A polypeptide and its use in the preparation of an anti-neurodegenerative disease drug

Abstract

The application provides a polypeptide and application thereof in preparation of an anti-neurodegenerative disease drug, and belongs to the technical field of biological medicine, and specifically provides a polypeptide with a neuroprotective effect, wherein the amino acid sequence of the polypeptide is SEQ ID NO. 2 or SEQ ID NO. 3; a composition containing a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 and / or SEQ ID NO. 3; application of the polypeptide or the composition in preparation of a product with a neuroprotective function; and the polypeptide provided by the application can be used in preparation of an anti-neurodegenerative disease drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide and its application in the preparation of drugs for treating neurodegenerative diseases. Background Technology

[0002] Parkinson's disease (PD) is a progressive neurodegenerative disease that primarily affects the elderly. Its main pathological features include the loss of dopaminergic neurons in the substantia nigra and abnormal aggregation of α-synuclein in Lewy bodies, which in turn leads to motor symptoms such as resting tremor, bradykinesia, and muscle rigidity, as well as non-motor symptoms such as cognitive impairment and autonomic dysfunction. The disease progressively worsens over time.

[0003] Currently, there is no cure for Parkinson's disease in clinical practice. Conventional treatments such as levodopa and dopamine receptor agonists can only relieve clinical symptoms, have significant side effects with long-term use, and cannot stop the progression of the disease. Therefore, there is an urgent clinical need to develop safe, effective, and multi-target neuroprotective drugs.

[0004] Marine organisms are a vast and largely untapped natural resource of bioactive peptides. Food-derived bioactive peptides have attracted widespread attention due to their high safety, good biocompatibility, ability to cross the blood-brain barrier, and multi-target mechanisms of action, as well as their neuroprotective potential.

[0005] Rapana venosa is rich in various nutrients and bioactive substances, possessing extremely high edible and medicinal value. Existing studies have shown that dietary intake of Rapana venosa can significantly improve lipid metabolism and enhance the body's antioxidant capacity. Rapana venosa protein and its enzymatically hydrolyzed peptides exhibit various biological activities, including antioxidant, anti-inflammatory, immunomodulatory, and anti-tumor effects. However, the efficacy and mechanism of action of Rapana venosa active peptides in Parkinson's disease models remain unclear. Whether they can exert anti-Parkinson's disease effects by integrating and regulating oxidative stress and metabolic disorders requires further systematic research. Furthermore, there is a lack of peptide drugs with clear anti-Parkinson's disease efficacy developed based on Rapana venosa in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a polypeptide and its application in the preparation of drugs for treating neurodegenerative diseases.

[0007] One objective of this invention is to provide three novel active polypeptides derived from *Rhodotorula rubra* with excellent anti-Parkinson's disease function and their pharmaceutical compositions, clarifying their neuroprotective activity and mechanism of action. Another objective of this invention is to provide methods for preparing these polypeptides and their applications in the pharmaceutical and health product fields.

[0008] The technical solution of the present invention is as follows: A polypeptide with neuroprotective effects, wherein the amino acid sequence of the polypeptide is SEQ ID NO.2 or SEQ ID NO.3.

[0009] SEQ ID NO.2: FLVKLPMFM; SEQ ID NO.3: SDSLSEILIS.

[0010] A composition comprising a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.3.

[0011] Preferably, the composition further contains mannitol.

[0012] Preferably, the composition further contains a pharmaceutically acceptable carrier, diluent, excipient, cosolvent, or sustained-release excipient.

[0013] The use of a polypeptide or the above composition in the preparation of a product having neuroprotective function, wherein the amino acid sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0014] Preferably, the product includes pharmaceuticals or health foods.

[0015] The use of a polypeptide or the above composition in the preparation of a medicament for the prevention and / or treatment of neurodegenerative diseases, wherein the amino acid sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0016] Preferably, the neurodegenerative disease is Parkinson's disease.

[0017] A method for preparing a polypeptide, comprising any one of the following schemes: Option 1: Enzymatic hydrolysis separation and purification method, including the following steps: (1) Preparation of protein hydrolysate of *Rhodotorula pulveratum*: Using *Rhodotorula pulveratum* tissue as raw material, the protein hydrolysate of *Rhodotorula pulveratum* was obtained by homogenization, stepwise enzymatic hydrolysis with pepsin-trypsin, inactivation centrifugation, and freeze drying. (2) Ultrafiltration fractionation: The enzymatic hydrolysate was fractionated using a polyethersulfone ultrafiltration membrane to retain components with a molecular weight <3 kDa; (3) Gel chromatography purification: The components obtained in step (2) were further purified using a Sephadex G-25 gel filter column. The components in the elution peak were collected to obtain the target peptide mixture. (4) Mass spectrometry identification and single peptide preparation: The peptide sequence was identified by ultra-high performance liquid chromatography-tandem mass spectrometry, and the target peptide was prepared. The amino acid sequence of the target polypeptide is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; Option 2: Solid-phase chemical synthesis method, using Fmoc solid-phase synthesis method, using Rink Amide resin, stepwise linking the amino acid sequences corresponding to SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; after synthesis, the peptide is cleaved from the resin with cleaving fluid, and the crude peptide is collected by ether precipitation and centrifugation; the crude peptide is purified by reversed-phase high-performance liquid chromatography to obtain the target peptide.

[0018] Beneficial effects 1. Excellent neuroprotective activity and clear in vivo efficacy: The three peptides provided by this invention can significantly alleviate MPTP-induced damage to dopaminergic neurons in the midbrain of zebrafish, as well as cerebral vascular structural disorder and loss, and reverse behavioral disorders such as decreased motor ability and abnormal photosensitivity in Parkinson's disease models. The efficacy is comparable to that of the positive control drug levodopa, and the improvement effect on some indicators is even better. The peptides provided by this invention can be used to prepare drugs against neurodegenerative diseases.

[0019] 2. Clear multi-target mechanism of action, capable of intervening in the core process of disease: The peptides provided by this invention can target and bind to key targets of Parkinson's disease, human dopa decarboxylase (DDC), human α-synuclein (α-syn), and human monoamine oxidase B (MAO-B), with binding affinity significantly superior to levodopa; at the same time, it can achieve multi-dimensional regulation from four core pathological pathways: oxidative stress, neuroinflammation, apoptosis, and metabolic disorders, making up for the shortcomings of existing drugs that can only relieve symptoms and cannot stop disease progression.

[0020] 3. High safety and good drug-like properties: The polypeptides provided by this invention are food-derived marine bioactive peptides with good biocompatibility. They can cross the blood-brain barrier and do not have the significant side effects caused by long-term use of conventional chemical drugs, thus possessing good potential for clinical translation.

[0021] 4. Mature preparation process, suitable for industrial scale-up: The peptides provided by this invention can be prepared on a large scale by proteolytic enzyme digestion and fractional purification of Mycosinusoids, or high-purity single peptides can be obtained efficiently by solid-phase synthesis. The process is stable and controllable, and the industrialization threshold is low. Attached Figure Description

[0022] Figure 1 Figure 1 shows the results of detecting the neuroprotective effects of the protein hydrolysate and purified peptide components of *Rhodotorula buergerianum* on dopaminergic neurons in zebrafish. Figure (A) shows a representative fluorescence image of the midbrain region of vmat2:GFP transgenic zebrafish, (B) shows the statistical analysis results of the length of dopaminergic neuron regions, (C) shows the elution curve of the <3 kDa component after Sephadex G-25 gel filtration chromatography, (D) shows a representative fluorescence image of the midbrain region of zebrafish after treatment with the gel chromatography purified component, and (E) shows the statistical analysis results of the length of the dopaminergic neuron regions after treatment with the gel chromatography purified component.

[0023] Figure 2 The results of the detection to identify the protective effect of the obtained active peptides on zebrafish dopaminergic neurons are shown in the figure. Figure (A) shows a representative fluorescence map of the midbrain region of vmat2:GFP transgenic zebrafish, and (B) shows the statistical analysis results of the length of dopaminergic neuron regions.

[0024] Figure 3 The results show the molecular docking of the target peptide with key targets of Parkinson's disease, specifically the docking results of target 1JS3 (dopa decarboxylase DDC). Each set of results includes an overall structure diagram, a magnified view of a local area, and a two-dimensional interaction diagram.

[0025] Figure 4 The results show the molecular docking of the target peptide with key targets of Parkinson's disease, specifically the docking results of target 3Q25 (α-synuclein). Each set of results includes an overall structure diagram, a magnified view of the local area, and a two-dimensional interaction diagram.

[0026] Figure 5 The results show the molecular docking of the target peptide with key targets of Parkinson's disease, specifically the docking results of target 4A79 (monoamine oxidase B MAO-B). Each set of results includes an overall structure diagram, a magnified view of the local area, and a two-dimensional interaction diagram.

[0027] Figure 6 The figure shows the results of detecting the protective effect of gradient concentration target peptides on zebrafish dopaminergic neurons. Figure (A) shows a representative fluorescence map of the midbrain region of vmat2:GFP transgenic zebrafish, and (B) shows the statistical analysis results of the length of dopaminergic neuron regions.

[0028] Figure 7 The results of the detection of the protective effect of the target peptide on the blood vessels of zebrafish, a model of Parkinson's disease; In the figure, (A) is a representative fluorescence image of Fli1:GFP transgenic zebrafish, and (B) is the statistical result of the number of intact blood vessels in the brain.

[0029] Figure 8 The results of the detection of the effect of the target peptide on the improvement of behavioral abnormalities in juvenile zebrafish model of Parkinson's disease are shown in the figure. Figure (A) shows the analysis results of the total movement distance of zebrafish in each group, (B) shows the detection results of the average swimming speed of zebrafish juveniles in each group, (C) shows the movement trajectory of zebrafish, (D) shows the change results of the average swimming speed of zebrafish juveniles in response to environmental stimuli, and (E) shows the statistical analysis results of the total movement distance of zebrafish under alternating light and dark conditions.

[0030] Figure 9 The figure shows the results of detecting the effects of three target peptides on the activity of superoxide dismutase (SOD), a biochemical indicator related to Parkinson's disease.

[0031] Figure 10 The figure shows the results of detecting the effects of three target peptides on the activity of glutathione peroxidase (GSH-Px), a biochemical indicator related to Parkinson's disease.

[0032] Figure 11 The figure shows the results of detecting the effects of three target peptides on the activity of catalase (CAT), a biochemical marker related to Parkinson's disease.

[0033] Figure 12 The figure shows the effect of three target peptides on the content of malondialdehyde (MDA), a biochemical indicator related to Parkinson's disease.

[0034] Figure 13 The figure shows the results of detecting the effects of three target peptides on the activity of acetylcholinesterase (AChE), a biochemical marker related to Parkinson's disease.

[0035] Figure 14 The transcriptome sequencing results of zebrafish, a Parkinson's disease model, treated with three target peptides are shown, specifically a volcano map of differential gene expression distribution.

[0036] Figure 15 The transcriptome sequencing results of zebrafish model of Parkinson's disease treated with three target peptides are shown, specifically Venn diagrams of common and differentially expressed genes among the groups.

[0037] Figure 16 The transcriptome sequencing results of zebrafish, a Parkinson's disease model, treated with three target peptides are shown in the figure, specifically the GO functional enrichment analysis results of differentially expressed genes.

[0038] Figure 17 The transcriptome sequencing results of zebrafish, a Parkinson's disease model, treated with three target peptides are shown in the figure. Specifically, the KEGG pathway enrichment analysis results of differentially expressed genes are presented.

[0039] Figure 18 The results of the validation of the effect of the target peptide on the expression of Parkinson's disease-related genes are shown in the figure, specifically the detection results of the mRNA expression level of metabolism-related genes.

[0040] Figure 19The results of the validation of the effect of the target peptide on the expression of Parkinson's disease-related genes are shown in the figure, specifically the detection results of the mRNA expression level of genes related to oxidative stress.

[0041] Figure 20 The results of the validation of the effect of the target peptide on the expression of Parkinson's disease-related genes are shown in the figure, specifically the detection results of the mRNA expression levels of inflammation and apoptosis-related genes. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0043] Unless otherwise specified, all contents in the following embodiments are based on the prior art; the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0044] Example 1 Preparation of Rhodospirelae protease hydrolysates and isolation and purification of anti-Parkinson's disease active peptides 1. Preparation of tissue protein hydrolysate from *Rhodotorula purpureus* Fresh whole tissue from *Rhodotorula pulveratum* was collected and homogenized with phosphate-buffered saline (PBS) at a ratio of 1:10 (w / v) to pH 7.2. The pH was adjusted to 2.0 with 1 mol / L hydrochloric acid, and homogenized again under ice bath conditions. Pepsin was added at a substrate-enzyme ratio of 1:50 (w / w), and the mixture was incubated at 37°C for 2 hours to complete the first step of simulated gastrointestinal digestion. Subsequently, the pH was adjusted to 7.0 with 1 mol / L sodium hydroxide, and trypsin was added at a substrate-enzyme ratio of 1:50 (w / w), and the mixture was incubated at 37°C for another 2 hours. The enzymatic reaction was terminated by heating in a boiling water bath for 10 minutes. The mixture was centrifuged at 4°C and 10,000 × g for 20 minutes, and the supernatant was collected, lyophilized, and then stored in a suitable container. Store at 80℃ for later use.

[0045] The degree of protein hydrolysis (DH) was determined by o-phthalaldehyde (OPA) derivatization, and a protein hydrolysate of spirochetes with a degree of hydrolysis of 45.81% was finally obtained.

[0046] 2. Ultrafiltration fractionation and initial activity screening of enzymatic hydrolysate The supernatant from the hydrolysis of *Mycobacterium erythrocyte sedimentation* was fractionated using polyethersulfone ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 5 kDa, and 3 kDa. The fractions were passed sequentially through membranes at 0.25 MPa: first through a 10 kDa ultrafiltration membrane, yielding the >10 kDa fraction (Fr1); the permeate was then passed sequentially through 5 kDa and 3 kDa membranes, yielding the 5–10 kDa fraction (Fr2), the 3–5 kDa fraction (Fr3), and the <3 kDa fraction (Fr4). The four fractions were then lyophilized and concentrated. Store at 80℃.

[0047] The neuroprotective activity of each component was evaluated using an MPTP-induced zebrafish Parkinson's disease model. The results showed that, at the same concentration, the original enzyme hydrolysate and the Fr4 component had the strongest neuroprotective activity, which could effectively improve the morphological abnormalities of MPTP-induced dopaminergic neurons in zebrafish and promote the growth of dopaminergic neurons.

[0048] 3. Gel chromatography purification and activity rescreening Take the lyophilized Fr4 sample and prepare a 50 mg / mL solution with deionized water. Filter the solution through a 0.45 μm syringe filter. Load 5 mL of the sample into a Sephadex G-25 gel filter column (2.0 × 90 cm) pre-equilibrated with deionized water. Elute with deionized water at a flow rate of 30 mL / h, collecting 10 mL of eluent per tube. Measure the absorbance at 220 nm. Combine the peak components according to the elution curve to obtain two subfractions: Fr4-F1 and Fr4-F2.

[0049] Based on the activity evaluation using a zebrafish model, Fr4-F2 showed significantly better protective effects against dopaminergic neurons than Fr4-F1. Therefore, the Fr4-F2 fraction was selected for subsequent peptide identification. Figure 1 .

[0050] Example 2 Mass spectrometry identification, screening and solid-phase synthesis of bioactive peptides 1. Peptide sequence identification The peptides in the Fr4-F2 fraction were identified using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The raw mass spectrometry data were compared with the NCBInr database using Mascot 2.3 software. An expected value <0.05 was considered a valid identification. A total of 14 peptides (P1-P14) were identified, with amino acid lengths ranging from 7 to 10.

[0051] 2. Screening of bioactive peptides Molecular docking was performed against three key protein targets in Parkinson's disease (human dopa decarboxylase DDC, human α-synuclein α-synuclein, and human monoamine oxidase B MAO-B). Simultaneously, the BIOPEPUWM database was used to predict the antioxidant, neuropeptide, and metabolic regulatory activities of the peptides, and these activities were validated in vivo in zebrafish. (See [link to study]). Figure 2 Ultimately, three peptides with potent anti-Parkinson's disease activity were screened out: KSTELLI ( Figure 2 P6 in the middle), FLVKLPMFM ( Figure 2 P3 in SDSLSEILIS Figure 2 (P7 in the text).

[0052] 3. Solid-phase synthesis of polypeptides Shanghai Qiangyao Biotechnology Co., Ltd. was commissioned to synthesize the above three peptides using the Fmoc solid-phase synthesis strategy. The specific steps are as follows: Using Rink Amide resin, a stepwise coupling reaction was performed according to the target amino acid sequence to complete peptide chain elongation. After synthesis, the peptide was cleaved from the resin using a cleavage buffer (TFA / TIS / H2O, 95:2.5:2.5), precipitated with anhydrous diethyl ether, and collected by centrifugation. The crude peptide was purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), and mass spectrometry confirmed that the molecular weight was consistent with the theoretical value, finally obtaining the target peptide with a purity >95% for subsequent experiments.

[0053] The amino acid sequence of the polypeptide is as follows: SEQ ID NO.1: Lys-Ser-Thr-Glu-Leu-Leu-Ile (KSTELLI); SEQ ID NO.2: Phe-Leu-Val-Lys-Leu-Pro-Met-Phe-Met (FLVKLPMFM); SEQ ID NO. 3: Ser-Asp-Ser-Leu-Ser-Glu-Ile-Leu-Ile-Ser (SDSLSEILIS).

[0054] Example 3 Molecular docking experiment between target peptide and key targets of Parkinson's disease The binding interaction between the target peptide and key targets of Parkinson's disease was verified using AutoDock 4.2.6 software. The specific steps are as follows: 1. Download the target crystal structures from the RCSB protein database: human dopa decarboxylase (PDB: 1JS3), human α-synuclein (PDB: 3Q25), and human monoamine oxidase B (PDB: 4A79); construct the three-dimensional structures of the three peptides and optimize them by minimizing energy; use PyMOL 2.6 and AutoDockTools-1.5.7 to preprocess the receptor protein and ligand peptide, respectively.

[0055] 2. Set up semi-flexible docking, run 50 simulations independently for each complex group, and select the model with the lowest binding free energy and the most stable conformation for binding mode analysis.

[0056] 3. Experimental results are shown below. Figure 3 , Figure 4 , Figure 5The three peptides exhibited excellent binding affinity to three key targets of Parkinson's disease, significantly superior to the positive control drug levodopa. FLVKLPMFM, in particular, achieved stable binding to all three targets through a synergistic hydrogen bond network, extensive hydrophobic interactions, and π-type interactions, demonstrating the best binding specificity. SDSLSEILIS exhibited the most complete hydrogen bond binding network with α-synuclein. KSTELLI demonstrated outstanding binding ability to regulate inflammation and apoptosis-related targets.

[0057] Example 4 Pharmacodynamic experiments of the target peptide on a zebrafish model of MPTP-induced Parkinson's disease This embodiment uses a zebrafish model to systematically verify the in vivo anti-Parkinson's disease efficacy of three target peptides. The zebrafish rearing and drug administration regimen is as follows: The Tg (vmat2:GFP) and Tg (fli1:GFP) transgenic zebrafish and AB wild-type zebrafish were provided by the Drug Screening Platform of the Institute of Biology, Shandong Academy of Sciences. Those skilled in the art can purchase adult zebrafish from the National Zebrafish Resource Center and breed them independently to obtain zebrafish juveniles of the corresponding strains for testing, or entrust the Drug Screening Platform of the Institute of Biology, Shandong Academy of Sciences to conduct activity evaluation tests.

[0058] Zebrafish embryo acquisition: Female and male zebrafish were raised separately, with alternating periods of 14 hours of light and 10 hours of darkness. They were fed artificial pellet food and newly hatched Artemia nauplii at regular intervals. Healthy, sexually mature zebrafish were placed in a mating tank at a 1:1 ratio of females to males. Fertilized eggs were obtained the following day between 9 and 10 am. After disinfection and washing, the fertilized eggs were transferred to zebrafish embryo culture medium (containing 5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4) and cultured under controlled light at 28°C.

[0059] Zebrafish juveniles were used as the research subject. Embryos 10–12 h post-fertilization were cultured in zebrafish embryo culture medium, with 0.003% phenylthiourea (PTU) added to inhibit pigment deposition. At 24 h post-fertilization, the juveniles were randomly divided into three groups: a blank control group, an MPTP model group (50 μM MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a levodopa positive control group (6 μg / mL levodopa + 50 μM MPTP solution), and three peptide treatment groups (12.5, 25, and 50 μg / mL peptide solution + 50 μM MPTP solution). Ten zebrafish were used in each group, with three replicates. The zebrafish in each experimental group were placed in a light incubator (28℃), and the embryo culture medium containing the corresponding drug was changed daily, allowing the embryos to continue developing for 3–4 days for subsequent pharmacodynamic experiments.

[0060] 1. Verification of the protective effect of dopaminergic neurons Tg (vmat2:GFP) juveniles that developed to 4 dpf were used in this experiment.

[0061] Ten tails were randomly selected from each group, and fluorescence imaging was performed using a Zeiss microscope to quantitatively analyze the length of GFP-labeled dopaminergic neuron regions.

[0062] The results are shown below. Figure 6 MPTP treatment can significantly shorten the length of dopaminergic neurons in zebrafish. p <0.0001); All three target peptides can significantly alleviate midbrain dopaminergic neuron damage caused by MPTP and reverse neuronal length shortening. Among them, KSTELLI (K7) and FLVKLPMFM (F9) have slightly better improvement effects than SDSLSEILIS (S10) and are comparable to the positive control drug levodopa.

[0063] 2. Verification of cerebrovascular protective effect Tg (fli1:GFP) transgenic zebrafish juveniles that developed to 4 dpf were used in this experiment.

[0064] Ten zebrafish were randomly selected from each group, and fluorescence imaging was performed using a Zeiss microscope to quantitatively analyze the vascular defects in the brain of GFP-labeled zebrafish.

[0065] The results are shown below. Figure 7 MPTP exposure causes significant loss and structural disorder of blood vessels in the zebrafish brain; intervention with peptides at gradient concentrations of 12.5, 25, and 50 μg / mL can effectively improve MPTP-induced vascular damage, and the protective effect shows a clear dose-dependent effect; among them, KSTELLI (K7), SDSLSEILIS (S10) and high concentration FLVKLPMFM (F9) have even better cerebral vascular repair effects than the positive control drug.

[0066] 3. Verification of behavioral improvement effects Wild-type AB zebrafish juveniles with a development of 5 dpf were used for behavioral testing.

[0067] (1) Routine movement ability test: Juvenile fish were individually transferred into a 48-well plate and placed in a fully automatic behavior tracker. After acclimatization for 15 minutes, their movement behavior was recorded for 20 minutes. The total swimming distance and average swimming speed were analyzed using ZebLab software. The results are shown in […]. Figure 8 The total movement distance and average swimming speed of zebrafish in the MPTP model group were significantly reduced; treatment with the three peptides could significantly reverse the decline in mobility caused by MPTP and restore normal mobilization function in zebrafish.

[0068] (2) Phototaxis experiment: Three cycles of "10 min darkness - 10 min light" were set up, with a total duration of 60 min. The movement response of zebrafish to sudden changes in light intensity was tracked and analyzed. The results are shown in […]. Figure 8 The MPTP model group showed a highly significant decrease in the ability of zebrafish to respond to external environmental stimuli. p <0.0001); All three peptides can enhance the kinetic activity of zebrafish in dark environments and restore their photosensitivity. Among them, FLVKLPMFM and SDSLSEILIS have more prominent effects on the recovery of kinetic function under alternating light and dark stimulation.

[0069] 4. Detection of Parkinson's disease-related biochemical indicators Wild-type AB zebrafish juveniles were used as experimental subjects. Fifty zebrafish juveniles were collected from each group after 3 days of drug administration and modeling. They were homogenized on ice, centrifuged at 10000×g for 10 min at 4℃, and the supernatant was collected. The activities and contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), and acetylcholinesterase (AChE) were determined using commercially available assay kits.

[0070] The results are shown below. Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 Compared with the blank control group, MPTP treatment significantly reduced SOD and CAT activities, while increasing MDA, GSH-Px and AChE levels. All three peptides could reverse the abnormal changes of the above indicators. Among them, SDSLSEILIS had the most comprehensive regulatory effect on oxidative stress-related indicators, while KSTELLI and FLVKLPMFM had better inhibitory effects on inflammation- and apoptosis-related AChE activities.

[0071] Example 5 Transcriptomic analysis and molecular mechanism verification of the anti-Parkinson's disease effect of the target peptide 1. Transcriptome sequencing analysis Wild-type AB zebrafish juveniles were used as experimental subjects. The zebrafish were grouped and treated as in Example 4. Fifty zebrafish juveniles were collected from each group after 3 days of drug administration and modeling. Three biological replicates were set up for each group, and transcriptome sequencing was performed. p Differentially expressed genes (DEGs) were screened based on a criterion of <0.05 and |log2 fold change|>1, and GO functional enrichment analysis, KEGG pathway enrichment analysis, and gene set enrichment analysis (GSEA) were performed.

[0072] The results are shown below. Figure 14 , Figure 15 , Figure 16 , Figure 17Compared with the MPTP model group, the FLVKLPMFM treatment group identified 618 differentially expressed genes, the KSTELLI treatment group identified 1459 differentially expressed genes, and the SDSLSEILIS treatment group identified 359 differentially expressed genes. The intersection of the four comparison sets yielded 9 core common differentially expressed genes, which are involved in key biological processes such as immune inflammatory response, apoptosis, and metabolism.

[0073] Enrichment analysis results showed that the common mechanism by which the three peptides exert their anti-Parkinson's disease effects is the regulation of metabolism, oxidative stress, inflammatory response and apoptosis; among them, KSTELLI is more inclined to anti-inflammatory and immune regulation, FLVKLPMFM focuses on metabolic pathway regulation, and SDSLSEILIS focuses on the regulation of neural signal transduction.

[0074] 2. Molecular mechanism verification by qRT-PCR Total RNA was extracted from samples from the same batch using transcriptome sequencing, and cDNA was synthesized by reverse transcription. Real-time quantitative PCR was used to verify the mRNA expression levels of differentially expressed genes related to metabolism, oxidative stress, inflammation, and apoptosis. β-actin was used as an internal reference gene, and the relative gene expression level was calculated using the 2^(-ΔΔCt) method.

[0075] The results are shown below. Figure 18 , Figure 19 , Figure 20 All three peptides significantly antagonized the abnormal expression of MPTP-induced metabolism-related genes (α-syn, g6pca2, acsl5, etc.), oxidative stress-related genes (nrf2, keap1, gpx4a, etc.), and inflammation and apoptosis-related genes (il-1β, cxcl8a, caspase3, etc.), consistent with transcriptome sequencing results, clarifying the molecular mechanism of action of the three peptides against Parkinson's disease.

[0076] Example 6 Preparation of anti-Parkinson's disease drug compositions This embodiment provides an injectable pharmaceutical composition containing a target polypeptide, with the following formulation: Take 2g of the FLVKLPMFM polypeptide shown in SEQ ID NO.2, add 18g of mannitol, add water for injection to 1000 mL, stir to completely dissolve the material, filter through a 0.22 μm microporous membrane for sterilization, fill into vials, partially stopper, freeze dry, then stopper and cap to prepare an injectable powder injection, each vial containing 10mg of polypeptide.

[0077] This drug composition can be administered intravenously for the clinical treatment of Parkinson's disease and exhibits good stability and bioavailability.

[0078] The polypeptides provided by this invention can be used to prepare products with neuroprotective functions and can be used to prepare drugs for neurodegenerative diseases, such as Parkinson's disease.

Claims

1. A polypeptide with neuroprotective effects, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO.2 or SEQ ID NO.

3.

2. A composition, characterized in that, A polypeptide containing amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.

3.

3. The composition according to claim 2, characterized in that, The composition also contains mannitol.

4. The composition according to claim 2, characterized in that, The composition also contains a pharmaceutically acceptable carrier, diluent, excipient, cosolvent, or sustained-release excipient.

5. The use of a polypeptide or the composition according to any one of claims 2-4 in the preparation of a product having neuroprotective function, wherein the amino acid sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

6. The application as described in claim 5, characterized in that, The products include pharmaceuticals or health foods.

7. The use of a polypeptide or the composition according to any one of claims 2-4 in the preparation of a medicament for the prevention and / or treatment of neurodegenerative diseases, wherein the amino acid sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

8. The application as described in claim 7, characterized in that, The neurodegenerative disease mentioned is Parkinson's disease.

9. A method for preparing a polypeptide, characterized in that, Including any of the following options: Option 1: Enzymatic hydrolysis separation and purification method, including the following steps: (1) Preparation of protein hydrolysate of *Rhodotorula pulveratum*: Using *Rhodotorula pulveratum* tissue as raw material, the protein hydrolysate of *Rhodotorula pulveratum* was obtained by homogenization, stepwise enzymatic hydrolysis with pepsin-trypsin, inactivation centrifugation, and freeze drying. (2) Ultrafiltration fractionation: The enzymatic hydrolysate was fractionated using a polyethersulfone ultrafiltration membrane to retain components with a molecular weight <3 kDa; (3) Gel chromatography purification: The components obtained in step (2) were further purified using a Sephadex G-25 gel filter column. The components in the elution peak were collected to obtain the target peptide mixture. (4) Mass spectrometry identification and single peptide preparation: The peptide sequence was identified by ultra-high performance liquid chromatography-tandem mass spectrometry, and the target peptide was prepared. The amino acid sequence of the target polypeptide is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; Option 2: Solid-phase chemical synthesis method, using Fmoc solid-phase synthesis method, using Rink Amide resin, stepwise linking the amino acid sequences corresponding to SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; after synthesis, the peptide is cleaved from the resin with cleaving fluid, and the crude peptide is collected by ether precipitation and centrifugation; the crude peptide is purified by reversed-phase high-performance liquid chromatography to obtain the target peptide.

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