Application of SAFit2 in preparation of reagent for improving pancreas islet dysfunction

Abstract

The invention relates to the technical field of biology, in particular to application of SAFit2 in preparation of a reagent for improving pancreas islet dysfunction. The invention discovers that SAFit2 has a remarkable improvement effect on insulin secretion dysfunction in a high-glucose and high-fat induced islet injury model. Experiments show that in a high glucose (33 mM) combined palmitic acid (500 [mu] M) injury model, the SAFit2 can significantly improve the insulin secretion function of the pancreas islet. Specifically, the SAFit2 is pretreated, the glucose stimulated insulin secretion (GSIS) index of the pancreas islet and the intracellular insulin content are remarkably increased, insulin secretion under high glucose stimulation is promoted, and the SAFit2 is proved to have the effect of promoting the insulin secretion function of the pancreas islet beta cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of SAFit2 in the preparation of agents to improve pancreatic islet dysfunction in diabetes. Background Technology

[0002] More than 400 million people worldwide suffer from diabetes and its complications, placing an increasing burden on public health. Over 90% of these cases are type 2 diabetes mellitus (T2DM), characterized by peripheral insulin resistance and insufficient insulin secretion. Glucose toxicity and lipid toxicity affect insulin secretion by impairing β-cell function or causing loss of functional β-cell mass. Therefore, developing new therapies to improve β-cell function is crucial for the treatment of type 2 diabetes.

[0003] Existing technologies document that SAFit2 can reduce paclitaxel-induced mechanosensitive responses in mice and improve paclitaxel-induced neuropathic pain by reducing peripheral sensitivity and resolving neuroinflammation. SAFit2 can also reduce alcohol consumption in stressed men, but not in women. The paper "Application of SAFit2 and Culture Medium in Promoting Differentiation of Human Induced Pluripotent Stem Cells into Dopaminergic Neurons" (CN114525241A) describes the role of SAFit2 in promoting the differentiation of human induced pluripotent stem cells into dopaminergic neurons.

[0004] However, there are currently no reports in existing technologies regarding the effects of SAFit2 on pancreatic insulin secretion function. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of SAFit2 in the preparation of reagents to improve pancreatic islet dysfunction.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of SAFit2 in the preparation of reagents to improve pancreatic islet dysfunction; SAFit2 promotes insulin secretion function of pancreatic β cells; SAFit2 improves pancreatic insulin secretion dysfunction induced by high glucose and high lipid levels; High glucose and high lipid induction was performed using glucose and palmitic acid at a concentration of 33 mmol / L and a palmitic acid concentration of 500 µmol / L for 48 h.

[0007] Preferably, the concentration of SAFit2 used is 1 µmol / L.

[0008] The beneficial effects of this invention are: This invention reveals that SAFit2 significantly improves insulin secretion dysfunction in a high-glucose, high-fat-induced pancreatic islet injury model. Experiments show that in a high-glucose (33 mM) combined with palmitic acid (500 μM) injury model, SAFit2 significantly improves pancreatic islet insulin secretion function. Specifically, SAFit2 pretreatment significantly increases the glucose-stimulated insulin secretion (GSIS) index and intracellular insulin content in the islets, promoting insulin secretion under high-glucose stimulation, demonstrating that SAFit2 has an effect on promoting insulin secretion function in pancreatic β-cells. Attached Figure Description

[0009] 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.

[0010] Figure 1 SAFit2 improves pancreatic islet function in mice induced by high glucose and high fat intake. Detailed Implementation

[0011] This invention provides the application of SAFit2 in the preparation of reagents to improve pancreatic islet dysfunction.

[0012] In this invention, the SAFit2 is a commercially available product purchased from Aobious, USA, AOB6548.

[0013] In this invention, SAFit2 preferably promotes insulin secretion function of pancreatic β-cells. In this invention, the concentration of SAFit2 used is preferably 1 µmol / L. In this invention, SAFit2 preferably improves pancreatic insulin secretion dysfunction induced by high glucose and high lipid intake. In this invention, glucose and palmitic acid are preferably used for high glucose and high lipid intake induction, wherein the glucose concentration is preferably 33 mmol / L, the palmitic acid concentration is preferably 500 µmol / L, and the induction time is preferably 48 h.

[0014] 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

[0015] The effect of SAFit2 on pancreatic dysfunction induced by high glucose combined with palmitic acid Materials and Methods

[0016] Mouse islet isolation and culture: 8-10 week old C57BL / 6 mice were euthanized by cervical spondylosis and placed supine on the operating table of a surgical microscope. The abdominal skin was disinfected with 75% alcohol. A V-shaped incision was made in the abdomen to fully expose the abdominal cavity. Under the microscope, the duodenum was located, and the junction of the duodenum and common bile duct was found and clamped with hemostatic forceps. The hepatic hilum was fully exposed, and the common bile duct was freed. A 25G syringe needle was connected to a 5 mL syringe, 5 mL of collagenase P solution was drawn up, and the tip of the needle was bent at a 15° angle and inserted into the common bile duct. 4 mL of collagenase P solution was injected to fully perfuse the pancreas. The pancreas was bluntly dissected from the duodenum, stomach, and spleen. The entire pancreas was cut off and placed in a 50 mL sterile centrifuge tube and placed on ice. Pancreas digestion: The centrifuge tube containing the mouse pancreas was placed in a 37°C water bath for 12 min. Then, the centrifuge tube was quickly removed, wiped with 75% alcohol, and transferred to a clean bench. 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum was added to stop digestion. The centrifuge tube was quickly manually shaken 40 times. 30 mL of 1640 solution containing 10% fetal bovine serum at 4°C was added, and the mixture was centrifuged at 1000 r / min for 1 min at 4°C. Discard the supernatant, add 10 mL of pre-cooled RPMI-1640 solution containing 10% fetal bovine serum, and resuspend using a sterile Pasteur pipette. Filter the suspension through a 40-mesh sieve. Combine the suspensions of 3-4 pancreas in a centrifuge tube, centrifuge at 1000 r / min for 1 min at 4°C, and discard the supernatant. Islet purification: Resuspend the above precipitate in 15 mL of Histopaque-1077 solution, resuspend using a sterile Pasteur pipette, and slowly add 10 mL of RPMI-1640 culture medium solution along the wall of the centrifuge tube. Once the solution in the centrifuge tube separates into layers, centrifuge at 2400 r / min for 20 min at 25°C. Slowly remove the centrifuge tube; the islets will be visible suspended in the lower Histopaque-1077 solution layer. Using a power pipette, transfer the Histopaque-1077 solution containing islets from the lower layer to another 50 mL sterile centrifuge tube. Add 35-40 mL of pre-chilled 1640 solution containing 10% fetal bovine serum (FBS), and centrifuge at 1000 rpm for 1 min at 4°C. Islets will precipitate after centrifugation. Wash once more with pre-chilled 1640 solution containing 10% FBS. Finally, transfer the islets to a cell culture dish and further purify them manually under an optical microscope. Culture in 1640 solution containing 10% FBS until ready for use. Experimental grouping and treatment:

[0017] Normal control group: 5.5 mM glucose medium; High glucose and high fat injury group: 33 mM glucose + 500 μM palmitic acid, treated for 48 h; SAFit2 treatment group: 1 µM SAFit2 pretreatment for 1 h, followed by 33 mM glucose + 500 μM palmitic acid treatment for 48 h; Each group had 3 replicates, and each replicate contained approximately 10 islets of similar size. Glucose-stimulated insulin secretion (GSIS) experiment:

[0018] After 48 h of treatment, the islets were pre-incubated for 1 h with low-glucose Krebs-Ringer buffer (KRB).

[0019] The islets were incubated for 1 hour in KRB containing 1.67 mM (low glucose) and 16.7 mM (high glucose) glucose, respectively.

[0020] Collect the supernatant and use a mouse insulin ELISA kit to detect insulin concentration.

[0021] Calculate the glucose stimulation index (GSI) = high glucose insulin secretion / low glucose insulin secretion, relative insulin secretion and intracellular insulin content (all normalized to pancreatic protein levels).

[0022] Insulin content measurement: After the GSIS experiment, total pancreatic protein was extracted using high-efficiency RIPA cell lysis buffer.

[0023] Total protein content was detected using a BCA kit.

[0024] (4) Data analysis: Statistical analysis was performed using Graphpad Prism 10 software, and a p-value < 0.05 was considered statistically significant. Experimental results

[0025] High glucose and high lipid intake significantly inhibited insulin secretion from pancreatic islet cells, while SAFit2 pretreatment significantly improved insulin secretion impairment induced by high glucose and high lipid intake, manifested as a significant increase in insulin secretion levels under high glucose concentrations. Furthermore, the intracellular insulin content in the SAFit2 pretreatment group was significantly higher than that in the high glucose and high lipid treatment group. These results indicate that SAFit2 can improve pancreatic islet cell dysfunction caused by high glucose and high lipid intake.

[0026] 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. Use of SAFit2 in the preparation of a reagent for improving islet dysfunction; the SAFit2 promotes the insulin secretion function of islet β cells; the SAFit2 improves high-glucose high-fat-induced islet insulin secretion dysfunction; high-glucose high-fat induction is performed using glucose at a concentration of 33 mmol / L and palmitic acid at a concentration of 500 µmol / L, and the induction is performed for 48 h; the SAFit2 is used at a concentration of 1 µmol / L.

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