A wheat peptide with neuroprotective effects, its preparation method and applications

Abstract

This invention relates to the field of biomedical technology, and particularly to a wheat peptide with neuroprotective effects, its preparation method, and its applications. The wheat peptide with neuroprotective effects has the amino acid sequence shown in SEQ ID NO:1. This neuroprotective wheat peptide can penetrate the blood-brain barrier and exert a protective effect against nerve cell damage, especially in gene-deficient AD models, with an in vivo half-life of approximately 6-8 hours, exhibiting good pharmacokinetic characteristics and excellent biosafety. The application of the above-mentioned neuroprotective wheat peptide in the preparation of neuroprotective drugs, which can prevent and / or alleviate nerve damage associated with neurodegenerative diseases, and have the effects of protecting nerve cells, resisting oxidative stress damage, and improving cognitive function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a wheat peptide with neuroprotective effects, its preparation method, and its applications. Background Technology

[0002] Neurodegenerative diseases, such as Alzheimer's disease (AD), are characterized by oxidative stress, β-amyloid (Aβ) deposition, and neuronal apoptosis. Currently, there are no drugs clinically capable of effectively halting or reversing disease progression. Bioactive peptides, due to their high activity, low toxicity, and good biocompatibility, show great potential in neuroprotection. However, most candidate peptides suffer from poor in vivo stability, difficulty crossing the blood-brain barrier (BBB), or low bioavailability. Wheat peptides (WPs) are a mixture of small-molecule peptides obtained by enzymatic hydrolysis of wheat germ proteins, and previous studies have shown that they possess physiological activities such as antioxidant activity. However, wheat peptides studied using current techniques still cannot cross the blood-brain barrier and exert a protective effect against neuronal cell damage, especially in gene-deficient AD models. Summary of the Invention

[0003] In response to the problems raised in the background art, the purpose of this invention is to propose a wheat peptide with neuroprotective effects that can penetrate the blood-brain barrier and exert a protective effect against nerve cell damage, especially in gene-deficient AD models. It also has an in vivo half-life of about 6-8 hours, good pharmacokinetic characteristics, and excellent biosafety.

[0004] Another objective of this invention is to provide a method for preparing the above-mentioned wheat peptides with neuroprotective effects.

[0005] Another objective of this invention is to propose the application of the aforementioned neuroprotective wheat peptides in the preparation of neuroprotective drugs, which can prevent and / or alleviate nerve damage associated with neurodegenerative diseases, and have the effects of protecting nerve cells, resisting oxidative stress damage, and improving cognitive function.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0007] A wheat peptide WPs8 with neuroprotective effects, the amino acid sequence of which is shown in SEQ ID NO:1;

[0008] The amino acid sequence of the wheat peptide WPs8 is GHHWPLPP (Gly-His-His-Trp-Pro-Leu-Pro-Pro).

[0009] The method for preparing the wheat peptide with neuroprotective effect includes the following steps:

[0010] Wheat germ protein was enzymatically hydrolyzed, and the hydrolysate was separated by membrane separation to obtain a component with a molecular weight of less than 3 kD. The component was purified by gel chromatography, followed by separation by reversed-phase high-performance liquid chromatography, and identified by LC-MS / MS. The target peptide with the amino acid sequence GHHWPLPP was obtained by screening based on non-toxicity, non-allergenicity, PeptideRanker score, and free radical scavenging ability score.

[0011] To further explain, the operation of purifying the component by gel chromatography includes: purifying the component by passing it through a Sephadex G-25 gel column, with the following purification parameters: detection wavelength of 220 nm, ultrapure water as the mobile phase, and a flow rate of 1 mL / min.

[0012] To further explain, the selection criteria included non-toxicity, non-allergenicity, a PeptideRanker score greater than 0.93, and a free radical scavenging ability score greater than 0.64.

[0013] The application of the neuroprotective wheat peptide in the preparation of a neuroprotective drug, wherein the neuroprotective drug comprises an effective dose of wheat peptide and a pharmaceutically acceptable carrier or excipient, and the amino acid sequence of the wheat peptide is shown in SEQ ID NO:1.

[0014] Furthermore, the neuroprotective drug is a drug that has the functions of protecting nerve cell function, resisting oxidative stress, and improving learning, memory, and cognitive functions.

[0015] To further explain, the neuroprotective drug is used to prevent and / or alleviate nerve damage associated with neurodegenerative diseases.

[0016] To further clarify, the neurodegenerative disease mentioned is Alzheimer's disease.

[0017] To further clarify, the neuroprotective drug is an oral formulation, and the effective dose is 10 to 90 mg per kilogram of body weight per day.

[0018] Specifically, the dosage form is prepared as follows: Weigh 150 mg of wheat peptide WPs8, dissolve it in purified water and bring the volume to 10 mL to prepare a high-dose WPs8 solution of 15 mg / mL; take 1 mL of the 15 mg / mL WPs8 solution and dilute it with purified water to 3 mL to prepare a medium-dose WPs8 solution; take 1 mL of the 15 mg / mL WPs8 solution and dilute it with purified water to 9 mL to prepare a low-dose WPs8 solution.

[0019] Preferably, the effective dose is 30 mg per kilogram of body weight per day.

[0020] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0021] 1. Clear neuroprotective effect: In vitro experiments have confirmed that the wheat peptide WPs8 of the present invention can significantly improve the survival rate of PC12 cells in the H2O2-induced oxidative stress model.

[0022] 2. Improvement of behavioral deficits: In APP / PS1 transgenic AD model mice, wheat peptide WPs8 can significantly improve the new object recognition index, shorten the escape time in the water maze, and increase the number of times the original platform is crossed, proving that it effectively improves the learning and memory ability of the model mice.

[0023] 3. Excellent intrabrain delivery capability: In vivo imaging experiments have shown that wheat peptide WPs8 can cross the blood-brain barrier and accumulate in brain tissue. Its in vivo half-life is about 6-8 hours, and it has good pharmacokinetic characteristics.

[0024] 4. Excellent biosafety: Acute toxicity tests showed that wheat peptide WPs8 had no significant adverse effects on the weight gain, major organ coefficients, and liver and kidney tissue morphology of experimental mice, confirming its high biosafety.

[0025] 5. Multiple protective mechanisms: Wheat peptide WPs8 can significantly increase antioxidant indicators (such as SOD and GSH-Px) in brain tissue, and this has been confirmed by brain tissue pathological sections (Nissl staining) to effectively reduce neuronal damage morphology. Attached Figure Description

[0026] Figure 1 This is the total ion chromatogram of wheat peptides from Example 1 of the present invention.

[0027] Figure 2 This is a secondary mass spectrum of the target peptide WPs8 (GHHWPLPP) in Example 1 of the present invention.

[0028] Figure 3 This is a high-performance liquid chromatogram of the target peptide WPs8 (GHHWPLPP) in Example 1 of the present invention, showing the purity determination.

[0029] Figure 4 This is a graph showing the protective effect of WPs8 against oxidative stress damage in PC12 cells. Figure 4 (a) is a cell morphology observation image (micrograph, where the arrows indicate oxidative damage to cells). Figure 4 (b) in the figure is a graph showing the effect of WPs8 on the survival rate of PC12 cells; Figure 4 (c) in the figure is the survival rate detection of PC12 cells induced by H2O2 using WPs8; Figure 4 (d) in the figure is a graph showing the detection of intracellular reactive oxygen species (ROS) levels.

[0030] Figure 5 This is a schematic diagram of gene identification in APP / PS1 transgenic mice.

[0031] Figure 6 This is a graph showing the effect of WPs8 intervention on the novel object recognition behavior of APP / PS1 mice. Figure 6 (a) in the diagram is a schematic of the mouse's exploration path during the testing phase. Figure 6 (b) in the figure is a bar chart of the recognition index of each group of mice (core result).

[0032] Figure 7 This is a graph showing the effect of WPs8 intervention on the water maze navigation ability of APP / PS1 mice. Figure 7 (a) in the diagram is a schematic of the mouse swimming path during the testing phase. Figure 7 (b) in the figure is a graph showing the change in average escape latency with the number of training days. Figure 7 (c) in the figure shows the escape latency results for each group of mice on day 5 (the end of the training period). Figure 7 (d) in the figure shows the results of the control index of the average swimming speed of mice in each group.

[0033] Figure 8 This is a graph showing the effect of WPs8 intervention on the spatial exploration ability of APP / PS1 mice in a water maze. Figure 8 (a) in the diagram is a schematic of the mouse swimming path during the testing phase. Figure 8 (b) in the figure shows the number of times the mouse crossed the original platform position. Figure 8 (c) in the figure shows the result of the time the mouse spent in the target quadrant (the quadrant where the original platform was located).

[0034] Figure 9 This is a statistical graph showing the effects of WPs8 on mouse body weight and organ coefficients. Figure 9 (a) in the figure shows the results of the mouse's weight change. Figure 9 (b), (c), (d), (e), and (f) in the figure are the results of the mouse heart coefficient, liver coefficient, spleen coefficient, lung coefficient, and kidney coefficient, respectively.

[0035] Figure 10 This is a graph showing the effect of WPs8 on mouse liver histopathology (HE staining).

[0036] Figure 11 This is a graph showing the effect of WPs8 on mouse kidney tissue pathology (HE staining).

[0037] Figure 12 This is an in vivo imaging and tissue distribution analysis of WPs8 in mice. Figure 12 (a) in the figure is a time-series image of a whole-body live mouse imaging system. Figure 12 (b) in the middle is... Figure 12 (a) shows a magnified view of the head region and brain fluorescence images at specific time points. Figure 12 (c) in the graph shows the change in fluorescence intensity in the mouse brain over time. Figure 12 Image (d) in the image is an isolated brain tissue image. Figure 12 Image (e) in the image is an ex vivo image of the major organs. Figure 12 (f) in the middle is... Figure 12 A quantitative bar chart of the fluorescence intensity of brain tissue was plotted after quantitative statistics were performed on (d) in the figure.

[0038] Figure 13 This is a graph showing the effect of WPs8 on serum antioxidant levels in APP / PS1 mice. Figure 13 (a) in the figure is a graph showing the detection of superoxide dismutase (SOD) activity. Figure 13 (b) in the figure is a graph showing the detection of glutathione peroxidase (GSH-Px) activity. Figure 13 (c) in the figure is a graph showing the detection level of malondialdehyde (MDA).

[0039] Figure 14 This is a diagram showing the effect of WPs8 on the morphology of brain cells in APP / PS1 mice (Nissl staining). Detailed Implementation

[0040] To facilitate understanding of the present invention, a more complete description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0042] The cell and animal sources used in the following examples are as follows:

[0043] Cell Name: Rat adrenal pheochromocytoma cells PC-12 (highly differentiated), Catalog Number: TCR 9, Source: Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee;

[0044] Animal name: APP / PS1 mice and littermate control mice; genetic background: C57BL / 6J; source: Beijing Huafukang Biotechnology Co., Ltd.

[0045] Example 1: Preparation and identification of wheat peptide WPs8 with neuroprotective effects.

[0046] Defatted wheat germ powder was mixed with 0.9% NaCl solution at a ratio of 1:10 and shaken at 30°C for 1.5 h. The mixture was then centrifuged, and the supernatant was freeze-dried to obtain wheat germ protein. The pH of the wheat germ protein aqueous solution (5%) was adjusted to 2.5 with 1 mol / L HCl, and pepsin (2000 U / mL) was added. The mixture was shaken at 37°C for 2 h, then the pH was adjusted to 7.5, and trypsin (100 U / mL) was added for 2 h. A fraction with a molecular weight below 3 kD was retained using an ultrafiltration membrane. This fraction was then purified using a Sephadex G-25 gel column (detection wavelength 220 nm; mobile phase: ultrapure water; flow rate 1 mL / min). Preparative reversed-phase high-performance liquid chromatography (RT-HPLC, column: BHE C18; wavelength: 220 nm; mobile phase: 0.1% trifluoroacetic acid water and 0.1% trifluoroacetic acid acetonitrile; flow rate: 2 mL / min) was used for separation, and the target peak was collected and freeze-dried. The obtained sample was dissolved in water, then subjected to reductive alkylation and desalting. The amino acid sequence of the peptide was identified by LC-MS / MS (scan range 100-1500 m / z, primary resolution 120000, secondary resolution 15000, followed by database comparison). Figure 1 The image shows the total ion current chromatogram of wheat peptides. The components of the target enzymatic hydrolysate, separated by chromatography, were introduced into a mass spectrometer. The mass spectrometer scanned the ion currents, and the total ion current chromatogram was plotted with ion intensity as the ordinate and time as the abscissa. Subsequently, each peptide was screened for non-toxicity, non-allergenicity, a Peptide Ranker score greater than 0.93, and a free radical scavenging ability score greater than 0.64 to obtain the target peptide. This peptide had a Peptide Ranker activity prediction score of 0.939304 and a free radical scavenging score of 0.64669687, and was predicted to be non-toxic and non-allergenic. Its amino acid sequence is GHHWPLPP, and it is named WPs8.

[0047] like Figure 2 The image shows the secondary mass spectrum of the target peptide WPs8 (GHHWPLPP). During the mass spectrometry scan, a primary spectrum is generated, followed by fragmentation of the top 20 abundance / response peptides, which are then recorded by the mass spectrometer to generate the secondary mass spectrum. Figure 3 The purity determination of the target peptide WPs8 (GHHWPLPP) is shown in the high performance liquid chromatogram. The main peak time is 9.647 min and the main area ratio is >95%, indicating that WPs8 has high purity.

[0048] 1. Study on the protective effect of WPs8 against oxidative stress damage in PC12 cells.

[0049] A PC12 cell oxidative stress model induced by H2O2 was established. The experiment was divided into a normal control group, an H2O2 damage model group, a WPs8 pretreatment group with different concentrations (50 μM, 100 μM and 200 μM), and a GSH (glutathione) positive control group with different concentrations (50 μM, 100 μM and 200 μM).

[0050] Specifically, the cell model was rat adrenal pheochromocytoma cells PC-12; the injury model was established by treating PC12 cells with H2O2 to create an oxidative stress injury model, as H2O2 can directly induce the production of large amounts of reactive oxygen species (ROS) in cells, leading to apoptosis and simulating the oxidative stress environment in neurodegenerative diseases; the intervention measures were: cells were pretreated with different concentrations of WPs8 and then co-cultured with H2O2; the positive control was: cells were pretreated with different concentrations of GSH (a known endogenous antioxidant) and then co-cultured with H2O2.

[0051] (1) Cell morphology observation: such as Figure 4 As shown in (a), cell morphology observations in the H2O2 model group revealed typical apoptotic / necrotic morphologies such as shrinkage, rounding, synaptic retraction, and even floating. WPs8 effect: Compared to the H2O2 model group, cells treated with WPs8, especially the 100 μM and 200 μM groups, exhibited more relaxed morphology, better adhesion, and relatively intact synaptic structures, closely resembling the normal control group. This provides the most direct morphological evidence for the protective effect of WPs8. The morphological characteristics of GSH-treated cells were similar to those of WPs8-treated cells.

[0052] (2) Detection of PC12 cell viability by WPs8 (without H2O2 induction): such as Figure 4 As shown in (b), there was no significant difference in cell survival rate between the control group and the control group treated with different concentrations of WPs8 / GSH, indicating that WPs8 has no toxic side effects on cells.

[0053] (3) Detection of WPs8 on the survival rate of H2O2-induced PC12 cells (e.g., CCK-8 assay): Figure 4 As shown in (c), the cell survival rate in the H2O2 model group was significantly reduced, down to about 50% of that in the normal control group. However, the pretreatment with WPs8 / GSH could reverse the H2O2-induced decrease in survival rate in a concentration-dependent manner. As the concentration of WPs8 increased, the cell survival rate gradually recovered, demonstrating that WPs8 can resist cell death caused by oxidative stress.

[0054] (4) Intracellular reactive oxygen species (ROS) levels: such as Figure 4As shown in (d), the intracellular fluorescence intensity of the H2O2 model group was significantly enhanced when detected using the DCFH-DA fluorescent probe, indicating a surge in ROS levels. In contrast, the fluorescence intensity of intracellular ROS levels in the WPs8-treated cells was significantly lower than that in the H2O2 model group, indicating that WPs8 has a strong free radical scavenging ability and can directly neutralize H2O2 or other free radicals derived from H2O2 in the cells, thus alleviating oxidative stress at its source.

[0055] It is evident that WPs8 significantly improved the morphological pathological changes in PC12 cells induced by H2O2, demonstrating good efficacy. Furthermore, compared to the H2O2 model group, the cell survival rate in the WPs8 pretreatment group was significantly increased (p<0.05). WPs8 significantly improved the survival rate of nerve cells under oxidative stress and alleviated cell membrane damage, indicating that WPs8 has a clear cytoprotective effect. The core protective mechanism of WPs8 lies in its strong antioxidant activity, which can directly scavenge ROS and reduce intracellular oxidation levels. These results demonstrate that WPs8 possesses direct antioxidant activity and can effectively protect nerve cells from oxidative stress damage.

[0056] 2. Construction and validation of the APP / PS1 transgenic mouse model.

[0057] A vector carrying the human APP gene and a mutant PS1 gene was microinjected into fertilized eggs of C57BL / 6J mice, which were then transplanted into pseudopregnant mothers to obtain offspring mice. Genomic DNA was extracted from the tails of mice at 3-4 weeks of age, and PCR was performed using specific primers. Mice that amplified specific bands for APP and PS1 were identified as positive transgenic mice and used for subsequent experiments. Figure 5 As shown, in the genotyping of transgenic mice, APP (approximately 300 bp) and PS1 (approximately 600 bp) were used to confirm the presence of the exogenously introduced human APP gene and the mutant PS1 gene in the mouse genome, respectively. Figure 5 Mice numbered 1, 2, 4, 6, 11, 12, 13, 17, 20, and 21 are APP / PS1 transgenic mice, while the rest are littermate control mice.

[0058] 3. Study on the effects of WPs8 on the behavioral and physiological indicators of APP / PS1 mice.

[0059] APP / PS1 positive mice were randomly divided into a model group and low, medium, and high dose WPs8 groups (10, 30, 90 mg / kg / d). Wild-type mice were used as a normal control group. After 14 weeks of gavage administration, behavioral tests were performed. The experiment included the following groups:

[0060] WT: Wild-type mice + solvent control (baseline control group);

[0061] APP / PS1: APP / PS1 transgenic model mice + solvent control (disease model group);

[0062] APP / PS1 + WPs8-L / M / H: APP / PS1 transgenic mice + low, medium, and high doses of WPs8 intervention groups (10, 30, 90 mg / kg / d).

[0063] APP / PS1 + Don: APP / PS1 transgenic mice + Don (Donepezil) positive control group (1 mg / kg / d). Donepezil is a drug for treating Alzheimer's disease (AD) and can clearly improve the cognitive function of patients.

[0064] (1) Effect of WPs8 intervention on the new object recognition behavior of APP / PS1 mice.

[0065] Experimental Procedure: Familiarization Phase: Place mice in an open space containing two identical objects A, allowing them to explore freely for a period of time; Testing Phase: Replace one of the familiar objects A with a new object B, and allow the mouse to explore freely again; Key Metric: Recognition Index, typically calculated as the time the mouse spends exploring the new object during the testing phase. Total exploration time (time spent exploring new objects) +Exploring Familiar Objects Time The proportion of ) Because mice with normal memory function spend more time exploring new objects, their RI values ​​are higher.

[0066] like Figure 6 As shown in (a), mice in the WT group, the WPs8 effective dose group, and the Don group showed a clear hotspot of activity around the new object B, while the exploration path of mice in the APP / PS1 group was more randomly distributed between the two objects, without showing a clear preference for the new object. This provides intuitive spatial localization evidence for behavioral differences.

[0067] like Figure 6As shown in (b), the RI value of the WT group was significantly higher than the random level (0.5), indicating that wild-type mice have normal recognition and memory abilities. The RI value of the APP / PS1 group was significantly lower than that of the WT group, around 0.5, indicating that the recognition and memory function of APP / PS1 mice was impaired, and they could not effectively distinguish between new and familiar objects. The RI value of the WPs8 intervention group (especially the medium-dose WPs8) was significantly higher than that of the APP / PS1 group (p<0.01), and essentially recovered to a level not significantly different from the WT group. This effect should show some dose dependence (WPs8L effect was not significant, WPs8M effect was the best, and WPs8H effect was slightly lower than WPs8M). The effect of the Don group was comparable to that of WPs8H. This demonstrates that WPs8 can effectively reverse cognitive impairment in APP / PS1 mice.

[0068] This demonstrates that WPs8 effectively improves recognition and memory impairment in APP / PS1 transgenic mice, providing strong in vivo evidence for improved cognitive function in AD model mice. WPs8 intervention, particularly a medium dose of 30 mg / kg / day, significantly improved the recognition index in APP / PS1 mice, bringing their memory performance close to that of normal wild-type mice. This behavioral improvement forms a logical loop with the antioxidant stress protection observed in PC12 cells. Through its antioxidant properties, WPs8 protects neurons in brain regions such as the hippocampus, thereby improving cognitive function dependent on these brain regions.

[0069] (2) Effect of WPs8 intervention on the water maze navigation ability of APP / PS1 mice.

[0070] Water maze experiment design: The task was to navigate and locate a platform underwater. Mice needed to use visual cues on the walls surrounding the pool to learn and memorize the spatial location of the platform. The key performance indicators are as follows:

[0071] Escape latency: The time required from being placed in the pool to finding and climbing onto the platform. This is the most direct indicator of spatial learning ability. Animals with strong learning abilities will have a significantly shorter latency period as the number of training days increases.

[0072] Swimming path: The movement trajectory of a mouse in a pool. Effective learning is represented by a direct path from the starting point to the platform.

[0073] Animal grouping: Consistent with the new object recognition experiment, WT group, APP / PS1 group, APP / PS1 + WPs8 dose groups, and APP / PS1 + Don group were set up.

[0074] like Figure 7As shown in (a), after 5 days of training, the WT group exhibited a "straight-line" or "directional" trajectory directly from the starting point to the platform, indicating that they had formed clear spatial memory. The APP / PS1 group showed a "random search" or "circling" trajectory, wandering outside the platform quadrant, with a chaotic swimming path, indicating impaired spatial learning ability and inability to effectively memorize the platform location. The WPs8 intervention group (especially the medium-dose WPs8M): the efficiency and directionality of the swimming path should be significantly better than the model control group, and closer to the "directional" trajectory of wild-type mice. This visually demonstrates the improvement of spatial learning strategies by WPs8. The Don group also showed significantly better path performance than the model control group.

[0075] like Figure 7 As shown in (b) of the curves illustrating the change in mean escape latency over training days, the WT group exhibits a steep decreasing curve, with the latency significantly shortening as the number of training days (Day 1 to Day 5), demonstrating strong learning ability. The APP / PS1 group shows a relatively flat curve, with a slow decrease in latency, and the latency remains significantly longer than that of the WT group throughout the training period, indicating a severe deficiency in its spatial learning ability. For the WPs8 intervention groups, the key results lie in the slope and final position of the curves. The curves for the effective dose groups of WPs8 (especially WPs8M) lie between the WT and APP / PS1 groups, and their decreasing slope is closer to that of the WT group. This indicates that WPs8 treatment significantly accelerates the learning process of APP / PS1 mice. The curve for the Don group also lies between the WT and APP / PS1 groups.

[0076] like Figure 7 As shown in (c), the escape latency on day 5 (the end of training) was used to assess the final learning outcome. The results showed that the WT group had the shortest latency, while the APP / PS1 group had the longest. The WPs8 intervention groups (especially WPs8M) had significantly shorter latency than the APP / PS1 group (p<0.01). This indicates that, after treatment, APP / PS1 mice were able to locate the platform quickly and accurately at the end of training, just like normal mice.

[0077] like Figure 7 As shown in (d), the results for the control index of average swimming speed showed no significant difference in average swimming speed among the groups of mice. This ruled out the possibility that the difference in escape latency was due to the drug affecting motor ability, physical strength, or anxiety levels. Since if the mice in the WPs8 group ran faster, their shorter latency might be due to "running faster" rather than "remembering better," the results showed that consistent speed was necessary to attribute the difference in latency to improved cognitive function. The overall conclusion is that the orientation and navigation experiment provides strong, quantitative behavioral evidence that WPs8 can improve spatial learning ability in AD model mice.

[0078] The results above show that WPs8 not only helped APP / PS1 mice achieve better results in the final test (see...) Figure 7 (c) In particular, it improves the entire learning process (see (c)). Figure 7 (b) WPs8, by restoring the learning curve to normal, specifically improved the spatial learning ability of APP / PS1 transgenic mice. Spatial learning ability is highly dependent on the integrity of the hippocampus and is associated with neuroprotective mechanisms. This improvement is corroborated by the antioxidant protection observed in previous in vitro experiments and the subsequent improvement in neuronal morphology observed in brain tissue sections. This indicates that WPs8 rescues higher cognitive functions mediated by hippocampal neurons by protecting them. This experiment further reinforced 30 mg / kg / day as the preferred effective dose for improving cognitive function.

[0079] (3) Effects of WPs8 intervention on the spatial exploration ability of APP / PS1 mice in water maze.

[0080] Space exploration experimental design: On the second day after the positioning and cruising training (day 6), the hidden platform was removed, allowing the mice to swim freely in the water for a set time (60 seconds). Key performance indicators are as follows:

[0081] Number of times the mouse traversed the original platform location: the number of times the mouse swam across the area where the original platform was located. This is the gold standard for measuring memory accuracy.

[0082] Time spent in the target quadrant (the quadrant where the original platform was located): The time the mouse spends exploring the target quadrant, which reflects the mouse's memory of the approximate area of ​​the platform.

[0083] Swimming path diagram: visually shows whether the mouse's search strategy is focused on the original platform area.

[0084] This task does not depend on motor ability or learning speed; it purely tests the mouse's long-term memory of spatial location and is closely related to the function of the hippocampus.

[0085] like Figure 8As shown in (a), the swimming paths of the WT group mice densely covered and repeatedly crossed the original platform area, exhibiting a "purposeful" search pattern. The paths of the APP / PS1 group were scattered, with mice randomly wandering throughout the pool or swimming along the pool wall (wall-seeking behavior), rarely or never crossing the original platform area, indicating that they failed to form or retain a stable memory of the platform location. The search paths of the WPs8 intervention group (especially the medium-dose WPs8M) were clearly concentrated in the target quadrant and repeatedly crossed the original platform location, a search strategy similar to the WT group. The search paths of the Don group also tended to be concentrated in the target quadrant, and the search paths also crossed the original platform location. This intuitively demonstrates that WPs8 treatment helped the APP / PS1 mice recover their spatial location memory.

[0086] like Figure 8 As shown in (b), the number of times the original platform location was traversed is the most critical indicator of memory accuracy. The WT group had the most traversals. The APP / PS1 group had significantly fewer traversals than the WT group. The results of the WPs8 intervention group showed that the effective dose of WPs8 had significantly more traversals than the APP / PS1 group, especially the WPs8M group (p<0.01), which was close to the level of the WT group. This directly demonstrates that mice treated with WPs8 had a clearer and more accurate "cognitive map" of the platform location in their minds. Furthermore, the WPs8L and WPs8H groups had similar numbers of traversals, followed by the Don group.

[0087] like Figure 8 As shown in (c), the time spent in the target quadrant reflects the strength of memory for the approximate location of the platform. The results showed that if mice remembered roughly which quadrant the platform was in, they spent more time searching there. The WT group spent significantly more time in the target quadrant than the random level (26 s). The proportion of time spent in the APP / PS1 group was close to or only slightly higher than 14 s, indicating fuzzy memory. The proportion of time spent in the target quadrant in the WPs8M group was significantly higher than that in the APP / PS1 group and the random level (p<0.01). This suggests that even with slightly less precise memory, WPs8 can still help mice remember the approximate location of the platform.

[0088] Overall Conclusion: This spatial exploration experiment provides decisive evidence that WPs8 can improve and protect spatial reference memory in AD model mice. Complementing the orientation navigation (learning process), the spatial exploration results show that WPs8 not only helps APP / PS1 mice learn faster, but more importantly, it helps mice stably store the learned spatial information, forming long-term memory. Therefore, it confirms the improving effect of WPs8 on long-term memory and effectively enhances the spatial reference memory retention capacity of APP / PS1 transgenic mice. Strongly correlated with hippocampal function: The loss of reference memory is a core behavioral manifestation of early hippocampal damage in AD. The results of this experiment strongly demonstrate that WPs8 treatment has a significant protective and improving effect on hippocampal function in AD model mice. Furthermore, a complete chain of behavioral evidence was constructed: the three behavioral tests of new object recognition (recognition memory), orientation navigation (spatial learning), and spatial exploration (spatial memory) comprehensively demonstrate the remarkable efficacy of WPs8 in improving the overall cognitive function of APP / PS1 mice from different dimensions and through mutual corroboration.

[0089] 4. Evaluation of the biosafety and tissue distribution of WPs8.

[0090] (1) Statistical analysis of the effects of WPs8 on mouse body weight and organ coefficient.

[0091] Objective: To systematically evaluate the subacute or subchronic toxicity of WPs8. Body weight and organ coefficients are the most basic and important indicators in preclinical drug safety evaluation.

[0092] The testing indicators are as follows:

[0093] Changes in body weight: Reflects the potential effects of the test substance on the animal's overall health, growth and development, metabolism and appetite.

[0094] Organ coefficient (organ index): the ratio of the weight of a particular organ to the body weight. An abnormal increase or decrease in this index may indicate pathological changes such as edema, hyperplasia, hypertrophy, or atrophy of the organ (the organ coefficient is calculated by weighing the major organs).

[0095] Animal groups: WT: wild-type control group, APP / PS1: transgenic model control group, APP / PS1 + WPs8-L / M / H: transgenic mice + low, medium and high dose WPs8 groups (10, 30, 90 mg / kg / d), APP / PS1 + Don group (1 mg / kg / d).

[0096] like Figure 9As shown in (a), the dynamic weight change curves display the average weight change of mice in all groups over the entire experimental period (within 16 weeks of drug administration). The weight gain curves of the WT group and the APP / PS1 group largely overlap. This indicates that the APP / PS1 transgene itself did not cause significant growth and developmental abnormalities or cachexia in the mice, providing a good model basis for observing drug effects. Key findings: The weight gain curves of all WPs8 intervention groups (WPs8L, WPs8M, WPs8H) highly overlapped with the curves of the APP / PS1 group and the WT group, with no significant differences. This indicates that during the long drug administration period of several weeks, even at the highest dose (90 mg / kg / d), WPs8 did not have any observable negative impact on the overall growth, nutritional status, and metabolism of the mice. In addition, the weight gain curve of the Don group also highly overlapped with the curves of the APP / PS1 group and the WT group.

[0097] like Figure 9 As shown in (b), (c), (d), (e), and (f), there were no significant differences in organ coefficients between the WT group and the APP / PS1 group. The coefficients for the heart, liver, spleen (the spleen is an important immune organ and is sensitive to toxicity), lung, and kidney in all WPs8 intervention groups were not statistically significantly different from those in the APP / PS1 group, WT group, and Don group. This provides strong evidence that WPs8 did not cause pathological hypertrophy or atrophy of major organs.

[0098] The aforementioned body weight and organ coefficient data provide primary, quantitative macroscopic evidence for the superior biocompatibility of WPs8: No systemic toxicity: WPs8 does not affect the normal growth, development, or overall health of laboratory animals. No organ toxicity: WPs8 does not cause abnormal weight in major organs, preliminarily ruling out toxic risks to core organs such as the liver and kidneys. Supporting effective dose: At the preferred effective dose (30 mg / kg / day) and even higher doses, which demonstrate significant neuroprotection and behavioral improvement, WPs8 still exhibits an extremely high safety window, crucial for its future application development.

[0099] (2) Effects of WPs8 on mouse liver histopathology (HE staining).

[0100] Experimental objective: Same as before, liver tissue from mice in the study of the effects of the above behavioral and physiological indicators was stained with hematoxylin and eosin (HE). Based on the macroscopic organ coefficient analysis, we directly observed and evaluated whether WPs8 caused damage to liver tissue from the microscopic cell morphology level.

[0101] Method: Hematoxylin-eosin (H&E) staining. This is the standard method for observing the basic morphological structure of tissues. It can clearly show structures such as hepatocyte cords, hepatic sinusoids, central veins, and portal areas, and can identify typical lesions such as cellular edema, fatty degeneration, inflammatory cell infiltration, and necrosis.

[0102] like Figure 10 As shown, the WT group (wild-type control group), serving as a normal control, exhibited typical healthy liver structure. Hepatocytes were arranged in a radial cord pattern (hepatic cords) centered on the central vein, with a clear and orderly structure. Hepatocytes were uniform in size, with homogeneous cytoplasm and large, round nuclei located in the center of the cell. The hepatic sinusoids were clearly defined, without abnormal material deposition or inflammatory cell aggregation. The APP / PS1 group (transgenic model control group) served as a negative control, used to rule out whether the APP / PS1 transgene itself would cause liver lesions. The results showed that its liver tissue structure was essentially consistent with the WT group, with no specific pathological changes observed. This demonstrates that the Alzheimer's-like neuropathological changes induced by the APP / PS1 transgene were not accompanied by spontaneous liver damage. The Don group also showed essentially the same results as the WT group. Therefore, any subsequent liver morphological changes observed in the WPs8 group can be attributed to the effect of WPs8, rather than defects in the model itself. The APP / PS1 + WPs8-L / M / H groups (WPs8 dose intervention groups) consisted of liver sections from the low, medium, and high dose groups, which were compared with the APP / PS1 and WT groups. Key findings revealed that the liver tissue morphology and structure of all WPs8 intervention groups, including hepatic cord arrangement, hepatocyte morphology, nuclear status, and hepatic sinusoidal structure, were essentially consistent with the APP / PS1 and WT groups, with no significant differences. Specific negative indicators included: no hepatocyte edema: cells were uniform in size, and no vacuolar degeneration (watery degeneration) was observed in the cytoplasm. No fatty degeneration: no large number of vacuoles in the cytoplasm (caused by the dissolution of lipid droplets). No inflammatory cell infiltration: no large aggregation of inflammatory cells such as lymphocytes and neutrophils was observed in the portal areas or hepatic sinusoids. No necrotic areas: hepatocyte structure was intact, and no signs of necrosis such as nuclear pyknosis, fragmentation, or dissolution were observed. No pathological hyperplasia or fibrosis was observed. In summary, Figure 9The liver HE staining results shown provide the most direct and compelling microscopic morphological evidence for the liver safety of WPs8. Confirmation of no hepatotoxicity: This experimental result indicates that even with long-term administration at high doses (90 mg / kg / day), WPs8 did not cause any observable pathological damage to liver tissue. Corroboration with macroscopic data: This result is consistent with the aforementioned macroscopic data showing no significant change in liver coefficients, forming a complete chain of evidence of "no abnormalities in macroscopic indices and no damage to microstructures," greatly enhancing the reliability of the conclusions. Support for high safety profile: The liver is the core organ for detoxification and metabolism. Its good tolerance to WPs8 suggests that WPs8 may have a low metabolic burden and good safety profile in vivo, providing an important guarantee for its further development and application.

[0103] (3) Effects of WPs8 on mouse kidney histopathology (HE staining).

[0104] Experimental objective: Same as before, take kidney tissue from mice in the study of the effects of the above behavioral and physiological indicators and perform HE staining to directly observe and evaluate whether WPs8 causes damage to kidney tissue at the microscopic level, focusing on the structural integrity of glomeruli and renal tubules.

[0105] Method: Hematoxylin-eosin (H&E) staining. Used to clearly visualize the cortical and medullary structures of the kidney, especially the morphology of the glomeruli, the condition of the renal tubular epithelial cells, and whether there are lesions in the interstitium.

[0106] like Figure 11As shown, the WT group (wild-type control group), serving as a normal control, exhibited typical healthy kidney structure, with normal glomerular size, clear cellular structure, open capillary loops, and no atrophy or hypertrophy. The renal tubular (especially the proximal convoluted tubule) epithelial cells were neatly arranged and plump, with clear brush borders, no casts or abnormal deposits within the lumen, and no inflammatory cell infiltration or fibrosis in the renal interstitium. The APP / PS1 group (transgenic model control group) showed similar characteristics to the liver. This group served as a key control for assessing the origin of kidney lesions, and its kidney tissue structure was essentially consistent with the WT group, with no specific pathological changes observed. This further demonstrates that the APP / PS1 transgene itself does not cause spontaneous kidney damage, providing a clean background for subsequent attribution analysis. The Don group also showed essentially the same characteristics as the WT group. The APP / PS1 + WPs8-L / M / H groups (WPs8 dose intervention groups) were compared with the two control groups in terms of kidney morphology. Key finding: No significant differences were observed in the kidney tissue morphology and structure of any of the WPs8 intervention groups compared to the APP / PS1 group and the WT group. Specific negative indicators (with a focus on structure): Glomeruli: Normal size and shape, no increased cell count, no basement membrane thickening, no cystic dilation or atrophy. Tubular damage: Intact tubular epithelial cells, no swelling (edematous degeneration), no vacuolar formation (fatty degeneration or vacuolar degeneration), no sloughing necrosis. Clean lumen, no protein casts or crystals. Interstitial lesions: No edema, inflammatory cell infiltration, or fibrous tissue proliferation in the renal interstitium.

[0107] Figure 11 The HE staining results of the kidneys shown provide crucial microscopic morphological evidence for the renal safety of WPs8. Confirmation of no nephrotoxicity: This result indicates that, under long-term administration, WPs8 and its potential metabolites do not cause functional or organic damage to the kidneys, a core excretory organ. The intact structure of the glomeruli and tubules indicates that their filtration and reabsorption functions are unaffected. Completion of the core safety evidence chain: This result, along with HE staining of the liver (… Figure 10 ) and body weight and organ coefficient ( Figure 9 The results together constitute a complete and robust safety evidence system. It demonstrates that at the experimental dose, WPs8 does not macroscopically affect overall growth and organ weight, nor microscopically damage the cellular structure of major metabolic organs (liver) and excretory organs (kidneys).

[0108] (4) In vivo imaging and tissue distribution of WPs8 in mice.

[0109] Experimental Objectives: To visualize and demonstrate the absorption, distribution, and clearance of WPs8 at the whole-animal level. To determine whether WPs8 can cross the blood-brain barrier and accumulate in brain tissue. To assess its distribution in major organs and preliminarily determine its potential target organs and safety.

[0110] Methods: WPs8 was labeled with a near-infrared fluorescent dye (Cy7) and administered to normal C57 mice via gavage. Observations were performed at different time points using an in vivo imaging system. After the experiment, major organs and brain tissue were removed for ex vivo imaging to obtain higher resolution distribution information.

[0111] Detection indicators: intensity, location, and changes over time of fluorescence signal.

[0112] like Figure 12 As shown in (a), the whole-body in vivo imaging time series plots display whole-body fluorescence images of a group of mice at different time points (30 min, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours, and 48 hours after injection). Early stage (30 min): Strong fluorescence signals are mainly concentrated in the heart and major blood vessel areas, indicating that the drug rapidly enters the systemic circulation. Mid-stage (1-4 hours): Systemic signal weakens, but relatively clear fluorescence accumulation appears in the head region and abdomen (liver and kidney areas). Late stage (8 hours): Systemic signal significantly weakens, but a weak signal remains in the head region. Dynamic significance: This time series plot visually demonstrates the dynamic process of WPs8's distribution from the bloodstream to all tissues and its gradual metabolic clearance over time.

[0113] like Figure 12 As shown in (b), close-up images of brain regions during live imaging. Figure 12 The magnified or specific time-point fluorescence images of the head region in (a) show that at specific time points (1-2 hours) after gavage, a clear fluorescence signal can be observed in the head region, which provides the most direct in vivo evidence that WPs8 can enter the brain.

[0114] like Figure 12 As shown in (c), the curve of brain fluorescence intensity changing over time is displayed. The curve of brain region fluorescence signal intensity changing over time is quantitatively analyzed. As shown in the figure, the curve shows a rapid rise phase (distribution phase) followed by a slow decline phase (elimination phase). From the curve, it can be concluded that the half-life of WPs8 in vivo is 6-8 hours. The long half-life means that the time window for WPs8 to act in vivo is ideal, and frequent dosing is not required.

[0115] like Figure 12 As shown in (d) above, a fluorescence image of the intact brain tissue removed after the experiment shows clear fluorescence signals observed in the entire brain tissue or specific brain regions (such as the cortex and hippocampus). This ultimately confirms that WPs8 does indeed penetrate the blood-brain barrier and distribute throughout the brain parenchyma, rather than merely remaining in the blood vessels of the brain.

[0116] like Figure 12As shown in (e), the ex vivo imaging of major organs involves simultaneous fluorescence imaging of the heart (H), liver (L), spleen (S), lung (LU), and kidney (K). The kidneys and liver exhibit the strongest signals. Strong kidney signal suggests that WPs8 may be primarily excreted through the kidneys; strong liver signal indicates that the liver also participates in its metabolism / clearance. Other organs show relatively weaker signals. This helps assess its tissue distribution specificity and potential risk of non-targeted accumulation.

[0117] like Figure 12 As shown in (f), the quantitative bar chart of brain tissue fluorescence intensity is... Figure 12 The bar chart is a quantitative statistical analysis of the fluorescence intensity of organs in (d) and the graph shows that... Figure 12 (d) provides quantitative evidence, clearly showing the relative fluorescence intensity values ​​of isolated brain tissue, indicating that WPs8 is enriched in the brain.

[0118] Figure 12 The study provides compelling pharmacokinetic evidence for WPs8 as a neuroprotective candidate: The most crucial finding is its ability to be delivered into the brain. Experiments clearly demonstrate that WPs8 can effectively cross the blood-brain barrier and remain stable in the brain for a period of time, possessing ideal pharmacokinetic characteristics for a central nervous system protective agent. This overcomes the challenge of many large-molecule drugs being unable to enter the brain, providing a prerequisite for its direct action on the central nervous system. The study also elucidates the in vivo dynamic process, revealing the systemic distribution pattern of WPs8 (rapid distribution, hepatic and renal accumulation) and clearance pathway (possibly renal excretion). Furthermore, it provides an ideal half-life: a 6-8 hour half-life indicates that WPs8 has moderate stability in vivo, neither being eliminated too quickly nor causing toxicity due to accumulation, making it highly suitable as a preventative or therapeutic agent. Finally, its efficacy and safety are corroborated: its brain distribution is consistent with observed behavioral improvements and neuroprotective effects; its distribution in the liver and kidneys is supported by histological results showing no toxicity in the liver and kidneys.

[0119] (5) Effect of WPs8 on serum antioxidant indicators in APP / PS1 mice.

[0120] Experimental objective: To quantitatively assess the changes in serum oxidative stress levels in AD model mice after WPs8 intervention, and to reveal its core mechanism of action from the perspectives of endogenous antioxidant defense system and lipid peroxidation damage.

[0121] Detection indicators (inferred from routine life science experiments): Superoxide dismutase (SOD): responsible for scavenging superoxide anion free radicals ( Glutathione peroxidase (GSH-Px) is the body's first and most important antioxidant defense. Its activity directly reflects the body's ability to scavenge free radicals. GSH-Px specifically catalyzes the reduction of hydrogen peroxide (H2O2) and lipid peroxides by reduced glutathione (GSH), thereby protecting the integrity of cell membrane structure and function. Malondialdehyde (MDA) is the end product of lipid peroxidation; its content can indirectly reflect the severity of free radical attack on cells and is a classic biomarker for measuring oxidative damage.

[0122] like Figure 13 As shown, the activities of SOD and GSH-Px in the APP / PS1 group (model validity verification) were significantly lower than those in the WT-Con group. The MDA content was significantly higher in the APP / PS1 group, indicating that the AD model successfully simulated the key pathophysiological feature of oxidative stress. The APP / PS1 transgene led to abnormal Aβ deposition, which in turn triggered severe oxidative stress, significantly depleting endogenous antioxidant enzymes (SOD, GSH-Px) and causing severe cell membrane lipid peroxidation damage (elevated MDA). This constitutes the pathological basis for the action of WPs8. The comparison between the WPs8 intervention group and the APP / PS1 group (pharmacodynamics and mechanism core) on the effects on SOD and GSH-Px activities: The results showed that the SOD and GSH-Px activities in the WPs8 intervention group, especially the medium and high dose groups (GP8M, GP8H), were significantly higher than those in the APP / PS1 group, exhibiting a certain dose-dependent trend. This suggests that WPs8 does not merely act as an exogenous antioxidant to directly scavenge free radicals; its more important role may be to activate or enhance the body's own antioxidant defense system. By enhancing the activity of these key antioxidant enzymes, nerve cells are endowed with a stronger and more durable ability to resist oxidative damage. Effect on MDA levels: The MDA levels in the WPs8 intervention group, especially the medium and high dose groups, were significantly lower than those in the APP / PS1 group. This is direct evidence that WPs8 produces the ultimate protective effect. The decrease in MDA levels demonstrates that due to WPs8 intervention, lipid peroxidation in serum was effectively inhibited, and the structural integrity of the cell membrane was protected.

[0123] Figure 13The antioxidant data shown indicate that after intervention with WPs8 (30, 90 mg / kg / d), the activities of SOD and GSH-Px in the brain tissue of APP / PS1 mice were significantly increased, while the MDA content was significantly decreased (p<0.05 or p<0.01). This advances the neuroprotective effect of WPs8 from phenomenological description (behavioral improvement) and morphological observation (pathological sections) to the explanation of its molecular mechanism. The core mechanism of action is elucidated: WPs8 exerts its neuroprotective effect through a dual pathway against oxidative stress, enhancing endogenous defense by upregulating the activity of key antioxidant enzymes such as SOD and GSH-Px, and mitigating oxidative damage by effectively reducing the level of MDA, the end product of lipid peroxidation. A complete causal chain is constructed: the data connects upstream Aβ pathology with downstream neuronal damage and behavioral defects, clearly demonstrating how WPs8, by intervening in the key intermediate link of "oxidative stress," breaks the vicious cycle of the disease, ultimately achieving neuroprotection and behavioral improvement.

[0124] (6) Effect of WPs8 on brain cell morphology in APP / PS1 mice (Nissl staining).

[0125] Nissl staining is a specific staining method in neuropathology that clearly displays Nissl bodies within neuronal cell bodies. Nissl bodies are aggregates of rough endoplasmic reticulum and free ribosomes, and are core structures for protein synthesis in neurons, directly reflecting the functional activity and health status of neurons.

[0126] Effects of WPs8 on brain cell morphology in APP / PS1 mice (Nissl staining) Objective: To further evaluate the protective effect of WPs8 on neuronal functional status at the subcellular structural level, with a focus on whether the neuronal ability to synthesize proteins is maintained.

[0127] Principle: Nissl staining (commonly toluidine blue or tar violet) can stain Nissl bodies into dark blue patchy or granular structures that aggregate in the cytoplasm, while the cell nucleus remains negative.

[0128] Observation indicators: number, size and density of Nissl bodies: the cytoplasm of healthy neurons is filled with a large number of dense Nissl bodies; distribution of Nissl bodies: Nissl bodies are evenly distributed under normal conditions.

[0129] "Nissl body dissolution" is a typical early sign of neuronal damage (such as oxidative stress, poisoning, and energy metabolism disorders). It is characterized by a decrease in the number of Nissl bodies, light staining, and disappearance, with the cytoplasm staining becoming lighter and only vacuolar structures remaining.

[0130] Figure 14The images are presented as microscopic photographs of neurons in brain tissue and highly pathogenic regions such as the hippocampus. The WT group (wild-type control group) is a normal control group, with neurons arranged neatly and densely. The cytoplasm of each neuron is filled with numerous, deeply stained, and evenly distributed blue-purple Nissl bodies, with clear cell outlines and prominent nucleoli. This indicates that the neurons in wild-type mice are in a state of vigorous functional activity. The APP / PS1 group (transgenic model control group) exhibits typical AD-related neuronal damage morphology, with a reduced number of neurons, sparse arrangement, and widespread "Nissl body dissolution" in surviving neurons: the number of Nissl bodies in the cytoplasm is significantly reduced, the staining is lighter, the structure is blurred, or even completely disappears, making the cytoplasm appear empty and pale, and the cells shrink and have irregular shapes. This functionally demonstrates that the APP / PS1 transgene causes severe neuronal dysfunction and damage, with severely impaired protein synthesis capacity, which is directly related to the observed cognitive impairment. Compared with the APP / PS1 group, the WPs8 intervention groups (especially the medium-dose WPs8) showed significant improvement in neuronal morphology compared to the APP / PS1 group (WPs8 intervention groups at various doses). Specific protective indicators included: a greater number of neurons, denser arrangement, and reduced neuronal loss; a significantly increased number of Nissl bodies in the neuronal cytoplasm, with deeper staining and clearly distinguishable morphology, approaching the level of the WT group; and fuller neuronal contours with a more normal morphology. This indicates that WPs8 treatment effectively reversed the "Nissl body dissolution" process in the APP / PS1 mouse model. In other words, WPs8 protected the structural integrity of the rough endoplasmic reticulum and ribosomes within neurons, maintaining their efficient protein synthesis function. This is the cellular biological basis for its ability to improve learning and memory. Figure 14 The Nissl staining results shown provide morphological evidence from a functional perspective for the neuroprotective effects of WPs8, confirming its protective effect on neuronal function. More importantly, WPs8 can effectively maintain the protein synthesis function of neurons, protecting them from functional damage. A neuron that can synthesize proteins normally is a functional and highly plastic neuron. Furthermore, the Nissl staining results form a closed loop with antioxidant mechanisms and behavioral improvements: upstream mechanism: WPs8 increases SOD and GSH-Px activity and reduces MDA (… Figure 13 This clears oxidative stress; downstream effects: the reduction of oxidative stress directly protects delicate organelles such as Nissl bodies from damage. Figure 14 Final phenotype: Functionally functioning neurons form the cellular basis of learning and memory, thus manifesting as improved behavior. Figure 6 ,7,8).

Claims

1. A wheat peptide having a neuroprotective effect, characterized by, The amino acid sequence of the wheat peptide is shown as SEQ ID NO:

1.

2. The method for preparing wheat peptides with neuroprotective effects as described in claim 1, characterized in that, The method comprises the following steps: The wheat germ protein is subjected to enzymatic hydrolysis, the enzymatic hydrolysis product is subjected to membrane separation to obtain a component with a molecular weight less than 3 kD, the component is subjected to gel chromatography purification, followed by reverse phase high performance liquid chromatography separation, and the target peptide segment with the amino acid sequence of GHHWPLPP is obtained through LC-MS / MS identification combined with non-toxicity, non-allergy, PeptideRanker score and free radical scavenging capacity score screening.

3. The method for preparing wheat peptides with neuroprotective effects according to claim 2, characterized in that, The operation of subjecting the component to gel chromatography purification comprises: subjecting the component to Sephadex G-25 gel column purification, and the purification process parameters comprise: a detection wavelength of 220 nm, ultrapure water as a mobile phase, and a flow rate of the mobile phase of 1 mL / min.

4. The method for preparing wheat peptides with neuroprotective effects according to claim 2, characterized in that, The screening is combined with non-toxicity, non-allergy, a PeptideRanker score greater than 0.93 and a free radical scavenging capacity score greater than 0.

64.

5. The wheat peptide of claim 1 in the preparation of a neuroprotective drug for preventing and / or relieving nerve damage related to Alzheimer's disease, wherein the neuroprotective drug comprises an effective dose of the wheat peptide of claim 1 and a pharmaceutically acceptable carrier or excipient.

6. Use according to claim 5, characterized in that, The neuroprotective drug is a drug with the functions of protecting nerve cells, resisting oxidative stress and improving learning and memory cognitive function.

7. Use according to claim 5, characterized in that, The dosage form of the neuroprotective drug is an oral preparation, and the effective dose is 10-90 mg per kilogram of body weight per day.

8. Use according to claim 5, characterized in that, The effective dose is 30 mg per kilogram of body weight per day.

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