A polypeptide composition for preventing, delaying or assisting intervention in the progression of neurodegenerative diseases, its preparation method and use

Abstract

This invention discloses a polypeptide composition for preventing, delaying, or adjuvant intervention in the progression of neurodegenerative diseases, comprising polypeptide A (SEQ ID NO.1) and polypeptide B (SEQ ID NO.2) obtained from extracts / enzymatic hydrolysis products of the head tissue of the high-altitude planarian. Both polypeptides exhibit synergistic neuroprotective effects, inhibiting abnormal αsynuclein aggregation, reducing neuroinflammation, improving oxidative stress and mitochondrial dysfunction, and protecting against dopaminergic neuronal-related damage. The preferred weight ratio of polypeptide A to polypeptide B is 1:1, with a total mass fraction of 5%–30%. Pharmaceutical excipients can be added to prepare injections, lyophilized powder injections, etc. The preparation method includes dissolution, pH adjustment, sterile filtration, and lyophilization. This composition can be used to prepare drugs, drug candidates, or functional compositions for delaying the pathological progression of neurodegenerative diseases such as Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide composition for preventing, delaying or assisting in the intervention of the progression of neurodegenerative diseases, its preparation method and application. Background Technology

[0002] Neurodegenerative diseases are a group of diseases characterized by progressive damage, dysfunction, and loss of neurons in the central nervous system. They mainly include Parkinson's disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, and amyotrophic lateral sclerosis (ALS). Parkinson's disease is a common neurodegenerative disease, characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, decreased dopamine levels in the striatum, and abnormal aggregation of α-synuclein to form Lewy bodies. This is accompanied by neuroinflammatory responses, oxidative stress, mitochondrial dysfunction, and synaptic damage.

[0003] Currently, clinical treatment for Parkinson's disease primarily focuses on symptom relief, with commonly used medications including levodopa, dopamine receptor agonists, monoamine oxidase B inhibitors, and catechol-O-methyltransferase inhibitors. While these drugs can alleviate motor symptoms to some extent, they are generally insufficient to halt disease progression, and long-term use can lead to fluctuations in efficacy, dyskinesia, psychiatric symptoms, and gastrointestinal adverse reactions. Therefore, developing novel bioactive substances with neuroprotective, anti-neuroinflammatory, mitochondrial function-improving, and abnormal protein aggregation-inhibiting effects has become a crucial direction in early intervention and disease progression slowing research for Parkinson's disease.

[0004] Peptides, due to their small molecular weight, well-defined structure, diverse activities, strong targeting, and ease of chemical synthesis or biological preparation, show promising application prospects in the prevention and treatment of neurological diseases. Existing research indicates that certain biologically derived or artificially designed bioactive peptides can intervene in the pathological processes related to neurodegenerative diseases by regulating the release of inflammatory factors, alleviating neuronal damage, inhibiting abnormal protein aggregation, and improving oxidative stress and mitochondrial dysfunction. However, existing peptide candidates are mostly derived from common plant and animal materials, microorganisms, or animal venom, and screening and evaluation often focus on a single activity indicator. Technical solutions for obtaining short peptide compositions with well-defined sequences through extraction / enzymatic hydrolysis of specific biological tissues with regenerative and neurorepair characteristics, and for systematically evaluating multiple key pathological aspects of Parkinson's disease, remain relatively limited.

[0005] Planarians from the high plateau possess strong tissue regeneration, nerve repair, and environmental adaptation capabilities. Their head tissues may contain bioactive molecules or short peptide precursors related to neuroprotection, stress resistance, tissue regeneration, and cellular homeostasis. Based on the regenerative and adaptive characteristics of this organism, screening bioactive short peptides with neuroprotective potential from extracts / enzymatic hydrolysates of planarian head tissues may provide a new source of candidate substances for early prevention, progression slowing, and adjunctive intervention in neurodegenerative diseases. Compared with directly using complex tissue extracts, obtaining short peptide compositions with well-defined sequences, synthetic preparation capabilities, and controllable quality is more conducive to the development of subsequent drug candidates or functional compositions. Furthermore, Parkinson's disease involves multiple interrelated pathological processes, including abnormal α-synuclein aggregation, neuroinflammation, oxidative stress, mitochondrial dysfunction, and dopaminergic neuron-related damage. Candidates with single short peptides or single mechanisms of action often struggle to simultaneously cover the aforementioned complex pathological network and meet multiple requirements such as anti-aggregation, anti-inflammation, anti-oxidation, mitochondrial protection, and neuroprotection. Therefore, there is an urgent need to develop a polypeptide composition with a clear source, well-defined sequence, and the ability to be prepared into multiple dosage forms, which can play a complementary role in multiple Parkinson's disease-related pathological processes, for the prevention, progression slowing, and adjunctive intervention of neurodegenerative diseases such as Parkinson's disease. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide composition for preventing, delaying, or assisting in the intervention of neurodegenerative diseases, its preparation method, and its application. The polypeptide composition is obtained through multi-level screening from extracts / enzymatic hydrolysis products of the head tissue of the high-altitude planarian. It can intervene in multiple pathological processes associated with Parkinson's disease, such as abnormal α-synuclein accumulation, neuroinflammation, oxidative stress, mitochondrial dysfunction, and dopaminergic neuron-related damage. This addresses the problems of existing short peptide candidates having relatively limited sources and screening indicators, making it difficult to simultaneously address multiple pathological interventions and achieve synergistic effects through combination.

[0007] In a first aspect, the present invention provides a polypeptide composition for preventing, delaying or assisting in the intervention of the progression of neurodegenerative diseases, the composition comprising polypeptide A and polypeptide B, wherein the amino acid sequence of polypeptide A is shown in SEQ ID NO.1 and the amino acid sequence of polypeptide B is shown in SEQ ID NO.2.

[0008] In a second aspect, the present invention also provides the use of the polypeptide composition in the preparation of medicaments, drug candidates or functional compositions for the prevention, delay or adjunctive intervention of Parkinson's disease-related pathological progression.

[0009] Furthermore, the drug also includes one or more of the following pharmaceutically acceptable excipients, carriers, diluents, stabilizers, buffers, osmotic pressure regulators, preservatives, and surfactants.

[0010] Furthermore, the drug is an injection, a lyophilized powder for injection, a nasal preparation, a nasal drop, a nasal spray, or a sustained-release microsphere preparation.

[0011] Furthermore, in the drug, the weight ratio of polypeptide A to polypeptide B is 1:1.

[0012] Furthermore, in the drug, the total mass fraction of peptide A and peptide B is 5%-30%.

[0013] Thirdly, the present invention also provides a method for preparing a polypeptide composition for preventing, delaying or assisting in the intervention of neurodegenerative diseases, comprising the following steps: S1, dissolving the polypeptide A and the polypeptide B in water for injection, purified water or buffer solution; S2, adding pharmaceutically acceptable excipients and mixing evenly; S3, adjusting the pH to 4.0-8.0; S4, performing sterile filtration and dispensing; S5, performing lyophilization to obtain the polypeptide composition for preventing, delaying or assisting in the intervention of neurodegenerative diseases.

[0014] The beneficial effects of this invention are as follows: This invention employs polypeptides A and B, obtained from the head tissue extract / enzymatic hydrolysis products of the high-altitude planarian, to form a polypeptide composition. The amino acid sequences of both polypeptides have been determined, providing a novel short peptide combination scheme for intervention in the pathological processes related to neurodegenerative diseases. The high-altitude planarian possesses strong tissue regeneration, nerve repair, and environmental adaptability capabilities, and its head tissue extract / enzymatic hydrolysis products can serve as a specific source for screening neuroprotective short peptides.

[0015] Unlike candidate short peptides obtained based on only a single activity indicator, this invention utilizes α-synuclein abnormal aggregation inhibition, LPS-induced neuroinflammation inhibition, and MPP... + This study comprehensively screened multiple pathological aspects of Parkinson's disease, including protection against dopaminergic neuron-related damage, improvement of oxidative stress, and restoration of mitochondrial function. The results showed that peptides A and B are not short peptides with completely overlapping effects: peptide A was superior in inhibiting the release of inflammatory factors, while peptide B was superior in reducing oxidative stress and improving mitochondrial function. When used in combination at a 1:1 mass ratio, both peptides were superior to either peptide alone in restoring neuronal vitality, improving oxidative stress, and restoring mitochondrial function, demonstrating complementary functions and synergistic effects.

[0016] This invention further clarifies the weight ratio and total mass fraction of peptide A and peptide B, which is beneficial for improving the certainty of composition, reproducibility of preparation, and feasibility of quality control. The composition can be prepared as an injection, lyophilized powder for injection, nasal preparation, nasal drops, nasal spray, or sustained-release microsphere formulation, providing a new peptide composition and formulation development scheme for the prevention, progression delay, and adjunctive intervention of neurodegenerative diseases such as Parkinson's disease. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort, wherein: Figure 1 MPP in Embodiment 3 of the present invention + Western blot results of TH and DAT protein expression in an induced SH-SY5Y cell injury model. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0021] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0022] Example 1 Preparation of head tissue extracts / enzymatic hydrolysates from the plateau planarian 1. Preparation of materials and instruments 1.1 Experimental Materials: Plateau planarian samples collected from freshwater environments in high-altitude areas were selected. The samples were morphologically identified as planarians, characterized by a flat, soft body shape, a distinct anterior head region, visible eyespots, and visible ciliary movement. Healthy, agile individuals with a body length of 1.5–2.0 cm were selected for the experiment. Physiological saline, PBS buffer (pH 7.4), trypsin, penicillin-streptomycin solution, BCA protein quantification kit, and magnesium sulfate were used. All reagents were analytical grade or bio-grade, and ultrapure water was used in the experiments.

[0023] 1.2 Experimental instruments: stereomicroscope, high-speed refrigerated centrifuge, laminar flow hood, sterile dissecting needle, curved ophthalmic forceps, sterile culture dish, homogenizer, 0.22 μm sterile filter membrane, 10 kDa ultrafiltration tube, 3 kDa ultrafiltration tube, freeze dryer, electronic balance and pH meter.

[0024] 2. Temporary rearing and pretreatment of planarians in the high-altitude region 2.1 The collected planarians from the plateau were placed in sterile culture dishes, and physiological saline containing penicillin-streptomycin antibiotics was added. They were then temporarily incubated at 18–22℃ under low light conditions for 24 h. No food was fed during this period to minimize interference from intestinal contents in the subsequent extraction process.

[0025] 2.2 After temporary rearing, gently rinse the planarians three times with sterile saline. After each rinse, gently blot dry the surface of the planarians with sterile filter paper, then transfer them to a new sterile culture dish. Add an appropriate amount of PBS buffer and a small amount of magnesium sulfate to the culture dish for mild anesthesia until the planarians are relaxed and their activity decreases, in order to facilitate subsequent dissection.

[0026] 3. Isolation of head tissue from *Planaria gracilis* (a type of planarian worm) in the plateau region 3.1 After anesthetizing, the planarian is placed under a stereomicroscope. The planarian is gently pressed and fixed with a sterile dissecting needle. Using the planarian's eyespot and the triangular head region at the front end as anatomical landmarks, the head tissue is separated using curved ophthalmic forceps. During the separation process, body tissue should be avoided as much as possible.

[0027] 3.2 The entire separation process was performed in a clean bench. The separated head tissue was immediately transferred to pre-cooled PBS buffer and gently rinsed twice to remove any residual body tissue and impurities from the surface. The surface liquid was then gently blotted dry with sterile filter paper and stored at -80°C for later use.

[0028] 4. Preparation of head tissue extracts / enzymatic hydrolysates 4.1 Take the frozen head tissue of the planarian from the plateau and add pre-cooled PBS buffer at a mass-to-volume ratio of 1:10, g / mL. Place the sample in a homogenizer and homogenize at 4°C for 10–30 s each time, cooling in an ice bath at intervals. Repeat 3–5 times to obtain the head tissue homogenate.

[0029] 4.2 Add trypsin to the homogenate to a final concentration of 0.05%, and hydrolyze at 37°C for 1–4 h, preferably 2 h. Gently mix every 30 min during hydrolysis to promote the full release of tissue proteins and the formation of short peptide components.

[0030] 4.3 After enzymatic digestion, the sample was placed in a 95℃ water bath for 5 min to inactivate trypsin, and then rapidly cooled on ice. The treated homogenate was placed in a high-speed refrigerated centrifuge and centrifuged at 4℃ and 12000 rpm for 20 min, and the supernatant was collected. The precipitate was resuspended in an equal volume of pre-cooled PBS buffer, homogenized again, centrifuged again, and the supernatants from both reactions were combined to obtain the head tissue extraction / enzymatic digest crude extract.

[0031] 5. Purification and concentration of extract / enzymatic hydrolysis products 5.1 The obtained crude extract was filtered through a 0.22 μm sterile filter membrane to remove insoluble particles and the risk of microbial contamination, thus obtaining a sterile crude extract.

[0032] 5.2 Add the sterile crude extract to a 10 kDa ultrafiltration tube and centrifuge at 4°C. Collect the permeate below 10 kDa to remove large molecular weight proteins and most protease components. Further, the permeate below 10 kDa can be treated with a 3 kDa ultrafiltration tube to enrich low molecular weight short peptide components.

[0033] 5.3 The collected short peptide enrichment solution was freeze-dried at low temperature in a freeze dryer to obtain a white or off-white loose powder of head tissue extract / enzymatic hydrolysis product from the plateau planarian. The obtained powder was sealed and stored at -20℃ or -80℃ for later use.

[0034] Example 2 Isolation, purification and sequence identification of candidate short peptides derived from the head tissue of *Planaria gracilis*. 1. Preparation of materials and instruments 1.1 Experimental Materials The dry powder of head tissue extract / enzymatic hydrolysis product of the plateau planarian prepared in Example 1, PBS buffer, pH 7.4, chromatographic grade acetonitrile, 0.1% chromatographic grade formic acid, polypeptide standard containing internal standard, protease inhibitor mixture and C18 solid phase extraction column.

[0035] In some embodiments, the protease inhibitor mixture includes PMSF, aprotinin, and EDTA-Na2; wherein PMSF is preferably added before use to achieve a final concentration of 1 mM in the sample system.

[0036] 1.2 Experimental Apparatus High performance liquid chromatograph or ultra-high performance liquid chromatograph (equipped with ultraviolet detector), MALDI-TOF-MS, LC-MS / MS, high-speed refrigerated centrifuge, clean bench, sterile centrifuge tubes, pipettes, 3 kDa ultrafiltration tubes, C18 solid phase extraction column and freeze-drying device.

[0037] In some implementations, an amino acid sequencer may be used as an auxiliary confirmation device, selected only when the abundance of the candidate short peptide is high and mass spectrometry analysis indicates that its N-terminus is not blocked.

[0038] 2. Dissolution and pretreatment of extract / enzymatic hydrolysis product powder The qualified head tissue extract / enzymatic hydrolysis product powder of the plateau planarian obtained in Example 1 was dissolved in pre-cooled PBS buffer containing protease inhibitors under low temperature conditions to prepare a crude peptide extract. The crude extract was centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant was collected.

[0039] The obtained supernatant was treated with a 3 kDa ultrafiltration tube and centrifuged at 4°C and 8000 rpm for 30 min to remove larger protein molecules. The ultrafiltration filtrate was collected for subsequent processing. The filtrate was then desalted and preliminarily enriched using a C18 solid-phase extraction column. Impurities were preferably washed away using a low-concentration acetonitrile system, followed by elution of peptide components using a higher-concentration acetonitrile system. The eluent was collected and concentrated or lyophilized to obtain a peptide-enriched sample suitable for subsequent chromatographic separation.

[0040] 3. Preliminary screening and purification of candidate peptides 3.1 Reversed-phase high-performance liquid chromatography separation and purification The above-mentioned peptide-enriched samples were separated and purified using a C18 reversed-phase chromatographic column. Preferably, a gradient elution method was used, with 0.1% formic acid aqueous solution as phase A and 0.1% formic acid acetonitrile solution as phase B.

[0041] In some embodiments, the gradient elution program may be set as follows: 0-5 min, 5%B; 5-30 min, 5%-40%B; 30-35 min, 40%-90%B; 35-40 min, 90%-5%B; with a flow rate of 1 mL / min and a column temperature of 30°C.

[0042] The preferred method for detection is to use dual wavelength monitoring at 214 nm and 280 nm. Elution peaks are collected according to different retention times and concentrated separately to obtain multiple peptide-enriched components.

[0043] 3.2 Molecular weight screening MALDI-TOF-MS was used for initial molecular weight screening of each peptide enrichment fraction. In some embodiments, CHCA was used as the matrix, and an internal standard was used for quality correction. Based on the detection results, oligopeptide fractions with apparent molecular weights in the range of 0.8-3.0 kDa were screened, and five candidate short peptide fractions were obtained, named candidate peptide 1, candidate peptide 2, candidate peptide 3, candidate peptide 4, and candidate peptide 5, respectively.

[0044] In some implementations, three technical replicates are set for each candidate component to improve the reliability of molecular weight screening results.

[0045] 3.3 Complex Purification Process The above-mentioned candidate short peptide components were further purified according to the chromatographic conditions described in 3.1 until they reached the purity requirements suitable for sequence identification.

[0046] 4. Sequence identification of candidate short peptides The purified candidate short peptide fractions were subjected to molecular weight verification and sequence analysis using LC-MS / MS. Preferably, HCD fragmentation mode was used to collect parent ion and secondary fragment information, and the candidate short peptides were sequenced in combination with de novo sequencing results.

[0047] In some implementations, the results of high-resolution mass spectrometry data, secondary fragment spectra, retention time characteristics, and comparison with synthetic standards can be further combined to confirm the sequence attribution and improve the reliability of the sequence attribution.

[0048] The identified candidate short peptides were chemically synthesized, and the synthesized standards were compared with the corresponding peaks in the head tissue extracts / enzymatic hydrolysis products of the plateau planarian worm by LC retention time, primary mass spectrometry molecular weight, and secondary fragment spectra. When the synthesized standards and the corresponding candidate short peptides in the samples were substantially consistent with the above indicators, their sequence assignments were confirmed.

[0049] In this embodiment, the characterization sequences of five candidate short peptides were obtained through preliminary identification as follows. Among them, candidate peptide 3 and candidate peptide 4 were subsequently identified as peptide A and peptide B of the present invention through activity screening, and are therefore numbered SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0050] Table 1. Peptide Sequences

[0051] Example 3 Synthesis, activity screening and functional evaluation of candidate peptides The candidate peptides in Table 1 were synthesized using the following method: The standard Fmoc solid-phase peptide synthesis method was employed. Using Rink Amide resin as a carrier, Fmoc-protected amino acids were sequentially coupled from the C-terminus to the N-terminus according to the amino acid sequences of the candidate peptides in Table 1. The coupling activation system was HBTU / HOBt / DIEA, and deprotection was performed using a 20% piperidine / DMF solution. After the complete sequence synthesis was completed, the peptides were cleaved from the resin using a TFA / TIS / H2O (95:2.5:2.5) cleavage buffer. The obtained crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and then lyophilized to obtain pure peptides. HPLC analysis showed a purity ≥95%; mass spectrometry (ESI-MS or MALDI-TOF-MS) confirmed that the molecular weight was consistent with the theoretical value.

[0052] The synthesized peptides were screened as follows (each experiment was repeated at least 3 times independently, and data were expressed as mean ± standard deviation; one-way ANOVA was used for comparisons among multiple groups, and corresponding post-hoc tests were used for pairwise comparisons between groups; P < 0.05 indicated statistical significance): 1. Screening for α-Synuclein's inhibitory activity against aberrant aggregation The inhibitory activity of candidate peptides against abnormal α-synuclein aggregation was evaluated using an in vitro α-synuclein aggregation detection model. The final concentrations of α-synuclein protein and heparin sodium (5 μmol / L as aggregation inducer) in the incubation system were 200 μmol / L. The system was incubated at 37 °C in a shaking incubator at 100 r / min for 72 h. The following control groups were set up: a buffer background control group (ThT and buffer only), a solvent control group (containing α-synuclein and heparin sodium), a positive control group (tea polyphenols, final concentration 10 μmol / L), a sham enzymatic digestion product control group (without using planarian tissue, only trypsin of the same concentration and batch as in Example 1 was dissolved in PBS, and the products collected in steps 4.2 to 5.3 of Example 1 were completely repeated), and peptide autofluorescence interference control group 1 (ThT and candidate peptide 1), peptide autofluorescence interference control group 2 (ThT and candidate peptide 2), peptide autofluorescence interference control group 3 (ThT and candidate peptide 3), peptide autofluorescence interference control group 4 (ThT and candidate peptide 4), and peptide autofluorescence interference control group 5 (ThT and candidate peptide 5). After incubation, the aggregation inhibition rate was detected by ThT fluorescence method (final ThT concentration 20 μmol / L, excitation wavelength 440 nm, emission wavelength 485 nm). The results are shown in Table 2.

[0053] Table 2 Results of α-Synuclein aggregation inhibition activity assay

[0054] As shown in Table 2, the fluorescence intensity of the autofluorescence control was slightly higher than that of the buffer group, but much lower than that of the positive control group, and did not substantially interfere with the calculation of the inhibition rate of candidate peptides. The inhibition rate of the pseudo-enzymatic digest was only 0.3%, which was not significantly different from that of the solvent control group and had no inhibitory activity. The fluorescence intensity of candidate peptides 3 and 4 and the positive control group was significantly lower than that of the solvent control group (P<0.01), while there was no significant difference between candidate peptides 1, 2, and 5 (P>0.05). There was no statistically significant difference between candidate peptides 3 and 4 and the positive control group (P>0.05). Therefore, candidate peptides 3 and 4 were selected for further screening because their inhibition rates were close to 60%, while the remaining three peptides were eliminated because their inhibition rates were below 20%.

[0055] 2. Neurocytotoxicity detection The cytotoxicity of BV2 and SH-SY5Y cells after treatment with different concentrations of candidate peptides for 24 h was evaluated using the LDH release assay, and relative cell viability was calculated based on the LDH release rate. Relative cell viability was calculated using the following formula: Relative cell viability (%) = 100% Cytotoxicity (%), with the blank control group set at 100%. Results are shown in Table 3: Table 3. Results of cytotoxicity assays of candidate peptides against BV2 and SH-SY5Y cells.

[0056] As shown in Table 3, candidate peptides 3 and 4 did not exhibit significant cytotoxicity against BV2 and SH-SY5Y cells within the range of 5–20 μmol / L, and the relative cell viability remained above 90%. After treatment with 40 μmol / L, the relative cell viability of some cells dropped below 90%, suggesting that higher concentrations may have some impact on cell state. Therefore, considering both safety and activity screening results, 20 μmol / L was selected as the working concentration for subsequent functional evaluation.

[0057] 3. Evaluation of neuroinflammation suppression (LPS-induced BV2 microglia inflammation model) BV2 cells were cultured in DMEM high-glucose medium containing 10% FBS at 37°C and 5% CO2 until confluence reached 70%-80%. After induction with LPS (final concentration 1 μg / mL) for 24 h, candidate peptides 3 and 4 (final concentration 20 μmol / L) or the positive control minocycline (final concentration 10 μmol / L) were added, and the cells were cultured for another 24 h. The supernatant was collected, and the levels of IL-1β and TNF-α were detected by ELISA. The results are shown in Table 4.

[0058] Table 4. Results of neuroinflammation suppression assay (IL-1β and TNF-α levels)

[0059] As shown in Table 4, the levels of inflammatory factors in each treatment group were significantly lower than those in the model group (P<0.05 or P<0.01). The levels of IL-1β and TNF-α in the candidate peptide 3 group were significantly lower than those in the candidate peptide 4 group (P<0.05), indicating that candidate peptide 3 has stronger anti-inflammatory activity.

[0060] 4. Evaluation of Dopaminergic Neuron Protection (MPP) + (Induced neuronal injury model) After seeding and culturing SH-SY5Y cells to a confluence of 70%-80%, MPP was added. +Cells were induced for 24 h at a final concentration of 500 μmol / L, followed by the addition of candidate peptides 3 and 4 (final concentration 20 μmol / L) or the positive control selegiline (final concentration 5 μmol / L). After another 24 h of culture, cell viability was detected by CCK-8 assay, apoptosis rate was detected by Annexin V-FITC / PI double staining, and TH and DAT protein expression was detected by Western blot. The results are shown in Table 5.

[0061] Table 5. Evaluation results of dopaminergic neuron protection (SH-SY5Y cells)

[0062] As shown in Table 5, all the above indicators in each treatment group were significantly improved compared with the model group (P<0.01). The improvement in cell viability, apoptosis rate, and TH / DAT expression in candidate peptide group 4 was slightly better than that in candidate peptide group 3, but the differences in some indicators between the two groups were not statistically significant (cell viability P=0.08, TH expression P=0.06). There was no significant difference between the positive control group and candidate peptide group 4 (P>0.05).

[0063] 5. Evaluation of the synergistic effect of joint applications Candidate peptide 3 and candidate peptide 4 were used in a 1:1 ratio in combination with MPP. + The induced cell damage inhibition rate or cell viability recovery rate were used as efficacy indicators. Three single-drug groups (candidate peptide 3, candidate peptide 4, and a fixed-ratio combination group of candidate peptide 3 and candidate peptide 4) were established. The concentrations of the single-drug groups were 5, 10, 20, and 40 μmol / L, respectively; the total concentration of the combination group was the sum of the concentrations of candidate peptide 3 and candidate peptide 4, with total concentrations of 5, 10, 20, and 40 μmol / L. For example, with a total concentration of 20 μmol / L, the combination group consisted of 10 μmol / L candidate peptide 3 + 10 μmol / L candidate peptide 4. Each group was used to treat MPP. + After damaging the SH-SY5Y cell model, the Fa value was calculated based on the cell viability recovery rate or damage inhibition rate, and the CI value was calculated using CompuSyn software. The results are shown in Table 6.

[0064] Table 6. Synergistic Index (CI) of Candidate Peptides 3 and 4 in Combined Application

[0065] Note: The total concentration of the combined group is the sum of the concentrations of candidate peptide 3 and candidate peptide 4. For example, a total concentration of 20 μmol / L in the combined group means 10 μmol / L of candidate peptide 3 + 10 μmol / L of candidate peptide 4.

[0066] As shown in Table 6, with increasing concentration, the Fa value of the combined group increased from 0.38 to 0.96, while the Fa values ​​of the single-drug groups of candidate peptide 3 and candidate peptide 4 were the highest at 0.73 and 0.66, respectively. The CI values ​​at all concentration points were less than 1, suggesting that the combined use of candidate peptide 3 and candidate peptide 4 at a 1:1 ratio has a synergistic effect in restoring cell viability.

[0067] 6. Evaluation of oxidative stress and improvement in mitochondrial function Cells via MPP + After damage and peptide treatment, SOD activity, MDA content, ROS level (DCFH-DA probe), mitochondrial membrane potential (JC-1 probe) and ATP level were measured. The results are shown in Tables 7 and 8.

[0068] Table 7 Results of Oxidative Stress Indicators

[0069] Note: The total concentration of 20 μmol / L in the 1:1 combination group represents candidate peptide 3 10 μmol / L + candidate peptide 4 10 μmol / L.

[0070] Table 8 Results of mitochondrial functional index detection

[0071] Note: The total concentration of 20 μmol / L in the 1:1 combination group represents candidate peptide 3 10 μmol / L + candidate peptide 4 10 μmol / L.

[0072] The results in Tables 7 and 8 show that all indicators in each treatment group were significantly improved compared to the model group (P<0.05 or P<0.01). Regarding antioxidant and mitochondrial protection, all indicators in candidate peptide group 4 were significantly better than those in candidate peptide group 3 (P<0.05). The 1:1 combination group showed even better results than any single peptide group (P<0.05).

[0073] As the results above show, this invention does not screen candidate short peptides based solely on a single cell viability indicator, but rather performs a stratified evaluation around multiple pathological stages related to Parkinson's disease. Both candidate peptide 3 and candidate peptide 4 exhibit strong inhibitory activity against α-synuclein aberrant aggregation, but they show different advantages in subsequent functional evaluations: candidate peptide 3 showed stronger inhibitory effects on IL-1β and TNF-α in the LPS-induced BV2 cell inflammation model; candidate peptide 4 showed stronger inhibitory effects in MPP... +The ameliorative effects on oxidative stress and mitochondrial dysfunction were more pronounced in the induced SH-SY5Y cell injury model. Furthermore, the combined application of the two peptides at a 1:1 mass ratio showed superior results compared to a single peptide in restoring cell viability, improving oxidative stress, and restoring mitochondrial function, with a CI value less than 1, suggesting a synergistic effect based on functional complementarity rather than simple additive effects. Therefore, candidate peptide 3 was designated as peptide A, candidate peptide 4 as peptide B, and the combination of the two was adopted as the core technical solution of this invention.

[0074] Example 4 Preparation of Lyophilized Powder Injection of Polypeptide A and Polypeptide B Composition Prescription: Polypeptide A 10 mg, Polypeptide B 10 mg, Mannitol 180 mg (as excipient), add water for injection to 2 mL.

[0075] step: S1. Dissolve polypeptide A, polypeptide B and mannitol in water for injection (about 1.8 mL) and stir until completely dissolved.

[0076] S2. Add water for injection to 2.0 mL and mix well.

[0077] S3. Adjust the pH to 6.5 ± 0.2 using 0.1 M NaOH or HCl.

[0078] S4. Sterilize by filtration through a 0.22 μm sterile filter membrane, and dispense into 2 mL sterile vials, each containing 1.0 mL (containing 5 mg of polypeptide A + 5 mg of polypeptide B).

[0079] S5. After partially plugging, place the product into a freeze dryer and freeze-dry it according to the following procedure: pre-freezing (-45℃, 3 h), first drying (-25℃, 24 h, vacuum degree <20 Pa), and second drying (30℃, 6 h). After the freeze dryer is finished, plug and cap it to obtain the freeze-dried powder injection.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide composition for preventing, delaying, or assisting in the intervention of neurodegenerative diseases, characterized in that, The mixture includes polypeptide A and polypeptide B, wherein the amino acid sequence of polypeptide A is shown in SEQ ID NO.1 and the amino acid sequence of polypeptide B is shown in SEQ ID NO.

2.

2. The use of a polypeptide composition as described in claim 1 for preventing, delaying or assisting in the intervention of neurodegenerative diseases in the preparation of a medicament for preventing, delaying or assisting in the intervention of Parkinson's disease.

3. The application according to claim 2, characterized in that, The drug also includes one or more of the following pharmaceutically acceptable excipients, carriers, diluents, stabilizers, buffers, osmotic pressure regulators, preservatives, and surfactants.

4. The application according to claim 2, characterized in that, The drug is an injection, a lyophilized powder for injection, a nasal preparation, a nasal drop, a nasal spray, or a sustained-release microsphere preparation.

5. The application according to claim 2, characterized in that, In the drug, the weight ratio of polypeptide A to polypeptide B is 1:

1.

6. The application according to claim 5, characterized in that, In the drug, the total mass fraction of polypeptide A and polypeptide B is 5%-30%.

7. A method for preparing a polypeptide composition as described in claim 1 for preventing, delaying, or assisting in the intervention of neurodegenerative diseases, characterized in that, Includes the following steps: S1. Dissolve the polypeptide A and the polypeptide B in water for injection, purified water or buffer solution; S2. Add pharmaceutically acceptable excipients and mix thoroughly; S3. Adjust the pH to 4.0-8.0; S4. Perform sterilization filtration and repackage; S5. Perform freeze drying to obtain the polypeptide composition used for preventing, delaying or assisting in the intervention of neurodegenerative diseases.

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