Use of nanoparticles derived from zingiber officinale in the preparation of a medicament for reducing the neurotoxicity caused by alpha-syn

Abstract

The application discloses application of ginger-derived nanoparticles in preparation of a medicine for reducing nerve toxicity caused by alpha-Syn, and belongs to the technical field of biological medicine. The application first finds that the ginger-derived nanoparticles can reduce droplet formation and fusion in a manner of regulating liquid-liquid phase separation of alpha-Syn, thereby inhibiting alpha-Syn amyloid aggregation, and can prevent the formation of fiber seeds to accelerate alpha-Syn amyloid aggregation. Finally, the alpha-Syn fiber-induced mitochondrial damage is reduced, thereby protecting nerve cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of ginger-derived nanoparticles in the preparation of drugs that reduce neurotoxicity caused by α-Syn. Background Technology

[0002] Parkinson's disease is the second leading cause of neurodegenerative disease worldwide, characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. The main component of Lewy bodies is α-synuclein, and the abnormal aggregation of this protein is considered a core event in the onset and progression of the disease. Recent studies have shown that α-synuclein can form protein-rich droplets through abnormal liquid-liquid phase separation, a process that accelerates its conversion into toxic amyloid fibrils.

[0003] In recent years, a growing body of research has shown that liquid-liquid phase separation (LLPS) is a crucial initial step in the pathological aggregation of proteins such as α-Syn. Under specific conditions, α-Syn can form highly concentrated liquid condensates, which provide a breeding ground for protein misfolding and fibrillation, accelerating the formation of amyloid deposits. Therefore, intervening in the LLPS process of α-Syn holds promise for inhibiting its abnormal aggregation at its source, providing important insights for developing new strategies to treat α-Syn-related neurodegenerative diseases such as Parkinson's disease. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ginger-derived nanoparticles in the preparation of drugs that reduce the neurotoxicity caused by α-Syn, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of ginger-derived nanoparticles in the preparation of drugs that reduce neurotoxicity caused by α-synuclein.

[0006] The second technical solution of the present invention, the method for preparing the ginger-derived nanoparticles, includes the following steps: (1) Juice the ginger, and centrifuge the juice at low speed and high speed in sequence to remove the precipitate. Collect the supernatant and filter it through a microporous membrane in sequence. (2) The filtrate obtained in step (1) is subjected to ultracentrifugation, and the precipitate is collected to obtain the ginger-derived nanoparticles.

[0007] The third technical solution of this invention is the application of ginger-derived nanoparticles in the preparation of drugs that inhibit α-synuclein liquid-liquid phase separation.

[0008] The fourth technical solution of this invention is the application of ginger-derived nanoparticles in the preparation of drugs that inhibit α-synuclein pre-fiber-forming amyloid aggregation.

[0009] The fifth technical solution of the present invention is a drug for reducing neurotoxicity caused by α-synuclein, comprising an effective amount of ginger-derived nanoparticles.

[0010] Based on the above technical solution, the present invention has the following technical effects: This invention reveals for the first time the regulatory role of plant-derived nanoparticles on α-synuclein liquid-liquid phase separation and screens out ginger-derived nanoparticles with significant inhibitory effects. This invention elucidates the unique molecular mechanism by which GDNP binds to α-Syn through electrostatic interactions, reducing its droplet fluidity and inhibiting droplet fusion. This invention confirms that GDNP can effectively block the transformation of α-Syn from phase-separated droplets into toxic amyloid fibers. This invention demonstrates the significant neuroprotective effect of GDNP in cell models and provides a novel, widely available, easily prepared, and highly biocompatible candidate therapeutic agent for Parkinson's disease. Attached Figure Description

[0011] Figure 1 To explore technical routes for regulating α-Syn fiber toxicity using GDNP.

[0012] Figure 2 GDNP inhibits the liquid-liquid phase separation of α-Syn. A: Fluorescence images of α-Syn aggregates in the presence of GDNP and WDNP recorded by laser confocal microscopy. B: ImageJ analysis of α-Syn droplet size in the presence of different small molecules under LLPS conditions. C: Centrifugation sedimentation experiment to determine the amount of protein in the supernatant and precipitate after centrifugation. D: ImageJ analysis of the supernatant to sedimentation ratio in different centrifugation sedimentation treatment groups.

[0013] Figure 3 GDNP inhibits the formation of amyloid aggregates in α-Syn fibers, while WDNP has no such inhibitory effect. A: ThT signal detection of the effect of different concentrations of GDNP on α-Syn fiber formation at different time points. B: ThT signal detection of the effect of different concentrations of WDNP on α-Syn fiber formation at different time points. C: TEM observation of the effect of 100 μg / mL GDNP and WDNP on α-Syn fiber formation. Scale bar: 250 nm.

[0014] Figure 4GDNP inhibits the promoting effect of PFF on α-Syn fiber formation in vitro. A: Confocal imaging analysis of the regulatory role of GDNP in PFF-induced α-Syn fiber formation. Scale bar: 5 μm. B: Image J quantitative analysis of ThS fluorescence values ​​of GDNP during PFF-induced α-Syn fiber formation.

[0015] Figure 5 GDNP can inhibit the promoting effect of PFF on α-Syn filament formation within cells. A: Fluorescence imaging analysis of the number of inclusion body spots in PC12 cells after co-incubation with PFFs from different treatment groups. Scale bars are 1 mm and 500 μm. B: Image J quantitative analysis of the number of inclusion body spots in PC12 cells after co-incubation with PFFs from different treatment groups.

[0016] Figure 6 Protective effects of GDNP and WDNP against α-Syn fibroblast cytotoxicity. A and B: MTT assay to determine the effect of GDNP (A) and WDNP (B) on PC12 cell viability. Experiments were independently repeated four times. C: MTT assay to determine the neurotoxicity of PFF fibers prepared from GDNP and WDNP groups on PC12 cells. Experiments were independently repeated four times. D: Calcein-AM live cell staining assay to demonstrate the neurotoxicity of PFF fibers prepared from GDNP and WDNP groups on PC12 cells. Scale bar: 20 μm. The concentrations of GDNP and WDNP were both 100 μg / mL, and the incubation time of PFF and PC12 cells was 24 h. E: Image J: Quantitative count of live cells in Image D. Data are presented as mean ± standard deviation, and all experiments were independently repeated three times.

[0017] Figure 7 Rescue of mitochondrial damage caused by α-Syn fibers by GDNP and WDNP. The concentrations of GDNP and WDNP were both 100 μg / mL, and the scale bar was 5 μm. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides the application of ginger-derived nanoparticles in the preparation of drugs that reduce neurotoxicity caused by α-synuclein.

[0020] In some specific implementations, the particle size of the ginger-derived nanoparticles is 50-200 nm.

[0021] This invention also provides a method for preparing the ginger-derived nanoparticles, comprising the following steps: (1) Juice the ginger, and centrifuge the juice at low speed and high speed in sequence to remove the precipitate. Collect the supernatant and filter it through a microporous membrane in sequence. (2) The filtrate obtained in step (1) is subjected to ultracentrifugation, and the precipitate is collected to obtain the ginger-derived nanoparticles.

[0022] In some specific implementations, the conditions for low-speed centrifugation in step (1) include: centrifugation at 1000 g for 10 min and centrifugation at 3000 g for 20 min; the conditions for high-speed centrifugation include: centrifugation at 10000 g for 30 min; and the pore sizes of the microporous filter membrane are 1.2 μm, 0.8 μm and 0.45 μm, respectively. The conditions for ultracentrifugation in step (2) include: centrifugation at 150,000 g for 1.5 h.

[0023] This invention also provides the application of ginger-derived nanoparticles in the preparation of drugs that inhibit α-synuclein liquid-liquid phase separation.

[0024] This invention also provides the application of ginger-derived nanoparticles in the preparation of drugs that inhibit α-synuclein prefibrillation-induced amyloid aggregation.

[0025] This invention also provides a drug for reducing neurotoxicity caused by α-synuclein, comprising an effective amount of ginger-derived nanoparticles.

[0026] In some specific implementation schemes, pharmaceutically acceptable carriers are also included.

[0027] This application is the first to discover that ginger-derived nanoparticles can reduce droplet formation and fusion by regulating α-Syn liquid-liquid phase separation, thereby inhibiting α-Syn amyloid aggregation and preventing the formation of fibrous seeds that accelerate α-Syn amyloid aggregation. Ultimately, this reduces α-Syn fiber-induced mitochondrial damage, thus protecting nerve cells.

[0028] Example 1 Plant-derived nanoparticles: Ginger was sliced ​​and juiced, and goji berries were juiced directly. The plant juices were centrifuged sequentially at 1000 g for 10 min, 3000 g for 20 min, and 10000 g for 30 min, removing the precipitate after each centrifugation. The plant juices were then passed sequentially through PES (polyethersulfone) microporous membranes of 1.2 μm, 0.8 μm, and 0.45 μm. The supernatant was then ultracentrifuged at 150000 g for 1.5 h to precipitate the plant-derived nanoparticles.

[0029] The precipitate was resuspended in PBS and filtered through a 0.22 μm PES (polyethersulfone) microporous membrane. The plant-derived nanoparticles were then precipitated again by ultracentrifugation at 150,000 g for 1.5 h. After resuspending, the precipitate was removed by centrifugation at 10,000 g for 5 min. The supernatant contained ginger nanoparticles (GDNP) and wolfberry nanoparticles (WDNP). The final suspension was stored at -80 °C for later use. The ginger nanoparticles were natural nanoparticles with a diameter of 50-200 nm and a saucer-like structure.

[0030] 1. GDNP inhibits α-Syn LLPS, while WDNP (goji berry-derived nanoparticles) has no effect. Experimental procedure: AB: First, prepare 40% PEG-8000 using protein buffer (25 mM Tris-HCl, pH=7.4). Mix EGFP-α-Syn (Furlong RA, Narain Y, Rankin J, Wyttenbach A, Rubinsztein DC. Alpha-synuclein overexpression promotes aggregation of mutant huntingtin. Biochem J. 2000 Mar 15;346 Pt 3(Pt 3):577-81. PMID: 10698681; PMCID: PMC1220887.) with α-synuclein (α-Syn) at a molar ratio of 2:98 to form a protein mixture. Then, blend the protein mixture with the phase separation inducer PEG-8000 and adjust to the target concentration: α-Syn 200 μM, PEG-8000 20% (w / v). The solvent system is 25 mM Tris-HCl containing 50 mM NaCl. Tris-HCl (pH=7.4) buffer solution was then used. Subsequently, 3 μL of the above mixture was dropped onto the surface of a clean glass slide, and the slide was placed under a Nikon A1 confocal microscope system. Images were acquired and analyzed using a 100x objective lens.

[0031] CD: Centrifugation sedimentation analysis: Prepare a 50 μL LLPS α-Syn system, add GDNP or WDNP, centrifuge at 16,000 g for 10 min at room temperature, and collect the supernatant into a new EP tube. Gently wash the precipitate with 50 μL of protein buffer, and then resuspend the precipitate with 50 μL of protein buffer. Take 10 μL of supernatant and precipitate resuspended into two new EP tubes, add 10 μL of 2× SDS Loading Dye to each tube, and denature in a 95℃ metal bath for 5 min. Perform electrophoresis, and stain and destain the gel after electrophoresis.

[0032] Note: The LLPS system consists of 200 μM α-Syn, 20% (w / v) PEG-8000, and a 25 mM Tris-HCl (pH=7.4) buffer solution containing 50 mM NaCl. The concentrations of GDNP and WDNP are 100 μg / mL.

[0033] Experimental results are as follows Figure 2 As shown, laser confocal microscopy results indicate that under LLPS-induced conditions, the GDNP co-treatment group significantly inhibited the formation of α-Syn droplet condensates, specifically manifested as a significant reduction in droplet size; while no significant inhibitory effect was observed in the WDNP-treated group. Figure 2 (AB). Similarly, centrifugation sedimentation experiments yielded the same results: the supernatant contained protein monomers, while the precipitate consisted mostly of proteins present in the droplets. It was found that the supernatant treated with GDNP contained significantly more protein than the precipitate group, indicating that GDNP effectively prevents the formation of α-Syn phase-separated droplets. Figure 2 Medium CD).

[0034] 2. GDNP inhibits the formation of amyloid deposits by LLPS using α-Syn. Experimental procedure: AB: The sample containing α-Syn droplets, or with added GDNP or WDNP, was thoroughly mixed with thioflavone T (ThT) solution to achieve a final protein concentration of 5 μM, a final ThT concentration of 40 μM, and a final GDNP or WDNP concentration of 100 μg / mL. Then, 50 μL of the mixture was added to an opaque 96-well plate and detected using a BioTek Synergy HT multi-mode microplate reader. The excitation wavelength was set to 440 nm, and the emission fluorescence intensity was collected at 485 nm. ThT specifically binds to the β-sheet structure of proteins and produces characteristic fluorescence; therefore, by monitoring the dynamic changes in fluorescence signal, the kinetic characteristics of the transformation of α-Syn into amyloid aggregates during phase separation can be quantitatively analyzed. Each experiment was performed in triplicate, with sampling times at 0, 12, 24, 36, and 48 h.

[0035] C: Prepare α-Syn protein solutions with a final concentration of 200 μM and α-Syn protein solutions containing 100 μg / mL GDNP or WDNP, respectively. Incubate these solutions at 37℃ for 48 h to prepare α-Syn fiber samples. Add 10 μL of sample to a copper mesh and let it stand for 1 min. Remove the sample with filter paper. Immerse the copper mesh in 10 μL of 3% water-saturated uranium acetate staining solution for 1 min, and remove the uranium acetate staining solution with filter paper. Wash the copper mesh twice with ddH2O, remove the ddH2O with filter paper, and allow the copper mesh to air dry. Load the copper mesh onto an H-7650 transmission electron microscope for observation and imaging.

[0036] Experimental results are as follows Figure 3 As shown, α-Syn promotes amyloid aggregation through the LLPS process, which is considered one of the important pathways for amyloid formation. To investigate the regulatory role of GDNP on the formation of α-Syn amyloid deposits under LLPS conditions, this invention used thioflavone T (ThT) fluorescence staining to monitor the formation of α-Syn amyloid aggregates during condensation. Under LLPS conditions, a strong ThT signal was detected after α-Syn incubation at 37℃ for 48 h, indicating that amyloid precipitate structures had formed in the aggregates. With the increase of GDNP concentration, the ThT signal intensity gradually decreased within the same time period, suggesting that GDNP inhibits the formation of α-Syn amyloid deposits in a concentration-dependent manner under LLPS conditions. Figure 3 (A). As a control, the effect of WDNP on this process was further investigated, and the results showed that the inhibitory effect of WDNP was weaker than that of GDNP (A). Figure 3 (B). Amyloid aggregates formed from droplet condensates were also observed using transmission microscopy, revealing that the GDNP-treated group formed significantly fewer flocculent fibrous structures than the WT and WDNP groups. Figure 3 (C)

[0037] 3. In vitro inhibition of the promoting effect of pre-formed fibrobrils (PFF) on α-Syn fibrobril formation. Experimental procedure: The purified α-Syn 5 mg / mL was placed in protein storage buffer [25 mM Tris (pH 7.4), 50 mM NaCl] and centrifuged at 12000 rpm for 2 min. The supernatant was incubated at 37 °C with constant stirring at 1000 rpm for 120 h to induce fibrillation. The obtained fibers were sonicated to generate PFF and stored at room temperature. The PFF was incubated with GDNP or WDNP under α-Syn LLPS conditions and stained with ThS (GDNP or WDNP final concentration 100 μg / mL), and then co-incubated for 12 h and 24 h. 5 μl samples were taken and observed by confocal fluorescence microscopy (A), and the fluorescence area of ​​ThS was counted (B).

[0038] Note: The α-Syn LLPS conditions were: 200 μM α-Syn, 20% PEG-8000, 50 mM NaCl, with a total volume of 20 μl; the concentrations of GDNP and WDNP were both 100 μg / mL, the final ThS concentration was 0.0125%, and the PFF concentration was 5 μM.

[0039] Experimental results are as follows Figure 4 As shown, pre-formed fibrous structures (PFFs) of α-Syn can act as seeds, recruiting endogenous normal α-Syn from cells, inducing them to misfold and incorporate into the extended fibrous structure, thereby initiating and accelerating the pathological amyloid aggregation process. To investigate whether GDNP inhibits the formation of α-Syn amyloid aggregates in the presence of PFFs, this invention conducted related experiments. ThS results showed that under in vitro LLPS conditions, with PFFs and α-Syn co-incubated for 12 h, GDNP effectively inhibited the formation of α-Syn amyloid aggregates; when the incubation time was extended to 24 h, GDNP still showed a stronger inhibitory effect compared to WDNP (AB). The experiment demonstrates that GDNP can effectively inhibit the promoting effect of PFFs on α-Syn fiber formation under in vitro LLPS conditions.

[0040] 4. GDNP can inhibit the promoting effect of PFF on α-Syn filament formation within cells. Experimental procedure: Purified α-Syn 5 mg / mL was mixed with 100 μg / mL GDNP or WDNP [total volume 200 μL: 70 μL α-Syn monomer (14.5 mg / mL) + 20 μL GDNP / WDNP (1 mg / mL) + 10 μL 1M NaCl (final concentration 50 mM) + 100 μL protein buffer (protein buffer component: 25 mM Tris-HCl)]. The mixture was then incubated at 1000 rpm and 37°C for 120 h to induce fibrosis. The resulting fibers were then sonicated to generate PFF. PFF was diluted to 100 nM with serum-free DMEM medium. PFF was transfected into PC12 cells stably expressing α-Syn-A53T-EGFP using Lipo2000. 24 h after transfection, the punctate structures within the cells were observed under a fluorescence microscope, and the data were statistically analyzed and plotted.

[0041] The method for constructing PC12 cells stably expressing α-Syn-A53T-EGFP is as follows: 1. Construction of mutant plasmids: α-Syn-A53T and EGFP fragments were amplified by primer PCR, and then these two fragments were ligated into the SHC003BSD-DelGFP-FLAG vector to construct a plasmid expressing the α-Syn-A53T-EGFP mutant.

[0042] α-Syn A53T sequence (SEQ ID NO.1): ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGACAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCA GTGGGTGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCC.

[0043] EGFP sequence (SEQ ID NO.2): ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG.

[0044] Construction method of SHC003BSD-DelGFP-FLAG: Using the SHC003BSD-DelGFP vector (Wang S, Crisman L, Miller J, Datta I, GulbransonDR, Tian Y, Yin Q, Yu H, Shen J. Inducible Exoc7 / Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis. J BiolChem. 2019 Dec 27;294(52):19988-19996. doi: 10.1074 / jbc.RA119.010821IF: 3.9Q2 . Epub 2019 Nov 18. PMID: 31740584; PMCID: PMC6937574.) as a template, primers were designed, and a FLAG tag was introduced by PCR to form SHC003BSD-DelGFP-FLAG.

[0045] 2. Transfection of PC12 cell line: Virus preparation: The A53T-EGFP-SHC003BSD-FLAG vector and three packaging plasmids: CMV-VSVG, PsPAX, and pAdvantage were mixed in a specific ratio, incubated with PEE25 for 20 minutes, and then transfected into 239T cells. The cell supernatant was collected to obtain the packaged lentivirus.

[0046] PC12 cell transfection: The prepared lentivirus was mixed with protamine sulfate, a transfection promoter, and then added to PC12 cells for infection. After passage, cells resistant to blisterin were selected, which were PC12 cells stably expressing α-Syn-A53T-EGFP.

[0047] Experimental results are as follows Figure 5 As shown, PFFs prepared under various conditions were added to the cells and incubated for 24 h. The results showed that PFFs formed in the presence of GDNP failed to effectively induce the formation of punctate α-Syn inclusion bodies in PC12 cells; while PFFs formed under WDNP treatment, similar to the control group, still promoted the formation of α-Syn inclusion bodies. In conclusion, GDNP can not only effectively inhibit PFF-induced α-Syn amyloid aggregate formation in vitro, but also inhibit its induction of α-Syn inclusion body formation in PC12 cells by reducing the generation of PFFs from α-Syn.

[0048] 5. GDNP reduces neurotoxicity of nerve cells caused by α-Syn fibers. Experimental procedure: α-Syn fiber preparation: Purified α-Syn (100 μM) was mixed with 100 μg / mL GDNP or WDNP [the system consisted of 200 μL α-Syn monomer (1000 μM) + 20 μL GDNP / WDNP (1 mg / mL) + 10 μL 1M NaCl (final concentration 50 mM) + 150 μL protein buffer (protein buffer component was 25 mM Tris-HCl)], and then incubated at 1000 rpm and 37 °C for 120 h to induce fibrosis.

[0049] AC: Seed PC12 cells into 96-well plates, adding 100 μL of cell suspension to each well, at a cell density of approximately 1 × 10⁻⁶ cells / well. 4 96-well plates were incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. Different concentrations of GDNP or WDNP (AB) were added; or α-Syn fibers formed in the presence of 100 μg / mL GDNP or WDNP were added, and incubation continued for another 24 h. 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 h. MTT was reduced to purple formazan by the living cells. The supernatant was aspirated, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader.

[0050] DE: PC12 cells were inoculated at 2.5 × 10⁻⁶. 5 Cells were seeded in 24-well plates and allowed to adhere for 12 h. The next day, 5 μM of α-Syn fibers formed after treatment with GDNP or WDNP were added, and the cells were cultured at 37°C for 24 h. 250 μl of Calcein AM / PI was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed three times with PBS, and the staining effect was observed under a fluorescence microscope. The number of viable cells was counted using ImageJ.

[0051] Experimental results are as follows Figure 6 As shown, direct exposure to α-Syn fibers is considered a key factor in causing mitochondrial dysfunction and cell death. Based on this, this invention further investigates whether GDNP can alleviate α-Syn-induced neuronal cell damage. MTT assay results showed that neither GDNP nor WDNP treatment alone produced significant toxicity to PC12 cells. Figure 6(AB). To assess its effect on α-Syn fiber toxicity, GDNP or WDNP was co-incubated with α-Syn for 5 days to prepare toxic α-Syn fibers, which were then added to PC12 cells and co-incubated for 24 h. MTT results showed that compared with the control group and the WDNP treatment group, the fibers formed in the GDNP treatment group had significantly reduced toxicity to PC12 cells (AB). Figure 6 (C) Calcein-AM live cell staining experiments further showed that the number of live cells in the GDNP-treated group was significantly higher than that in the WDNP-treated group and the control group. Figure 6 middle DE).

[0052] 6. GDNP reduces mitochondrial damage to nerve cells caused by α-Syn fibers. Experimental procedure: Purified α-Syn (100 μM) was mixed with 100 μg / mL GDNP or WDNP [the system consisted of 200 μL α-Syn monomer (1000 μM), 20 μL GDNP / WDNP (1 mg / mL), 10 μL 1M NaCl (final concentration 50 mM), and 150 μL protein buffer (protein buffer component was 25 mM Tris-HCl)]. The mixture was then incubated at 1000 rpm and 37°C for 120 h to induce fibrosis. PC12 cells were cultured at 3 × 10⁻⁶ cells per well. 4 The α-Syn filaments were spread into 6-well plates and cultured for 24 h after cell adhesion. The filaments from step 1 were added to PC12 cells, maintaining a final concentration of 5 μM, and the cells were cultured at 37°C for 24 h. A 3 mM stock solution of JC-10 dye was prepared using 500 μL of DMSO and stored at -20°C in the dark. The stock solution was diluted to 10 μM using antibiotic-free DMEM medium. After filament treatment, 500 μL of JC-10 working solution was added to each well of the PC12 cells, and the cells were treated in the dark for 30 min. The cells were then washed three times with PBS to remove the dye, followed by Hoechst dye treatment for 10 min to stain the nuclei. The cells were then washed three times with PBS to remove the dye again. JC-10 staining within the cells was observed using a laser confocal microscope. The distribution and intensity of green and red fluorescence within the cells were observed using the Alexa 488 channel and mCherry channel. Fluorescence intensity was statistically analyzed using ImageJ.

[0053] Experimental results are as follows Figure 7 As shown, mitochondrial functional impairment manifests as a decrease in membrane potential, and the degree of membrane potential decrease is positively correlated with the toxicity of α-Syn fibers. Experimental results showed that the toxicity of α-Syn fibers formed in the GDNP-treated group was significantly lower than that in the WDNP group and the control group, while the degree of mitochondrial membrane potential salvage was similar to that in the blank control group. Figure 7 Based on the above experimental results, it can be concluded that GDNP can reduce the toxic effects of α-Syn on nerve cells by inhibiting the formation of α-Syn amyloid aggregates, while WDNP did not show a significant inhibitory effect.

[0054] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of ginger-derived nanoparticles in the preparation of drugs that reduce neurotoxicity caused by α-synuclein.

2. The application according to claim 1, characterized in that, The nanoparticles derived from ginger have a particle size of 50-200 nm.

3. The method for preparing ginger-derived nanoparticles as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Juice the ginger, and centrifuge the juice at low speed and high speed in sequence to remove the precipitate. Collect the supernatant and filter it through a microporous membrane in sequence. (2) The filtrate obtained in step (1) is subjected to ultracentrifugation, and the precipitate is collected to obtain the ginger-derived nanoparticles.

4. The preparation method according to claim 3, characterized in that, The conditions for low-speed centrifugation in step (1) include: centrifugation at 1000 g for 10 min and centrifugation at 3000 g for 20 min; the conditions for high-speed centrifugation include: centrifugation at 10000 g for 30 min; the pore sizes of the microporous filter membrane are 1.2 μm, 0.8 μm and 0.45 μm respectively; The conditions for ultracentrifugation in step (2) include: centrifugation at 150,000 g for 1.5 h.

5. Application of ginger-derived nanoparticles in the preparation of drugs that inhibit α-synuclein liquid-liquid phase separation.

6. Application of ginger-derived nanoparticles in the preparation of drugs that inhibit α-synuclein prefibrillation-induced amyloid aggregation.

7. A drug for reducing neurotoxicity caused by α-synuclein, characterized in that, It contains an effective amount of ginger-derived nanoparticles.

8. The medicament according to claim 7, characterized in that, It also includes pharmaceutically acceptable carriers.

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