK1, and ACSL1-MN transfection promoted the K63 ubiquitination modification of TBK1 more than ACSL1-WT transfection.

[0155] according to Figure 6 As shown in middle f, overexpression of ACSL-WT increased the affinity of MIB1 and Nrdp1 for TBK1. ACSL1 can bind to MIB1 and Nrdp1 to regulate the phosphorylation of TBK1.

[0156] 6.3. To study the effect of Nrdp1 on ACSL1-mediated TBK1 ubiquitination and phosphorylation, this example designed an siRNA to knock down Nrdp1 expression in cells. The nucleotide sequence of the siRNA is a double-stranded RNA molecule shown in SEQ ID NO: 5-6 and is named si-Nrdp1.

[0157] SEQ ID NO: 5: 5'-CCUUCAGAACCUGGAGGAGTT-3';

[0158] SEQ ID NO:6: 5'-CUCCUCCAGGUUCUGAAGGTT-3'.

[0159] The negative control sequence is:

[0160] NC: 5'-UUCUCCGAACGUGUCACGUTT-3' and 5'-ACGUGACACGUUCGGAGAATT-3'.

[0161] Subsequently, NC and si-Nrdp1 were transfected into BV-2 cells, respectively. Subsequently, cells transfected with the corresponding small interfering RNA were transfected with Vector expression vector or ACSL1-WT expression vector for 48 h, and cells were collected for western blotting experiments, e.g. Figure 6 As shown in Figure 3, knockdown of Nrdp1 inhibited the phosphorylation of TBK1 and the K63 ubiquitination of TBK1. In addition, NC or si-Nrdp1 small interfering gene was transferred into BV-2 cells, and the cells transfected with the corresponding vectors were injected with LPS injection (a mixed solution obtained by dissolving LPS in PBS, with an LPS concentration of 100 ng / ml) to induce an inflammatory response in microglia. PBS buffer was injected as a control group. After 18 hours, microglia were collected for immunofluorescence staining and the relative expression levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 were analyzed. Figure 6 As shown in (i), knockdown of Nrdp1 significantly inhibited the proinflammatory response of microglia induced by LPS treatment.

[0162] Example 7 P65 can bind to the ACSL1 promoter region to promote transcription

[0163] 7.1. ACSL1 can activate the NF-κb (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway, thereby regulating immune and inflammatory responses. To verify whether NF-κb regulates ACSL1 transcription, in this example, BV-2 cells were cultured with TNFα (TNFα concentration in cell culture medium was 20 ng / ml). Cells were harvested at 15 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours, and the expression level of ACSL1 was measured after cell lysis. The results are shown in Figure 2. Figure 7 As shown in middle b, it can be seen that under TNFα induction, the expression level of ACSL1 increases with time.

[0164] BV-2 cells were cultured with TNFα (TNFα concentration in cell culture medium was 20 ng / ml), and cells were harvested at 3 h, 6 h, and 12 h. The cells were lysed to collect proteins and subjected to Western blot analysis. The analysis results were shown in Table 1. Figure 7 As shown in middle c, it can be seen that with the increase of time, p-p65 S536 The expression level gradually increased, indicating that TNFα activated the NF-κB pathway in a time-dependent manner, and the expression level of ACSL1 protein gradually increased.

[0165] 7.2. To verify whether TNFα-induced ACSL1 expression can be mediated by p65 activation, this example provides siRNAs that target and knock down p65 expression. The nucleotide sequences of the siRNAs are double-stranded RNA molecules shown in SEQ ID NOs: 7-10.

[0166] SEQ ID NO:7: 5'-AGAAGACAUUGAGGUGUAUTT-3';

[0167] SEQ ID NO:8: 5'-AUACACCUCAAUGUCUUCUTT-3';

[0168] SEQ ID NO:9: 5'-GAAGAAGAGUCCUUUCAAUTT-3';

[0169] SEQ ID NO: 10: 5'-AUUGAAAGGACUCUUCUUC-3'.

[0170] siRNA was transfected into BV-2 cells using a transfection kit (purchased from Thermo Fisher Scientific, catalog number: L3000015). BV-2 cells were then divided into two groups, one of which was added with TNFα (the concentration of TNFα in the cell culture medium was 20 ng / ml), and the other group was not added with TNFα. After the cells were cultured for another 12 h, the cells were collected and subjected to Western blot analysis. The analysis results are shown in FIG. Figure 7 As shown in Figures 5 and 6, it can be seen that the expression level of ACSL1 can be effectively reduced by reducing the expression of p65.

[0171] 7.3. JSH-23 is an inhibitor of NF-κB pathway activity, which inhibits NF-κB pathway activity by inhibiting the nuclear translocation of NF-κB p65. BV-2 cells were treated with TNFα alone or in combination with JSH-23 (purchased from Sellerk, Catalog No.: S7351) (JSH-23 concentration: 10 μM or 30 μM; TNFα concentration: 20 ng / ml) for 12 hours. After the treatment, the cells were harvested and lysed, and proteins were collected for Western blot analysis. The results were as follows: Figure 7 As shown in fg. Figure 7 As shown in Figure 5, ACSL1 expression can be effectively reduced by inhibiting the transcriptional activity of NF-κB.

[0172] Example 8 Effect of targeting TBK1 on inhibiting the progression of PD neuroinflammation.

[0173] 8.1、As Figure 8 As shown in (a), a total of 40 C57BL / 6J mice aged 6-8 weeks were selected and divided into four groups, with 10 mice in each group. Among them, the first group of mice were injected with PBS solution, followed by intraperitoneal injection of GSK8612 drug solvent for 7 consecutive days, named PBS+Vehicle; the second group of mice were injected with PBS solution, followed by intraperitoneal injection of 10 mg / kg / d of GSK8612 every day for 7 consecutive days, named PBS+GSK8612; the third group of mice were injected once with 1 μl of LPS in PBS solution (LPS concentration of 500 ng / ul) in the substantia nigra area using a stereotaxic apparatus, followed by intraperitoneal injection of GSK8612 drug solvent for 7 consecutive days, named LPS+Vehicle; the fourth group of mice were injected with 1 μl of LPS in PBS solution, followed by intraperitoneal injection of 10 mg / kg / d of GSK8612 every day for 7 consecutive days, named LPS-GSK8612.

[0174] The brain tissues of the substantia nigra of the mice in the above groups were collected and immunofluorescence staining of IBA1+ cells and TH cells was performed. The staining results are shown in Figure 2. Figure 8 As shown in bd. Figure 8 It can be seen from the data that the TBKK1 inhibitor GSK8612 can significantly reduce the number of IBA1+ cells in the substantia nigra region of mice and increase the number of TH-positive cells, thereby reducing the inflammatory response of mouse microglia and promoting the regulation of the inflammatory process.

[0175] 8.2. The substantia nigra brain tissues of the four groups of mice were lysed using RIPA protein lysis buffer (purchased from Beyotime Biotech, Cat. No.: P0013C) and subjected to protein immunoblotting analysis. The analysis results are as follows: Figure 8 As shown in middle ef, it can be seen that injection of GSK8612 can effectively alleviate neuroinflammation.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Inhibition ACSL1 Use of a gene-expressed substance in the preparation of a drug for alleviating and / or treating Parkinson's disease, characterized in that: inhibition ACSL1 The gene-expressed substance is selected from at least one of A1) to A6): A1) Targeted Inhibition ACSL1 RNA molecules expressed by genes; A2) a DNA molecule encoding the RNA molecule described in A1); A3) an expression cassette containing the DNA molecule described in A2); A4) a recombinant vector containing the DNA molecule described in A2), or a recombinant vector containing the expression cassette described in A3); A5) a recombinant microorganism containing the DNA molecule described in A2), or a recombinant microorganism containing the expression cassette described in A3), or a recombinant microorganism containing the recombinant vector described in A4); A6) A modified RNA molecule according to A1); Targeted inhibition ACSL1 The RNA molecule expressed by the gene is siRNA, and the siRNA is selected from one of A11) to A12): A11) Targeted Inhibition ACSL1 The nucleotide sequences of the gene-expressed siRNAs are SEQ ID NO: 1 and SEQ ID NO: 2; A12) Targeted Inhibition ACSL1 The nucleotide sequences of the gene-expressed siRNAs are SEQ ID NO: 3 and SEQ ID NO: 4; inhibition ACSL1 The gene-expressing substance alleviates and / or treats Parkinson's disease in at least one of the following aspects: B1) Reduce the pro-inflammatory response of microglia; B2) inhibit excessive accumulation of lipid droplets in microglia; B3) weaken PD neuroinflammation; B4) attenuates microglial dopamine neurotoxicity; B5) Inhibits dopamine neuron death in PD progression.

2. A pharmaceutical composition, characterized in that Including the inhibition described in claim 1 ACSL1 Gene expression substance and pharmaceutically acceptable carrier.

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