Application of walnut peptide in preparation of medicine for treating or preventing cognitive impairment of diabetes

Abstract

The invention discloses application of walnut peptide in preparation of a medicine for treating or preventing cognitive impairment of diabetes mellitus. The preparation process of the walnut peptide is as follows: after walnut protein is subjected to enzymolysis by alkaline protease, the molecular weight cut-off is 1t; and separating by using a 3kDa ultrafiltration tube to obtain the walnut peptide. The walnut peptide can effectively reduce the blood sugar level of a diabetic model mouse, regulate the disorder effect of blood fat, relieve brain tissue oxidative damage and hippocampal neuroinflammation damage of the diabetic model mouse, and improve learning and memory damage. The compound can be used for preparing medicines for treating or preventing cognitive impairment of diabetes mellitus.

Description

Technical Field

[0001] The invention relates to the field of medical applications, and in particular to application of a walnut peptide in preparing a drug for treating or preventing diabetic cognitive impairment. Background Art

[0002] The relationship between diabetes and cognitive impairment is complex. With the aging of the population, the incidence of diabetes is increasing year by year, and the cognitive impairment caused by it is also increasing. Compared with the general population, the risk of cognitive impairment in diabetic patients is 1.5-2.5 times higher. The brain of diabetic patients is in a state of hyperglycemia for a long time, which leads to necrosis of brain nerve cells, oxidative stress and neuroinflammatory response in the brain, thus affecting cognitive function. Studies have shown that cognitive dysfunction in patients with type 2 diabetes is related to atrophy of the hippocampal structure caused by chronic hyperglycemia, oxidative stress, inflammation and cholinergic dysfunction. Its abnormality can increase the level of reactive oxygen species (ROS) in the body and release a large number of inflammatory factors, such as interleukin-1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor (TNF), etc., which increase inflammation and insulin resistance, thereby causing neuroinflammation and cognitive impairment.

[0003] Diabetic cognitive impairment is a type of neurodegenerative disease characterized by decreased language and judgment ability, accompanied by symptoms such as apathy, dull expression, emotional disorders and unstable motor coordination, especially learning and memory impairment. Currently, biguanides, sulfonylureas, thiazolidinediones, dipeptidyl peptidase 4 inhibitors (DPP-4) and glucagon-like peptide-1 (GLP-1) agonists commonly used in clinical practice are all used to improve diabetes and cognitive impairment, but they are not effective in improving learning and memory impairment. Compared with chemical drugs, the main advantages of natural products are effectiveness, few side effects and low cost. Food-derived bioactive peptides have rich biological activity and are a functional factor with great development potential. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides an application of walnut peptide in the preparation of a drug for treating diabetic cognitive impairment. The present invention hydrolyzes walnut cake and uses alkaline protease to prepare walnut peptide (MW < 3kDa). Through animal behavioral experiments, biochemical indicators, histopathological morphological observations and transcriptome sequencing, it is found that walnut peptide (MW < 3kDa) can improve cognitive impairment in type II diabetes.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows: an application of walnut peptide in the preparation of a drug for treating or preventing diabetic cognitive impairment.

[0006] Preferably, the walnut peptide has a molecular weight of less than 3 KDa.

[0007] As a further description of the above scheme: the production process of the walnut peptide is as follows: after the walnut protein is hydrolyzed by alkaline protease, the walnut peptide is separated by an ultrafiltration tube with a molecular weight cutoff of <3kDa. Alkaline protease is an endoenzyme with a wide range of specificity and high efficiency of hydrolysis. Therefore, the present invention uses alkaline protease to hydrolyze walnut protein, and the hydrolysis degree is the highest.

[0008] As a further description of the above scheme: the walnut peptide can effectively reduce the blood sugar level of diabetic model mice, regulate the disorder of blood lipids, reduce the oxidative damage of brain tissue and neuroinflammatory damage in the hippocampus of diabetic model mice, and has the effect of improving learning and memory impairment.

[0009] The present invention uses alkaline protease enzymatic extraction to obtain walnut peptides (MW < 3kDa). Based on in vivo experiments, growth indicators, animal behavior tests, biochemical indicators, histopathological morphological observations and transcriptome analysis were used to explore the effect of walnut peptides (MW < 3kDa) on improving cognitive impairment in diabetic model mice. The main research contents and results are as follows:

[0010] The effect of walnut peptide on the behavioral ability of diabetic cognitive impairment model mice. Through behavioral tests such as water maze, rotarod, open field, and new object recognition, the results showed that the escape latency of the walnut peptide group to find the exit was significantly shortened, the rotarod residence time and new object recognition index were significantly improved, and the number of mice entering the central area increased. That is, walnut peptide can improve the behavioral ability of diabetic cognitive impairment model mice.

[0011] Effect of walnut peptide on glucose metabolism indexes in diabetic cognitive impairment model mice. The results of blood indexes and liver tissue pathology sections showed that walnut peptide can effectively inhibit the weight loss of diabetic mice induced by high fat and high sugar, reduce the blood glucose level of diabetic mice induced by high fat and high sugar, significantly reduce the content of TG, TC, and LDL-C, significantly increase the level of HDL-C, and reduce liver damage in diabetic model mice, indicating that walnut peptide can improve the glucose metabolism level of diabetic cognitive impairment model mice.

[0012] Effect of walnut peptide on oxidative stress in brain tissue of diabetic cognitive impairment model mice. Based on the observation of brain tissue oxidation level and hippocampal pathological morphology, the results showed that walnut peptide can significantly increase the activity of antioxidant enzymes (CAT, SOD and GSH-px) and reduce MDA content by improving the oxidative stress state of brain tissue. At the same time, through hematoxylin-eosin staining (HE) and Nissl staining, it was observed that walnut peptide can effectively reduce the damage of the hippocampus of diabetic model mice, reduce the number of nuclear condensations near the dentate gyrus (DG), hippocampal CA1 (cornu ammonis CA1) and hippocampal CA3 (cornu ammonis CA3) regions, reduce neuronal apoptosis, and increase the number of neurons, indicating that walnut peptide can effectively reduce the oxidative damage caused by diabetes and improve the antioxidant capacity of tissues, indicating that walnut peptide can effectively reduce oxidative damage in brain tissue of diabetic model mice.

[0013] Based on transcriptomic analysis, walnut peptides regulate the hippocampus-related signaling pathways in the brains of diabetic cognitive impairment model mice to improve cognitive impairment. The results showed that the Gene Ontology (GO) annotation analysis showed that it was mainly involved in inflammation and immune-related biological processes after walnut peptide dietary intervention; the Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis results showed that it mainly affected the tumor necrosis factor (TNF) and interleukin 17 (IL17) inflammation-related signaling pathways; after qPCR verification, it was consistent with the results of transcriptomic regulation of differentially regulated genes, proving that the transcriptomic results were accurate, indicating that walnut peptides have a potential mechanism for improving neuroinflammation in diabetic model mice by regulating TNF and IL17 signaling pathways.

[0014] Compared with the prior art, the present invention has the following beneficial effects: walnut peptide can effectively reduce the blood sugar level of diabetic model mice, regulate the disorder of blood lipids, reduce the oxidative damage of brain tissue and the damage of neuroinflammation in the hippocampus of diabetic model mice, and improve the effect of learning and memory damage. This study preliminarily explored the mechanism of action of walnut meal protein peptide in improving cognitive impairment in diabetic model mice, and the walnut peptide can be used to develop foods or drugs that assist in improving memory function. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 For glucose tolerance test in mice;

[0016] Figure 2 Fasting blood glucose of mice in each group;

[0017] Figure 3 The weight of mice in each group;

[0018] Figure 4 It is the open field motion trajectory diagram;

[0019] Figure 5 is the number of times entering the central area (A), and the time spent in the central area (B); Note: Different lowercase letters in the figure indicate significant differences at the 0.05 level;

[0020] Figure 6 This is the motion trajectory diagram during the exploration period of the new object recognition experiment;

[0021] Figure 7 This is the motion trajectory diagram during the test period of the novel object recognition experiment;

[0022] Figure 8 The effect of walnut peptide on the content of TG, TC, LDL-C and HDL-C in diabetic model mice;

[0023] Fig. 9 Pathological changes of liver tissues in mice of each group;

[0024] Fig.10 The effect of walnut peptide on the content of SOD, MDA, CAT and GSH-px in diabetic model mice;

[0025] Fig.11 HE staining shows the effect of walnut peptide on hippocampal neurons in diabetic mice;

[0026] Fig.12 Nissl staining showed the effect of walnut peptide on hippocampal neurons in diabetic mice. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0028] Table 1 Abbreviations and symbols

[0029]

[0030]

[0031] Example 1 Preparation of walnut peptide

[0032] Extraction and preparation of walnut peptide

[0033] First, the walnut meal was dried at 55°C to constant weight and then crushed and sieved to 150 mesh. The walnut meal was defatted with n-hexane, and walnut protein was extracted and separated by alkali dissolution and acid precipitation. 500g defatted walnut powder was weighed and dissolved in ultrapure water (solid-liquid ratio 1:15w / v), and the pH of the mixed solution was adjusted to 9.0 with 1mol / L NaOH solution, magnetically stirred at 45°C for 1.5h, centrifuged at 4°C (4000rpm, 30min), and the supernatant was adjusted to pH 4.5-5.0 with 1mol / L HCl solution. After standing at room temperature for 1h, the precipitate was separated by centrifugation at 4°C (4000rpm, 30min), dissolved with distilled water with a pH value of 4.5, washed twice with distilled water, and centrifuged at 4°C (4000rpm, 15min). The precipitate was redispersed in distilled water, adjusted to pH 7 with 1mol / L NaOH solution, dialyzed with distilled water at 4°C, and freeze-dried for use. The freeze-dried walnut protein was dissolved in ultrapure water, mixed thoroughly to obtain a solution with a substrate concentration of 2%, added with 2% alkaline protease, and the pH of the mixed solution was adjusted to 9.0 with 1 mol / L NaOH, and hydrolyzed at 55°C for 3h. After the hydrolysis was completed, the mixture was heated in boiling water for 10min to inactivate the enzyme. After the enzymatic solution was cooled to room temperature, it was centrifuged (4°C, 4000rpm, 30min), and the supernatant was collected and freeze-dried for later use.

[0034] Preparation of walnut peptide (MW < 3KDa)

[0035] The walnut peptides were separated using an ultrafiltration tube with a molecular weight cutoff of <3 kDa and the lower fraction of the ultrafiltration tube was collected and freeze-dried to obtain walnut peptides with MW <3 KDa.

[0036] Example 2 Establishment of Type II Diabetes Model Mouse Model

[0037] STZ was dissolved in sterile citrate buffer (pH 4.5, 0.1 M) at 4°C, stored at low temperature and away from light, and used immediately after preparation within 10 minutes of preparation.

[0038] 120 SPF male ICR mice, 5 weeks old, weighing 24-28g, were raised in the animal laboratory of Yunnan Key Laboratory of Precision Nutrition and Personalized Food Manufacturing, and approved by the Animal Ethics Committee of Yunnan Agricultural University. 12 mice were selected as the blank control group and fed with ordinary feed, and the rest of the mice were fed with 60% high-fat and high-sugar feed for 4 consecutive weeks. In the 5th week, the mice fed with high-fat and high-sugar feed were intraperitoneally injected with 60mg / kg STZ solution prepared with citric acid buffer for 2-3 times, fasting but not water for 12h, blood was collected from the tail tip, and the fasting blood glucose of the mice was tested with a blood glucose meter. Mice with blood glucose values ​​≥11.1mmol / L were selected as successful models, and mice with low blood glucose were targeted for STZ injection once in the 6th week, stabilized for one week, and glucose tolerance test was performed.

[0039] The mice were fasted for 12 hours, and the fasting blood glucose of the mice was measured to obtain the blood glucose value at 0 min. Then, 0.01 mL / 10 g of 20% (w / v) glucose solution was gavaged into the mice, and the blood glucose values ​​of the mice were measured at 30 min, 60 min, 90 min, and 120 min after gavage. The oral glucose tolerance test (OGTT) curve was drawn with the blood glucose value as the ordinate and the time as the abscissa.

[0040] like Figure 1 As shown, compared with the blank group, the serum glucose content in the diabetes modeling group was significantly increased from 0 to 120 min (P<0.001), which is consistent with the research results of existing literature, indicating that the diabetes model was successfully constructed.

[0041] After the mice were intraperitoneally injected with STZ and stabilized, it was observed that the mice in the modeling group showed "three more and one less phenomenon": polyuria, polyphagia, polydipsia, and weight loss. Mice with FBG greater than 11.1mmol / L were recorded as successful model construction. Among the 120 mice, 96 mice were successfully built, with a success rate of 80.0%. Subsequent experiments showed that diabetic mice had cognitive dysfunction, so the mouse model constructed in this example is also a diabetic cognitive impairment model.

[0042] Example 3 Application of walnut peptide in the preparation of drugs for treating or preventing diabetic cognitive impairment

[0043] After the diabetic model was established, the subjects were randomly divided into 8 groups according to blood glucose index and body weight (blank group: CON group; model group: T2DM group; donepezil group: DON group; walnut protein group: WMP group; walnut enzymatic solution group: WME group; walnut peptide low-dose group: WP-L group; walnut peptide medium-dose group: WP-M group; walnut peptide high-dose group: WP-H group). The subjects were intragastrically administered with donepezil (3.0 mg / kg), walnut protein (450 mg / kg), walnut enzymatic solution (450 mg / kg), and walnut peptide (low dose: 50 mg / kg, medium dose: 150 mg / kg, and high dose: 450 mg / kg) for 5 weeks. The blank group and the model group were intragastrically administered with sterile water, with a volume of 0.01 mL / 10 g, for 5 consecutive weeks.

[0044] Effects of walnut peptide on FBG in diabetic model mice

[0045] Figure 2The effect of walnut peptide on fasting blood glucose in diabetic model mice during oral gavage was studied. After the injection of STZ was stable, the mice were randomly divided according to body weight and blood glucose data. The gavage intervention was started in the 5th week. The fasting blood glucose of the intervention group decreased significantly in the 6th week. With the continuous intervention of walnut peptide and donepezil, the FBG of diabetic model mice was dynamically measured. It was found that compared with the CON group, the FBG level of the T2DM group was significantly increased (P<0.05); compared with the T2DM group, the FBG level of the oral gavage intervention group showed a downward trend, showing a significant difference (P<0.05). It shows that the WMP group, WME group, WP-L group, WP-M group, and WP-H group can stabilize blood glucose during the feeding of high-sugar and high-fat feed. At the 10th week, the intervention groups with the same dose (450 mg / kg) were compared. The results showed that the WP-H group was better than the WMP group and the WME group in stabilizing blood glucose. Therefore, considering the dose, the dose of walnut protein and walnut enzymatic hydrolysis is equivalent to the high-dose group of walnut peptide, so the group with oral gavage of walnut peptide has a better effect. It is further confirmed that walnut peptides are low molecular weight peptides with good activity and easy digestion and absorption. Compared with the WP-L group and the WP-M group, the WP-H group has a more obvious trend of lowering blood sugar, which indicates that the stability of blood sugar is dependent on the dose of walnut peptides, and the stability of blood sugar is dose-dependent.

[0046] Effect of walnut peptide on body weight of diabetic model mice

[0047] Dynamic measurement of body weight of diabetic mice revealed that Figure 3 As shown in the data, compared with the CON group, the body weight of mice in the T2DM group was reduced. At the 10th week, there was a significant difference in body weight between the T2DM group (35.84±0.85g) and the CON group (39.06±1.25g) (P<0.05), and the "three more and one less" symptoms of T2DM mice became more and more obvious. Compared with the T2DM group, during the oral gavage intervention period, the body weight of the DON group, WMP group, WME group, WP-L group, WP-M group, and WP-H group was reduced, but there was no significant difference (P>0.05).

[0048] Effects of walnut peptide on the automatic recording of water maze time in diabetic model mice

[0049] The water maze test was automatically recorded on the first day of the third week after the gavage intervention. The experiment lasted for 5 days, of which the first 4 days were for positioning and navigation experiments, and the fifth day was for spatial test experiments. Positioning and navigation experiment method: training was performed 3 times a day, each time the mouse was placed in the water from the entrance of the maze, and then the timing was started, and a bright green stick was used to guide the mouse to find the exit of the maze, and the time required for the mouse to enter the water and find the exit of the maze was recorded, that is, the escape latency. If the exit is not successfully found within 120 seconds, the mouse is led ashore and allowed to rest for 60 seconds. At this time, the escape latency is 120 seconds. Spatial test method: on the 5th day, the mouse was placed in the water from the entrance of the maze without using a stick to guide it, and the mouse was allowed to find the exit in the maze, and the time required for the mouse to reach the exit was recorded.

[0050] As shown in Table 2, on the first day of training, the escape latency of the T2DM group (117.88±3.18s) was significantly longer than that of the CON group (50.38±4.81s) (P<0.05), indicating that the learning and memory ability of the T2DM group declined seriously. The escape latency of the DON group (62.88±5.74s), WMP group (104.75±10.44s), WME group (89.25±15.39s), WP-L group (94.50±8.17s), WP-M group (83.63±10.88s), and WP-H group (78.00±10.47s) was significantly lower than that of the T2DM group (117.88±3.18s) (P<0.05). On the fifth day of training, the DON group (33.63±7.17s) took a shorter time than the other groups, followed by the WP-H group (36.00±1.77s). This study found that high-dose walnut peptides (450mg / kg) have a better effect on improving cognitive impairment in diabetic model mice.

[0051] Table 2 Effect of escape latency in water maze test

[0052]

[0053] Effects of walnut peptide on rotarod time in diabetic mice

[0054] The rotarod test was performed on the 6th day of each week after the gavage intervention, for a total of 5 weeks. The experiment was divided into a training period and a test period. On the first day, the training time was 1 min, the speed was 10 rpm, every 30 min, and then 5 min, the speed was from 410 rpm to 4010 rpm. During the training period, once the mouse fell from the rotarod instrument, it was immediately restored to its original position to ensure that each mouse ran the full time. On the second day after the training period, the mouse was placed in the rotarod device, the rotarod speed was set to 4-40 rpm, and the time and speed of the mouse falling from the rotarod device within 5 min were recorded. Each mouse was tested 3 times, each time with an interval of no less than 1 h, and the average of the final results was taken.

[0055] After oral gavage intervention, as shown in Table 3, at the beginning of the first week of rotarod, the rotarod residence time of the T2DM group (15.80±2.65s) was significantly lower than that of the CON group (33.56±2.13s) (P<0.05). In the next 4 weeks, the rotarod residence time of the T2DM group was also significantly lower than that of the CON group (P<0.05), indicating that diabetes can cause deterioration of central coordination ability and reduce the sense of balance. Compared with the T2DM group, the rotarod residence time in the DON group (50.11±5.14s) at week 4 was significantly higher than that in the T2DM group (20.34±4.35s) (P<0.05), while that in the WMP group (27.28±7.14s), WME group (27.70±5.19s), WP-L group (21.05±5.41s), WP-M group (26.46±7.79s) and WP-H group (32.65±9 .80s) There was an increasing trend in the first 4 weeks of rotarod time, but there was no significant difference (P>0.05), indicating that each dose group had no significant effect on central coordination ability; in the 5th week, the rotarod residence time of the DON group (43.64±11.31s) and the WP-H group (41.71±8.45) was significantly higher than that of the T2DM group (29.88±8.81s) (P<0.05), indicating that DON and WP-H can promote the sense of balance of diabetic mice.

[0056] Table 3 Effects of walnut peptide on the rotarod time of diabetic model mice

[0057]

[0058]

[0059] Effects of walnut peptide on the frequency of open field activity in diabetic model mice

[0060] The open field test can be used to detect the autonomous exploration, motor ability and nervous fear state of mice in a new environment. The open field test device is a 45cm×45cm×45cm cube box. The mouse is placed in the open field box with its head facing the box wall. An automatic camera and related software are used to record the free exploration of the mouse for 5 minutes, and calculate its moving distance, residence time and number of shuttles in the center position, and statistically process the data.

[0061] Depend on Figure 4 As shown in the figure, compared with the CON group, the movement trajectory of the T2DM group was significantly reduced in the route through the central area. Compared with the T2DM group, after oral gavage intervention, the movement trajectory of the diabetic model mice increased in all the oral gavage intervention groups, among which the DON group had the largest increase, followed by the WP-H group. The experimental results show that walnut peptides can improve the spatial memory and exploration ability of diabetic model mice. Figure 5 It can be seen that compared with the CON group, the number of times mice entered the central area and the time they stayed in the central area in the T2DM group were significantly reduced (P<0.05). Compared with the T2DM group, the number of times mice entered the central area and the time they stayed in the central area in the DON group and WP-H group were significantly increased (P<0.05), and the number of times mice entered the central area and the time they stayed in the central area in the WMP group, WME group, WP-L group, and WP-M group increased, but there was no significant difference (P>0.05); this shows that DON and WP-H can promote the autonomous exploration ability of diabetic model mice.

[0062] Effects of walnut peptide on novel object recognition ability in diabetic model mice

[0063] After the administration, a new object recognition experiment was conducted to detect the spatial learning and memory level of mice. There were four white new object recognition boxes made of acrylic resin. Day 1 adaptation period: Put the mouse in the new object recognition box and let it move freely for 5 minutes to adapt to the environment. Day 2 exploration period: Put two identical cylinders in the box, the distance between the cylinder and the side wall of the box is 10cm, and then put the mouse in the box with its back to the cylinder, and let the mouse explore freely in the box for 5 minutes. Day 3 test period: Each cylinder in the upper left position was replaced with a new cone, and the automatic camera and software were used to record the exploration time of the mouse on each object, and the number, time and movement trajectory of the mouse exploring the new and old objects within 5 minutes. After the test was completed, the mouse feces was immediately cleaned and disinfected with 75% alcohol to avoid odor interference. The recognition index (RI) calculation formula is: discrimination index = (new object exploration time-old object exploration time) / (new object exploration time + old object exploration time) × 100%.

[0064] The results of the effects on the novel object recognition ability of each experimental group are shown in Table 4. Compared with the CON group (68.08±5.54%), the novel object recognition index of the T2DM group (32.10±3.06%) decreased, and the difference was statistically significant (P<0.05), indicating that the cognitive impairment model was successfully established; compared with the T2DM group (32.10±3.06%), the novel object recognition index of the WMP group (36.10±4.34%) and the WME group (43.09±7.16%) showed an increasing trend, but there was no significant difference (P>0.05); the novel object recognition index of the DON group (54.32±1.88%), the WP-L group (34.51±1.27%), the WP-M group (50.80±6.14%) and the WP-H group (53.36±8.63%) were significantly higher than those of the T2DM group (P<0.05). Figure 6 , Figure 7As shown in the figure, in the exploration period and test period, compared with the CON group, the spontaneous activity ability of the T2DM group was very weak, and they only explored the edge, and the number of explorations of new and old objects was very small; compared with the T2DM group, after oral gavage intervention, the spontaneous activity ability, memory ability and exploration ability of the diabetic model mice were improved, and the mice began to increase their activity tracks in the surrounding area and the middle area, and the number of explorations of new and old objects gradually increased. The results showed that WMP, WME, WP-L, WP-M, and WP-H had a certain regulatory effect on the spontaneous activity ability, memory ability, and exploration ability of diabetic model mice. Among them, the new object recognition index of the WP-H group (53.36±8.63%) and the WP-M group (50.80±6.14%) was significantly higher than that of the WMP group (36.10±4.34%), the WME group (43.09±7.16%), and the WP-L group (34.51±1.27) (P<0.05). It shows that WP-H and WP-M have a good effect on improving the spontaneous activity, memory and exploration ability of diabetic model mice.

[0065] Table 4 Effects of novel object recognition test on each group of mice

[0066]

[0067] Effects of walnut peptide on glucose and lipid metabolism in diabetic mice

[0068] Effects of walnut peptide on serum parameters of diabetic mice

[0069] Clinical and basic research shows that glucose and lipid metabolism disorders can aggravate diabetic cognitive dysfunction. Figure 8 As shown in the results, compared with the contents of TG, TC, LDL-C and HDL-C in the CON group, the contents of TG, TC and LDL-C in the T2DM group were significantly increased (P<0.05), and the content of HDL-C was significantly decreased (P<0.05); compared with the T2DM group, the contents of TG, TC and LDL-C in the DON group, WMP group, WME group, WP-L group, WP-M group and WP-H group were significantly decreased (P<0.05), and the level of HDL-C was significantly increased (P<0.05). The results showed that WMP, WME and WP intervention had a certain regulatory effect on the blood lipid level of diabetic mice.

[0070] Effect of walnut peptide on H&E staining of liver in diabetic mice

[0071] Increased glucose production in the liver may lead to memory and cognitive impairment in diabetic patients. To explore the role of liver dysfunction in cognitive impairment in diabetes, dietary intervention was used to observe the effects of pathological changes in liver tissue of diabetic mice in each experimental group. Fig. 9As shown in the figure, the liver lobule structure in the CON group liver tissue was complete and clear, the liver cell plates were neatly arranged, the cytoplasm was rich, and the portal area separated the adjacent liver lobules, which was easy to identify. The liver lobule structure in the T2DM group liver tissue was fuzzy, the liver cells became larger and round, accompanied by ballooning, the complete structure of the cell membrane was destroyed, the boundaries were unclear, and some cells were diffusely fatty and vacuolar degeneration, and a large number of inflammatory cells were infiltrated. Compared with the T2DM group, the liver cells in the WMP group and WP-L group only had small nuclear remnants, the cytoplasm was lightly stained and difficult to distinguish from the surrounding liver cells, and a small number of inflammatory cells were infiltrated. The liver lobule structure in the DON group, WME group, WP-M group, and WP-H group was clear, the liver cells were neatly arranged, and some liver cells were larger. The results showed that walnut peptide intervention can reduce liver damage in diabetic mice.

[0072] Effect of walnut peptide on the oxidative level of brain tissue in diabetic mice

[0073] Memory impairment caused by diabetes is related to oxidative stress in brain tissue. Oxidative stress is caused by the accumulation of ROS in the body far exceeding the load capacity of its antioxidant defense system. A large amount of ROS can cause neuronal damage by inducing lipid peroxidation, ultimately leading to cognitive impairment. Studies have found that diabetic cognitive impairment can enhance oxidative stress in the brain by reducing the activity of antioxidant enzymes (CAT, SOD and GSH-px) and increasing MDA content. Therefore, it can be seen from the experimental results that the intervention of walnut peptides can significantly reverse diabetes-induced cognitive impairment.

[0074] like Fig.10 As shown in A, compared with the CON group (198.46±14.82U / mgprot), the T2DM group (72.22±1.58U / mgprot) significantly reduced the activity of SOD in the brain tissue of mice (P<0.05). Compared with the T2DM group, the intervention of the DON group (162.51±3.50U / mgprot), WMP group (123.68±5.58U / mgprot), WME group (138.96±4.95U / mgprot), WP-L group (150.83±7.58U / mgprot), WP-M group (151.86±11.08U / mgprot), and WP-H group (151.34±2.50U / mgprot) significantly increased the activity level of SOD (P<0.05).

[0075] like Fig.10As shown in B, the MDA content in the brain tissue of mice in the T2DM group (11.93±0.13mmol / mgprot) was significantly higher than that in the CON group (5.31±0.09mmol / mgprot) (P<0.05). Compared with the T2DM group (11.93±0.13mmol / mgprot), the MDA content in the brain tissue of mice in the DON group (6.63±0.15mmol / mgprot), WP-L group (8.90±0.25mmol / mgprot), WP-M group (7.10±0.17mmol / mgprot), and WP-H group (7.49±0.26mmol / mgprot) was significantly reduced (P<0.05).

[0076] like Fig.10 As shown in C, compared with the CON group (39.02±2.01mmol / mgprot), the T2DM group (22.42±1.94mmol / mgprot) significantly reduced the CAT content in the mouse brain tissue (P<0.05). Compared with the T2DM group, the intervention of the DON group (33.09±1.13mmol / mgprot), WP-M group (27.42±2.84mmol / mgprot), and WP-H group (27.35±2.05mmol / mgprot) significantly increased the CAT content (P<0.05).

[0077] like Fig.10 As shown in D, compared with the CON group (132.92±4.09mmol / mgprot), the T2DM group (62.81±2.75mmol / mgprot) significantly reduced the activity of GSH-px in the brain tissue of mice (P<0.05). Compared with the T2DM group, the intervention of the DON group (109.48±4.71mmol / mgprot), WME group (82.73±2.27mmol / mgprot), WP-L group (85.66±5.57mmol / mgprot), WP-M group (90.74±6.01mmol / mgprot), and WP-H group (90.54±5.40mmol / mgprot) could significantly increase the activity of GSH-px (P<0.05).

[0078] The study showed that the protective ability of WP-L, WP-M and WP-H groups on the brain tissue of diabetic mice with cognitive impairment was significantly stronger than that of WMP and WME groups, and was dose-dependent. Therefore, it is speculated that walnut peptides may improve cognitive impairment by regulating the oxidative stress pathway in the brain of diabetic mice.

[0079] Effects of walnut peptide on hippocampal neurons in diabetic mice

[0080] H&E staining

[0081] The hippocampus plays a vital role in the process of cognition and memory, and its pathology is closely related to memory deficits. In order to further clarify the effect of walnut peptide on improving cognitive impairment in diabetic model mice, H&E staining was performed to detect the effect of walnut peptide on the neuronal morphology of diabetic cognitive impairment mice ( Fig.11 ). In the CON group, there were no abnormalities in the hippocampal neurons and glial cells in the CA1, CA2, CA3, and DG regions. The arrangement was concentrated and neat with clear layers, and the nuclear membrane and nucleolus were clearly visible. In the T2DM group, the number of hippocampal neurons in the CA1, CA2, CA3, and DG regions was small and the arrangement was irregular, the gaps were enlarged, some neurons were swollen and deformed, the nuclei were dissolved and condensed, the nucleolus morphology was not obvious, the nuclear membrane was not clear, or even disappeared. Compared with the T2DM group, there was no significant change in the CA2 region among the six groups of DON, WMP, WME, WP-L, WP-M, and WP-H. The DON, WMP, WME, WP-L, WP-M, and WP-H groups effectively reduced the looseness of the cells near the CA1, CA3, and DG regions of the hippocampus and changed the number of nuclear condensations. Among them, the CA1, CA3, and DG regions of the hippocampus in the DON and WP-H groups were arranged in a concentrated and neat manner, and the cell morphology was relatively full and complete.

[0082] Nissl stain

[0083] Nissl staining is used to detect Nissl bodies, which are markers of mature neurons and can be used to detect neuronal apoptosis. Fig.12 As shown in the figure, in the CON group, it was observed that in the CA1, CA2, CA3 and DG regions of the hippocampus, the mice hippocampal neurons were neatly arranged and compactly distributed, and the intracellular Nissl bodies were filled. Compared with the CON group, the T2DM group had obvious neuronal damage and apoptosis, neuronal loss and scattered distribution, and morphological abnormalities such as nuclear pyknosis in the CA1 and DG regions of the hippocampus. Compared with the T2DM group, there was no significant change in the CA2 region among the six groups of DON, WMP, WME, WP-L, WP-M and WP-H. The results of Nissl staining in the CA1 and DG regions showed that compared with the T2DM group, the neuronal apoptosis in the DON, WMP, WME, WP-L, WP-M and WP-H groups was significantly reduced, the neurons were neatly arranged, and the Nissl bodies were clear and obvious. The DON, WMP, WME, WP-L, WP-M and WP-H groups improved the neuronal cell morphology in the CA1, CA3 and DG regions of the hippocampus of diabetic mice and increased the number of neurons.

[0084] In order to further understand how walnut peptide regulates and improves the mechanism of cognitive impairment in diabetic model mice, transcriptome sequencing analysis was performed on the hippocampus of diabetic model mice. The present invention statistically processes the raw data obtained by sequencing, performs quality control, and evaluates its rationality and reliability through expression analysis. Differential expression analysis is performed on genes in different groups to obtain differential genes, count differential genes, and cluster analysis is performed on different differential genes. In order to further study the intelligence mechanism of walnut peptide, GO annotation analysis is performed to compare the functional differences between samples in different groups. Through KEGG enrichment analysis, the signal pathways associated with it are further screened out. The quality of sequencing data was evaluated, and the results showed that all evaluation indicators of 18 samples met the quality requirements of gene expression level and functional analysis. KEGG enrichment results showed that the high-dose walnut peptide group (WP-H) mainly affected inflammation-related signaling pathways. After qPCR verification, it was consistent with the results of up-regulation and down-regulation of differentially regulated genes in transcriptome regulation, which further verified the results of transcriptome. TNF signaling pathway and IL17 signaling pathway are the potential mechanisms for walnut peptide to regulate diabetic cognitive impairment. In the future, more evidence is still needed to reveal that walnut peptide improves the learning and memory function of diabetic mice through inflammatory pathways.

[0085] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. Use of walnut peptide in the preparation of a drug for treating or preventing diabetic cognitive impairment.

2. The use of the walnut peptide according to claim 1 in the preparation of a drug for treating or preventing diabetic cognitive impairment, characterized in that: The molecular weight of the walnut peptide is less than 3KDa.

3. The use of the walnut peptide according to claim 1 in the preparation of a drug for treating or preventing diabetic cognitive impairment, characterized in that: The preparation process of the walnut peptide is as follows: walnut protein is enzymatically hydrolyzed with alkaline protease, and then separated with an ultrafiltration tube with a molecular weight cutoff of <3kDa to obtain the walnut peptide.

4. The use of the walnut peptide according to claim 1 in the preparation of a drug for treating or preventing diabetic cognitive impairment, characterized in that: The walnut peptide can effectively reduce the blood sugar level of diabetic model mice, regulate the disorder of blood lipids, reduce the oxidative damage of brain tissue and the damage of neuroinflammation in the hippocampus of diabetic model mice, and has the effect of improving learning and memory impairment.

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