Use of recombinant protein DEL-1 in preparation of a drug for improving insulin secretion function of human islets

Abstract

The application relates to the technical field of biological pharmacy, in particular to application of recombinant protein DEL-1 in preparation of a drug for improving insulin secretion function of human islets. The application finds that the recombinant protein DEL-1 has a significant improvement effect on insulin secretion dysfunction in various human islet injury models. Experiments show that in a high-sugar combined palmitic acid injury model and an inflammatory cytokine injury model, DEL-1 can significantly improve the insulin secretion function of human islets. Specifically, compared with an injury group, after DEL-1 treatment, the glucose stimulation index of human islets and the relative secretion amount of insulin under high-sugar stimulation are both significantly increased, which proves that DEL-1 has a clear effect of promoting insulin secretion function of human islet beta cells.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to the application of recombinant protein DEL-1 in the preparation of drugs that improve the function of human pancreatic insulin secretion. Background Technology

[0002] With the continued rise in the global incidence of type 2 diabetes, finding effective treatments to protect and improve pancreatic β-cell function has become an important topic in diabetes research. Pancreatic β-cell dysfunction is one of the core mechanisms of type 2 diabetes pathogenesis, mainly manifested as glucose-stimulated insulin secretion (GSIS) deficiency and reduced insulin synthesis.

[0003] Currently, drugs used clinically to improve β-cell function mainly include sulfonylureas, GLP-1 receptor agonists, and DPP-4 inhibitors. However, these drugs have some limitations; for example, sulfonylureas may accelerate β-cell dysfunction, and GLP-1 receptor agonists require injection. Therefore, developing novel, highly effective, and low-toxicity drugs to improve pancreatic islet function has significant clinical importance and application prospects.

[0004] DEL-1 (Developmental endothelial locus-1), also known as EDIL3 (EGF-like repeats and discoidin I-like domains 3), is a secreted extracellular matrix glycoprotein containing three epidermal growth factor (EGF)-like repeat sequences and two coagulation factor V / VIII homologous domains. Previous studies have shown that DEL-1 / EDIL3 plays an important role in angiogenesis, inflammation regulation, cell adhesion, and apoptosis. There are currently no reports on recombinant protein DEL-1 improving human pancreatic insulin secretion function. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of recombinant protein DEL-1 in the preparation of drugs that improve human pancreatic insulin secretion function. The recombinant protein DEL-1 provided by this invention improves human pancreatic insulin secretion function. This invention targets the core problem of type 2 diabetes—impaired human pancreatic insulin secretion function—and provides a novel treatment strategy.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of recombinant protein DEL-1 in the preparation of drugs that improve the insulin secretion function of human pancreatic islets.

[0008] Preferably, the amino acid sequence of the recombinant protein DEL-1 is shown in SEQ ID No. 1.

[0009] Preferably, the concentration of the recombinant protein DEL-1 used is 1000 ng / ml.

[0010] Preferably, the recombinant protein DEL-1 promotes insulin secretion from human pancreas.

[0011] Preferably, the dosage form of the drug includes an injection, wherein the concentration of recombinant protein DEL-1 in the injection is 0.1~1 mg / ml.

[0012] Preferably, the recombinant protein DEL-1 improves insulin secretion dysfunction induced by high glucose and high lipid levels in humans.

[0013] Preferably, high glucose and high lipid induction is performed using glucose and palmitic acid, wherein the concentration of glucose is 33 mmol / L, the concentration of palmitic acid is 500 µmol / L, and the induction time is 24 h.

[0014] Preferably, the recombinant protein DEL-1 improves inflammatory cytokine-induced insulin secretion dysfunction in human pancreatic islets.

[0015] Preferably, IL-1β, TNF-α and IFN-γ are used in combination for induction, wherein the concentration of IL-1β is 10 ng / ml, the concentration of TNF-α is 25 ng / ml, the concentration of IFN-γ is 100 ng / ml, and the induction time is 24 h. Beneficial effects

[0016] This invention reveals that recombinant protein EDIL3 (DEL-1) significantly improves insulin secretion dysfunction in various human islet injury models. Experiments show that in both high glucose (33 mM) combined with palmitic acid (500 μM) injury models and inflammatory cytokine (10 ng / ml IL-1β + 25 ng / ml TNF-α + 100 ng / ml IFN-γ) injury models, DEL-1 (1000 ng / ml) significantly improves insulin secretion function in human islets. Specifically, after DEL-1 treatment, the glucose-stimulated insulin secretion (GSIS) index and relative insulin secretion in human islets significantly increased, demonstrating that DEL-1 has a clear effect on promoting insulin secretion function in human pancreatic β cells.

[0017] Compared with existing human pancreatic islet function protection drugs, the present invention has the following outstanding advantages:

[0018] High efficacy: DEL-1 (1000 ng / ml) can significantly improve insulin secretion function in the injury model;

[0019] Safety: As an endogenously expressed protein, DEL-1 has good biocompatibility and few adverse reactions;

[0020] Broad spectrum: DEL-1 has a wide range of applications, improving pancreatic dysfunction caused by different mechanisms.

[0021] Novelty: The application of DEL-1 as a drug to improve the insulin secretion function of human pancreas has not been reported before, representing a completely new treatment approach.

[0022] These technological advantages mainly stem from the unique structural features and biological functions of the DEL-1 protein, including its multiple roles in cell survival, functional maintenance, and microenvironment regulation.

[0023] Terminology Explanation:

[0024] DEL-1 / EDIL3: Developmental endothelial locus-1 / EGF-like repeats and discoidin I-like domains 3;

[0025] GSIS: Glucose-Stimulated Insulin Secretion, an important indicator for evaluating β-cell function;

[0026] IEQ: Islet Equivalent, a standard unit for assessing the number of islets. 1 IEQ is equivalent to the volume of a standard islet with a diameter of 150 μm.

[0027] IL-1β: Interleukin-1β, an important pro-inflammatory cytokine;

[0028] TNF-α: Tumor necrosis factor-α, a major pro-inflammatory cytokine;

[0029] IFN-γ: Interferon-γ, an inflammatory factor, often works synergistically with IL-1β and TNF-α to damage β cells. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0031] Figure 1 The effect of DEL-1 on improving insulin secretion dysfunction in human pancreatic islets induced by high glucose combined with palmitic acid;

[0032] Figure 2This study aimed to improve the effect of DEL-1 on insulin secretion dysfunction induced by inflammatory cytokines in human pancreas. Detailed Implementation

[0033] This invention provides the use of recombinant protein DEL-1 in the preparation of drugs that improve human pancreatic insulin secretion function. In this invention, the amino acid sequence of the recombinant protein DEL-1 is shown in SEQ ID No. 1. In this invention, the preferred concentration of the recombinant protein DEL-1 is 1000 ng / ml. In this invention, the recombinant protein DEL-1 preferably promotes human pancreatic insulin secretion.

[0034] SEQ ID No. 1:

[0035] DICDPNPCENGGICLPGLADGSFSCECPDGFTDPNCSSVVEVASDEEEPTSAGPCTPNPCHNGGTCEISEAYRGDTFIGYVCKCPRGFNGIHCQHNINECEVEPCKNGGICTDL VANYSCECPGEFMGRNCQYKCSGPLGIEGGIISNQQITASSTHRALFGLQKWYPYYARLNKKGLINAWTAAENDRWPWIQINLQRKMRVTGVITQGAKRIGSPEYIKSYKIAYSN DGKTWAMYKVKGTNEDMVFRGNIDNNTPYANSFTPPIKAQYVRLYPQVCRRHCTLRMELLGCELSGCSEPLGMKSGHIQDYQITASSIFRTLNMDMFTWEPRKARLDKQGKVNA WTSGHNDQSQWLQVDLLVPTKVTGIITQGAKDFGHVQFVGSYKLAYSNDGEHWTVYQDEKQRKDKVFQGNFDNDTHRKNVIDPPIYARHIRILPWSWYGRITLRSELLGCTEEE.

[0036] In this invention, the recombinant protein DEL-1 preferably improves insulin secretion dysfunction induced by high glucose and high lipid levels in human pancreas. This invention preferably uses glucose and palmitic acid for high glucose and high lipid induction, wherein the glucose concentration is 33 mmol / L, the palmitic acid concentration is 500 µmol / L, and the induction time is 24 h.

[0037] In this invention, the recombinant protein DEL-1 preferably improves inflammatory cytokine-induced insulin secretion dysfunction in human pancreas. This invention preferably uses IL-1β, TNF-α, and IFN-γ for induction, with IL-1β at a concentration of 10 ng / ml, TNF-α at a concentration of 25 ng / ml, and IFN-γ at a concentration of 100 ng / ml, and the induction time being 24 h.

[0038] The recombinant DEL-1 protein used in this invention can preferably be prepared by the following method:

[0039] 1. Construct an expression vector containing the full-length coding sequence of human DEL-1, and select a eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro with a suitable promoter and selection marker;

[0040] 2. The constructed expression vector was transfected into HEK293T cells, and cell lines stably expressing DEL-1 were obtained by selection with puromycin;

[0041] 3. Culture the stable cell line in serum-free medium for 48-72 hours and collect the culture supernatant containing secretory DEL-1 protein;

[0042] 4. Purify DEL-1 protein by affinity chromatography (using an affinity column conjugated with an anti-His tag antibody);

[0043] 5. The recombinant DEL-1 protein was further purified using gel filtration chromatography to obtain a purity >95%;

[0044] 6. Dialyze the purified DEL-1 protein into PBS buffer (pH 7.4), filter it through a sterile filter membrane, aliquot it, and store it at -80°C.

[0045] The recombinant DEL-1 protein preparation method in this invention can use different expression systems (such as HEK293T cells, CHO cells or baculovirus-insect cell system), as well as different expression vectors and purification methods, as long as biologically active DEL-1 protein can be obtained.

[0046] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example 1

[0047] The effect of DEL-1 on improving human pancreatic dysfunction induced by high glucose combined with palmitic acid

[0048] Materials and Methods

[0049] Human islet isolation and culture:

[0050] Human islets were isolated from the pancreas of a healthy donor (a brain-dead organ donor with no history of diabetes).

[0051] The steps for isolating and purifying human pancreatic islets are as follows:

[0052] (1) Obtaining human pancreas: Human pancreas is obtained from organ donation from deceased clinical patients, with informed consent and approval from the medical ethics committee;

[0053] (2) Pretreatment of pancreas: Remove excess fat and lymph nodes around the pancreas on a sterile operating table in a GMP laboratory, soak and wash with collagenase solution, amphotericin B solution and Hank's balanced salt solution, and cut the pancreas into two parts.

[0054] (3) Pancreatic perfusion and digestion: The main pancreatic duct is located on the cut surface and a trocar is inserted. After being fixed with sutures, Liberase MTF C / F digestive enzymes are perfused for 15 minutes using an automatic perfusion machine.

[0055] (4) Digestion monitoring: The digestion temperature was controlled at 37°C. Mechanical and collagenase digestion was performed. After 5 minutes of digestion, samples were taken every 1 minute to assess the degree of digestion. Digestion was stopped when the islets of Langerhans separated from the exocrine tissue and the exocrine tissue acini became smaller.

[0056] (5) Purification preparation: Collect the digestion product into cell culture medium containing human albumin, centrifuge and wash 2-4 times, add 150mL UW solution, and place on an ice box for 30 minutes;

[0057] (6) Purification procedure: Pancreatic islets were isolated and purified by Cobe 2991 cell washing machine and density gradient centrifugation.

[0058] The purified human islets were cultured in CMRL 1066 medium containing 10% fetal bovine serum and 5.5 mM glucose and incubated at 37°C and 5% CO2 for 24 hours to allow the islets to adapt to the in vitro environment.

[0059] Experimental grouping and treatment:

[0060] Normal control group: 5.5 mM glucose medium;

[0061] High glucose and high fat injury group: 33 mM glucose + 500 μM palmitic acid, treated for 24 hours;

[0062] DEL-1 treatment group: 33mM glucose + 500μM palmitic acid + 1000ng / ml DEL-1, treated for 24 hours;

[0063] Each group had 3 replicates, and each replicate contained approximately 100 islets of similar size (IEQ).

[0064] Glucose-stimulated insulin secretion (GSIS) experiment:

[0065] After 24 hours of treatment, the islets were pre-incubated for 1 hour with low-glucose Krebs-Ringer buffer (KRB).

[0066] The islets were incubated for 1 hour each in KRB containing 1.67 mM (low glucose) and 16.7 mM (high glucose) glucose.

[0067] The islets of Langerhans were removed, and the islet cells were lysed using high-efficiency RIPA cell lysis buffer.

[0068] The supernatants from high- and low-glucose incubation and islet lysate were collected separately. Insulin secretion levels and intracellular insulin content under high- and low-glucose stimulation were detected using a human insulin ELISA kit. Finally, total protein content was detected using a BCA kit.

[0069] Calculate the glucose stimulation index (the ratio of insulin secretion concentration stimulated by high glucose / low glucose), relative insulin secretion (normalized to islet protein content), and intracellular insulin content (the ratio of absolute intracellular insulin content to total islet protein).

[0070] Data analysis: Statistical analysis was performed using Graphpad Prism 10 software. A p-value < 0.05 was considered statistically significant.

[0071] result:

[0072] Glucose stimulation index (ratio of insulin secretion stimulated by high glucose to low glucose):

[0073] Normal control group: 4.52±0.82;

[0074] High sugar and high fat injury group: 0.68±0.008 (significantly reduced by 85.0% compared with the control group);

[0075] DEL-1 treatment group: 0.97±0.14 (significantly improved by 42.6% compared with the injury group);

[0076] Insulin secretion under hypoglycemic stimulation (ng / μg / h):

[0077] Normal control group: 0.048±0.001;

[0078] High sugar and high fat damage group: 0.078±0.019;

[0079] DEL-1 treatment group: 0.089±0.008;

[0080] Insulin secretion under high glucose stimulation (ng / μg / h):

[0081] Normal control group: 0.228±0.043;

[0082] High sugar and high fat injury group: 0.053±0.015 (significantly reduced by 76.8% compared with the control group);

[0083] DEL-1 treatment group: 0.085±0.006 (significantly improved by 60.4% compared with the injury group);

[0084] Intracellular insulin level (ng / μg):

[0085] Normal control group: 37.18±5.75;

[0086] High sugar and high fat injury group: 21.79±5.70 (41.39% lower than the control group);

[0087] DEL-1 treatment group: 26.05±1.25 (19.6% higher than the damage group, and 70.1% of the normal level recovered);

[0088] These results indicate that DEL-1 (1000 ng / ml) treatment significantly improves high glucose and high fat-induced insulin secretion dysfunction in human pancreas, not only increasing the glucose stimulation index but also restoring sensitivity to glucose stimulation (increasing insulin secretion stimulated by high glucose) and increasing insulin reserves.

[0089] Figure 1 Example 1: After 24 hours of treatment with high glucose and high fat or high glucose and high fat + DEL-1, the glucose stimulation index (GSI) of donor human islets was measured in different groups. Figure 1 (Left) Relative insulin secretion under high and low glucose stimulation ( Figure 1 (in the middle) and intracellular insulin content ( Figure 1 (Right). Example 2

[0090] The effect of DEL-1 on improving insulin secretion dysfunction induced by inflammatory cytokines in human pancreatic islets

[0091] Materials and Methods

[0092] Human islet isolation and culture: Same as in Example 1.

[0093] Experimental grouping and treatment:

[0094] Normal control group: blank standard culture medium;

[0095] Inflammatory factor injury group: 10 ng / ml IL-1β + 25 ng / ml TNF-α + 100 ng / ml IFN-γ, treated for 24 hours;

[0096] DEL-1 treatment group: 10 ng / ml IL-1β + 25 ng / ml TNF-α + 100 ng / ml IFN-γ + 1000 ng / ml DEL-1, treated for 24 hours;

[0097] Each group had 3 replicates, and each replicate contained approximately 100 islets of similar size (IEQ).

[0098] Glucose-stimulated insulin secretion (GSIS) experiment: Same as Example 1.

[0099] Data analysis: Statistical analysis was performed using Graphpad Prism 10 software. A p-value < 0.05 was considered statistically significant.

[0100] result:

[0101] Glucose stimulation index (ratio of insulin secretion stimulated by high glucose to low glucose):

[0102] Normal control group: 7.02±2.52;

[0103] Inflammatory factor injury group: 1.19±0.20 (significantly reduced by 83.0% compared to the control group);

[0104] DEL-1 treatment group: 2.57±0.77 (116.0% higher than the damage group, and 36.7% of the normal level).

[0105] Insulin secretion under hypoglycemic stimulation (ng / μg / h):

[0106] Normal control group: 0.024±0.011;

[0107] Inflammatory factor injury group: 0.037±0.012 (an increase of 54.2% compared with the control group, indicating increased basal secretion);

[0108] DEL-1 treatment group: 0.032±0.013 (13.5% lower than the damage group);

[0109] Insulin secretion under high glucose stimulation (ng / μg / h):

[0110] Normal control group: 0.148±0.009;

[0111] Inflammatory factor injury group: 0.043±0.007 (significantly reduced by 70.9% compared with the control group);

[0112] DEL-1 treatment group: 0.075±0.004 (significantly higher than the injury group by 74.4%).

[0113] Intracellular insulin level (ng / μg):

[0114] Normal control group: 18.69±8.89;

[0115] Inflammatory factor injury group: 13.29±4.60 (28.89% lower than the control group);

[0116] DEL-1 treatment group: 19.75±15.25 (48.61% higher than the damage group, returning to normal level).

[0117] These results indicate that DEL-1 treatment (1000 ng / ml) significantly improved inflammatory cytokine-induced insulin secretion dysfunction in human pancreatic islets, including improvements in glucose stimulation index, insulin secretion under high glucose stimulation, and insulin reserves. This suggests that DEL-1 has a protective effect against insulin secretion dysfunction in human pancreatic islets caused by different mechanisms.

[0118] Figure 2 Example 2: After treatment with inflammatory factors or inflammatory factors + DEL-1 for 24 hours, the glucose stimulation index (GSI) of donor human islets was measured in different groups. Figure 2 (Left) Relative insulin secretion under high and low glucose stimulation ( Figure 2 (in the middle) and intracellular insulin content ( Figure 2 (Right).

[0119] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of recombinant protein DEL-1 in the preparation of drugs that improve human pancreatic insulin secretion function; characterized in that, The improvement of human pancreatic insulin secretion function refers to the improvement of impaired human pancreatic insulin secretion function in type II diabetes. It improves human pancreatic insulin secretion function by increasing the glucose stimulation index, restoring sensitivity to glucose stimulation, and increasing insulin levels. The amino acid sequence of the recombinant protein DEL-1 is shown in SEQ ID No. 1; The recombinant protein DEL-1 was used at a concentration of 1000 ng / ml; The recombinant protein DEL-1 promotes insulin secretion from human pancreas. The drug is available in the form of an injection, wherein the concentration of recombinant protein DEL-1 in the injection is 0.1 to 1 mg / ml.

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