Application of recombinant protein DEL-1 in preparation of drugs for resisting pancreatic beta cell senescence

Abstract

The invention relates to the technical field of medicines, in particular to application of a recombinant protein DEL-1 in preparation of a medicine for resisting pancreatic beta cell senescence. Within the concentration range of 400-1000 ng / ml, the DEL-1 can reduce the high glucose and high fat induced pancreatic beta cell SA-beta-Gal positive rate by 39.5% or above, and the anti-aging effect is remarkable.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of recombinant protein DEL-1 in the preparation of drugs for the treatment of pancreatic β-cell aging. Background Technology

[0002] With the increasing aging of the global population, the prevention and treatment of age-related diseases has become an important research direction in the biomedical field. Pancreatic β-cells are the main cell type in the endocrine portion of the pancreas responsible for the synthesis and secretion of insulin, and the decline in their function and number is one of the core mechanisms in the pathogenesis of type 2 diabetes. Studies have shown that the aging process of pancreatic β-cells involves multiple biological processes, including increased oxidative stress, activated inflammatory responses, weakened autophagy, and increased apoptosis.

[0003] Currently, drug development strategies for combating pancreatic β-cell aging mainly focus on antioxidants, anti-inflammatory drugs, and autophagy activators. However, clinical efficacy is not ideal, and long-term use may lead to side effects. Therefore, finding new, highly effective, and low-toxicity drugs to combat pancreatic β-cell aging 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. However, the role of DEL-1 / EDIL3 in pancreatic β-cell senescence and its potential anti-aging effects have not yet been reported. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of recombinant protein DEL-1 in the preparation of drugs against pancreatic β-cell aging. This invention targets pancreatic β-cell aging, a key aspect of the pathogenesis of type 2 diabetes, and offers 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 against pancreatic β-cell aging.

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

[0009] Preferably, the model of pancreatic β-cell senescence is constructed using glucose and palmitic acid.

[0010] Preferably, the concentration of glucose used is 33 mmol / L, and the concentration of palmitic acid used is 500 μmol / L.

[0011] Preferably, the construction time is 48 hours.

[0012] Preferably, the dosage form of the drug includes an injection solution, wherein the content of recombinant protein DEL-1 in the injection solution is 0.1 to 1 mg / ml.

[0013] This study found that DEL-1 (EDIL3) is highly expressed in normal pancreatic β-cell regions, suggesting it may play a crucial role in maintaining normal pancreatic β-cell function and physiological state. However, during the development and progression of diabetes, the expression level of DEL-1 in pancreatic tissue is significantly downregulated, and this downregulation is positively correlated with β-cell dysfunction and senescent phenotype. This phenomenon indicates that pancreatic local homeostasis is disrupted during the course of diabetes, and DEL-1 (EDIL3) is likely an important factor in maintaining this local homeostasis. The reduced expression of DEL-1 in the pancreatic microenvironment may be one of the causes, rather than a result, of β-cell senescence and dysfunction, providing a theoretical basis for DEL-1 as a potential therapeutic target.

[0014] Based on the known biological functions of DEL-1, including its anti-inflammatory, pro-angiogenic, and cell adhesion-regulating effects, and the newly discovered changes in DEL-1 expression patterns in pancreatic β-cells, this invention hypothesizes that DEL-1 may have a protective effect against pancreatic β-cell senescence. In particular, DEL-1 interacts with integrin αvβ3 through its RGD sequence, potentially affecting pancreatic β-cell survival and function. Furthermore, DEL-1's role in regulating inflammatory responses may also be related to its potential anti-aging effects, as inflammation is a key characteristic and driver of aging.

[0015] In this context, supplementing with exogenous DEL-1 may help restore the homeostasis of the pancreatic islet microenvironment, reverse or slow down the aging process of β cells, and ultimately improve insulin secretion function, which constitutes the core theoretical basis of this invention.

[0016] Therefore, this invention aims to explore the effects of DEL-1 on pancreatic β-cell senescence and its potential application prospects, providing new targets and strategies for the prevention and treatment of type 2 diabetes.

[0017] This invention reveals that recombinant protein DEL-1 (EDIL3) significantly inhibits pancreatic β-cell senescence induced by high glucose and high lipid levels. Experiments show that in a pancreatic β-cell senescence model constructed using 33 mM glucose combined with 500 μM palmitic acid, DEL-1 inhibits β-cell senescence in a dose-dependent manner. Specifically, when the DEL-1 concentration was gradually increased from 0 ng / ml to 100, 200, 400, 800, and 1000 ng / ml, the SA-β-Gal staining positivity rate (a marker of senescence) showed a significant decreasing trend, indicating that DEL-1 has a clear anti-pancreatic β-cell senescence effect.

[0018] Beneficial effects:

[0019] High efficacy: Within the concentration range of 400-1000 ng / ml, DEL-1 can reduce the SA-β-Gal positivity rate of pancreatic β cells induced by high glucose and high lipid by more than 39.5%, showing significant anti-aging effects;

[0020] Safety: As an endogenously expressed protein, DEL-1 has good biocompatibility and few adverse reactions;

[0021] Novelty: The application of DEL-1 as an anti-pancreatic β-cell aging drug 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 EGF-like domain-mediated cell signal transduction, its coagulation factor-like domain-mediated cell adhesion regulation, and the anti-inflammatory effects of the RGD sequence interacting with integrins. Attached Figure Description

[0023] 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.

[0024] Figure 1 SA-β-Gal staining of βTC-6 cells after treatment with recombinant protein DEL-1 (orange arrows indicate blue-green senescent cells);

[0025] Figure 2 The results show the improvement of βTC-6 cell senescence by different concentrations of DEL-1. Detailed Implementation

[0026] This invention provides the application of recombinant protein DEL-1 in the preparation of drugs for treating pancreatic β-cell senescence. In this invention, the amino acid sequence of the recombinant protein DEL-1 is shown in SEQ ID No. 1, and is as follows:

[0027] DICDPNPCENGGICLPGLADGSFSCECPDGFTDPNCSSVVEVASDEEEPTSAGPCTPNPCHNGGTCEISEAYRGDTFIGYVCKCPRGFNGIHCQHNINECEVEPCKNGGICTDL VANYSCECPGEFMGRNCQYKCSGPLGIEGGIISNQQITASSTHRALFGLQKWYPYYARLNKKGLINAWTAAENDRWPWIQINLQRKMRVTGVITQGAKRIGSPEYIKSYKIAYSN DGKTWAMYKVKGTNEDMVFRGNIDNNTPYANSFTPPIKAQYVRLYPQVCRRHCTLRMELLGCELSGCSEPLGMKSGHIQDYQITASSIFRTLNMDMFTWEPRKARLDKQGKVNA WTSGHNDQSQWLQVDLLVPTKVTGIITQGAKDFGHVQFVGSYKLAYSNDGEHWTVYQDEKQRKDKVFQGNFDNDTHRKNVIDPPIYARHIRILPWSWYGRITLRSELLGCTEEE.

[0028] The recombinant DEL-1 protein used in this invention can be prepared by the following method:

[0029] 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;

[0030] 2. The constructed expression vector was transfected into HEK293T cells, and cell lines stably expressing DEL-1 were obtained by selection with puromycin;

[0031] 3. Culture the stable cell line in serum-free medium for 48-72 hours and collect the culture supernatant containing secretory DEL-1 protein;

[0032] 4. Purify DEL-1 protein by affinity chromatography (using an affinity column conjugated with an anti-His tag antibody);

[0033] 5. The recombinant DEL-1 protein was further purified using gel filtration chromatography to obtain a purity >95%;

[0034] 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.

[0035] 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.

[0036] In this invention, the pancreatic β-cell senescence model is preferably constructed using glucose and palmitic acid. The glucose concentration is preferably 33 mmol / L, and the palmitic acid concentration is preferably 500 μmol / L. The construction time is preferably 48 hours. The drug dosage form includes an injection, wherein the recombinant protein DEL-1 content in the injection is 0.1–1 mg / ml.

[0037] 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.

[0038] Example 1

[0039] Materials and Methods

[0040] 1. Cell culture: Mouse pancreatic β-cell line βTC-6 cells were used and cultured in commercial DMEM medium containing 10% fetal bovine serum (containing 5.5 mM glucose and 1% penicillin-streptomycin). All cells were cultured in a 37°C, 5% CO2 incubator.

[0041] 2. Aging model construction: βTC-6 cells were exposed to a high-glucose, high-fat environment (33mM glucose + 500μM palmitic acid) for 48 hours to induce β-cell senescence.

[0042] 3. DEL-1 treatment: The treatment group was induced with senescence and the addition of different concentrations of recombinant DEL-1 protein (0, 100, 200, 400, 800, 1000 ng / ml) to the culture medium and cultured for 48 h; while the normal control group was cultured for 48 h after the culture medium was replaced with fresh medium. Each group had 3 replicates.

[0043] 4. Senescence assessment: 48 hours after senescence induction, cell senescence was detected using the SA-β-Gal staining kit (Beyotime, #9860), and the proportion of SA-β-Gal positive cells was calculated.

[0044] 5. Data Analysis: The proportion of SA-β-Gal positive cells was quantitatively analyzed using ImageJ software, and statistical analysis was performed using Graphpadprism 10 software. P < 0.05 was considered statistically significant.

[0045] result

[0046] After 48 hours of high-sugar, high-fat treatment (33 mM glucose + 500 μM palmitic acid), the positive rate of SA-β-Gal in βTC-6 cells significantly increased to 40.5 ± 3.1%, while that in the normal control group (5.5 mM glucose) was only 8.2 ± 1.5%, indicating that a β-cell senescence model was successfully constructed.

[0047] The positive rates of SA-β-Gal in different DEL-1 treatment groups are as follows:

[0048] 0 ng / ml (aging model group): 40.5 ± 3.1%;

[0049] 100 ng / ml: 36.1 ± 5.6% (a decrease of 10.9% compared to the aging model group);

[0050] 200 ng / ml: 32.0 ± 3.9% (a decrease of 21.0% compared to the aging model group);

[0051] 400 ng / ml: 24.5 ± 3.7% (a decrease of 39.5% compared to the aging model group);

[0052] 800 ng / ml: 18.6 ± 2.1% (a decrease of 54.1% compared to the aging model group);

[0053] 1000 ng / ml: 15.7 ± 2.5% (a decrease of 61.2% compared to the aging model group).

[0054] The results showed that DEL-1 significantly inhibited pancreatic β-cell senescence induced by high glucose and high lipid levels, and this inhibitory effect was clearly dose-dependent. The anti-aging effect was most significant at concentrations of 400 ng / ml and above, with a reduction in SA-β-Gal positivity exceeding 39.5%.

[0055] 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. Application of recombinant protein DEL-1 in the preparation of drugs against pancreatic β-cell aging.

2. The application according to claim 1, characterized in that, The amino acid sequence of the recombinant protein DEL-1 is shown in SEQ ID No.

1.

3. The application according to claim 1, characterized in that, The model of pancreatic β-cell senescence was constructed using glucose and palmitic acid.

4. The application according to claim 3, characterized in that, The glucose concentration used is 33 mmol / L, and the palmitic acid concentration used is 500 μmol / L.

5. The application according to claim 3, characterized in that, The construction time was 48 hours.

6. The application according to claim 1, characterized in that, The drug is available in the form of an injection, wherein the content of recombinant protein DEL-1 in the injection is 0.1–1 mg / ml.

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