Use of a walnut-derived peptide MDP-TY in the preparation of a medicament or food for preventing or treating diabetic encephalopathy

Abstract

The application of a walnut-derived peptide MDP-TY in the preparation of a medicine or food for preventing or treating diabetic encephalopathy belongs to the technical field of biotechnology. The amino acid sequence of the walnut-derived peptide MDP-TY is TWLPYPR. The walnut-derived peptide MDP-TY can significantly alleviate the weight loss of type 2 diabetic mice, and significantly reduce the food intake, water intake, fasting blood glucose and improve glucose tolerance abnormality of the model mice. The walnut-derived peptide MDP-TY can significantly reduce the TC, TG, LDC-C and NEFA levels in the serum of the model mice, and improve the HDL-C level, thereby improving the blood lipid metabolism disorder state of the T2DM mice. The walnut-derived peptide MDP-TY can improve the liver and kidney tissue pathological damage related to T2DM, increase the complexity of the hippocampal neuron dendritic spine of the T2DM mice, and improve the synaptic structure damage of neurons caused by hyperglycemia. The walnut-derived peptide MDP-TY can be used for manufacturing a medicine for preventing, alleviating or treating diabetic encephalopathy, and can also be used for preparing a food with the function of assisting in improving the nerve damage caused by hyperglycemia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a walnut-derived peptide MDP-TY (TWLPYPR) in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy. Background Technology

[0002] Diabetes mellitus is a systemic metabolic disease characterized by chronic hyperglycemia, and it has become one of the most serious threats to human health worldwide. The latest data released by the International Diabetes Federation (IDF) shows that the number of people with diabetes globally has reached 589 million, making it a significant challenge in global public health. Diabetic encephalopathy (DE) is a major central nervous system complication of diabetes. Its pathogenesis involves multiple aspects, including insulin resistance, hyperglycemia-induced oxidative stress, inflammatory responses, nerve damage, and energy metabolism disorders. These factors interact to lead to serious consequences such as cognitive decline and memory loss. Currently, prevention of diabetic encephalopathy mainly focuses on controlling blood glucose levels with medications such as metformin and GLP-1 receptor agonists. However, clinical practice shows that even when these indicators are controlled within ideal ranges, a considerable number of diabetic patients still experience cognitive impairment, and the toxic side effects of these medications gradually become apparent. This indicates that existing prevention strategies have significant limitations, and there is an urgent need to find new and more effective prevention and intervention measures. Summary of the Invention

[0003] The present invention aims to provide the application of a walnut-derived peptide MDP-TY (TWLPYPR) with tyrosine (Y) residues as the key active site in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy. The walnut-derived peptide MDP-TY is designed based on the sequence characteristics of active peptides derived from walnut protein and obtained through artificial solid-phase peptide synthesis. Its amino acid sequence is Thr-Trp-Leu-Pro-Try-Pro-Arg (TWLPYPR). For details on the preparation of this peptide, please refer to Chinese Invention Patent 202311161773.3.

[0004] Furthermore, the diabetic encephalopathy mentioned above is diabetic encephalopathy caused by type 2 diabetes.

[0005] The dosage form of the drug is one of the following: tablets, capsules, granules, powders, sprays, films, suppositories, nasal drops, or pills.

[0006] The drug carrier is selected from one or more of lactose, mannitol, microcrystalline cellulose, starch, hydroxypropyl methylcellulose, povidone, croscarmellose sodium, magnesium stearate, talc, polyethylene glycol, chitosan, sodium alginate, cyclodextrin, poloxamer, and Tween 80.

[0007] Furthermore, the application of the aforementioned walnut-derived peptide MDP-TY in the preparation of foods that help improve nerve damage caused by hyperglycemia is provided.

[0008] The food mentioned is one of the following: health food, functional food, general food, dietary supplement, special dietary food, and special medical food.

[0009] The food additives are selected from one or more of the following: maltodextrin, corn starch, white sugar, xylitol, erythritol, xanthan gum, gum arabic, sodium alginate, agar, pectin, food-grade hydroxypropyl methylcellulose, magnesium stearate, micronized silica gel, citric acid, honey, and purified water.

[0010] The walnut-derived peptide MDP-TY (TWLPYPR) provided by this invention can be applied to improve glucose and lipid metabolism disorders and related cognitive impairments. It can significantly alleviate weight loss in type 2 diabetes mellitus (T2DM) mice induced by a high-fat diet (HFD) combined with intraperitoneal injection of streptozotocin (STZ), and significantly reduce food intake, water intake, fasting blood glucose, and improve glucose tolerance in model mice. Regarding lipid regulation, walnut-derived peptide MDP-TY can significantly reduce serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDC-C), and free fatty acids (NEFA) in model mice, while increasing serum levels of high-density lipoprotein cholesterol (HDL-C), thereby improving the lipid metabolism disorder in T2DM mice. Furthermore, walnut-derived peptide MDP-TY can also improve T2DM-related liver and kidney histopathological damage, increase the complexity of dendritic spines in hippocampal neurons in T2DM mice, and improve neuronal synaptic structural damage caused by hyperglycemia. Therefore, walnut-derived peptide MDP-TY can be used to manufacture drugs for the prevention, relief or treatment of diabetic encephalopathy, and can also be used to prepare food products that help improve nerve damage caused by hyperglycemia, showing good application prospects and industrialization value. Attached Figure Description

[0011] Figure 1 This is a graph showing the change in mouse body weight provided in Example 2 of the present invention;

[0012] Figure 2 This is a graph showing the changes in the diet of mice provided in Example 2 of the present invention;

[0013] Figure 3 This is a graph showing the changes in water intake in mice as provided in Example 2 of the present invention;

[0014] Figure 4 This is a fasting blood glucose chart of mice provided in Example 2 of the present invention;

[0015] Figure 5 This is a graph of oral glucose tolerance in mice provided in Example 2 of the present invention;

[0016] Figure 6 This is a graph showing the mouse serum LDL-C content provided in Example 3 of the present invention;

[0017] Figure 7 This is a graph showing the TC content in mouse serum provided in Example 3 of the present invention;

[0018] Figure 8 This is a graph showing the TG content in mouse serum provided in Example 3 of the present invention;

[0019] Figure 9 This is a graph showing the NEFA content in mouse serum provided in Example 3 of the present invention;

[0020] Figure 10 This is a graph showing the HDL-C content in mouse serum provided in Example 3 of the present invention;

[0021] Figure 11 This is a representative HE staining image of mouse liver provided in Example 4 of the present invention;

[0022] Figure 12 This is a representative HE staining image of mouse kidneys provided in Example 4 of the present invention;

[0023] Figure 13 This is the mouse blank control group positioning and navigation test path diagram provided in Example 5 of the present invention;

[0024] Figure 14 This is a path diagram for the mouse model group positioning and navigation test provided in Embodiment 5 of the present invention;

[0025] Figure 15 This is the route map of the mouse MDP-TY group positioning and navigation test provided in Example 5 of the present invention;

[0026] Figure 16 This is a statistical chart of the escape latency of three groups of mice provided in Example 5 of the present invention;

[0027] Figure 17 This is a statistical chart of the effective movement distance of three groups of mice provided in Example 5 of the present invention;

[0028] Figure 18 This is a morphological diagram of mouse hippocampal dendritic spines provided in Embodiment 6 of the present invention;

[0029] Figure 19 This is a diagram showing the number of dendritic spines in the mouse hippocampus provided in Embodiment 6 of the present invention. Detailed Implementation

[0030] 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 merely illustrative and not intended to limit the invention.

[0031] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1: Effects of walnut-derived peptide MDP-TY on body weight, food intake, water intake, fasting blood glucose, and oral glucose tolerance in type 2 diabetic mice

[0033] Animal experiments were conducted with the approval of our laboratory animal ethics committee and in accordance with the relevant guidelines of the European Commission's "Guidelines for the Care and Use of Laboratory Animals". Six-week-old SPF-grade male C57B / 6J mice (purchased from Speford Biotechnology Co., Ltd.) were used in the experiments. Mice were divided into a control group, a model group, and an MDP-TY group. The T2DM mouse model was induced using HFT combined with STZ. The entire experiment was conducted in an SPF-grade animal facility, with the ambient temperature controlled at 22–25°C and the relative humidity at 40%–60%, using a 12-hour light / dark cycle. The diet used included maintenance feed and D12492 high-fat diet (both purchased from Speford Biotechnology Co., Ltd.). Animal cages, bedding, and water bottles were all autoclaved (121°C, 20 min). Sterile ultrapure water was used for drinking water to minimize the impact of environmental microbial contamination on the modeling effect.

[0034] After one week of acclimatization with a maintenance diet, all mice were fasted for 8 hours with free access to water. Blood samples were then collected via tail tip sampling, and fasting blood glucose levels were measured using a glucometer. Mouse weight was also recorded. Mice were randomly assigned to groups based on body weight and fasting blood glucose levels to ensure no significant differences in initial body weight and fasting blood glucose levels among groups. The experimental mice were divided into a control group, a model group, and an MDP-TY group. After grouping, the control group was fed a maintenance diet, while the model and MDP-TY groups were fed a high-fat diet (D12492) to induce insulin resistance. Starting in the second week after the acclimatization period, the MDP-TY group was administered walnut-derived peptide MDP-TY via gavage daily. This involved preparing a saline solution of MDP-TY and administering 200 μL of this solution to each mouse daily, resulting in a daily dose of 60 mg / kg. The control and model groups received an equal volume of saline solution via gavage daily as a control. This gavage intervention continued until the end of the experiment, from week 2 to week 11.

[0035] STZ induction modeling was performed in week 6. Before modeling, all mice were fasted for 12 hours, with free access to water during the fasting period. After fasting, mice in the control group were intraperitoneally injected with 0.1 mol / L, pH 4.5 citrate buffer solution (purchased from Beijing Solarbio Biotechnology Co., Ltd.), with an injection volume of 200 μL per mouse, serving as a solvent blank control. Mice in the model group and MDP-TY group were intraperitoneally injected with STZ solution to selectively damage pancreatic β-cells and reduce insulin secretion, thereby inducing a persistent hyperglycemic state and establishing a T2DM mouse model. The STZ solution was prepared by dissolving STZ in 0.1 mol / L, pH 4.5 citrate buffer and was prepared immediately before use; then, each mouse was injected with 200 μL of STZ solution daily, resulting in a dosage of 60 mg / kg per mouse. Mice in the control group, model group, and MDP-TY group were injected once daily for 4 consecutive days. During the continuous injection period, mice in each group continued to be fed according to their corresponding group and had free access to water, while maintaining daily gavage intervention.

[0036] Following continuous injections, mice in the control group, model group, and MDP-TY group underwent acclimatization for 3 days. During this period, mice in each group continued to be fed according to their respective diets, received gavage intervention, and had free access to water. After acclimatization, all mice were fasted for 8 hours, with free access to water during the fasting period. Fasting blood glucose levels were measured by blood collection from the tail tip. When the fasting blood glucose level in the model group and MDP-TY group was ≥11.1 mmol / L, the T2DM mouse model was considered successfully established. Mice that did not reach the above blood glucose standard were given a low-dose STZ supplemental injection (30 mg / kg) according to the experimental design. Mice that still did not reach the modeling standard were not included in subsequent experimental statistical analysis.

[0037] Further evaluation of the effects of gavage intervention with walnut-derived peptide MDP-TY on mouse body weight, fasting blood glucose, food intake, and water intake was conducted, and the results are as follows: Figures 1 to 4 As shown. Figure 1As shown, during weeks 2 to 7 of the experiment, the body weight of mice in each group showed an increasing trend with the progress of the experiment, and the differences between groups were relatively small. After STZ modeling in week 6, the weight gain trend of mice in the model group was significantly inhibited. By week 8, the body weights of mice in the blank group, model group, and MDP-TY group were 29.12±1.97 g, 25.12±1.47 g, and 25.22±1.78 g, respectively. The body weight of mice in the model group was significantly lower than that in the blank group (P<0.05), indicating that the weight gain of T2DM model mice was inhibited after STZ induction. As the experiment progressed to week 11, the body weight of mice in the blank group continued to increase, from 29.12±1.97 g in week 8 to 30.32±2.21 g, while the body weight of mice in the model group remained at a low level, with a body weight of only 23.10±1.78 g in week 11, which was significantly lower than that of the blank group (P<0.05). Compared with the model group, the body weight of mice significantly improved after MDP-TY intervention. From week 8 to week 11, the body weights of mice in the MDP-TY group were 25.22±1.78 g, 25.63±1.95 g, 26.69±2.17 g, and 27.03±2.19 g, respectively, all significantly higher than those in the model group (P<0.05), and the trend of body weight change gradually approached that of the control group. These results indicate that the walnut-derived peptide MDP-TY can alleviate the suppressed body weight gain induced by STZ in T2DM mice and improve abnormal body weight changes in diabetic states.

[0038] like Figure 2 and Figure 3 As shown, the effects of gavage intervention with walnut-derived peptide MDP-TY on food intake and water consumption in T2DM model mice were further evaluated. During weeks 2 to 7 of the experiment, the overall food intake and water consumption of mice in each group showed a gradual increasing trend. The model group had higher food intake and water consumption than the control group, indicating that mice gradually developed diabetes-related metabolic abnormalities after HFD combined with STZ induction. Figure 2 As shown, after week 8, the food intake of mice in the model group increased significantly. The food intake in weeks 8, 9, 10 and 11 reached 77.94±14.09 g, 86.05±4.71 g, 74.70±5.80 g and 78.70±9.68 g, respectively, which were significantly higher than those in the control group (42.53±4.77 g, 45.49±4.41 g, 41.14±2.13 g and 41.93±4.68 g) at the same time point (P<0.05), showing a polyphagia phenotype associated with the T2DM model. Compared with the model group, the food intake of mice in the MDP-TY group decreased to 66.24±9.90 g, 66.16±10.88 g, 64.74±2.89 g and 67.59±7.99 g, respectively, with statistically significant differences (P<0.05), indicating that MDP-TY can alleviate the abnormal increase in food intake in T2DM mice.

[0039] like Figure 3 As shown, the water intake of mice in the model group further increased after STZ induction. From week 8 to week 11, the water intake of mice in the model group reached 215.11±90.19 mL, 267.29±84.45 mL, 162.10±29.90 mL and 159.10±44.28 mL, respectively, which were significantly higher than the levels of the blank group at the same time (55.26±6.64 mL, 52.30±14.04 mL, 55.47±6.99 mL and 65.95±12.19 mL, P<0.05), showing a clear polydrinking phenotype. Compared with the model group, the water intake of mice in the MDP-TY group was significantly reduced, at 123.81±63.21 mL, 79.96±14.21 mL, 83.69±14.56 mL and 79.49±21.88 mL respectively (P<0.05), indicating that walnut-derived peptide MDP-TY can improve the abnormally high water intake behavior in T2DM mice.

[0040] like Figure 4 As shown, after STZ induction modeling in week 6, compared with the control group, the fasting blood glucose level of mice in the model group was significantly increased (P<0.05), from 9.06±1.01 mmol / L before modeling to 9.43±2.04 mmol / L in week 7, and remained at a high level from week 8 to week 11. The fasting blood glucose level reached 12.69±3.95 mmol / L, 18.76±1.61 mmol / L, 19.90±2.70 mmol / L and 19.79±2.62 mmol / L in weeks 8, 9, 10 and 11, respectively, which were significantly higher than the control group at the same time (P<0.05), indicating that HFD combined with intraperitoneal injection of STZ successfully induced hyperglycemia in T2DM mice. Compared with the model group, mice in the MDP-TY group showed significantly lower fasting blood glucose levels after intervention with the walnut-derived peptide MDP-TY (P < 0.05). From week 8 to week 11, fasting blood glucose levels decreased to 5.78 ± 0.78 mmol / L, 7.09 ± 0.69 mmol / L, 7.64 ± 1.26 mmol / L, and 8.05 ± 0.99 mmol / L, respectively. These results indicate that intervention with the walnut-derived peptide MDP-TY during the establishment of the T2DM model can effectively alleviate STZ-induced hyperglycemia and improve the disordered glucose metabolism in T2DM mice.

[0041] The effect of walnut-derived peptide MDP-TY on glucose tolerance in type 2 diabetes mellitus (T2DM) mice was further evaluated using an oral glucose tolerance test. The specific method was as follows: After gavage intervention, mice in each group were selected for the oral glucose tolerance test. The grouping and treatment of experimental animals were as described above. Before the experiment, mice in each group were fasted for 8 hours, with free access to water during the fasting period. A glucose solution was then prepared at a dose of 2 g / kg body weight, and the gavage volume was calculated based on the weight of each group of mice. The glucose load was administered to the mice via gavage injection. Blood samples were collected at 0 min after glucose gavage and at 30 min, 60 min, 90 min, and 120 min after gavage, using a tail tip sampling method. Blood glucose levels were measured at each time point using a glucometer. Mice were fasted and deprived of water during the testing period. The results are as follows: Figure 5 As shown, compared with the control group, the blood glucose levels of mice in the model group were significantly increased at all time points after glucose gavage (P < 0.05). At 0 min, 30 min, 60 min, 90 min, and 120 min, the blood glucose levels of mice in the model group reached 19.56 ± 1.56 mmol / L, 31.25 ± 3.00 mmol / L, 28.52 ± 2.20 mmol / L, 23.63 ± 1.81 mmol / L, and 22.77 ± 2.18 mmol / L, respectively, which were significantly higher than the levels in the control group at the same time points, indicating that the glucose clearance capacity of the T2DM model mice was decreased and there was obvious glucose intolerance. Compared with the model group, the blood glucose levels of mice in the MDP-TY group were significantly decreased at all detection time points after glucose loading (P < 0.05). At 0 min, 30 min, 60 min, 90 min, and 120 min, the blood glucose levels in the MDP-TY group mice were 15.07±1.56 mmol / L, 25.14±3.00 mmol / L, 22.69±2.20 mmol / L, 18.82±1.81 mmol / L, and 17.61±2.18 mmol / L, respectively. The rate of blood glucose elevation was significantly reduced, indicating improved glucose tolerance. These results demonstrate that continuous use of walnut-derived peptide MDP-TY during the development of type 2 diabetes mellitus (T2DM) can alleviate abnormal glucose metabolism under hyperglycemic conditions and improve glucose tolerance in T2DM model mice.

[0042] Example 2: Effects of walnut-derived peptide MDP-TY on lipid metabolism in type 2 diabetic mice

[0043] The experimental animals were grouped and treated as in Example 1. After successful modeling in week 6, blood was collected from the orbital venous plexus of mice in each group. After standing at room temperature for 2 h, the blood was centrifuged at 4 ℃ and 4000 r / min for 15 min, and the upper serum was collected and stored for later use.

[0044] 1. Determination of LDL-C

[0045] The LDL-C level in serum was detected using an LDL-C assay kit purchased from Nanjing Jiancheng Biotechnology Institute (catalog number A113-1-1). A 96-well plate was used for the assay. 2.5 μL of distilled water was added to the blank wells, 2.5 μL of LDL-C standard was added to the standard wells, and 2.5 μL of serum sample diluted 1:10 with physiological saline was added to the sample wells. Then, 180 μL of reagent one from the kit was added to each well. After gentle vortexing, the wells were incubated at 37°C for 5 min, and the absorbance of each well was measured at 550 nm using a microplate reader, recorded as A1. Subsequently, 60 μL of reagent two from the kit was added to each well, and after gentle vortexing, the wells were incubated at 37°C for 10 min, and the absorbance of each well was measured again at 550 nm using a microplate reader, recorded as A2. The change in absorbance for each well was calculated using the formula ΔA = A2 - A1. Calculate serum LDL-C levels according to the kit instructions using the following formula:

[0046]

[0047] Among them, C 标准 The concentration of LDL-C standard (7.06 mmol / L), ΔA 样本孔 The difference between the absorbance values ​​measured in two sample wells is ΔA, which is the difference between sample well A2 and sample well A1. 空白孔 The difference between the absorbance values ​​measured in two blank wells is ΔA, which is the difference between the absorbance values ​​measured in blank well A2 and blank well A1. 标准孔 The difference between the absorbance values ​​measured in two standard wells is calculated as: standard well A2 - standard well A1.

[0048] 2. Determination of TG

[0049] The serum supernatant was collected, and the serum TG level was detected using a TG assay kit purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A110-1-1. A 96-well plate was used for the assay. 2.5 μL of distilled water was added to the blank wells, 2.5 μL of TG standard was added to the standard wells, and 2.5 μL of serum sample diluted 1:10 with physiological saline was added to the sample wells. Then, 250 μL of the working solution provided with the kit was added to each well. After gentle shaking to mix, the plate was incubated at 37 ℃ for 10 min, and the absorbance of each well was measured at 500 nm using a microplate reader. The serum TG content was calculated according to the kit instructions using the following formula:

[0050]

[0051] Where C 标准 The concentration of TG standard is 2.64 mmol / L, A样本孔 A represents the absorbance value measured at 500 nm for the sample well, and A represents the absorbance value measured at 500 nm for the blank well. 标准孔 The absorbance value is the standard well measured at a wavelength of 500 nm.

[0052] 3. Measurement of total cholesterol (TC)

[0053] The upper layer of serum was collected, and the T-CHO level in the serum was detected using a TC assay kit purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A111-1-1. A 96-well plate was used for the assay. 2.5 μL of distilled water was added to the blank wells, 2.5 μL of T-CHO standard was added to the standard wells, and 2.5 μL of serum sample diluted 1:10 with physiological saline was added to the sample wells. Then, 250 μL of the working solution provided with the kit was added to each well. After gentle shaking to mix, the plate was incubated at 37 ℃ for 10 min, and the absorbance of each well was measured at 500 nm using a microplate reader. The serum TC content was calculated according to the kit instructions using the following formula:

[0054]

[0055] Among them, C 标准 The concentration of TC standard is 7.06 mmol / L, A 样本孔 A represents the absorbance value measured at 500 nm for the sample well, and A represents the absorbance value measured at 500 nm for the blank well. 标准孔 The absorbance value is the standard well measured at a wavelength of 500 nm.

[0056] 4. Determination of free fatty acids (NEFA)

[0057] The upper layer of serum was collected, and the NEGA level in the serum was detected using an NEFA assay kit purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A042-2-1. A 96-well plate was used for the assay. 4 μL of distilled water was added to the blank wells, 4 μL of NEFA standard was added to the standard wells, and 4 μL of serum sample diluted 1:10 with physiological saline was added to the sample wells. Then, 200 μL of reagent one from the kit was added to each well. After gentle shaking and mixing, the wells were incubated at 37 ℃ for 5 min, and the absorbance of each well was read using a microplate reader at a main wavelength of 546 nm and a secondary wavelength of 600 nm, recorded as A1. Next, 50 μL of reagent two from the kit was added to each well, and after gentle shaking and mixing, the wells were incubated at 37 ℃ for 5 min, and the absorbance of each well was read again using a microplate reader, recorded as A2. The change in absorbance of each well was calculated using the formula ΔA = A2 - A1. The serum NEFA content was calculated according to the kit instructions using the following formula:

[0058]

[0059] Among them, C 标准 The concentration of NEFA standard (1.00 mmol / L), ΔA 样本孔 The difference between the absorbance values ​​measured in two sample wells is ΔA, which is the difference between sample well A2 and sample well A1. 空白孔 The difference between the absorbance values ​​measured in two blank wells is ΔA, which is the difference between the absorbance values ​​measured in blank well A2 and blank well A1. 标准孔 The difference between the absorbance values ​​measured in two standard wells is calculated as: standard well A2 - standard well A1.

[0060] 5. Determination of HDL-C

[0061] The upper layer of serum was collected, and the HDL-C level in the serum was detected using an HDL-C assay kit purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A112-1-1. A 96-well plate was used for the assay. 2.5 μL of distilled water was added to the blank wells, 2.5 μL of LDL-C standard was added to the standard wells, and 2.5 μL of serum sample diluted 1:10 with physiological saline was added to the sample wells. Then, 180 μL of reagent one from the kit was added to each well. After gentle shaking and mixing, the wells were incubated at 37°C for 5 min, and the absorbance of each well was measured at 550 nm using a microplate reader, recorded as A1. Subsequently, 60 μL of reagent two from the kit was added to each group, and after gentle shaking and mixing, the wells were incubated at 37°C for 10 min, and the absorbance of each well was measured again at 550 nm using a microplate reader, recorded as A2. The change in absorbance of each well was calculated using the formula ΔA = A2 - A1. Calculate serum HDL-C levels according to the kit instructions using the following formula:

[0062]

[0063] Among them, C 标准 The concentration of LDL-C standard (1.64 mmol / L), ΔA 样本孔 The difference between the absorbance values ​​measured in two sample wells is ΔA, which is the difference between sample well A2 and sample well A1. 空白孔 The difference between the absorbance values ​​measured in two blank wells is ΔA, which is the difference between the absorbance values ​​measured in blank well A2 and blank well A1. 标准孔 The difference between the absorbance values ​​measured in two standard wells is calculated as: standard well A2 - standard well A1.

[0064] The test results are as follows Figures 6 to 10 As shown in the figure, compared with the control group, the serum levels of LDL-C, TC, TG, and NEFA in the model group mice were significantly increased (P < 0.05), while the HDL-C level was significantly decreased (P < 0.05), indicating that the T2DM model mice induced by HFD combined with intraperitoneal injection of STZ had significant lipid metabolism disorders. However, compared with the model group, the serum levels of LDL-C, TC, TG, and NEFA in the MDP-TY group mice were significantly decreased to 2.65 ± 1.14 mmol / L, 3.25 ± 0.99 mmol / L, 1.16 ± 0.23 mmol / L, and 0.93 ± 0.27 mmol / L, respectively (P < 0.05). At the same time, the serum high-density lipoprotein cholesterol (HDL-C) level significantly recovered to 3.19 ± 0.63 mmol / L (P < 0.05). These results indicate that the walnut-derived peptide MDP-TY can significantly improve lipid metabolism disorders in T2DM mice.

[0065] Example 3: Effects of walnut-derived peptide MDP-TY on liver and kidney in type 2 diabetic mice observed by HE staining

[0066] The grouping and processing of experimental animals were the same as in Example 1. After successful modeling, mice were euthanized, and liver and kidney tissues were quickly removed. The tissue surface was gently rinsed with physiological saline to remove blood and impurities, and the surface moisture was blotted dry with filter paper. The liver and kidney tissues were then placed in 40 g / L paraformaldehyde fixative (purchased from Wuhan Sewell Biotechnology Co., Ltd.) and fixed at room temperature for 24 h. Fixation refers to using the fixative to cause cross-linking or coagulation of tissue proteins, thereby maintaining the original morphological structure of the tissue and reducing autolysis, putrefaction, and morphological changes during subsequent slide preparation. After fixation, the liver and kidney tissues were placed in a dehydration box and subjected to the following steps sequentially: "gradient ethanol dehydration, clearing, paraffin embedding, sectioning, baking, dewaxing and rehydration, hematoxylin-eosin staining, dehydration, clearing, and mounting." The specific steps are as follows:

[0067] (1) Dehydration treatment: The fixed liver and kidney tissues were placed in 75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, and 95% ethanol for 1 h.

[0068] (2) Dehydration treatment with anhydrous ethanol: In order to further remove residual water from the tissue, anhydrous ethanol is divided into anhydrous ethanol I and anhydrous ethanol II; the liver and kidney tissues after dehydration treatment in step (1) are immersed in anhydrous ethanol I for 30 min, and then immersed in anhydrous ethanol II for 30 min.

[0069] (3) Transparency treatment: The liver and kidney tissues treated in step (2) were immersed in an ethanol-xylene solution for 10 min (anhydrous ethanol and xylene were mixed in a volume ratio of 1:1) to promote the transition of the tissues from the ethanol system to the xylene system and reduce the morphological changes caused by the rapid solvent conversion; then xylene was divided into xylene I and xylene II, and the liver and kidney tissues were then immersed in xylene I for 10 min and xylene II for 10 min each, respectively, to make the tissues fully transparent;

[0070] (4) Paraffin infiltration, embedding and sectioning: Paraffin is divided into paraffin I, paraffin II and paraffin III. The liver and kidney tissues obtained in step (3) are placed in paraffin I, paraffin II and paraffin III respectively for 1 h each to fully infiltrate the tissues; after the paraffin infiltration is completed, the tissues are embedded in paraffin using an embedding machine to obtain paraffin blocks of liver and kidney tissues; then the paraffin blocks are sectioned using a paraffin microtome with a section thickness of 4 μm to obtain tissue sections.

[0071] (5) Baking, dewaxing and rehydration treatment: The tissue sections obtained in step (4) were baked in a 60 ℃ oven for 1 h. After baking, the tissue sections were dewaxed and hydrated. The specific operation was as follows: The tissue sections were soaked in xylene I and xylene II for 10 min each to remove the paraffin in the tissue sections. Then, they were placed in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol in sequence for 5 min each to gradually restore the tissue sections to the hydrated state. Finally, they were rinsed with distilled water for 2 min to restore the tissue sections to the water content suitable for staining.

[0072] (6) Hematoxylin-eosin staining treatment: After rehydration, the tissue sections were stained in hematoxylin staining solution for 5 min to stain the cell nuclei; after staining, the tissue sections were stained in 1% hydrochloric acid ethanol differentiation solution to remove the excess hematoxylin dye bound in the cell nuclei and make the cell nuclei staining clearer; then the tissue sections were stained in 0.1% lithium carbonate blue solution to change the color of the cell nuclei from purple-red to blue and enhance the nuclear staining effect; after the blue treatment, the tissue sections were rinsed with distilled water and then stained in eosin staining solution for 5 min to stain the cytoplasm and extracellular matrix.

[0073] (7) Dehydration, clearing and mounting: After staining, the tissue sections were dehydrated, cleared and mounted. The specific operation was as follows: the tissue sections were placed in 75% ethanol for 2 min, 85% ethanol for 2 min, 95% ethanol for 3 min and anhydrous ethanol I and anhydrous ethanol II for 5 min each to gradually remove the water from the tissue sections; then, the tissue sections were placed in xylene I and xylene II for 5 min each to make the tissue sections fully clear; after clearing, a neutral resin mounting medium was dropped on the surface of the tissue sections and covered with a coverslip for mounting. After the mounting medium was fully cured, the pathological morphological changes of the liver and kidney tissues of mice in each group were observed and recorded using an optical microscope.

[0074] The effects of walnut-derived peptide MDP-TY on the pathological morphology of liver tissue in T2DM mice are as follows: Figure 11 As shown, the liver tissue structure of mice in the blank control group was basically normal, with relatively intact hepatocyte morphology, regular arrangement of hepatic cords, and clear sinusoidal structure. No obvious fatty degeneration, cellular edema, or inflammatory cell infiltration was observed. In the model group, the liver tissue structure of mice was impaired, with disordered arrangement of hepatic cords, varying degrees of hepatocyte swelling, loose cytoplasm with obvious vacuolar changes and fatty degeneration, and localized hepatocyte degeneration, indicating significant liver pathological damage in T2DM mice. Compared with the model group, the degree of liver tissue damage in the MDP-TY group was significantly reduced, with relatively intact hepatocyte morphology, regular arrangement of hepatic plates, no obvious dilation or compression of hepatic sinusoids, no obvious abnormalities in the portal canal, and no obvious inflammatory cell infiltration. These results indicate that the walnut-derived peptide MDP-TY can improve liver tissue structural damage in T2DM mice and alleviate liver pathological changes related to hyperglycemia and lipid metabolism disorders.

[0075] The effects of walnut-derived peptide MDP-TY on the pathological morphology of kidney tissue in T2DM mice are as follows: Figure 12As shown, the kidney tissue of the control group mice was structurally intact, with a clear Bowman's capsule structure, normal morphology of renal tubular epithelial cells, and uniform cytoplasmic staining, without obvious inflammatory cell infiltration. The kidney tissue of the model group showed obvious pathological changes, with disordered arrangement of some renal tubules, varying degrees of swelling of renal tubular cells, loose cytoplasm with vacuolar degeneration, and local tubular structural abnormalities, indicating significant renal pathological damage in T2DM mice. Compared with the model mice, the degree of renal tissue pathological damage in the MDP-TY group mice was significantly reduced, the glomerular structure was more intact, the morphology of renal tubular epithelial cells was more complete, the degree of vacuolar degeneration and tissue structural disorder was significantly reduced, and no obvious inflammatory cell infiltration was observed. These results indicate that the walnut-derived peptide MDP-TY can improve the structural damage of kidney tissue in T2DM mice and alleviate renal pathological changes related to glucose and lipid metabolism disorders.

[0076] Example 4: Effects of walnut-derived peptide MDP-TY on Morris water maze behavioral test in T2DM mice

[0077] The experimental animals were grouped and treated as in Example 1. After successful modeling, the spatial learning and memory abilities of the mice in each group were evaluated using the Morris water maze navigation test. All water mazes were circular pools with a diameter of 120 cm. Warm water was added to the pools during the experiment, and the water temperature was controlled at 20±2 ℃. The pools were divided into four quadrants: NE, NW, SE, and SW, and fixed visual references were placed around the pools. A platform with a diameter of 65 mm was placed in the SW quadrant, with its surface approximately 1 cm below the water surface, preventing the mice from directly observing its location. During the experiment, the mice were gently placed into the water from the NE quadrant, facing the pool wall. After entering the water, their swimming trajectory was recorded using a water maze behavioral video tracking and analysis system, and the time required for them to find and climb the hidden platform within 120 seconds was recorded as the escape latency period. After reaching the platform, the mice were allowed to remain on it for 10 seconds to help them form a memory of the platform's spatial location before being returned to their cages. For mice that failed to find the platform within 120 seconds, the experimenters gently guided them to the platform and allowed them to remain there for 10 seconds; their escape latency was recorded as 120 seconds. After the experiment, a water maze behavioral video tracking and analysis system was used to analyze behavioral indicators such as the mice's swimming trajectory, escape latency, and effective distance traveled. The movement trajectories of the mice in the positioning and navigation experiment for each group are shown below. Figures 13 to 17 As shown.

[0078] like Figures 13 to 15As shown, the control group mice were able to develop a relatively clear spatial exploration strategy in the navigation experiment, with relatively concentrated movement trajectories and a tendency to search the platform area. Conversely, the model group mice exhibited more dispersed movement trajectories, with swimming paths mostly distributed along the pool edge and in non-target areas, demonstrating obvious marginalized and ineffective search characteristics, indicating impaired spatial localization and learning abilities in T2DM mice. Compared to the model, the MDP-TY group mice showed a significantly more concentrated movement trajectory towards the platform area, with fewer ineffective detours, indicating that the walnut-derived peptide MDP-TY can improve the learning and memory abilities of T2DM mice.

[0079] like Figure 16 and Figure 17 As shown, the effect of intervention with walnut-derived peptide MDP-TY on spatial learning and memory abilities in T2DM model mice was further evaluated. Figure 16 As shown, compared with the control group, the escape latency of mice in the model group was significantly increased (P < 0.05), from 54.75 ± 40.00 s in the control group to 117.04 ± 5.38 s, indicating that the time required for T2DM model mice to find hidden platforms during the navigation test increased, and their spatial learning ability was significantly reduced. Compared with the model group, the escape latency of mice in the MDP-TY group was significantly shortened to 36.93 ± 20.07 s (P < 0.05), indicating that the walnut-derived peptide MDP-TY can improve the spatial localization learning ability of T2DM model mice.

[0080] like Figure 17 As shown, compared with the control group, the effective movement distance related to the target area in the model group mice was significantly reduced, decreasing from 6.99±4.29 cm in the control group to 1.21±1.57 cm (P<0.05), indicating that T2DM status leads to a weakened spatial memory ability of mice in the target platform area. Compared with the model group, the effective movement distance in the MDP-TY group mice was significantly increased to 7.77±3.73 cm (P<0.05), indicating that the walnut-derived peptide MDP-TY can improve the spatial memory retention ability of T2DM model mice. These results indicate that continuous administration of MDP-TY during the development of T2DM can improve hyperglycemia-related spatial learning and memory impairment, and has a potential protective effect against diabetes-related cognitive impairment.

[0081] Example 5: Effects of walnut-derived peptide MDP-TY on dendritic spines of hippocampal neurons in type 2 diabetic mice

[0082] The grouping and treatment methods for experimental animals were the same as in Example 1. After successful model establishment, mice were sacrificed, and brain tissue was quickly extracted, with the hippocampus isolated. The hippocampal tissue was treated in Golgi fixative for at least 48 hours to reduce autolysis and morphological changes during subsequent slide preparation, facilitating observation using Golgi staining. After fixation, the brain tissue was cut into approximately 3 mm thick pieces, rinsed with physiological saline, and placed in EP tubes. Golgi staining solution was added to completely submerge the brain tissue, and the tissue was treated in a cool, ventilated, and dark environment for 14 days. The staining solution was changed after 48 hours of soaking, and then every 3 days thereafter, for a total of 14 days. After Golgi staining, the tissue was sequentially cleaned, softened, dehydrated, sectioned, developed, fixed, and mounted, following these steps:

[0083] (1) Tissue cleaning treatment: The brain tissue stained with Golgi dye was removed and rinsed with distilled water three times to remove residual dye from the tissue surface;

[0084] (2) Tissue softening treatment: The brain tissue obtained in step (1) was placed in a 14 mol / L glacial acetic acid solution to completely immerse the brain tissue and left at room temperature overnight to soften the tissue. After softening, the tissue was rinsed three times with distilled water to remove residual glacial acetic acid from the tissue surface.

[0085] (3) Sucrose dehydration protection treatment: The brain tissue treated in step (2) was placed in a 30% w / v sucrose solution for dehydration protection until the tissue was fully infiltrated; the sucrose dehydration protection treatment was used to reduce tissue morphological damage during the sectioning process;

[0086] (4) Sectioning and mounting: The brain tissue processed in step (3) was sectioned using a microtome, and the section thickness was 100 μm; the obtained tissue sections were then mounted on gelatin slides and dried overnight in the dark.

[0087] (5) Ammonia treatment: The tissue sections obtained in step (4) were soaked in 28% ammonia solution for 15 min to promote the staining and development of Golgi staining solution, and then rinsed with distilled water for 1 min to remove residual ammonia.

[0088] (6) Fixing: The tissue sections treated with ammonia in step (5) were immersed in 5% w / v sodium thiosulfate solution for 15 min to fix and stabilize the silver chromium deposits formed by Golgi staining and enhance the color development of neuronal cell bodies and dendritic structures. After fixing, the tissue sections were rinsed in distilled water for 3 min to remove residual fixing solution;

[0089] (7) Mounting: After the tissue sections processed in step (6) are dried, glycerol gelatin mounting medium is added and covered with coverslips for mounting; after the mounting medium has solidified, it can be used for subsequent microscopic observation and image acquisition;

[0090] (8) Image acquisition and dendritic complexity analysis: Hippocampal neuron images were observed and acquired using CaseViewer 2.2 scanning software, and Sholl analysis was performed using Image-Pro Plus 6.0 software. In the specific analysis, neurons with intact cell bodies, clear dendritic structures, and minimal background interference were selected as the analysis objects. Concentric circles were established with the intact neuron cell body as the center and with a spacing of 10 μm. The number of intersections between the dendrites and each concentric circle was counted, and parameters such as the dendritic extension distance and total dendritic length were recorded to evaluate the effect of MDP-TY on the dendritic branching complexity of hippocampal neurons in T2DM mice.

[0091] The results of Golgi staining and Sholl analysis are as follows: Figure 18 and Figure 19 As shown. Figure 18 In the study, significant differences were observed in the morphology of hippocampal neurons in the control group and the model group. The hippocampal neurons in the control group exhibited intact cell bodies, clear boundaries, and extensive dendritic extension. Compared to the control group, the model group showed significantly damaged dendritic structures in hippocampal neurons, with reduced dendritic branches and a narrower extension range, indicating that type 2 diabetes mellitus (T2DM) leads to a decrease in the integrity of hippocampal neuronal dendritic structures. Compared to the model group, the MDP-TY group showed significantly improved hippocampal neuronal dendritic structures, with expanded dendritic extension range and an overall neuronal morphology closer to that of the control group, suggesting that the walnut-derived peptide MDP-TY can alleviate T2DM-induced damage to hippocampal neuronal dendritic structures.

[0092] like Figure 19As shown, Sholl analysis was further used to evaluate the complexity of dendritic branching in hippocampal neurons of each group of mice. Compared with the control group, the number of dendritic intersections in the model group mice was significantly reduced within different radii, and the Sholl curves showed an overall decrease. Within the analysis range, the number of dendritic intersections in the control group mice reached a maximum of approximately 14.26±2.49, while the maximum number of dendritic intersections in the model group mice was only 1.67±0.47, indicating that T2DM status leads to a reduction in the number of dendritic branches and a significant decrease in complexity of hippocampal neurons. Compared with the model group, after MDP-TY intervention, the number of dendritic intersections in different radii of mice increased significantly, and the Sholl curves shifted upwards overall. The maximum number of dendritic intersections in the MDP-TY group recovered to 7.33±4.19, which was significantly higher than that in the model group (P<0.05), indicating that the walnut-derived peptide MDP-TY can improve the abnormal dendritic branching structure of hippocampal neurons caused by T2DM. The above results indicate that the walnut-derived peptide MDP-TY can improve the dendritic branching complexity of hippocampal neurons in T2DM model mice and improve the damage to neuronal dendritic structure under hyperglycemic conditions, suggesting that it has a potential protective effect against diabetes-related cognitive impairment.

[0093] Examples 2 to 5 show that the walnut-derived peptide MDP-TY of the present invention can improve glucose and lipid metabolism disorders, liver and kidney tissue pathological damage, learning and memory impairment, and hippocampal neuronal synaptic structure damage in T2DM mice. It can be used to prepare drugs for the prevention, relief, or treatment of diabetic encephalopathy, and can also be used to prepare food with the function of assisting in improving nerve damage caused by hyperglycemia.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a walnut-derived peptide MDP-TY in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: The amino acid sequence of the walnut-derived peptide MDP-TY is TWLPYPR.

2. The application of the walnut-derived peptide MDP-TY as described in claim 1 in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: Diabetic encephalopathy is a type of diabetic encephalopathy caused by type 2 diabetes.

3. The application of the walnut-derived peptide MDP-TY as described in claim 1 or 2 in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: The dosage form of the drug is one of the following: tablets, capsules, granules, powders, sprays, films, suppositories, nasal drops, or pills.

4. The application of the walnut-derived peptide MDP-TY as described in claim 1 or 2 in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: The drug carrier is selected from one or more of lactose, mannitol, microcrystalline cellulose, starch, hydroxypropyl methylcellulose, povidone, croscarmellose sodium, magnesium stearate, talc, polyethylene glycol, chitosan, sodium alginate, cyclodextrin, poloxamer, and Tween 80.

5. The application of the walnut-derived peptide MDP-TY as described in claim 1 in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: Used to prepare food products that can help improve nerve damage caused by hyperglycemia.

6. The application of the walnut-derived peptide MDP-TY as described in claim 5 in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: The food is one of the following: health food, functional food, general food, dietary supplement, special dietary food, or special medical food.

7. The application of the walnut-derived peptide MDP-TY as described in claim 5 in the preparation of drugs or foods for the prevention or treatment of diabetic encephalopathy, characterized in that: The food additives are selected from one or more of the following: maltodextrin, corn starch, white sugar, xylitol, erythritol, xanthan gum, gum arabic, sodium alginate, agar, pectin, food-grade hydroxypropyl methylcellulose, magnesium stearate, micronized silica gel, citric acid, honey, and purified water.

Images