Preparation of Sipunculus nudus Hypoglycemic Peptide and Its Application in the Preparation of Drugs for Regulating Blood Glucose and Blood Lipids in Type 2 Diabetes

Abstract

The present invention discloses the preparation of Sipunculus nudus hypoglycemic peptide and its application in the preparation of drugs for regulating blood glucose and blood lipids in type 2 diabetes, belonging to the field of biomedical technology. The Sipunculus nudus polypeptide provided by the present invention can significantly reduce the blood glucose concentration of T2DM, regulate the level of insulin resistance, inhibit the levels of triglyceride and low-density lipoprotein cholesterol in the serum of T2DM, increase the level of high-density lipoprotein cholesterol, and at the same time has a protective effect on the pathological changes of pancreatic tissue. In addition, administration of Sipunculus nudus polypeptide can increase the activity of SOD in the serum and inhibit the expression of malondialdehyde, a lipid peroxidation marker. The Sipunculus nudus polypeptide can significantly improve the abnormal blood glucose and blood lipids in type 2 diabetes, and has no toxicity or side effects on animals and will not accumulate in animals. Therefore, the Sipunculus nudus polypeptide can be used to prepare drugs or foods for regulating blood glucose and blood lipids in type 2 diabetes.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the preparation of Sipunculus nudus hypoglycemic peptide and its application in the preparation of drugs for regulating blood glucose and blood lipids in type 2 diabetes mellitus. Background Art

[0002] Diabetes is an endocrine and metabolic disease caused by various etiologies, mainly characterized by chronic hyperglycemia and accompanied by abnormal metabolism of carbohydrates, fats, and proteins caused by insulin secretion and / or action defects, which seriously endangers human health. The most common type is type 2 diabetes mellitus (T2DM), accounting for more than 90% of diabetic patients. Epidemiological studies show that the number of global diabetes patients has increased from 108 million to 422 million, among which 103 million are in China, accounting for 24.4% of the global total. It is urgent to prevent and delay the occurrence and development of T2DM and its complications. Studies have found that in addition to hyperglycemia, most T2DM patients also have problems such as lipid metabolism disorders. Abnormal blood lipid levels for a long time will reduce insulin secretion, affect the physiological activity of insulin, lead to disorders of glucose and lipid metabolism, and at the same time will also cause atherosclerosis, coronary heart disease, etc. In addition, studies have found that oxidative stress is highly correlated with the occurrence and development of diabetes. Reactive oxygen free radicals and the oxidative reactions induced by them may be important factors leading to various chronic diseases such as diabetes and metabolic syndrome. Therefore, antioxidant therapy can effectively delay the progression of diabetes and reduce the occurrence of complications.

[0003] Sipunculus nudus, also known as "naked sipunculus", "sand worm", etc., has delicious meat and rich nutrition, and is known as the "marine cordyceps sinensis". According to "Chinese Marine Materia Medica", Sipunculus nudus tastes salty, is cold in nature, and belongs to the lung and kidney meridians, and has the effects of nourishing yin and reducing fire, clearing the lung and tonifying deficiency, promoting blood circulation and strengthening the body, and tonifying the kidney and beautifying the face. Sipunculus nudus polypeptide (SNP) is an enzymatic hydrolysis product obtained after the hydrolysis of Sipunculus nudus by trypsin. Modern pharmacological studies have found that SNP has the characteristics of scavenging free radicals, antioxidant, anti-inflammatory, anti-tumor, anti-thrombotic, improving memory, anti-radiation, anti-hypoxia, accelerating wound healing, and regulating immune function. However, there are few research reports on the improvement of diabetic symptoms by SNP, and the mechanism of its effect on insulin resistance in T2DM is still unclear. Summary of the Invention

[0004] The purpose of the present invention is to provide the preparation of Sipunculus nudus hypoglycemic peptide and its application in the preparation of drugs for regulating blood glucose and blood lipids in type 2 diabetes mellitus, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a sipunculus nudus polypeptide, which is prepared by enzymolysis of sipunculus nudus with trypsin and purification.

[0007] Another technical solution of the present invention is the application of the sipunculus nudus polypeptide in the preparation of a drug for treating type 2 diabetes.

[0008] A further technical solution of the present invention is a drug for treating type 2 diabetes, which includes the sipunculus nudus polypeptide.

[0009] Another technical solution of the present invention is the application of the sipunculus nudus polypeptide in the preparation of a drug for reducing the levels of triglyceride and low-density lipoprotein cholesterol in serum.

[0010] Another technical solution of the present invention is the application of the sipunculus nudus polypeptide in the preparation of a drug for increasing the level of high-density lipoprotein cholesterol in serum.

[0011] Another technical solution of the present invention is the application of the sipunculus nudus polypeptide in the preparation of a drug for improving pancreatic tissue lesions.

[0012] Another technical solution of the present invention is the application of the sipunculus nudus polypeptide in the preparation of a drug for increasing the activity of SOD in serum.

[0013] Another technical solution of the present invention is the application of the sipunculus nudus polypeptide in the preparation of a drug for inhibiting the synthesis of MDA.

[0014] Based on the above technical solutions, the present invention has the following technical effects:

[0015] The sipunculus nudus polypeptide provided by the present invention can significantly reduce the blood glucose concentration of T2DM, regulate the insulin resistance level, inhibit the levels of triacylglycerol (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum of T2DM, increase the level of high-density lipoprotein cholesterol (HDL-C), and at the same time has a protective effect on the pathological changes of pancreatic tissue. In addition, administration of the sipunculus nudus polypeptide can increase the activity of SOD in serum and inhibit the expression of lipid peroxidation marker malondialdehyde (MDA). The sipunculus nudus polypeptide (SRP) can significantly improve the blood glucose and lipid abnormalities of type 2 diabetes, and has no toxicity or side effects on animals and will not accumulate in animals. Therefore, the sipunculus nudus polypeptide can be used to prepare drugs or foods for regulating the blood glucose and lipids of type 2 diabetes. Description of the Drawings

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figure 1 It is the chromatogram of the molecular mass distribution of Sipunculus nudus polypeptides;

[0018] Figure 2 It is the ion current spectrum of UPLC-MS mass spectrometry of Sipunculus nudus polypeptides;

[0019] Figure 3 It is the improvement result of SNP on the water intake and food intake of T2DM mice. Among them, A is the water intake of mice; B is the food intake of mice; the results are expressed as mean ± SD (n = 6); compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, P < 0.001;

[0020] Figure 4 It is the regulation result of SNP on the body weight (A) and fasting blood glucose (B) of T2DM mice (n = 6); the results are expressed as mean ± SD (n = 6); compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, P < 0.001;

[0021] Figure 5 It is the improvement result of SNP on insulin resistance in T2DM mice (n = 5). Among them, A is the result chart of insulin content in the serum of mice in each group; B is the result chart of insulin resistance index in each group; compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, P < 0.001;

[0022] Figure 6 It is the regulation result of SNP on the blood lipid metabolism level in the serum of T2DM mice (n = 4). Among them, A is the concentration chart of triglycerides in the serum of mice in each group; B is the concentration chart of low-density lipoprotein in the serum of mice in each group; C is the concentration chart of high-density lipoprotein in the serum of mice in each group; compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group, #P < 0.05, ##P < 0.01, P < 0.001;

[0023] Figure 7Effects of SNP on oxidative indexes in serum of T2DM mice (n = 4). In the figure, A shows the results of superoxide dismutase activity in serum of mice in each group; B shows the results of malondialdehyde concentration in serum of mice in each group. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, P<0.001;

[0024] Figure 8 Improvement results of SNP on pancreatic tissue damage in T2DM mice (HE staining pictures). Detailed implementation manners

[0025] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation manners of the present invention.

[0026] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of this application are only exemplary.

[0029] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are intended to include but not limited to.

[0030] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified. The reagents or raw materials used are all purchased from commercial channels or have been made public unless otherwise specified.

[0031] An embodiment of the present invention provides a sipunculus nudus polypeptide, which is prepared by enzymatically hydrolyzing and purifying sipunculus nudus with trypsin.

[0032] In some specific embodiments, the enzymatic hydrolysis and purification of the sipunculus nudus with trypsin are specifically as follows:

[0033] The fresh sipunculus nudus is desanded, washed, and pulped. Deionized water is added according to a material ratio of 1:10, and trypsin is added for enzymatic hydrolysis. After cooling, centrifugation is performed, and the supernatant is taken.

[0034] Under the condition of an ice bath at 4°C, the supernatant is ultrafiltered through ultrafiltration membranes with molecular weights of 50 kDa and 2.5 kDa step by step. The ultrafiltration fraction with a molecular weight less than 2.5 kDa is retained and collected. This fraction is passed through a Millipore 4040 type reverse osmosis membrane, and the finally obtained filtrate is SNP.

[0035] In some specific embodiments, the conditions for the enzymatic hydrolysis are as follows: the dosage of trypsin is 4000 U / g, the pH is 7.5, the temperature is 45°C, and the time is 4 h.

[0036] In some specific embodiments, the centrifugation conditions are as follows: 10000 r / min, the temperature is 4°C, and the time is 20 min.

[0037] In some specific embodiments, the sipunculus nudus polypeptide is composed of 10 polypeptides, and their amino acid sequences are respectively: RQPRG, RATLPRM, RQPRG, RKDRL, MFKPMRK, RDPALRR, KCRP, MLMPKR, KPHRYRP, RATLPRM.

[0038] An embodiment of the present invention provides the application of the sipunculus nudus polypeptide in the preparation of a drug for treating type 2 diabetes.

[0039] An embodiment of the present invention provides a drug for treating type 2 diabetes, which includes the sipunculus nudus polypeptide.

[0040] Furthermore, it also includes pharmaceutically acceptable excipients.

[0041] An embodiment of the present invention provides the application of the sipunculus nudus polypeptide in the preparation of a drug for reducing the levels of triglyceride and low-density lipoprotein cholesterol in serum.

[0042] An embodiment of the present invention provides the application of the sipunculus nudus polypeptide in the preparation of a drug for increasing the level of high-density lipoprotein cholesterol in serum.

[0043] An embodiment of the present invention provides the application of the sipunculus nudus polypeptide in the preparation of a drug for improving pancreatic tissue lesions.

[0044] An embodiment of the present invention provides the application of the sipunculus nudus polypeptide in the preparation of a drug for increasing the SOD activity in serum.

[0045] An embodiment of the present invention provides the application of the sipunculus nudus polypeptide in the preparation of a drug for inhibiting MDA synthesis.

[0046] The materials and reagents used in this embodiment are as follows:

[0047] Fresh sipunculus nudus was purchased from Dongfeng Aquatic Product Market, Xiasha District, Zhanjiang City, Guangdong Province, mainly selecting sipunculus nudus produced in Xiaoliu Village, Caotan Town, Suixi County, Zhanjiang City; Trypsin (4000U / g, Nanning Pangbo Bioengineering Co., Ltd.); AX010-03-EK01 polyamide composite membrane (Xiamen Sida Membrane Technology Co., Ltd.); Streptozotocin (STZ) (batch number S8050, Beijing Solarbio Science & Technology Co., Ltd.); Malondialdehyde (MDA) kit, Superoxide Dismutase (SOD) kit (Quanzhou Ruixin Biotechnology Co., Ltd.); Serum Insulin (FINS) kit, Triglyceride (TG) kit, High-Density Lipoprotein (HDL-C) kit, Low-Density Lipoprotein (LDL-C) kit (Jiangsu Enzyme Immunoassay Industry Co., Ltd.); Hematoxylin and Eosin (HE) staining kit (Beyotime Biotechnology Co., Ltd.).

[0048] The instruments and equipment used in this embodiment are as follows: CH-209C food processor (Wanlida Group Co., Ltd.); 3H16RI intelligent high-speed refrigerated centrifuge (Huxi Instrument & Equipment Co., Ltd., Hunan); SPPM-44S(24S)-1 membrane separation system, 1812 spiral wound membrane equipment (Xiamen Sida Membrane Technology Co., Ltd.); Freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.); ICS-5000 ion chromatograph, CYTATION5 multifunctional microplate detection system (Biotek, USA); Mithras LB940 multifunctional microplate reader (Thermo, USA); Four-chamber integrated incubator (Jeiotech, Korea); Yuwell blood glucose tester, disposable alcohol cotton pads (Jiangsu Yuwell Medical Equipment Co., Ltd.); Disposable fingertip blood collection needles (Tianjin Huahong Technology Co., Ltd.); Paraffin embedding machine, microtome (Guangzhou Yuanqi Health Technology Co., Ltd.); Optical microscope (Olympus, Japan).

[0049] Example 1

[0050] Preparation of sipunculus nudus polypeptide

[0051] Enzymatic preparation of Sipunculus nudus polypeptides: Fresh Sipunculus nudus was washed to remove sand, weighed, preliminarily pulped, deionized water was added according to the material ratio of 1:10, the pH of the sample solution was adjusted to 7.5 with 0.1 mol / L NaOH, and trypsin was added for enzymatic hydrolysis (4000 U / g, 45 °C, 4 h). The enzyme was inactivated in a water bath at 90 °C for 10 min. After cooling, centrifugation was carried out (10000 r / min, 4 °C, 20 min), and the supernatant was taken for standby. Under the condition of ice bath at 4 °C, the enzymatic hydrolysate of Sipunculus nudus was ultrafiltered through ultrafiltration membranes with molecular weights of 50 kDa and 2.5 kDa step by step. The ultrafiltration fraction with <2.5 kDa was intercepted and collected, and this fraction was passed through a Millipore 4040 reverse osmosis membrane. The finally obtained filtrate was SNP, and after freeze-drying, SNP was stored at -80 °C in a refrigerator for standby.

[0052] Example 2

[0053] Determination of the molecular mass distribution of Sipunculus nudus polypeptides

[0054] The molecular weight distribution of Sipunculus nudus polypeptides was determined by high performance size exclusion chromatography (HPSEC). The chromatographic conditions were as follows: chromatographic column GE Superdex 30 Increase 10 / 300 GL, mobile phase was PBS buffer solution with a concentration of 0.05 mol / L and pH 7.2; elution speed was 0.5 mL / min; injection volume was 25 μL; detection wavelength was 220 nm. At the same time, glycine (molecular mass 75), triglycine (molecular mass 189), aprotinin (molecular mass 6500), and cytochrome C (molecular mass 12500) were used as molecular mass standards. With the retention volume (v) of these four peptides as the abscissa and the logarithm of molecular weight (LgM) as the ordinate, a standard curve was plotted. The regression equation was obtained: LgM = -0.2315v + 6.3412 (R 2 = 0.9949).

[0055] The molecular weight distribution of Sipunculus nudus polypeptides was determined by HPSEC. As Figure 1 shown in Table 1, the distribution of Sipunculus nudus polypeptides was mainly concentrated in the range of <2.5 kDa. Among them, small molecular peptides with <500 Da accounted for 94.96% of the total content, indicating that the enzymatic hydrolysis and ultrafiltration effect of Sipunculus nudus was good and the content of short peptides was high.

[0056] Table 1 Molecular mass distribution of Sipunculus nudus polypeptides

[0057]

[0058] Example 3

[0059] Analysis of Sipunculus nudus polypeptide segments:

[0060] (1) Purification of Sipunculus nudus polypeptides:

[0061] Take an appropriate amount of the sample and dissolve it in pure water to obtain a polypeptide solution. Add DTT solution to the appropriate amount of the solution sample to make its final concentration 10 mmol / L, and reduce it in a water bath at 56 °C for 1 h. Add IAA solution to make its final concentration 50 mmol / L, and react in the dark for 40 min. Use a self-packed desalting column for desalting treatment, and evaporate the solvent in a vacuum centrifugal concentrator at 45 °C.

[0062] (2) Separation of Sipunculus nudus polypeptides

[0063] Separate the purified Sipunculus nudus polypeptides by capillary ultra-high performance liquid chromatography (UPLC).

[0064] Use an analytical column: 150 μm i.d.×150 mm, packed with Acclaim PepMap RPLC C18, 3 μm. Separate the Sipunculus nudus polypeptides. During the separation process, mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% ACN; flow rate: 600 nL / min; analysis time: 60 min;

[0065] 75 - 78 min, 40% - 95%.

[0066] (3) Identification of Sipunculus nudus polypeptides

[0067] The separated peptide segments enter an electrospray - combined ion trap Orbitrap mass spectrometer Q Exactive TM HybridQuadrupole - Orbitrap TM Mass Spectrometer for on - line detection. The specific parameters are as follows:

[0068] Primary mass spectrometry parameters: Resolution: 70,000 AGC target: 3e6 Maximum IT: 60 ms Scan range: 300 to 1400 m / z;

[0069] Secondary mass spectrometry parameters: Resolution: 17,500 AGC target: 1e5 Maximum IT: 50 ms TopN: 20;

[0070] NCE / stepped NCE: 28.

[0071] (4) Database search

[0072] Search the target protein database with the original mass spectrometry files using Byonic. The search parameters are as follows:

[0073] Fixed modifications: Carbamidomethyl (C);

[0074] Variable modifications: Oxidation (M), Acetyl (Peptide N-term);

[0075] Enzyme: Trypsin;

[0076] Maximum Missed Cleavages: 3;

[0077] Peptide Mass Tolerance: 20 ppm;

[0078] Fragment Mass Tolerance: 0.02 Da.

[0079] Using mass spectrometry to identify the amino acid sequence of bioactive peptides is currently the most common method. After the Sipunculus nudus polypeptides are separated and purified and subjected to ESI / MS mass spectrometry, the ion current map of the mass spectrometry is as Figure 2 shown. Combining with the PEAKS software to deconvolute the mass spectrometry, the original data such as the first-order mass spectrometry and the second-order mass spectrometry are obtained to identify the amino acid sequence of the polypeptide. Finally, the amino acid sequence is compared with the known protein data Unipro to determine its origin. 41 polypeptides are identified by ESI / MS / MS, and their sequences are shown in Table 2.

[0080] Table 2 Polypeptide composition of Sipunculus nudus polypeptides

[0081]

[0082]

[0083]

[0084] Example 4

[0085] Screening of Sipunculus nudus hypoglycemic peptides:

[0086] Peptides identified by mass spectrometry with a score > 100 when compared with the Unipro database are peptides with a peptide sequence identification result confidence level > 95%. These peptides were first subjected to PeptideRanker bioactivity prediction at http: / / distilldeep.ucd.ie / PeptideRanker / . According to the reference, a PeptideRanker prediction value > 0.5 was considered to have good bioactivity potential. Subsequently, cell membrane permeability prediction was performed at http: / / distilldeep.ucd.ie / CPPpred / . According to the reference, a PeptideRanker prediction value > 0.5 was considered to have good cell membrane permeability. Finally, the AutoDockVina 1.1.2 software was used to perform molecular docking of these peptides with dipeptidyl peptidase-IV (DPP-IV, uniprotID: 2QT9). According to the software prediction results, a peptide with a binding energy < -6 Kcal / mol to DPP-IV was considered to have a tight binding.

[0087] Peptide Ranker and CPPpred predictions were performed on the distribution of 41 polypeptides identified by mass spectrometry. Then, the AutoDock Vina 1.1.2 software was used to perform molecular docking of these peptides with dipeptidyl peptidase-IV (DPP-IV, uniprot ID: 2QT9). A total of 10 small molecule peptides with hypoglycemic activity were screened out as shown in Table 3.

[0088] Table 3 Screening results of hypoglycemic peptides from Sipunculus nudus

[0089]

[0090] Example 5

[0091] 1. Improvement effect of Sipunculus nudus polypeptides on blood glucose and lipid abnormalities in type 2 diabetic mice

[0092] Forty 6-week-old male ICR mice were divided into 4 cages, with 10 mice in each cage. The mice were allowed to adapt to the new environment for one week to ensure that they could freely access food and water. First, the mice were fed a high-sugar and high-fat diet for 6 weeks. Starting from the 7th week, in addition to continuing the high-sugar and high-fat diet, the mice were fasted overnight once a week and then quickly intraperitoneally injected with a freshly prepared STZ solution (30 mg / kg) for 4 consecutive weeks. Normal control mice were intraperitoneally injected with an equal volume of citrate buffer. After modeling, the mice were bled by tail vein puncture to detect fasting blood glucose (FBG). An FBG ≥ 11.1 mmol / L was considered a successful T2DM model.

[0093] After that, the mice were divided into 4 groups according to the average body weight and blood glucose, with 10 mice in each group:

[0094] Blank control group: gavaged with normal diet + 0.9% normal saline;

[0095] Model group: gavaged with high-sugar and high-fat diet + 0.9% normal saline;

[0096] SNP low-dose group: gavaged with high-sugar and high-fat diet + low-dose SNP (100 mg / kg);

[0097] SNP high-dose group: gavaged with high-sugar and high-fat diet + high-dose SNP (400 mg / kg).

[0098] Continuous gavage administration was carried out for 6 weeks. From the 1st, 2nd, 3rd, 4th, 5th, and 6th weeks after the administration, the water intake and food intake of each group of mice were weighed at the same time every week. Body weight and fasting blood glucose measurement: The mice were fasted for 12 h at the 1st, 2nd, 3rd, 4th, 5th, and 6th weeks after the administration, and the body weight of the mice was weighed at the same time. The fasting blood glucose level of the mice was measured by using a blood glucose meter and a blood glucose test strip to collect blood from the tip of the tail.

[0099] 2. Detection of blood biochemical indexes

[0100] Blood was taken from the mouse eyeballs. After the blood was placed in a 4°C refrigerator and allowed to stand for 4 h, it was centrifuged for 10 min at a speed of 15,000 r / min using a centrifuge to obtain serum. Commercial kits were used to detect the concentrations of FINS, TG, HDL, LDL, and MDA in the serum, and commercial kits were used to detect the activity of superoxide dismutase SOD in the serum. The specific experimental operations were carried out according to the product instructions. The calculation formula for the insulin resistance index is as follows (HOMA-insulin resistance index, HOMA-IR) = cFBG × cFINS / 22.5.

[0101] 3. HE pathological staining of pancreatic tissue

[0102] After removing the mouse pancreas, the tissue was fixed in 4% paraformaldehyde. After 24 h, the pancreatic tissue was appropriately trimmed, dehydrated in a gradient of ethanol, embedded in paraffin, and the tissue was cut into about 4 μm thickness on a microtome. Staining was performed using an HE kit. The specific staining process of the sections was referred to the product instructions of the kit. The pathological changes of the pancreatic tissue were observed under an optical microscope.

[0103] Experimental results:

[0104] SNP improved the symptoms of polydipsia and polyphagia in T2DM mice:

[0105] Such as Figure 3As shown in Figure A, compared with the control group, the water intake of the mice in the model group increased significantly (P<0.05, P<0.01). After intervention with different doses of SNP, the water intake of the mice began to decrease at the 4th week of administration (P<0.01). After six weeks of intragastric administration of different doses of SNP, the mice recovered to the level of the normal control group. See Figure 3 In Figure B, the food intake of the mice in the model group continued to increase with time, while that of the blank group remained relatively stable (P<0.01). After administration of SNP, the food intake of the high-dose group decreased (P<0.01), suggesting that SNP can improve the symptoms of polyphagia and polydipsia in T2DM mice.

[0106] SNP can regulate the body weight and fasting blood glucose of T2DM mice:

[0107] See Figure 4 In Figure A, although the mice in the model group had polyphagia and polydipsia all the time, their body weight was significantly lower than that of the mice in the blank control group (P<0.05, P<0.01). Three weeks after administration of different doses of SNP to T2DM mice, the body weight increased significantly compared with that of the mice in the model group (P<0.05, P<0.01). Fasting blood glucose is the most basic and commonly used index for detecting diabetes, reflecting the function of pancreatic islet cells and the real-time blood glucose level of the body. As Figure 4 shown in Figure B, before administration intervention, the fasting blood glucose values of the diabetic mice were all greater than 11.1 mmol / L and were significantly higher than those of the blank control group (P<0.05), indicating that the T2DM model was successful. The fasting blood glucose level of the mice in the model group was significantly higher than that of the normal control group (P<0.01), and the blood glucose value remained at a very high level. However, the fasting blood glucose values of the mice in the SNP groups with different doses began to decrease at the 2nd week of intragastric administration (P<0.05, P<0.01) and continued until the 6th week. By the 6th week, the fasting blood glucose values of the SNP groups were significantly lower than those of the model group.

[0108] SNP can improve insulin resistance in T2DM mice:

[0109] The effect of SNP on insulin resistance is as Figure 5 shown. Compared with the blank group, the FINS and HOMA-IR of the mice in the model group were significantly increased (P<0.001). After administration of SNP, the FINS and HOMA-IR of the mice were significantly decreased (P<0.05, P<0.01, P<0.001). It shows that SNP has an improving effect on insulin resistance in T2DM mice.

[0110] SNP can regulate the blood lipid metabolism level in the serum of T2DM mice:

[0111] As Figure 6As shown, compared with the control group of mice, the concentrations of TG and LDL in the serum of the model group of mice were significantly increased (P<0.05, P<0.001). After treatment with low-dose SNP, the TG level in the serum of T2DM mice decreased significantly. Administration of low-dose and high-dose SNP both significantly reduced the LDL concentration in the serum of T2DM mice (P<0.01, P<0.001). On the contrary, compared with the control group of mice, the HDL level in the serum of the model group of mice was significantly decreased (P<0.001), and high-dose SNP administration significantly increased the HDL concentration in T2DM mice (P<0.001). By detecting the blood lipid metabolism level of mouse serum, it was found that SNP had an improving effect on the blood lipid metabolism disorder caused by diabetes.

[0112] SNP can improve the oxidative stress response in the serum of T2DM mice:

[0113] As Figure 7 shown in A, the activity of SOD in the serum of the model group of mice was significantly lower than that of the blank control group (P<0.001), and administration of low- and high-dose SNP significantly increased the SOD activity in T2DM mice (P<0.001). Figure 7 In B, the content of MDA in the serum of the model group of mice was significantly higher than that of the blank control group (P<0.01), while high-dose SNP administration could reduce the MDA level in T2DM mice (P<0.01). By detecting the indexes such as SOD and MDA in mouse serum, it was found that SNP could enhance the antioxidant ability of mice and reduce their oxidative stress level.

[0114] SNP can improve the damage of pancreatic tissue in T2DM mice:

[0115] As Figure 8 shown, there were no obvious lesions in the pancreatic tissue, islets and islet cells of the blank control group of mice. However, in the pancreatic tissue of the model group of mice, vacuolar degeneration of islets, disordered structure, obvious reduction and uneven distribution of islet cells, and blurred boundaries were visible, indicating that there was certain damage to the pancreatic tissue of diabetic mice. After administration of SNP, the morphology of the pancreatic tissue of mice improved, and the islet cells also increased significantly. It is suggested that SNP has an improving effect on the pathological damage of pancreatic tissue in T2DM mice.

[0116] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly explaining the present invention, and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of Sipunculus fangqie polypeptide in the preparation of a drug for treating type 2 diabetes, characterized in that: The Sipuncula sutchuenensis polypeptide is prepared by hydrolyzing and purifying Sipuncula sutchuenensis with trypsin; The Sipunculus quadripunctatus is hydrolyzed and purified by trypsin in detail as follows: Fresh Siempuri worms were cleaned and beaten after sand removal, deionized water was added at a material ratio of 1:10, trypsin was added for enzymatic hydrolysis, and the mixture was centrifuged after cooling to obtain the supernatant; the enzymatic hydrolysis conditions were as follows: the amount of trypsin was 4000 U / g, the pH was 7.5, the temperature was 45°C, and the time was 4h; the centrifugal conditions were as follows: 10,000 r / min, the temperature was 4°C, and the time was 20 min; Under ice bath conditions at 4 ℃, the supernatant was ultrafiltered step by step through 50 kDa and 2.5 kDa ultrafiltration membranes, and the ultrafiltration fractions <2.5 kDa were intercepted and collected. The fractions were passed through a Millipore 4040 reverse osmosis membrane, and the filtrate was finally obtained, which was the Sipunculus polypeptide.

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