Application of pancreatic-derived protein peptide in preparation of product for improving insulin resistance and repairing pancreatic function

Abstract

The invention discloses application of pancreatic protein peptide in preparation of products for improving insulin resistance and repairing pancreatic functions, and relates to the technical field of biological medicines. The pancreatic-derived protein peptide is extracted and purified from porcine pancreatic tissue through a specific enzymolysis process, the molecular weight is concentrated at 800-2000Da, and the pancreatic-derived protein peptide contains 12 essential amino acids. Experiments prove that the protein peptide can significantly improve insulin sensitivity and promote pancreatic beta cell proliferation through oral administration or intraperitoneal injection, and has no obvious toxic or side effect. The invention provides a novel treatment means for diseases related to type 2 diabetes and pancreatic function impairment, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of pancreatic protein peptide in preparation of a product for improving insulin resistance and repairing pancreatic function. BACKGROUND

[0002] Insulin resistance refers to a state in which the sensitivity of body tissues to insulin is reduced, and a normal dose of insulin produces a lower-than-normal biological effect. In order to maintain normal blood glucose levels, the pancreatic islet beta cells have to compensate by secreting more insulin, thereby causing hyperinsulinemia. Over time, the function of the islet beta cells is gradually impaired, and insulin secretion is insufficient, ultimately leading to diabetes. Insulin resistance is not only an important pathological basis for the onset of type 2 diabetes, but is also closely related to the occurrence and development of many diseases such as metabolic syndrome, cardiovascular disease, polycystic ovary syndrome, etc. For example, insulin resistance can lead to lipid metabolism disorders, increase the risk of atherosclerosis and cardiovascular disease, and increase the levels of triglycerides and low-density lipoprotein cholesterol in the blood, and decrease the level of high-density lipoprotein cholesterol in the blood; in patients with polycystic ovary syndrome, insulin resistance can cause hyperandrogenism, leading to menstrual disorders, infertility and other problems.

[0003] The pancreas is an important digestive and endocrine organ of the human body, and its impaired function directly affects the normal secretion of insulin. The islet beta cells in the pancreas are responsible for the synthesis and secretion of insulin, and when the beta cells are damaged by various factors (such as genetics, environment, immunity, etc.), the amount of insulin secretion is reduced or the secretion pattern is abnormal, and the body's demand for blood glucose regulation cannot be met, thereby leading to elevated blood glucose. In addition, exocrine pancreatic dysfunction can also indirectly affect glucose metabolism, because the digestive enzymes secreted by the pancreas are essential for the digestion and absorption of food, and abnormal digestion can lead to poor absorption of nutrients, affecting energy metabolism and blood glucose stability.

[0004] Current clinical intervention methods have significant defects: (1) Chemical drug therapy: although metformin can improve insulin resistance, it has no repair effect on pancreatic function, and 30% of patients experience gastrointestinal adverse reactions; glipizide and other secretagogues can easily cause hypoglycemia, and long-term use can accelerate beta cell apoptosis; (2) Lifestyle intervention: although diet control and exercise can delay disease progression, they cannot reverse damaged pancreatic tissue, and patient compliance is poor (long-term compliance rate is less than 25%); (3) Existing biological agents: insulin can only be used for replacement therapy and cannot improve the resistance state; glucagon-like peptide-1 (GLP-1) receptor agonists are expensive, require daily injection, and have limited effect on patients with severe pancreatic damage.

[0005] In summary, the prior art is difficult to achieve the dual goals of improving resistance and repair function at the same time, and there is an urgent need to develop new high-efficiency and high-safety bioactive substances. SUMMARY

[0006] The purpose of the present application is to provide an application of pancreatic protein peptide in the preparation of a product for improving insulin resistance and repairing pancreatic function, to clarify the role and mechanism of pancreatic protein peptide in improving insulin resistance and repairing pancreatic function, and to develop a convenient preparation based on the protein peptide to solve the core defects of the prior art that cannot improve resistance and repair function at the same time.

[0007] To achieve the above-mentioned purpose, the present application provides an application of pancreatic protein peptide in the preparation of a product for improving insulin resistance and repairing pancreatic function, wherein the pancreatic protein peptide is derived from pig pancreatic tissue and is prepared by a complex enzymatic hydrolysis-ultrafiltration process, has a molecular weight distribution of 800-2000 Da, and contains Val-Pro-Leu and Gly-Phe-Met active fragments in the amino acid sequence, with a purity of ≥95%.

[0008] Preferably, the complex enzymatic hydrolysis-ultrafiltration process specifically includes the following steps: S1, raw material pretreatment: taking fresh pig pancreas, removing fat tissue, homogenizing after ultra-high pressure treatment; S2, enzymatic hydrolysis: adding trypsin according to an enzyme-substrate ratio of 1:50, and then adding papain according to an enzyme-substrate ratio of 1:80, stepwise enzymatic hydrolysis, total enzymatic hydrolysis time 6h; S3, purification: the enzymatic hydrolysate is subjected to impurity removal by a 10kDa ultrafiltration membrane, and then the target component is enriched by a 3kDa ultrafiltration membrane, and the finished product is obtained by freeze-drying at-50℃ under a vacuum degree of 10Pa.

[0009] Preferably, in S2, the trypsin enzymatic hydrolysis temperature is 45℃ and the pH is 7.2.

[0010] Preferably, the papain enzymatic hydrolysis temperature is 50℃ and the pH is 6.5.

[0011] Preferably, the product is an insulin resistance improving drug, the administration dose is 100-200mg / kg·d, and the administration route is oral or intraperitoneal injection.

[0012] Preferably, the product is a pancreatic function repairing preparation, which can promote the proliferation of pancreatic islet beta cells, increase the insulin secretion amount, and reduce the levels of pancreatic tissue inflammatory factors TNF-α and IL-6.

[0013] Therefore, the application of pancreatic protein peptide in the preparation of a product for improving insulin resistance and repairing pancreatic function has the following beneficial effects: (1) Pancreatic protein peptides can achieve the dual goals of improving insulin resistance and repairing pancreatic function: on the one hand, they significantly improve insulin sensitivity by activating the AMPK signaling pathway. Animal experiments have shown that they can reduce the HOMA-IR index of insulin-resistant mice from 5.8 to 3.6, with a sensitivity improvement rate of up to 38.2%. On the other hand, they promote the proliferation and functional recovery of pancreatic β cells by upregulating the expression of the anti-apoptotic protein Bcl-2. They increase the number of β cells in pancreatic function-damaged mice by 68.9% and reduce the level of TNF-α, an inflammatory factor in pancreatic tissue, by 59.0%.

[0014] (2) Acute toxicity tests showed that mice administered 5000 mg / kg of the protein peptide orally did not experience any deaths or abnormal reactions within 14 days. Autopsies revealed no pathological damage to major organs such as the liver, kidneys, and pancreas, indicating that the pancreatic-derived protein peptide has high safety and meets the requirements for clinical application. (3) The composite enzymatic hydrolysis-ultrafiltration process is adopted, using fresh pig pancreas as raw material. Through stepwise enzymatic hydrolysis with trypsin and papain and enrichment by two-stage ultrafiltration membrane, the target peptide extraction rate reaches 18.2%, which is 114% higher than the traditional acid extraction method (8.5%). No chemical reagents are added throughout the process. The purity of the finished product is 96.5% as detected by HPLC. It still maintains a high purity of 95.1% after being sealed and stored at 4℃ for 6 months. Moreover, the raw materials are widely available and inexpensive, making it suitable for large-scale industrial production.

[0015] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0016] The technical solution of the present invention will be further described below through embodiments.

[0017] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention are clearly and completely described below through embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanations of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0018] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0019] Example 1 A method for preparing a pancreatic protein peptide, comprising the following steps: S1. Raw material preparation: Take 500g of fresh pig pancreas, remove the fatty tissue and cut into 1cm pieces. 3 Rinse the small pieces three times with 0.9% saline solution and drain.

[0020] S2. Ultra-high pressure treatment: Place the pancreatic block into an ultra-high pressure treatment vessel and maintain it at a pressure of 300MPa for 5 minutes. After taking it out, homogenize it with a tissue homogenizer at 8000r / min for 5 minutes.

[0021] S3. Enzymatic hydrolysis: Transfer the homogenate to a 5L enzymatic hydrolysis tank, add phosphate buffer (pH 7.2) to adjust the pH of the system, add 2000 U / mg trypsin at an enzyme-substrate ratio of 1:50, and hydrolyze at 45℃ for 3 hours. Then adjust the pH to 6.5 with 1 mol / L hydrochloric acid, add 800 U / mg papain at an enzyme-substrate ratio of 1:80, and continue enzymatic hydrolysis at 50℃ for 3 hours.

[0022] S4. Enzyme inactivation: After the enzymatic hydrolysis is completed, the hydrolysate is heated to 90°C and kept at this temperature for 15 minutes to inactivate the enzyme activity.

[0023] S5. Purification: After cooling to room temperature, use 0.22... Microporous membrane filtration was performed, and the filtrate was sequentially passed through 10kDa and 3kDa ultrafiltration membranes (operating pressure 0.2MPa, temperature 30℃), and the 3kDa permeate was collected.

[0024] S6. Drying: The permeate was placed in a freeze dryer and pre-frozen at -50°C for 2 hours, and then dried under a vacuum of 10 Pa for 12 hours to obtain 89.2 g of white powdery pancreatic protein peptides. The purity was determined to be 96.5% by high performance liquid chromatography (HPLC).

[0025] Molecular weight determination: High-performance gel filtration chromatography (GFC) was used, with standard molecular weight proteins (1000Da, 2000Da, 5000Da) as references. The molecular weight of the target peptide was found to be 1200Da, and the proportion of peptides in the 800-2000Da range reached 92.3%.

[0026] Amino acid sequence analysis: The Edman degradation method confirmed the presence of Val-Pro-Leu (VPL) and Gly-Phe-Met (GFM) active fragments, with the total content of 12 essential amino acids accounting for 42.6%.

[0027] Purity and stability: The purity was 96.5% as determined by HPLC (C18 column, mobile phase acetonitrile-water = 30:70, detection wavelength 220nm); after being stored in a sealed container at 4℃ for 6 months, the purity remained at 95.1%, with no degradation products generated.

[0028] Example 2 Animal studies demonstrating the improvement of insulin resistance by pancreatic protein peptides: Experimental animals: 60 SPF-grade male C57BL / 6 mice, weighing 20-22g, were acclimatized for 1 week.

[0029] Model establishment: An insulin resistance model was established by feeding the animal with a high-fat diet (60% fat content) for 8 weeks.

[0030] Grouping: Randomly divided into 4 groups (n=15): ① Normal control group: fed with normal feed and administered physiological saline by gavage; ② Model control group: fed a high-fat diet and administered physiological saline by gavage; ③ Low-dose group: fed with high-fat diet, and administered pancreatic protein peptides at 50 mg / kg·d by gavage; ④ High-dose group: fed with high-fat diet, and administered pancreatic protein peptides at 200 mg / kg·d by gavage; Dosing cycle: Administer the medication continuously for 4 weeks, monitoring relevant indicators weekly. ① Fasting blood glucose: The glucose oxidase method was used to detect the tail vein blood glucose of mice after fasting for 12 hours using a fully automated biochemical analyzer; ② Fasting insulin: Serum insulin levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit in mice; ③ Calculation of the Homeostasis Model Assessment of Insulin Resistance Index (HOMA-IR Index): ; Among them, fasting blood glucose is measured in mmol / L, and fasting insulin is measured in mU / L; ④ Insulin sensitivity improvement rate, calculated using the following formula: ; Among them, HOMA-IR1 refers to the HOMA-IR after administration to the model control group, and HOMA-IR2 refers to the HOMA-IR after administration to the experimental group.

[0031] The test results are shown in Table 1 below: Table 1. Effects of pancreatic protein peptides on HOMA-IR index and insulin sensitivity in insulin-resistant mouse models.

[0032] The data in Table 1 are expressed as mean ± standard deviation. Compared with the model control group, the high-dose group... P <0.01.

[0033] As shown in Table 1, pancreatic protein peptides can significantly reduce the insulin resistance index in a dose-dependent manner, with the high-dose group showing the best effect.

[0034] Example 3 Animal experiments on the repair of pancreatic function by pancreatic protein peptides: Experimental animals: SPF-grade male C57BL / 6 mice, weighing 20-22g. A pancreatic function impairment model (insulin secretion reduced by 50% compared to the normal group) was established by intraperitoneal injection of streptozotocin (STZ, 40mg / kg) combined with a high-fat diet.

[0035] Grouping: Randomly divided into 4 groups (n=12): ① Normal control group: physiological saline injection + normal feed; ② Model control group: STZ injection + high-fat diet + intraperitoneal injection of physiological saline; ③ Drug control group: STZ injection + high-fat diet + metformin 200mg / kg·d by gavage; ④ Protein peptide group: STZ injection + high-fat diet + intraperitoneal injection of pancreatic protein peptide 200mg / kg·d; Dosing cycle: 6 weeks, with pancreatic function indicators measured after the last dose.

[0036] ① Number of pancreatic β cells: Pancreatic tissue was collected after mouse sacrifice, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned (5 μm thick). Immunohistochemical staining was performed using the SP method. The primary antibody was rabbit anti-mouse insulin monoclonal antibody, and the secondary antibody was goat anti-rabbit IgG (DAB staining). Five pancreatic islet fields were randomly selected under a light microscope (×200), and the number of insulin-positive cells (brownish-yellow staining) was counted. The average number of cells / mm² was calculated. 2 ).

[0037] ②Insulin secretion: Pancreatic tissue was homogenized, centrifuged (3000 r / min, 10 min), and the supernatant was collected. The supernatant was detected by ELISA using a mouse insulin quantitative kit and an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance value at a wavelength of 450 nm was measured, and the concentration was calculated according to the standard curve.

[0038] ③ Pancreatic TNF-α level: The double-antibody sandwich ELISA method was used with a mouse TNF-α detection kit. The operation was strictly in accordance with the kit instructions. The detection wavelength was 450nm, and the concentration value was calculated by converting the standard curve.

[0039] The test results are shown in Table 2 below: Table 2 Results of pancreatic function indicators

[0040] Note: Compared with the model control group, the differences in all indicators of the protein peptide group were statistically significant. P <0.01).

[0041] As shown in Table 2, pancreatic protein peptides can significantly increase the number of pancreatic β cells, increase insulin secretion, and reduce the level of pancreatic inflammatory factors, and their effect on repairing pancreatic function is better than that of metformin.

[0042] Example 4 Forty SPF-grade ICR mice (half male and half female) were administered pancreatic protein peptides by gavage at a dose of 5000 mg / kg once, and the mice were observed for 14 consecutive days.

[0043] The results showed that none of the mice died, and their mental state, diet, and weight gain were normal. No pathological damage was found in the major organs such as the liver, kidneys, and pancreas after dissection.

[0044] Conclusion: The pancreatic protein peptides prepared in Example 1 have extremely low acute toxicity and high safety.

[0045] Example 5 A mouse pancreatic β-cell line MIN6 was cultured in vitro and treated with high glucose (30 mmol / L) for 48 h to establish an insulin secretion dysfunction model.

[0046] The study included a normal control group (low-glucose medium, 25 mmol / L), a model control group (high-glucose medium), a protein peptide group (high-glucose medium + 200 μg / mL pancreatic protein peptide), and an AMPK inhibitor group (high-glucose medium + 200 μg / mL protein peptide + 10 μmol / L Compound C), with six replicates per group. Cells and culture medium were collected after 48 hours of intervention for assay.

[0047] ①AMPK phosphorylation level and Bcl-2 protein expression: Western blotting was used to detect the protein expression. After collecting cells from each group, total protein was extracted with RIPA lysis buffer, and protein concentration was quantified by BCA method. 50 μg of protein was subjected to SDS-PAGE electrophoresis, transferred to PVDF membrane, and blocked with 5% skim milk for 1 h. Primary antibody incubation (rabbit anti-p-AMPK antibody, ab23875, rabbit anti-AMPK antibody, ab32047, rabbit anti-Bcl-2 antibody, ab182858, all purchased from Abcam) was performed at 4°C overnight, and secondary antibody incubation (goat anti-rabbit IgG, ab6721, Abcam) was performed at room temperature for 1 h. ECL chemiluminescence reagent was used for development, and the gray value of the bands was quantified using ImageJ software. The relative protein expression level was expressed as the gray value ratio of the target protein to the internal control protein (GAPDH, ab9485, Abcam).

[0048] ②Insulin secretion: Cell culture medium was collected and detected by ELISA. The absorbance at 450 nm was measured using a mouse insulin detection kit (product number: CSB-E08070m, Wuhan Huamei Biotechnology) and an enzyme-linked immunosorbent assay reader (model: Bio-Tek ELx800). Insulin concentration was calculated based on the standard curve.

[0049] The test results are shown in Table 3 below: Table 3. Effects of pancreatic protein peptides on AMPK signaling pathway and functional indices in MIN6 cells under high glucose stress.

[0050] Note: Compared with the model control group, P <0.01; AMPK is adenosine monophosphate activated protein kinase, and Bcl-2 is an anti-apoptotic protein.

[0051] It is known that pancreatic protein peptides can promote the recovery of insulin secretion function in pancreatic β cells damaged by high glucose by activating the AMPK signaling pathway (increasing phosphorylation level) and upregulating the expression of the anti-apoptotic protein Bcl-2. When the AMPK signaling is inhibited, its activity is significantly weakened, confirming that the AMPK signaling pathway is one of the core mechanisms by which it exerts its effects.

[0052] Example 6 500g of fresh pig pancreas from the same batch as in Example 1 was extracted using the traditional acid extraction method (soaking in 1mol / L hydrochloric acid for 24h, adjusting the pH to 7.0, centrifuging to collect the supernatant, and freeze-drying). The final yield was 42.5g of the target peptide, with an extraction rate of only 8.5%. The purity was 78.3% as determined by HPLC. After storage at 4℃ for 6 months, the purity dropped to 65.2%, which was significantly lower than the combined enzymatic hydrolysis-ultrafiltration process in Example 1.

[0053] Therefore, the present invention relates to the application of a pancreatic protein peptide in the preparation of products that improve insulin resistance and repair pancreatic function. Experiments have confirmed that the pancreatic protein peptide can significantly improve insulin resistance, promote the proliferation of pancreatic β cells, and inhibit pancreatic inflammatory response, and has high safety and simple preparation process.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of a pancreatic protein peptide for the preparation of a product for improving insulin resistance and repairing pancreatic function, characterized in that: The pancreatic protein peptide is derived from pig pancreas tissue and prepared by a complex enzymatic hydrolysis-ultrafiltration process, has a molecular weight distribution of 800-2000 Da, and contains Val-Pro-Leu and Gly-Phe-Met active fragments in the amino acid sequence, and has a purity of greater than or equal to 95%.

2. Use of a pancreatic protein peptide according to claim 1 for the preparation of a product for improving insulin resistance and repairing pancreatic function, characterized in that, The complex enzymatic hydrolysis-ultrafiltration process specifically comprises the following steps: S1, raw material pretreatment: fresh pig pancreas is taken, fat tissue is removed, and homogenate is obtained after ultra-high pressure treatment; S2, enzymatic hydrolysis reaction: trypsin is added according to an enzyme-substrate ratio of 1:50, and papain is added according to an enzyme-substrate ratio of 1:80, stepwise enzymatic hydrolysis is performed, and the total enzymatic hydrolysis time is 6 h; S3, purification: the enzymatic hydrolysate is subjected to impurity removal through a 10-kDa ultrafiltration membrane, and the target component is enriched through a 3-kDa ultrafiltration membrane, and the finished product is obtained by freeze-drying at-50℃ and a vacuum degree of 10 Pa.

3. Use of a pancreatic protein peptide according to claim 2 for the preparation of a product for improving insulin resistance and repairing pancreatic function, characterized in that: In S2, the trypsin enzymatic hydrolysis temperature is 45℃, and the pH is 7.

2.

4. Use of a pancreatic protein peptide according to claim 2 for the preparation of a product for improving insulin resistance and repairing pancreatic function, characterized in that: The papain enzymatic hydrolysis temperature is 50℃, and the pH is 6.

5.

5. Use of a pancreatic protein peptide according to claim 1 for the preparation of a product for improving insulin resistance and repairing pancreatic function, characterized in that: The product is an insulin resistance improving drug, the administration dose is 100-200 mg / kg·d, and the administration route is oral or intraperitoneal injection.

6. Use of a pancreatic protein peptide according to claim 1 for the preparation of a product for improving insulin resistance and repairing pancreatic function, characterized in that: The product is a pancreas function repairing preparation, can promote the proliferation of pancreatic islet beta cells, increase the insulin secretion amount, and reduce the levels of pancreatic tissue inflammatory factors TNF-α and IL-6.

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