Nostoc commune polypeptide and application thereof in preparation of medicine for preventing and treating neurodegenerative diseases

Abstract

This invention belongs to the field of small molecule peptides, specifically relating to a *Ge Xian Mi* peptide and its application in the preparation of drugs for the prevention and treatment of neurodegenerative diseases. Using *Ge Xian Mi* as raw material, this invention extracts, separates, and identifies a pure natural *Ge Xian Mi* peptide through enzymatic hydrolysis, ultrafiltration purification, and LC-MS / MS identification. The amino acid sequence of this *Ge Xian Mi* peptide is TQFYLR, and its molecular formula is C0. 39 H 58 N 10 O 10 With an average relative molecular mass of approximately 826.43 Da and a theoretical isoelectric point of pI=8.41, this is a hydrophilic polypeptide. This *Ge Xian Mi* polypeptide possesses anti-protein aggregation and neuroprotective functions, and has broad application prospects in the development of pharmaceuticals and other products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of small molecule polypeptides, and particularly relates to a Nostoc sphaeroides polypeptide and application thereof in preparation of a medicament for preventing and treating a neurodegenerative disease. BACKGROUND

[0002] Protein aggregation refers to a process in which a protein aggregates into an abnormal polymer or fiber in a cell or in vitro. This phenomenon is usually caused by abnormal structure of the protein, which cannot be correctly folded or maintained in a stable state, thereby tending to aggregate. Mis-sense mutations, oxidative modifications and errors in amino acid pairing during translation occurring in the aging process of the human body can all lead to changes in protein structure and weakening of cell repair ability, and thus protein aggregation, so that aging is both a cause of such diseases and exacerbation of which can make the self-repairing ability of the organism decrease and the symptoms of the disease worsen.

[0003] Toxicity caused by protein aggregation can make the organism in a stressed state: on the one hand, the spatial structure of the protein that occurs aggregation changes, the normal physiological function is damaged, and the life activity is blocked; on the other hand, the aggregation process produces aggregates, and common forms include amyloid fibers (such as β-amyloid in Alzheimer's disease) and neurofibrillary tangles (such as tau protein in Alzheimer's disease). The toxicity of protein aggregates mainly manifests in cytotoxicity (directly damaging the cell membrane, leading to dysfunction or death of cells, and interfering with normal protein function or enzyme activity), inflammatory response (activating the immune system and inflammatory pathways, further damaging tissues and exacerbating symptoms), and intracellular toxicity (accumulating in cells, interfering with the function of organelles such as endoplasmic reticulum and mitochondria, leading to oxidative stress and apoptosis). These toxic effects are closely related to various neurodegenerative diseases - neurodegenerative diseases refer to a general term for diseases in which the structure and function of neurons are chronically and progressively degraded or even die, leading to dysfunction, and their common feature is extracellular fiber amyloid deposition or intracellular abnormal protein fibril inclusion caused by protein aggregation, including Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis and prion disease; taking Alzheimer's disease as an example, the related Aβ oligomers (Amyloid Beta Oligomers) are formed by the polymerization of β-amyloid, and Aβ is a protein fragment produced by the cleavage of amyloid precursor protein (APP) in the brain of patients, Aβ oligomers are composed of 2 to 12 Aβ monomers and appear earlier than long-chain amyloid plaques, and are considered to be one of the toxic factors of Alzheimer's disease, which can interfere with the normal function of nerve cells, trigger oxidative stress, inflammatory response and synaptic damage, and ultimately lead to cognitive decline and neurodegeneration.

[0004] For this key pathogenic process, bioactive peptides—protein fragments composed of short-chain amino acids that can be extracted from food, microorganisms, or cells and exert antioxidant, anti-inflammatory, blood pressure-lowering, and immunomodulatory effects—have become an important research direction for anti-protein aggregation. Their intervention mechanisms mainly include: directly binding to abnormally folded proteins and inhibiting their formation of aggregates; helping to maintain correct protein folding and reducing misfolding and aggregation; promoting intracellular autophagy and protein degradation pathways and helping to clear accumulated abnormal aggregates; and protecting cells from aggregation-induced damage through antioxidant and anti-inflammatory effects.

[0005] However, the application of bioactive peptides in anti-protein aggregation research still faces many limitations: the synthesis process is complex, especially with high production costs under high purity and specific modification requirements; production requires multiple steps such as peptide synthesis, purification, folding, and modification, each of which may introduce instability or efficiency issues, making large-scale production difficult; many bioactive peptides are easily degraded or inactivated during storage and handling, requiring specific conditions to maintain stability, increasing complexity and cost; purifying bioactive peptides from complex mixtures is also a technical challenge, and purification efficiency may decrease during large-scale production, affecting product quality and consistency. Nevertheless, bioactive peptides still have great potential in anti-protein aggregation and require further research and optimization to overcome the above limitations, potentially providing new ideas for the treatment of protein aggregation-related diseases. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a Ge Xian Mi polypeptide that has the functions of anti-protein aggregation and neuroprotection.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned Ge Xian Mi polypeptide.

[0008] Another object of the present invention is to provide the application of the above-mentioned Ge Xian Mi polypeptide.

[0009] The objective of this invention is achieved through the following technical solution: A type of Ge Xian Mi polypeptide with the amino acid sequence: TQFYLR.

[0010] The molecular formula of the aforementioned Ge Xian Mi polypeptide is C 39 H 58 N 10 O 10 It has an average relative molecular mass of approximately 826.43 Da and a theoretical isoelectric point of pI=8.41, making it a hydrophilic polypeptide.

[0011] The preparation method of the aforementioned Ge Xian Mi polypeptide includes the following steps: The above-mentioned *Ge Xian Mi* polypeptides can be prepared directly through solid-phase synthesis, or by using *Ge Xian Mi* as a raw material and obtaining the above-mentioned *Ge Xian Mi* polypeptides through enzymatic hydrolysis, separation, extraction, and purification.

[0012] The enzymatic hydrolysis is preferably performed by sequentially using trypsin and papain.

[0013] The preferred specific operation for the enzymatic hydrolysis is as follows: Mix dried Nostoc commune powder with water, adjust the pH of the system to 7.5-8.0, add trypsin for enzymatic hydrolysis; then adjust the pH of the system to 6.0-7.0, add papain for enzymatic hydrolysis; after enzymatic hydrolysis, inactivate to obtain Nostoc commune protein hydrolysate.

[0014] The preferred dosage of trypsin is 10,000 to 60,000 U of trypsin per gram of dried Ge Xian Mi powder, and the preferred enzymatic hydrolysis conditions are 45°C for 4 hours.

[0015] The preferred dosage of papain is 10,000 to 60,000 U of papain per gram of dried kudzu root powder, and the preferred enzymatic hydrolysis conditions are 60°C for 4 hours.

[0016] The preferred specific operation for the separation and extraction is as follows: After cooling the enzyme hydrolysate of *Ge Xian Mi*, add 95% ethanol (volume fraction) to the solution until the ethanol concentration is 60-80%, and let it stand. Then remove the precipitate by vacuum filtration or centrifugation. Remove the ethanol from the supernatant by vacuum rotary evaporation and freeze-dry to obtain crude *Ge Xian Mi* polypeptide powder.

[0017] The purification process is preferably carried out by ultrafiltration, gel chromatography, or reversed-phase high-performance liquid chromatography (RP-HPLC), wherein RP-HPLC purification can obtain small molecule peptides with a purity of over 97%.

[0018] The ultrafiltration purification is preferably performed using an Ultracel-3 membrane to collect components with a molecular weight of less than 3 kDa.

[0019] The preferred method for purification using gel chromatography is to use a Sephadex G-25 dextran gel chromatography column, with primary deionized water as the eluent.

[0020] The application of the aforementioned Ge Xian Mi polypeptide in the preparation of products that resist protein aggregation.

[0021] The application of the aforementioned Ge Xian Mi polypeptide in the preparation of products with neuroprotective activity.

[0022] The application of the aforementioned Ge Xian Mi polypeptide in the preparation of drugs for the prevention and treatment of neurodegenerative diseases.

[0023] The neurodegenerative diseases mentioned include Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or prions.

[0024] A drug for preventing and treating neurodegenerative diseases, comprising at least one of the above-mentioned *Ge Xian Mi* polypeptide, a protease hydrolysate containing the above-mentioned *Ge Xian Mi* polypeptide, and a hydrolysate containing the above-mentioned *Ge Xian Mi* polypeptide as an active ingredient.

[0025] The principle of this invention: This invention uses *Gnaphalium affine* as raw material and extracts, separates, and identifies a pure natural *Gnaphalium affine* polypeptide through enzymatic hydrolysis, ultrafiltration purification, and LC-MS / MS identification. The amino acid sequence of this *Gnaphalium affine* polypeptide is TQFYLR, and its molecular formula is C2. 39 H 58 N 10 O 10 The average relative molecular mass is approximately 826.43 Da, and the theoretical isoelectric point is pI=8.41, classifying it as a hydrophilic polypeptide. This invention further utilizes the model organism *C. elegans* to discover that this *Ge Xin Mi* polypeptide possesses anti-protein aggregation and protein homeostasis regulating functions, and also exhibits neuroprotective activity, thus playing a role in preventing neurodegenerative diseases, as detailed below: (1) ASH neuron survival test: This test verifies that the anti-protein aggregation peptide can effectively inhibit the toxic aggregation of pathogenic proteins and protect ASH neurons from damage.

[0026] (2) PolyQ (polyglutamine) aggregation inhibition test: This test verifies that the anti-protein aggregation peptide has the effect of inhibiting polyQ aggregation.

[0027] (3) Experiment on regulating abnormal avoidance behavior: This experiment verifies that the anti-protein aggregation peptide can regulate abnormal avoidance behavior caused by ASH neuron damage.

[0028] In summary, the present invention demonstrates through the above experiments that the *Ge Xian Mi* polypeptide possesses anti-protein aggregation function, meaning it can prevent or slow down the aggregation of proteins into insoluble deposits within cells or in vivo. The advantages of this anti-protein aggregation *Ge Xian Mi* polypeptide include good biocompatibility, low potential side effects, the ability to inhibit abnormal protein folding, helping proteins fold correctly and preventing aggregation, and effectively regulating protein homeostasis, thereby achieving a neuroprotective effect.

[0029] The present invention has the following advantages and effects compared with the prior art: (1) This invention uses natural *Gynostemma pentaphyllum* as the target, and obtains *Gynostemma pentaphyllum* polypeptide components with neuroprotective activity through enzymatic hydrolysis and separation methods. Then, a single polypeptide is obtained through sequence identification, sequence comparison and polypeptide synthesis methods. Its amino acid sequence is Thr-Gln-Phe-Tyr-Leu-Arg (TQFYLR), and its molecular formula is C 39 H 58 N 10 O 10 It has an average relative molecular mass of approximately 826.43 Da and a theoretical isoelectric point of pI = 8.41, making it a hydrophilic polypeptide. This polypeptide can be isolated and purified from *Nostoc commune*, or it can be obtained through artificial synthesis.

[0030] (2) In this invention, the polyQ aggregation model AM141 nematode and the Huntington's disease model HA759 nematode were selected to conduct polyglutamine aggregation inhibition test and neuronal survival test, respectively, to study the neuroprotective effect of the nematode. The results showed that the nematode could significantly inhibit the aggregation of abnormal proteins and polyQ, reduce neuronal damage, protect neurons, improve neuronal survival rate, and regulate the abnormal avoidance behavior caused by ASH neuronal damage. Therefore, the nematode has a clear anti-protein aggregation effect and excellent neuroprotective activity.

[0031] (3) The Ge Xian Mi polypeptide provided by the present invention is derived from the enzymatic hydrolysate of the natural food Ge Xian Mi. It has a small molecular weight, is easy to absorb, and has a clear source. As a drug, it can improve the efficiency and accuracy of treatment, reduce the occurrence of immune reactions, and ensure its long-term efficacy and safety.

[0032] (4) The Ge Xian Mi polypeptide provided by the present invention has the functions of anti-protein aggregation and neuroprotection, and can be further used in the development of pharmaceuticals and other products, with broad application prospects. Attached Figure Description

[0033] Figure 1 This is a graph showing the results of the analysis of the effect of the gexianmi polypeptide Ns#001 on the survival rate of neurons in HA759 nematodes.

[0034] Figure 2 This is a statistical graph showing the number of polyQ aggregation points in *C. elegans* AM141 after treatment with *Ge Xian Mi* polypeptide Ns#001.

[0035] Figure 3 This is a diagram illustrating a chemoattractant behavior assay. From left to right, the images show: nematodes, hypertonic glycerol nematodes, sodium azide nematodes, and diacetyl attractants.

[0036] Figure 4 This is a graph showing the results of the chemical avoidance behavior of nematodes HA759 after treatment with Ge Xian Mi polypeptide Ns#001. DETAILED DESCRIPTION

[0037] The present invention is described in further detail below with reference to examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] The concentrated NA22 bacterial solution in the examples is food for Caenorhabditis elegans, and the preparation method thereof is: inoculating activated Escherichia coli NA22 into LB liquid medium for shaking culture, then centrifuging to collect the bacterial cells, which is the concentrated NA22 bacterial solution.

[0039] The N2, AM141 and HA759 Caenorhabditis elegans models are all from the Caenorhabditis Genetics Center, University of Minnesota, USA.

[0040] Example 1 Isolation and Identification of Nostoc sphaeroides Peptides 1. Enzymatic Isolation The Nostoc sphaeroides peptides are extracted by an enzymatic hydrolysis and alcohol precipitation method, and the specific method is as follows: (1) Nostoc sphaeroides is cultured according to a conventional method, collected, naturally dried, and then ground into powder to obtain Nostoc sphaeroides dry powder. 1 g of the Nostoc sphaeroides dry powder is resuspended in pure water at a ratio of 1:400 (g / mL) and mixed uniformly by magnetic stirring. A water bath is preheated to 45°C, the pH of the above mixed system is adjusted to 8.0 and then placed in the water bath. When the temperature of the system measured by a thermometer reaches 45°C, 24 mg of trypsin (solid, 2500 U / mg) is added to the solution to make the final concentration 150 U / mL, followed by enzymatic hydrolysis for 4 h; then the pH of the above mixed system is adjusted to 6.0, the temperature of the water bath is raised to 60°C, and when the temperature measured by the thermometer reaches 60°C, 10 mg of papain (solid, 6000 U / mg) is added to make the final concentration 150 U / mL, followed by enzymatic hydrolysis for 4 h; finally, a boiling water bath is applied for 10 min to terminate the enzymatic hydrolysis reaction.

[0041] (2) After the Nostoc sphaeroides protein enzymatic hydrolysate prepared in step (1) is cooled to room temperature, ethanol with a volume fraction of 95% is added while stirring, so that the final concentration of ethanol in the solution is 60% (V / V), and the mixture is placed at 4°C overnight. The precipitate is removed by vacuum filtration, and the solution is collected; the obtained solution is further evaporated under reduced pressure to obtain a crude solution of Nostoc sphaeroides peptides.

[0042] 2. Ultrafiltration Purification and Gel Chromatography Column Purification (1) The crude solution of Nostoc sphaeroides peptides prepared in step 1 is subjected to ultrafiltration using a centrifugal filter, and PES membrane ultrafiltration is used to obtain retentate fractions of Nostoc sphaeroides protein enzymatic hydrolysate with MW>5 kDa, 3 kDa<MW<5 kDa, and MW<3 kDa; the fraction with a molecular weight of less than 3 kDa is collected, and then passed through a 0.45 μm filter membrane to remove impurities.

[0043] (2) The peptide components after step (1) were further separated using a packed Sephadex G-25 dextran gel chromatography column. The peptides were eluted with primary deionized water at a flow rate of 1 mL / min and monitored at 280 nm using a 785 UV / VIS detector. The fractions of each absorption peak were collected at the same time, and a total of 4 peptide components were obtained. The 4 peptide components were freeze-dried to obtain the corresponding lyophilized powder of Ge Xian Mi peptide.

[0044] 3. LC-MS / MS identification The polypeptide composition and amino acid sequence of the freeze-dried Ge Xian Mi polypeptide powder obtained in step 2 were identified by LC-MS / MS.

[0045] 4. Database comparison and filtering Based on the neuroprotective peptide sequence information provided in the BIOPEP-UWM database, the *Gynostemma pentaphyllum* peptide sequence obtained in step 3 was compared with this database for virtual activity screening. The specific steps were as follows: Log in to the BIOPEP-UWM website, select the Bioactive peptides database, click the Analysis option, select Profile of potential biological activity in the tab, and then enter the *Gynostemma pentaphyllum* peptide sequence obtained in step 3 by liquid chromatography-mass spectrometry (LC-MS) for comparison. *Gynostemma pentaphyllum* peptide sequences with potential neuroprotective effects were selected for the following experiments. After screening, the *Gynostemma pentaphyllum* peptide with the sequence TQFYLR was obtained and named Ns#001. The molecular formula was calculated using a specialized peptide calculator to be C001. 39 H 58 N 10 O 10 The average relative molecular mass of the polypeptide was calculated to be approximately 826.43 Da using the online tool Expasy Compute pI / Mw, and the theoretical isoelectric point was pI=8.41, indicating that it is a hydrophilic polypeptide.

[0046] 5. Synthesis of Nostoc commune polypeptide The Ge Xian Mi polypeptide Ns#001 was prepared by Shanghai Taopu Biotechnology Co., Ltd. through solid-phase synthesis.

[0047] Example 2: Ge Xian Mi polypeptide Ns#001 has neuroprotective activity. The nematode model HA759 is a disease model of Huntington's disease (HD), capable of simulating the pathogenesis of HD. Simultaneously, through gene technology, it stably expresses green fluorescent protein (GFP), which not only facilitates the localization of ASH neurons but also allows for the assessment of neuronal survival status by observing GFP expression, providing an important observational window for studying the mechanisms of neuronal death in HD. Under strong drive from the osm-10 promoter, which is associated with osmotic sensing, this model specifically expresses the 150-glutamate-residue huntingtin protein Htn-Q150 in head ASH and ASI neurons, as well as tail PHA and PHB neurons. Htn-Q150 expression is most significant in ASH neurons, while expression is weaker in the other three types of neurons. Furthermore, the point-mutated pqe-1 gene enhances the sensitivity of ASH neurons to Htn-Q150 toxicity, accelerating the induction of Htn-Q150 toxicity. This results in the mass death of HA759 ASH neurons due to protein toxicity accumulation within approximately 3 days under 15°C culture conditions. When ASH neurons die, the GFP fluorescent spots attached to the ASH neurons near the nematode's head disappear immediately, decreasing from two spots to one or zero. Both of these cases are recorded as ASH neuron death. Only when the GFP fluorescence on two ASH neurons is normal simultaneously is the nematode considered alive. This invention uses HA759 as an example to study the neuroprotective activity of the nematode's peptide Ns#001. The specific method is as follows: 1. Culture HA759 nematodes to L4 stage using conventional methods; wash L4 stage HA759 nematodes three times with S. Medium solution to remove NA22 bacterial culture; then adjust the nematode density to 40-50 nematodes / 10 μL with S. Medium solution.

[0048] 2. Using a 96-well plate, add 10 μL of concentrated NA22 bacterial culture to each well (final OD per well). 570 The solution consisted of 1.5 μL of 5 mg / mL 5-fluorouracil (5-FUdR, which inhibits nematode oviposition), 2 μL of 5 mg / mL ampicillin (AMP, which inhibits the growth of other bacteria), 10 μL of worm suspension, and a polypeptide stock solution. The final polypeptide concentrations were set at 0, 0.5, 1, 2, and 4 mM. An equal volume of S. Medium solution was added to replace the polypeptide solution as a control group. Each well was brought to a final volume of 100 μL with S. Medium solution, and three replicates were performed for each concentration. The outermost ring was sealed with S. Medium solution to reduce water evaporation, and finally sealed with sealing film. The 96-well plate was incubated at 15°C and 120 rpm for 72 h using a constant temperature shaker.

[0049] 3. After 72 hours of culture, aspirate the nematodes and place them on an agarose mat. Fix and paralyze them, then photograph them using a fluorescence microscope, taking pictures from top to bottom and left to right. Use a microscope with 20x magnification and the blue fluorescence channel to obtain images showing green GFP fluorescent dots within the nematodes.

[0050] The results are as follows Figure 1 As shown, the survival rate of ASH neurons in the control group was approximately 57%. The survival rates of neurons treated with 0.5, 1, and 2 mM peptides were all improved, while the survival rate of the 4 mM peptide group was similar to that of the control group and did not effectively improve the survival of ASH neurons. The highest survival rate was observed at a peptide concentration of 1 mM, increasing the ASH neuron survival rate to approximately 68%, about 11% higher than the control group. Furthermore, treatment with the 1 mM peptide did not inhibit the normal growth and development of nematodes. These results indicate that the Ns#001 peptide can effectively inhibit the toxic accumulation of pathogenic proteins in neurons, protecting ASH neurons from damage and thus improving their survival rate.

[0051] In summary, 1 mM of *Gynostemma pentaphyllum* peptide effectively alleviates polyQ toxicity and aggregation while exhibiting optimal biocompatibility, verifying that 1 mM is the optimal dosage concentration for in vivo validation of *Gynostemma pentaphyllum* active peptide.

[0052] Example 3: Inhibitory effect of Ge Xian Mi polypeptide Ns#001 on polyglutamine (polyQ) aggregation The degree of protein aggregation can be evaluated by the number of aggregation points; an increase in the number of aggregation points indicates a deeper degree of aggregation. This experiment used the *C. elegans* polyQ aggregation model AM141 as the research subject. Immediately after the L1 stage, soluble Q40::YFP distribution was observed in the body wall muscle cells of AM141 nematodes. As they grow and develop, polyQ is expressed in the body wall muscle cells of AM141, gradually forming aggregation points from a diffuse state. When they reach adulthood, the AM141 nematode model will exhibit a phenotype of complete Q40::YFP aggregation. The effect of *Gexinmi* polypeptide Ns#001 on protein aggregation can be reflected by the change in the number of polyQ aggregation points. To determine the detection time of polyQ aggregation points, AM141 nematodes were cultured from the L1 stage to 24 h, 48 h, and 72 h, and the number of polyQ fluorescent aggregation spots in their bodies was measured. All nematode fluorescence images at 72 h were processed, and the distribution of polyQ protein aggregation points in the nematodes at this time was analyzed to evaluate the polyQ aggregation inhibitory efficacy of *Gexinmi* polypeptide Ns#001. The specific method is as follows: 1. The nematode AM141 was synchronized using conventional methods to obtain L1 stage larvae. The larvae were washed three times with S. Medium solution to remove NA22 bacterial solution. The nematode density was adjusted to 30-40 nematodes / 10 μL by adding S. Medium solution.

[0053] 2. Add 10 μL of concentrated NA22 bacterial culture to each well of a 96-well plate (final OD per well). 570 Add 2 μL of 5 mg / mL AMP solution (between 0.5 and 0.6 μg / mL), 30 μL of the insect fluid prepared in step 1, and the peptide sample stock solution for drug administration. The treatment group received 1 mM of *Ge Xian Mi* peptide Ns#001, while the control group received an equal volume of S. Medium solution. Then, bring the volume of each well to 100 μL with S. Medium solution. Each group has at least 9 replicates. Add 100 μL of S. Medium solution around each well for liquid sealing. Seal the edge of the plate cap with sealing film and mark the cap. Incubate the 96-well plate at 20°C and 120 rpm in a constant temperature shaker until the appropriate time point.

[0054] 3. At 24 h, 48 h, and 72 h of culture, respectively, three wells of culture medium from each group were transferred from a 96-well plate to 1.5 mL centrifuge tubes. The nematodes were washed with M9 buffer until the supernatant was clear and thoroughly mixed. The nematodes were then aspirated onto an agarose pad and anesthetized with sodium azide. Fluorescence images were taken using a high-content imaging system, and the number of fluorescent spots around the nematodes was counted.

[0055] The test results are shown in Figure 2 At 24 h of culture, 1 mM of *Gnaphalium affine* polypeptide Ns#001 did not inhibit polyglutamine aggregation. This may be because this period is not the peak phase for polyQ protein aggregation in the AM141 nematode model, and therefore, polypeptide treatment cannot inhibit polyQ protein aggregation at this time. However, polyQ protein aggregation was inhibited at 48 h and 72 h, with a particularly strong inhibitory effect at 72 h. Compared with the control group, treatment with this *Gnaphalium affine* polypeptide for 72 h reduced polyQ protein aggregation in somatic cells, thereby alleviating the neurodegenerative diseases in nematodes.

[0056] Example 5: The ability of Ge Xian Mi polypeptide Ns#001 to regulate abnormal avoidance behavior The transgenic *C. elegans* model HA759, associated with hypertonic cholangitis (HD), specifically expresses human huntingtin protein HtnQ150 (a 150-residue polyQ fragment) in four neuronal types: head (ASH and ASI), tail (PHA and PHB), under the regulation of the osm-10 promoter. PolyQ150 expression is most pronounced in ASH neurons, with weak expression in the other three neuronal types. Point mutations in the pqe-1 gene make ASH neurons more sensitive to polyQ150 toxicity—specifically, polyQ expression and aggregation toxicity can lead to ASH neuron death after approximately 3 days of culture at 15°C. ASH neurons control the nematode's sensory responses to toxic chemical stimuli, one of which is avoidance behavior to hypertonic solutions. The death of these neurons causes the nematode to lose its ability to avoid hypertonic solutions. Therefore, the ability of *Gnaphalium affine* peptides to alleviate behavioral abnormalities and the neuroprotective activity of HA759 nematodes can be detected through chemoavy behavior experiments on 8 M glycerol solution. The specific methods are as follows: 1. After culturing and synchronizing HA759 nematodes according to conventional methods, culture them to the L1 stage. Wash the L1 stage HA759 nematodes three times with S. Medium solution to remove NA22 bacterial culture; then adjust the nematode density to about 30-40 nematodes / 10 μL with S. Medium solution.

[0057] 2. Use 24-well plates with a total liquid volume of 700 μL per well. One well is used for each experimental group, and three plates are used per group (i.e., three replicates). For the treatment group, concentrated NA22 bacterial culture is added to each well (final OD per well). 570 AMP solution (0.5–0.6 mg / mL) and peptide sample stock solution were added to each well to ensure approximately 700 nematodes. The volume was then brought up to 700 μL with S. Medium solution, with a final AMP concentration of 100 μg / mL. The OD of the system was controlled after adding concentrated NA22 bacterial solution. 570 The concentration of Ns#001 peptide was between 0.5 and 0.6, and the final concentration (drugated concentration) was 1 mM. The liquid was mixed by shaking for 30 s, covered, and sealed. An S. Medium solution with an equal volume of the peptide sample stock solution was added as a control group. The mixture was incubated at 16°C for 3 days.

[0058] 3. Draw the nematodes into a test tube and wash three times to remove all the liquid from the top. Figure 3As shown, draw a line in the middle of a 12cm diameter bacterial culture dish using 8M glycerol, dividing it into N and T ends. At the T end, draw an anesthesia line using 50mM sodium azide, 1 cm from the glycerol line. Take half of the nematodes and evenly add them to the center of the N side of three culture dishes, parallel to the glycerol line. Each dish should contain approximately 100 nematodes. After the nematodes crawl out of the buffer solution, add 2 μL of nematode attractant (1% diacetyl (butanedione) solution) 1 cm from the edge of the T side. Figure 3 Immediately close the lid and place the plate in a 23°C incubator for 90 minutes.

[0059] 4. Count the number of nematodes in the regions on both sides of the glycerol line under a microscope, and label them as N and T respectively. The formula for calculating the chemical avoidance rate of HA759 nematodes is: AI = N / (T + N).

[0060] The test results are as follows Figure 4 Before peptide treatment, the chemoavage rate of HA759 nematodes was about 63%, while after peptide treatment, the chemoavage rate increased to about 73%, effectively increasing the survival rate of ASH. This indicates that the nematode Ns#001 peptide has a certain resistance to the neurotoxicity caused by polyQ aggregation and can effectively protect HA759 nematodes from chemoavage behavioral abnormalities caused by ASH neuronal damage.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type of Ge Xian Mi polypeptide, characterized in that... Its amino acid sequence is TQFYLR.

2. The method for preparing the *Ge Xian Mi* polypeptide according to claim 1, characterized in that... It includes the following steps: Ge Xianmi polypeptides can be prepared directly through solid-phase synthesis, or by using Ge Xianmi as a raw material and obtaining Ge Xianmi polypeptides through enzymatic hydrolysis, separation, extraction, and purification.

3. The method for preparing Ge Xian Mi polypeptide according to claim 2, characterized in that: The enzymatic hydrolysis was performed sequentially using trypsin and papain.

4. The method for preparing Ge Xian Mi polypeptide according to claim 3, characterized in that: The specific operation of the enzymatic hydrolysis is as follows: Mix dried Nostoc commune powder with water, adjust the pH of the system to 7.5-8.0, add trypsin for enzymatic hydrolysis; then adjust the pH of the system to 6.0-7.0, add papain for enzymatic hydrolysis; after enzymatic hydrolysis, inactivate to obtain Nostoc commune protein hydrolysate.

5. The method for preparing Ge Xian Mi polypeptide according to claim 2, characterized in that: The specific operations for separation and extraction are as follows: After cooling the enzyme hydrolysate of *Ge Xian Mi*, add 95% ethanol (volume fraction) to the solution until the ethanol concentration is 60-80%, and let it stand. Then remove the precipitate by vacuum filtration or centrifugation. Remove the ethanol from the supernatant by vacuum rotary evaporation and freeze-dry to obtain crude *Ge Xian Mi* polypeptide powder.

6. The method for preparing Ge Xian Mi polypeptide according to claim 2, characterized in that: The purification process includes ultrafiltration, gel chromatography, and reversed-phase high-performance liquid chromatography.

7. The use of the Ge Xian Mi polypeptide according to claim 1 in the preparation of products with anti-protein aggregation or neuroprotective activity.

8. The use of the Ge Xian Mi polypeptide according to claim 1 in the preparation of drugs for the prevention and treatment of neurodegenerative diseases.

9. The application according to claim 8, characterized in that: The neurodegenerative diseases mentioned are Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or prions.

10. A drug for preventing and treating neurodegenerative diseases, characterized in that... It contains the Ge Xian Mi polypeptide as described in claim 1.

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