Sipunculus nudus polypeptide capable of improving cognitive impairment as well as preparation method and application of sipunculus nudus polypeptide

Abstract

The invention relates to the technical field of bioactive peptides, in particular to sipunculus nudus polypeptide capable of improving cognitive impairment as well as a preparation method and application of the sipunculus nudus polypeptide. The sipunculus nudus polypeptide (SNP) prepared through enzymolysis can regulate the blood glucose level of type 2 diabetes mellitus, inhibit serum inflammation, improve cognitive impairment caused by type 2 diabetes mellitus and improve learning memory and space exploration ability. The sipunculus nudus polypeptide prepared through enzymolysis can activate a PI3K / Akt signal channel by inhibiting phosphorylation of JNK and P38 of an MAPK signal channel, promote expression of a brain-derived neurotrophic factor BDNF, inhibit expression of hippocampus Tau and neuronal cell apoptosis caused by type 2 diabetes mellitus and further resist related symptoms of cognitive impairment caused by type 2 diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioactive peptides, and particularly relates to a fan worm polypeptide for improving cognitive dysfunction and a preparation method and application thereof. BACKGROUND

[0002] Type 2 diabetes mellitus (T2DM) is a common and frequently-occurring disease in clinic, has a high incidence, and a wide population. If a patient with type 2 diabetes mellitus maintains a high blood glucose state for a long time, a series of serious complications are easily caused. Among them, type 2 diabetes mellitus cognitive dysfunction is one of these chronic complications, and has a relatively high incidence. According to statistics, about 60% to 70% of patients with diabetes mellitus are faced with mild to moderate cognitive dysfunction problems, and the specific performance is that the overall memory, attention, reasoning ability, learning ability and the like of the patient are impaired. Long-term high-sugar environment can cause Aβ deposition and Tau protein phosphorylation, and make nerve cells apoptosis, and affect the cognition of the patient. Once type 2 diabetes mellitus cognitive dysfunction (DCD) occurs, it is difficult to reverse, and is easily to become dementia, and brings a heavy burden to the family and society.

[0003] The possible pathogenesis of type 2 diabetes mellitus leading to cognitive dysfunction is insulin resistance, inflammatory response, oxidative stress, and glucose-lipid metabolic disorder. At present, although the medical field has not yet made a breakthrough in the development of special drugs for treating cognitive dysfunction, doctors often use some chemical synthetic drugs for degenerative neurological diseases as an alternative therapy. These drugs include cholinesterase inhibitors such as donepezil, galantamine and tacrine, calcium antagonists such as nimodipine, excitatory amino acid antagonists such as memantine, and neuroprotective regulators such as cerebrolysin. Although the mechanism of action of these drugs has been relatively clear, due to the lack of in-depth understanding of the pathogenesis of cognitive dysfunction, the therapeutic effect of these drugs is relatively single. Although they can improve the symptoms of patients to a certain extent, long-term use not only has high cost, but also may bring a series of side effects. Therefore, developing new, safe and efficient drugs for improving cognitive dysfunction has become a research hotspot.

[0004] Marine organisms are rich in bioactive substances with novel structure and diverse activities due to their unique living environment. Sipunculus nudus, commonly known as "sandworm", is a characteristic marine invertebrate in the southeast coastal areas of China. It has delicious meat and rich nutritional ingredients, and has the effects of anti-fatigue and immune regulation in traditional medicine. Recent studies have found that the body wall and coelomic fluid of Sipunculus nudus are rich in functional proteins and peptides, which have the potential of antioxidant, anti-inflammatory and neuroprotective. For example, Sipunculus nudus peptides have been confirmed to protect neurons by removing free radicals and inhibiting the release of inflammatory factors. However, there is no relevant report on the improvement of cognitive dysfunction caused by diabetes mellitus type 2, and the specific target and mechanism are not clear, and there is no related patent application report.

[0005] The Sipunculus nudus peptide prepared by the method has the characteristics of small molecular weight, high stability, health and non-toxicity, and can be developed into a new type of drug for improving serum inflammation or cognitive dysfunction caused by diabetes mellitus type 2, and can also be developed into a health food and functional food for improving learning and memory function. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a Sipunculus nudus peptide for improving cognitive dysfunction, and a preparation method and application thereof.

[0007] To achieve the above object, the present application realizes the following technical scheme:

[0008] The preparation method of the Sipunculus nudus peptide comprises the following steps: beating fresh Sipunculus nudus, adding deionized water, adjusting the pH to 7.5, adding protease for enzyme hydrolysis, 90℃ water bath inactivation for 10-30min, cooling and centrifugation, and taking the supernatant for standby use; under the condition of 4℃ ice bath, the Sipunculus nudus enzyme hydrolysate is gradually passed through 50kDa and 2.5kDa ultrafiltration membranes, the <2.5kDa ultrafiltration components are intercepted and collected, and the collected components are passed through a nanofiltration membrane to remove free amino acids and inorganic salts, and the intercepted liquid is the Sipunculus nudus peptide.

[0009] Preferably, the addition amount of the protease is 2% of the substrate, the enzyme hydrolysis temperature is 45℃, and the time is 3-4h; the centrifugation speed is 8000-10000r / min, the temperature is 4℃, and the time is 20min.

[0010] Correspondingly, a Sipunculus nudus peptide prepared by the preparation method.

[0011] Correspondingly, the Sipunculus nudus peptide is applied to the preparation of a drug for improving cognitive dysfunction.

[0012] Preferably, the Sipunculus nudus peptide is applied to the preparation of a drug for improving serum inflammation and cognitive dysfunction caused by diabetes mellitus type 2.

[0013] Preferably, the dosage of the S. nudgeroundis administered at 100-800 mg / kg·bw.

[0014] Correspondingly, the application of a S. nudgeroundis polypeptide in the preparation of a medicine or a pharmaceutical composition, wherein the medicine or the pharmaceutical composition comprises the S. nudgeroundis polypeptide, and the medicine or the pharmaceutical composition has the effect of improving serum inflammation and cognitive dysfunction caused by type 2 diabetes.

[0015] Preferably, fresh S. nudgeroundis is pulped, deionized water is added, the pH is adjusted to 7.5, protease is added for enzymolysis, and the enzyme is inactivated in a 90℃ water bath for 10-30 min; after cooling, centrifugation is performed, and the supernatant is taken for standby use; under the condition of ice bath at 4℃, the S. nudgeroundis enzymolysis solution is gradually passed through 50 kDa and 2.5 kDa ultrafiltration membranes, <2.5 kDa ultrafiltration components are intercepted and collected, and the components are passed through a nanofiltration membrane to remove free amino acids and inorganic salts, so as to obtain the intercepting liquid, that is, the S. nudgeroundis polypeptide.

[0016] Preferably, the addition amount of the protease is 2% of the substrate, the enzymolysis temperature is 45℃, the time is 3-4 h; the centrifugation speed is 8000-10000 r / min, the temperature is 4℃, and the time is 20 min.

[0017] Preferably, the dosage of the S. nudgeroundis polypeptide is 100-800 mg / kg.

[0018] The present application has the following beneficial effects:

[0019] 1. The S. nudgeroundis polypeptide is prepared by enzymolysis, and it is found that the S. nudgeroundis polypeptide can reduce the high blood sugar and high insulin level of type 2 diabetes mice, inhibit insulin resistance, inhibit the secretion of inflammatory factors IL-6, IL-1β and TNF-α in serum, and inhibit serum inflammation in a type 2 diabetes mouse model.

[0020] 2. The T2DM mouse model is constructed. During the 6-week administration period after the model is established, it is found through the water maze experiment detection that these T2DM mice have obvious cognitive dysfunction. After the T2DM mice are given SNP intervention treatment for 6 weeks, the learning and memory ability and the spatial exploration ability of the T2DM mice are improved, and the cognitive dysfunction symptoms of the T2DM mice are improved. In addition, the S. nudgeroundis polypeptide can inhibit the formation and accumulation of Aβ1-42 and Tau protein in the hippocampal tissue of the T2DM mice, inhibit cell apoptosis, and promote the secretion of neurotrophic factor BDNF, thereby reducing the damage of neurons caused by diabetes. It can be seen that the S. nudgeroundis polypeptide has a good improvement effect on the cognitive dysfunction and neuron damage caused by type 2 diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Effects of SNP on latency (a) and average speed (b) of T2DM mice (n=6), Note: compared with Control, ** P<0.01, *** P<0.001; compared with T2DM, ## P<0.01, ### P<0.001;

[0022] Figure 2 Effects of SNP on target quadrant (a) and platform times (b) of T2DM mice (n=6), Note: compared with Control, * P<0.05, ** P<0.01; compared with T2DM, # P<0.05, ## P<0.01;

[0023] Figure 3 Effects of SNP on IL-6 (a), IL-1β (b), TNF-α (c) contents of T2DM mice (n=4), Note: compared with Control, ** P<0.01, *** P<0.001; compared with T2DM, # P<0.05, ## P<0.01, ### P<0.001;

[0024] Figure 4 Effects of SNP on SOD (a), MDA (b) contents of T2DM mice (n=4), Note: compared with Control, * P<0.05, ** P<0.01; compared with T2DM, # P<0.05, ## P<0.01.

[0025] Figure 5 Effects of SNP on Aβ1-42 protein (a), Tau protein (b) contents of T2DM mice (n=4), Note: compared with Control, ** P<0.01, *** P<0.001; compared with T2DM, # P<0.05, ## P<0.01, ### P<0.001;

[0026] Figure 6The immunofluorescence results of each group of mice; Note: (a) Tau fluorescence map of hippocampal tissue CA1, CA3, DG region; (b) Tau fluorescence expression results of hippocampal tissue CA1 region; (c) Tau fluorescence expression results of hippocampal tissue CA3 region; (d) Tau fluorescence expression results of hippocampal tissue DG region; (e) BDNF fluorescence map of hippocampal tissue CA1, CA3, DG region; (f) BDNF fluorescence expression results of hippocampal tissue CA1 region; (g) BDNF fluorescence expression results of hippocampal tissue CA3 region; (h) BDNF fluorescence expression results of hippocampal tissue DG region; compared with Control, ** P<0.05, ** P<0.01, *** P<0.001; compared with T2DM, # P<0.05, ## P<0.01;

[0027] Figure 7 The expression of Caspase3, Bax and Bcl-2 proteins in the hippocampal tissue of each group of mice; Note: (a) Gray diagram of Caspase3, Bax and Bcl-2 proteins in hippocampal tissue, (b) Caspase3 protein expression results in hippocampal tissue, (c) Bax protein expression results in hippocampal tissue, (d) Bcl-2 protein expression results in hippocampal tissue, compared with Con, * P<0.05, ** P<0.01; compared with T2DM, # P<0.05, ## P<0.01;

[0028] Figure 8 The expression of MAPKA in the hippocampal tissue of each group of mice; Note: (a) Effect of SNP on MAPK signaling pathway, (b) Effect of SNP on p-JNK phosphorylation in MAPK signaling pathway, (c) Effect of SNP on p-P38 phosphorylation in MAPK signaling pathway, compared with Con, * P<0.05, * *P<0.01; compared with T2DM, # P<0.05;

[0029] Figure 9 The activation of PI3K / Akt signaling pathway in the hippocampal tissue of each group of mice; Note: (a) Effect of SNP on PI3K / Akt signaling pathway, (b) Effect of SNP on p-PI3K phosphorylation in PI3K / Akt signaling pathway, (c) Effect of SNP on p-Akt phosphorylation in PI3K / Akt signaling pathway, compared with Con, ** P<0.01; compared with T2DM, # P<0.05,## P<0.01. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0031] If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art.

[0032] The application discloses application of a sipunculid polypeptide in preparation of a medicine for improving cognitive dysfunction.

[0033] Further, the preparation process of the sipunculid polypeptide is as follows: fresh sipunculid is beaten into pulp, deionized water is added, pH is adjusted to 7.5, protease is added for enzymolysis, 90 DEG C water bath inactivation is performed for 10-30 min, after cooling, centrifugation is performed, and the supernatant is taken for standby use; under the condition of 4 DEG C ice bath, the sipunculid enzymolysis solution is gradually passed through 50 kDa and 2.5 kDa ultrafiltration membranes, and is passed through a nanofiltration membrane, free amino acids and inorganic salts are removed, and the cut-off liquid is the sipunculid polypeptide.

[0034] Further, the addition amount of the protease is 2% of the substrate (pulp of the sipunculid), the enzymolysis temperature is 45 DEG C, the time is 3-4 h, the centrifugation speed is 8000-10000 r / min, the temperature is 4 DEG C, and the time is 20 min.

[0035] The application further discloses application of a medicine or a pharmaceutical composition in preparation of a medicine for improving cognitive dysfunction caused by type 2 diabetes, and the medicine or the pharmaceutical composition comprises the sipunculid polypeptide.

[0036] The application will be further described below in combination with specific examples.

[0037] The main reagents used in the following examples are shown in Table 1.

[0038] Table 1 shows the reagents used.

[0039] Rabbit anti-mouse Tau polyclonal antibody Wuhan Seervel Biotechnology Co., Ltd. Rabbit anti-mouse BDNF polyclonal antibody Wuhan Seervel Biotechnology Co., Ltd. Secondary antibody goat anti-rabbit lgG Wuhan Seervel Biotechnology Co., Ltd. BCA kit Bi Yun Tian Biotechnology Co., Ltd. OCT freezing embedding agent Bi Yun Tian Biotechnology Co., Ltd. Aβ1-42 kit Quanzhou Ruixin Biotechnology Co., Ltd. Tau kit Quanzhou Ruixin Biotechnology Co., Ltd. IL-6 kit Zhanjiang Dingjie Biotechnology Co., Ltd. IL-1β kit Zhanjiang Dingjie Biotechnology Co., Ltd. TNF-α kit Zhanjiang Dingjie Biotechnology Co., Ltd. SOD kit Zhanjiang Dingjie Biotechnology Co., Ltd. MDA kit Zhanjiang Dingjie Biotechnology Co., Ltd.

[0040] Example 1

[0041] 1. Preparation of the sipunculid polypeptide

[0042] Fresh S. gigantea was washed and weighed, and then preliminary pulping was performed. Deionized water was added, and the pH of the sample solution was adjusted to 7.5 by adding 0.1 mol / L NaOH. Enzymatic hydrolysis was performed by adding protease (4000 U / g, 45°C, 4 h), and inactivation was performed by water bath at 90°C for 10-30 min. After cooling, centrifugation was performed (10000 r / min, 4°C, 20 min), and the supernatant was collected for use. Under the condition of ice bath at 4°C, the S. gigantea enzymatic hydrolysate was subjected to ultrafiltration by using 50 kDa and 2.5 kDa ultrafiltration membranes, respectively. The <2.5 kDa ultrafiltration component was collected and subjected to nanofiltration to remove free amino acids and inorganic salts, and the retentate was S. gigantea polypeptide. Subsequently, the S. gigantea polypeptide was freeze-dried and stored in a refrigerator at -80°C for use.

[0043] 2. Establishment of T2DM model and administration

[0044] Fifty 6-week-old male ICR mice were first adapted to a new environment for one week to ensure that the mice could freely eat food and drink water. Then, the mice were fed with high-sugar and high-fat feed for 8 weeks, and after fasting overnight, the mice were rapidly injected intraperitoneally with freshly prepared streptozotocin solution (30 mg / kg.bw; injection once a week; for 4 consecutive weeks). The normal control mice were injected intraperitoneally with an equal amount of citric acid buffer. After 12 weeks of modeling, the mice were subjected to tail vein puncture for blood sampling to detect fasting blood glucose (FBG), and FBG≥11.1 mmol / L was considered as successful modeling of T2DM mice. Then, the T2DM mice were divided into 5 groups according to the average of body weight and blood glucose, with 10 mice in each group. The blank control group: normal feed + 0.9% physiological saline gavage; the model group: high-sugar and high-fat feed + 0.9% physiological saline gavage; the S. gigantea polypeptide low-dose group (SNP-L): high-sugar and high-fat feed + S. gigantea polypeptide low-dose gavage (100 mg / kg.bw dose); the S. gigantea polypeptide medium-dose group (SNP-M): high-sugar and high-fat feed + S. gigantea polypeptide medium-dose gavage (400 mg / kg.bw dose); the S. gigantea polypeptide high-dose group (SNP-H): high-sugar and high-fat feed + S. gigantea polypeptide high-dose gavage (800 mg / kg.bw dose), and the gavage was continuously administered for 6 weeks.

[0045] 3. Results of administration of S. gigantea polypeptide (SNP)

[0046] 3.1 Morris water maze experiment

[0047] After 6 weeks of continuous administration, the mice were subjected to the Morris water maze experiment (place navigation experiment, spatial search experiment) to observe the learning and memory ability and spatial exploration ability of the mice, and to evaluate the improvement effect of S. gigantea polypeptide on the cognitive function impairment of T2DM mice.

[0048] The Morris water maze experiment equipment is a pool with a height of 50 cm and a width of 214 cm, and the bottom of the pool is black. The water depth in the pool is kept at 25 cm, and a circular platform with a height of 30 cm and a width of 12 cm is provided. In order to better locate and navigate, four equally spaced points are marked around the pool, thereby dividing the pool into east, south, west, and north quadrants. In order to accurately record the behavior of the mice in the water, an advanced video recording and acquisition system is installed directly above the pool.

[0049] Position navigation experiment: four water entry points are randomly selected outside the platform quadrant, and the mouse is slowly placed into the water with its head facing the pool wall during the experiment. The time taken by the mouse to find the underwater platform (i.e., escape latency) is recorded. If the mouse does not find the platform within 90 seconds, it is placed back on the platform and allowed to stay on the platform for 10 seconds, at which time the escape latency is recorded as 90 seconds. One water entry point is selected each time, and four times are performed in one day, and the average of the four recorded escape latencies is taken as the daily score. The position navigation test is performed continuously for four days.

[0050] Spatial exploration experiment: on the last day, the mouse is placed into the water from the opposite quadrant of the original platform, facing the pool wall, and the number of times and the time spent by the mouse in the original platform quadrant area in the pool within 90 seconds are observed and recorded.

[0051] 3.1.1 Effect of SNP on the behavior of T2DM mice in the Morris water maze test

[0052] 3.1.1.1 Effect of SNP on the position navigation of T2DM mice

[0053] As shown in Figure 1 a, on the first day of the Morris water maze behavior test, there was no significant difference in the escape latency of the mice in each group. On the second day of the test, the latency of the T2DM group was longer than that of the blank control group (P<0.01), indicating that the learning and memory ability of diabetic mice decreased significantly. After SNP treatment, the latency of the mice was shortened (P<0.01, P<0.001), indicating that SNP can improve the memory of mice. Figure 1 b, compared with the blank control group, the average speed of the T2DM group mice decreased (P<0.001), indicating that the learning and memory ability of diabetic mice decreased, and after SNP treatment, the average speed of the mice to find the target platform increased (P<0.01). This indicates that SNP can improve the memory of mice.

[0054] 3.1.1.2 Effect of SNP on the spatial exploration ability of T2DM mice

[0055] The results of the spatial exploration experiment are shown in Figure 2 a and Figure 2b shows that compared with the blank control group, the time of T2DM group mice in the target quadrant is shortened (P<0.01), and the number of crossing the platform is reduced (P<0.05), which indicates that the spatial exploration ability and learning and memory ability of T2DM mice are impaired, and certain cognitive function appears. After SNP administration intervention, the time of T2DM mice in the target quadrant is prolonged, and the number of crossing the platform is increased (P<0.01). It shows that SNP can improve the spatial exploration ability of T2DM mice.

[0056] 3.2 Specimen preparation

[0057] After the Morris water maze experiment, the mice were fasted for 12 hours without water.

[0058] Serum sample preparation: After the blood was placed in a 4°C refrigerator for 4 hours, it was centrifuged for 10 minutes at a speed of 15000 r / min to obtain serum, which was quickly placed in a -80 refrigerator for standby and used for subsequent biochemical detection and analysis.

[0059] Mouse hippocampal tissue sample preparation: After the mouse brain tissue was removed, the mouse brain tissue was quickly dissected on dry ice, and the hippocampal tissue was separated. The hippocampus of the same side was placed in a 4% paraformaldehyde solution for overnight fixation, and the room temperature sucrose gradient dehydration was performed: 20% sucrose overnight, and the next day 30% sucrose was replaced. After the tissue precipitated, the dehydrated tissue was taken out, the surface moisture was absorbed, and then it was placed in an OCT embedding box, and stored in a -80°C refrigerator for subsequent histological analysis. The other side of the hippocampus was placed in liquid nitrogen and quickly placed in a -80 refrigerator for standby and used for subsequent biochemical analysis.

[0060] 3.2.1 Determination of IL-6, IL-1β, TNF-α content in serum of T2DM mice as shown in Figure 3 a shows that the expression of IL-6 in the serum of the T2DM model group is higher than that of the blank control group (P<0.001), and after SNP administration treatment, the content of IL-6 in the SNP group mice is reduced (P<0.001). As shown in Figure 3 b shows that compared with the normal control group, the content of IL-1β in the serum of the T2DM model group is increased (P<0.01), and after SNP treatment, the secretion of IL-1β in the SNP group is reduced (P<0.05, P<0.01). Figure 3 c shows that compared with the blank control group, the expression of TNF-α in the serum of the T2DM model group is increased (P<0.01), and after SNP administration treatment, the content of TNF-α is reduced (P<0.01). Figure 3 It is indicated that SNP has an inhibitory effect on inflammation in T2DM mice.

[0061] 3.2.2 Determination of SOD, MDA content in serum of mice

[0062] The content of MDA and the activity of SOD in the serum of mice were detected by enzyme-linked immunosorbent assay, and the specific process was prior art.

[0063] More and more studies have proved that oxidative stress is closely related to the occurrence and development of T2DM, and people often use SOD and MDA to evaluate the level of oxidative stress. SOD reflects the body's ability to scavenge oxygen free radicals, and MDA is an important indicator of lipid peroxidation. Both of them can reflect the degree of oxidative stress damage to tissues and cells. For example Figure 4 As shown in Figure 8a, the activity of SOD in the serum of model mice was lower than that of the blank control group (P<0.05), and after the administration of SNP, the activity of SOD in T2DM mice was increased (P<0.05). Figure 4 As shown in Figure 8b, the content of MDA in the serum of model mice was higher than that of the blank control group (P<0.01), and the administration of SNP reduced the level of MDA in T2DM mice (P<0.01). Through the detection of SOD, MDA and other indicators in the serum of mice, it was found that SNP could inhibit the expression of inflammatory factors, enhance the activity of SOD, and reduce the concentration of MDA, which indicated that SNP could play a hypoglycemic effect through anti-inflammatory and antioxidant, that is, SNP could enhance the antioxidant capacity of T2DM mice and reduce the level of oxidative stress.

[0064] 3.2.3 Determination of the content of Aβ1-42 and Tau protein in the hippocampus of mice

[0065] First, weigh the appropriate amount of hippocampus tissue and add an appropriate amount of cell lysis solution (50 mmol / L Tris-HCl, 1.0 mmol / L EDTA, 150 mmol / L NaCl, 0.1% SDS). Then, cut the tissue into small pieces and prepare a homogenate in an ice water bath. Then, centrifuge at 12000 r / min for 10 min to separate the supernatant for subsequent determination. After the sample is prepared, use the BCA kit to determine the protein concentration, which will be used for subsequent calculation. In order to determine the optimal determination condition, the tissue homogenate supernatant is diluted to different concentrations for pre-experiment. Next, according to the instructions of the Aβ1-42 and Tau kit, add the homogenate supernatant to be tested and the reagents in the kit to the 96-well plate in the specified order. Then, measure the absorbance at 562 nm wavelength. Finally, plot the absorbance standard curve according to the measured OD value, and calculate the protein concentration of the sample according to the standard curve.

[0066] The results are shown in Figure 8a and Figure 8b. Figure 5The content of Aβ1-42 in the T2DM group was higher than that in the control group (P<0.01), indicating that the T2DM mice had β amyloid deposition. After the intervention of SNP administration, the expression of Aβ1-42 in the mice was reduced, indicating that SNP could inhibit the expression of Aβ1-42 protein in the hippocampus of the T2DM mice (P<0.05). Figure 5 The expression of Tau protein in the T2DM group in b was higher than that in the blank control group (P<0.001), and the expression of the mice was reduced after the administration of SNP (P<0.01, P<0.001). Figure 5 It was shown that the administration of SNP intervention had a good improvement effect on the deposition of β amyloid Tau protein induced by type 2 diabetes.

[0067] 3.3 Immunofluorescence staining

[0068] (1) The tissue was cut into a slice with a thickness of 4 μm by using a freezing microtome.

[0069] (2) The brain tissue was fixed on a glass slide according to the patch method, and then was placed in a 37℃ oven for 1.5 h, was placed in a slide box, and was stored in a-80℃ refrigerator.

[0070] (3) When staining, one brain slice was taken out after 6 slices were separated between each index, and was placed in a 37℃ oven for 30 min.

[0071] (4) After being rehydrated with PBS for 5 min, the slice was placed in citric acid for antigen repair. After being boiled for 10 min at high fire, it was taken out to cool at room temperature.

[0072] (5) 5 min x 3 times, 1 h of room temperature blocking.

[0073] (6) The brain tissue was immersed in an appropriate amount of primary antibody solution (5% BSA dilution, rabbit anti-mouse Tau, 1:200; rabbit anti-mouse BDNF, 1:100) and was incubated at 4℃ overnight.

[0074] (7) After the specimen was taken out, it was placed at room temperature for 30 min, the primary antibody solution was poured out, 3 times of PBS washing was performed, and each time was 5 min. The brain tissue was immersed in an appropriate amount of secondary antibody solution (5% BSA dilution, secondary antibody goat anti-rabbit lgG, 1:400), and was incubated at room temperature for 1 h in the dark.

[0075] (8) The secondary antibody solution was poured out, 3 times of PBS washing was performed, and each time was 5 min. The brain tissue was immersed in Dapi working solution (PBS dilution, 1:500), and was incubated at room temperature for 20 min in the dark.

[0076] (9) 3 times of PBS washing was performed, and each time was 5 min.

[0077] (10) The water in the tissue is absorbed by the water-absorbing paper, and the cover sheet is attached after the anti-fluorescence quenching sealing solution is used to seal the slice.

[0078] (11) Under the fluorescence microscope, 3 fields of view (x10) of the hippocampus of each slice were selected and photographed, and the average fluorescence value of each sample was analyzed using Image J software.

[0079] 3.3.1 Effect of SNP on the fluorescence expression of Tau and BDNF proteins in T2DM mice

[0080] As Figure 6 a. Compared with the blank control group, the fluorescence expression of Tau protein in the CA1 and CA3 regions of the hippocampus of T2DM mice increased (P<0.05), indicating that hyperglycemia had a damaging effect on the CA1 and CA3 neurons of the hippocampus of mice, and the expression of Tau protein in the hippocampus of mice decreased after SNP treatment (P<0.05, P<0.01). This indicates that SNP has an improving effect on brain damage caused by hyperglycemia. As Figure 6 e. The fluorescence expression intensity of BDNF in the CA1, CA3, and DG regions of the hippocampus of T2DM mice was lower than that of the blank control group (P<0.01, P<0.001), and the fluorescence intensity of BDNF in mice increased after SNP low, medium, and high administration (P<0.05), indicating that SNP can promote the expression of neurotrophic factors in the hippocampus of mice.

[0081] 3.4 Western Blotting experiment

[0082] An appropriate amount of hippocampal tissue was weighed and an appropriate amount of cell lysis solution (50 mmol / L Tris-HCl, 1.0 mmol / L EDTA, 150 mmol / L NaCl, 0.1% SDS) was added. Then, the tissue was cut and homogenized in an ice water bath. Then, centrifugation was performed at 12000 r / min for 10 min to separate the supernatant for subsequent determination. After the sample was prepared, the protein concentration was determined using a BCA kit. The extracted protein was added to the loading buffer and boiled at 95°C for 10 min to denature the protein. SDS-polyacrylamide gel was prepared, and the protein sample was loaded and electrophoresed. After electrophoresis, the membrane was transferred, and the transferred membrane was blocked with blocking solution for 30 min. Then, the primary antibody was added and incubated at 4°C overnight, and the washing solution was washed 3 times for 10 min each time. The secondary antibody was incubated at room temperature for 1 h, and then washed with washing solution for 3 times for 10 min each time. Finally, chemiluminescence solution was added for imaging, and the software provided by the system was used for band analysis.

[0083] 3.4.1 Effect of SNP on the expression of Caspase3, Bax, and Bcl-2 in T2DM mice

[0084] As shown in Figure 7 : Compared with the blank control group, the expression level of Caspase3 in the T2DM group was significantly increased (P<0.01), and the expression of Caspase3 in the hippocampus of T2DM mice was reduced after treatment with SNP (P<0.05). Compared with the blank control group, the expression of Bax in the hippocampus of T2DM group was increased (P<0.01), and the expression of Bax in mice was significantly reduced after SNP intervention (P<0.05, P<0.01). Compared with the blank control group, the expression of Bcl-2 in the T2DM group was reduced (P<0.05), and the expression of Bcl-2 in mice was increased after SNP intervention (P<0.05).

[0085] 3.4.2 Effect of SNP on the expression of MAPK in the hippocampus of T2DM mice

[0086] As shown in Figure 8 : Compared with the blank control group, the expression level of Caspase3 in the T2DM group was significantly increased (P<0.01), and the expression of Caspase3 in the hippocampus of T2DM mice was reduced after treatment with SNP (P<0.05). Compared with the blank control group, the expression of Bax in the hippocampus of T2DM group was increased (P<0.01), and the expression of Bax in mice was significantly reduced after SNP intervention (P<0.05, P<0.01). Compared with the blank control group, the expression of Bcl-2 in the T2DM group was reduced (P<0.05), and the expression of Bcl-2 in mice was increased after SNP intervention (P<0.05).

[0087] 3.4.2 Effect of SNP on the expression of MAPK in the hippocampus of T2DM mice

[0088] As shown in Figure 9 : Compared with the blank control group, the expression level of Caspase3 in the T2DM group was significantly increased (P<0.01), and the expression of Caspase3 in the hippocampus of T2DM mice was reduced after treatment with SNP (P<0.05). Compared with the blank control group, the expression of Bax in the hippocampus of T2DM group was increased (P<0.01), and the expression of Bax in mice was significantly reduced after SNP intervention (P<0.05, P<0.01). Compared with the blank control group, the expression of Bcl-2 in the T2DM group was reduced (P<0.05), and the expression of Bcl-2 in mice was increased after SNP intervention (P<0.05).

[0089] The above-described embodiments are only preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A method for preparing a Sipuncula polypeptide, characterized in that: Fresh Siegesbeckia spp. is pulped, deionized water is added, the pH is adjusted to 7.5, protease is added for enzymatic hydrolysis, the product is inactivated in a 90°C water bath for 10-30 minutes, the product is cooled and centrifuged, and the supernatant is collected for later use; in a 4°C ice bath, the Siegesbeckia spp. enzymatic hydrolyzate is passed through 50kDa and 2.5kDa ultrafiltration membranes step by step, the ultrafiltration fraction <2.5kDa is retained and collected, and the product is passed through a nanofiltration membrane to remove free amino acids and inorganic salts, thereby obtaining the retentate, which is the Siegesbeckia spp. polypeptide.

2. The preparation method according to claim 1, wherein: The added amount of the protease is 2% of the substrate, the enzymolysis temperature is 45° C., and the time is 3-4 hours; the centrifugal speed is 8000-10000 r / min, the temperature is 4° C., and the time is 20 minutes.

3. A Sipunculus spp. polypeptide prepared by the preparation method according to claim 1 or 2.

4. Use of the Sipunculus sphenotype polypeptide according to claim 3 in the preparation of a drug for improving cognitive dysfunction.

5. The use according to claim 4, characterized in that: The Sipunculus quadrilateral polypeptide is used in the preparation of a drug for improving serum inflammation and cognitive dysfunction caused by type 2 diabetes.

6. The use according to claim 4 or 5, characterized in that: The dosage of the Sipunculus quadrilateral polypeptide is 100-800 mg / kg·bw.

7. Use of a Sipunculus spp. polypeptide in the preparation of a medicine or a pharmaceutical composition, characterized in that: The medicine or pharmaceutical composition comprises Sipuncula spp. polypeptide, and has the effects of improving serum inflammation and cognitive dysfunction caused by type 2 diabetes.

8. The use according to claim 7, characterized in that: The preparation process of the Sipuncula polypeptide is as follows: fresh Sipuncula are beaten, deionized water is added, the pH is adjusted to 7.5, protease is added for enzymatic hydrolysis, the product is inactivated in a 90°C water bath for 10-30 minutes, the product is cooled and centrifuged, and the supernatant is collected for later use; in a 4°C ice bath, the Sipuncula hydrolyzate is passed through 50kDa and 2.5kDa ultrafiltration membranes step by step, the ultrafiltration fraction <2.5kDa is retained and collected, and the product is passed through a nanofiltration membrane to remove free amino acids and inorganic salts, thereby obtaining a retentate, which is the Sipuncula polypeptide.

9. The use according to claim 8, characterized in that: The added amount of the protease is 2% of the substrate, the enzymolysis temperature is 45° C., and the time is 3-4 hours; the centrifugal speed is 8000-10000 r / min, the temperature is 4° C., and the time is 20 minutes.

10. The use according to claim 7, characterized in that: The dosage of the Sipunculus quadrilateral polypeptide is 100-800 mg / kg.

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