Application of deferoxamine and deferoxone in preparation of biological agent for relieving or preventing hypoxia injury in pancreas islet in-vitro culture

Abstract

The invention relates to the technical field of biology, and particularly discloses application of deferoxamine and deferiprone in preparation of a biological agent for relieving or preventing hypoxia injury in pancreas islet in-vitro culture. Through combined treatment of deferoxamine and deferoxone, the accumulation amount of Fe < 2 + > and the accumulation amount of a lipid peroxidation product 4-HNE of in-vitro cultured pancreas islet are remarkably reduced, the situation that insulin secretion GSIS is remarkably weakened due to glucose stimulation is remarkably improved, and finally hypoxia injury is reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of deferoxamine and deferoxone in the preparation of biological agents that alleviate or prevent hypoxic damage in in vitro culture of pancreatic islets. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease caused by abnormal insulin secretion or impaired insulin action. The number of patients worldwide continues to rise, and its core pathological features are long-term hyperglycemia and the risk of multi-organ complications. Current clinical treatment primarily relies on medications such as insulin injections, metformin, or GLP-1 receptor agonists to control blood sugar. However, these methods can only slow disease progression and cannot reverse the disease. Although cutting-edge technologies such as stem cell therapy show curative potential, they are still in the research stage and lack standardized clinical application. Islet transplantation, as a potential cure, has established standardized procedures, but its widespread application is severely limited by bottlenecks such as donor shortages, post-transplant immune rejection, and low islet survival rates.

[0003] In vitro islet culture technology plays a crucial role in improving islet survival rates. Since islet transplantation relies on direct transplantation from donor islets, they often need to be isolated for several hours to several days after separation to maintain optimal islet activity and function, and depending on the patient's condition and transplantation plan. Because islets themselves lack the expression of antioxidant enzymes and have a high oxygen requirement, isolation can lead to hypoxic damage, which is one of the challenges of in vitro islet culture. Summary of the Invention

[0004] To explore a pathway to reduce hypoxic damage during islet culture, this invention provides the application of deferoxamine and deferoxone in the preparation of biological agents to alleviate or prevent hypoxic damage during islet culture. This invention significantly reduces Fe in cultured islets through combined treatment with deferoxamine (DFO) and deferoxone (DFP). 2+ The accumulation of glucose-induced insulin secretion GSIS and lipid peroxidation product 4-HNE significantly improved the situation of significantly weakened insulin secretion GSIS caused by glucose stimulation, and ultimately reduced pancreatic islet hypoxia damage.

[0005] This invention provides the use of deferoxamine and deferoxone in the preparation of biological agents that alleviate or prevent hypoxic damage in in vitro culture of pancreatic islets.

[0006] This invention significantly reduces Fe in cultured pancreatic islets through combined treatment with deferoxamine (DFO) and deferoxone (DFP). 2+ It reduces the accumulation of both the amount of 4-HNE (a lipid peroxidation product) and the amount of 4-HNE, and simultaneously helps to significantly improve the situation where insulin secretion GSIS is significantly weakened due to glucose stimulation, ultimately reducing pancreatic islet hypoxia damage.

[0007] Furthermore, the deferoxamine in the biological agent has a final concentration of 5 μM to 100 μM, and the deferoxone has a final concentration of 10 μM to 100 μM.

[0008] Furthermore, the solvent for the biological agent is DMSO.

[0009] Furthermore, the biological agent for alleviating or preventing hypoxic injury in in vitro culture of pancreatic islets includes the following steps: After purification and culture, the islets are placed in a complete culture medium containing the aforementioned biological agent for further culture, thereby reducing islet hypoxia damage. The volume ratio of the biological agent to the complete culture medium is 2:2000-2100.

[0010] Furthermore, the complete culture medium is based on RPMI-1640 medium, with 5.2mM to 5.5mM glucose, 1% to 2% penicillin-streptomycin antibiotics, and 8% to 10% fetal bovine serum added to every 500ml of the basic medium.

[0011] Furthermore, the culture conditions are: 35℃~37℃, 5% CO2 culture for 48~50 hours, with deferoxamine and deferoxone supplemented every 23h~24 hours.

[0012] Further, the islet purification and culture steps are as follows: the islets are placed in the complete culture medium and incubated at 35℃~37℃ and 5% CO2 for 25~30 minutes, and the supernatant is discarded.

[0013] Furthermore, the biological agent possesses the following functions: Reduce Fe 2+ The accumulation of 4-HNE, a lipid peroxidation product, reduces hypoxic damage to pancreatic islets during in vitro culture.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the application of deferoxamine and deferoxone in the preparation of biological agents to alleviate or prevent hypoxic injury to pancreatic islets in vitro. The invention significantly reduces Fe in cultured pancreatic islets through combined treatment with deferoxamine (DFO) and deferoxone (DFP). 2+ It reduces the accumulation of lipid peroxidation products, including 4-HNE, and also helps to significantly improve the situation where glucose-stimulated insulin secretion (GSIS) is significantly weakened.

[0015] This invention improves in vitro culture conditions by adding deferoxamine (DFO) and deferoxone (DFP), inhibits ferroptosis during short-term in vitro culture of islets, and helps reduce in vitro culture damage to islets, improve islet survival rate, and reduce loss of islet functional activity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Combined treatment with deferoxamine (DFO) and deferoxone (DFP) to improve the iron content of cultured pancreatic islets in vitro 2+ The cumulative effect; In the figure, A represents the fluorescence intensity of iron ions in in vitro cultured pancreatic islets under different treatments.

[0018] B represents the combined treatment of deferoxamine (DFO) and deferoxone (DFP) on the in vitro cultured pancreatic islets Fe... 2+ Statistical chart of the cumulative impact.

[0019] Figure 2 The effect of combined treatment with deferoxamine (DFO) and deferoxone (DFP) on the accumulation of 4-HNE, a lipid peroxidation product of cultured pancreatic islets, in vitro; In the figure, A is a 4-HNE laser confocal microscopy image of lipid peroxidation products of cultured pancreatic islets after combined treatment with deferoxamine (DFO) and deferoxone (DFP). B is a statistical graph showing the effect of combined treatment with deferoxamine (DFO) and deferoxone (DFP) on the accumulation of 4-HNE, a lipid peroxidation product of cultured pancreatic islets.

[0020] Figure 3 The effect of combined treatment with deferoxamine (DFO) and deferoxone (DFP) on insulin secretion GSIS. Detailed Implementation

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0022] Example 1: A method for short-term in vitro culture of pancreatic islets to alleviate hypoxia-induced damage.

[0023] I. Experimental Methods 1. Islet isolation and purification Preliminary purification and stabilization of isolated islets: Isolated mouse islets require preliminary purification and stabilization, specifically including: transferring the islets to RPMI-1640 basal medium containing 5.5 mM glucose, 10% (v / v) fetal bovine serum, and 2% (v / v) penicillin / streptomycin, stored at 4°C, and then incubating at 37°C and 5% CO2 for 30 minutes. The supernatant of the medium is discarded and replaced with an equal volume of freshly prepared complete medium of the same formulation for later use.

[0024] The purpose of the above preliminary purification and stabilization steps is: (a) The islets were transferred to RPMI-1640 basal medium containing 5.5 mM glucose, 10% (V / V) fetal bovine serum and 2% (V / V) penicillin and streptomycin to dilute and remove endogenous digestive enzymes released by the residual tissue fragments, thereby terminating their potential chemical damage to the islet structure.

[0025] (b) During the settling process, the difference in physical density between the islet tissue and the residual pancreatic tissue fragments is utilized to promote the natural settling of the tissue fragments, thereby achieving physical separation.

[0026] (c) Maintaining a constant temperature of 4°C during the separation process, and then suddenly switching to 37°C, may cause heat stress in the islets. This warming phase effectively prevents the islets from experiencing heat stress caused by sudden temperature changes, thereby maximizing the maintenance of the structural integrity and physiological function of the islets.

[0027] (d) Replace with an equal volume of freshly prepared complete culture medium of the same formula. This is intended to remove the culture medium that has been settled and diluted, thereby completing the initial purification of the islets and providing a clean initial environment for subsequent formal culture.

[0028] 2. Islet culture In this invention, isolated and purified mouse islets were randomly divided into three groups: a 0-hour baseline group, a 48-hour control group, and a 48-hour combined drug administration group. Each group had six biological replicates, and each biological replicate contained 50 morphologically intact islets.

[0029] After grouping, all islets were first transferred to drug-free complete culture medium and incubated at 37°C with 5% CO2 for 30 minutes to stabilize their condition. After incubation, they were cultured in 6-well plates according to the following protocol, with a culture volume of 2 mL per well:

[0030] 0-hour baseline group: Samples were collected immediately after the static treatment and used as the initial baseline for functional testing.

[0031] 48-hour control group: Islets were placed in 2 mL of drug-free complete culture medium and cultured continuously for 48 hours. No medium changes or replenishment were performed during this period.

[0032] 48-hour combined administration group: Initial administration: Islets were incubated in 2 mL of complete culture medium containing 5 μM deferoxamine (DFO) and 10 μM deferoxone (DFP). This culture medium was prepared by adding 2 μL of DMSO stock solution containing 5 mM DFO and 10 mM DFP to 2 mL of complete culture medium.

[0033] Preparation steps of DMSO mixed reservoir (DMSO solution): Dissolve DFO and DFP in DMSO until the final concentration of DFO is 5mM and the final concentration of DFP is 10mM.

[0034] Complete culture medium: 500 ml of RPMI-1640 medium, with 10 ml of penicillin-streptomycin antibiotics at a final concentration of 2% (V / V) and 50 ml of fetal bovine serum at a final concentration of 10% (V / V).

[0035] Mid-term replenishment: After 24 hours of culture, add 2 μL of the same batch of DMSO mixed stock solution (containing 5 mM DFO and 10 mM DFP) directly to each well of the original culture system, mix gently, and continue to culture for 48 hours.

[0036] All culture operations were carried out in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0037] 3. Detection Method (1) Method for detecting intracellular Fe²⁺ levels, specifically including the following steps: a. Sample preparation and inoculation: After the culture treatment is completed, each group of islet samples are independently transferred to a black transparent bottom 96-well plate, and one replicate sample (containing about 13 islets) is inoculated in each well, and 2 ml of complete culture medium is added.

[0038] b. Fluorescent probe loading: Add FerroOrange (F374, Dojin, Japan) fluorescent probe to each well to a final concentration of 1 μmol / L in the culture system, and incubate in a 37°C incubator in the dark for 30 minutes to specifically label Fe²⁺ in cells.

[0039] c. Nuclear counterstaining: After incubation, add blue fluorescent dye Hoechst 33342 (HY-15559, MCE) to each well to a final concentration of 5 μg / mL in the culture system, and continue incubation at 37°C in the dark for 10 minutes to label cell nuclei.

[0040] d. Fluorescence signal detection and imaging: After incubation, detection was performed using a Zeiss LSM 900 laser confocal microscope. Hoechst 33342 was excited with a 405nm laser, and fluorescence emission signals near 461nm were acquired; FerroOrange was excited with a 488nm laser, and fluorescence emission signals near 580nm were acquired. Intracellular Fe²⁺ levels were quantitatively assessed by analyzing the average fluorescence intensity of the 580nm channel within the islet region.

[0041] (2) Immunofluorescence detection of 4-HNE protein: The 4-HNE protein in the pancreatic islets was localized and semi-quantitatively analyzed by immunofluorescence, specifically including the following steps: a. Sample fixation and permeabilization: The cultured islets were fixed with 4% (w / v) paraformaldehyde solution at room temperature for 1 hour. After fixation, the samples were washed three times with phosphate-buffered saline (PBS) for 5 minutes each time. Subsequently, the islets were permeabilized with PBS solution containing 0.5% (v / v) Triton X-100 and incubated at room temperature for 1 hour.

[0042] b. Non-specific site blocking: After permeabilization, wash the sample three times with PBS for 5 minutes each time. Then, block the sample with PBS blocking solution containing 10% (v / v) goat serum at room temperature for 1 hour to block non-specific antibody binding.

[0043] c. Primary antibody incubation: After blocking, discard the blocking solution and add a mixed working solution of 4-HNE mouse monoclonal primary antibody (MA5-27570, Thermo) and Insulin rabbit monoclonal primary antibody (ab181547, Abcam) diluted with the blocking solution. Ensure that the antibody working solution completely covers the sample and incubate overnight (12-16 hours) at 4°C in the dark.

[0044] d. Secondary antibody incubation and nuclear staining: After primary antibody incubation, wash the sample three times with PBS for 15 minutes each time to completely remove unbound primary antibody. Then, add a mixed working solution of fluorescent secondary antibodies diluted with blocking buffer, corresponding to the species of 4-HNE and Insulin primary antibodies, ensuring complete coverage of the sample, and incubate overnight (12-16 hours) at 4°C in the dark. After secondary antibody incubation, wash the sample three times with PBS for 15 minutes each time in the dark. Then, add DAPI staining solution to stain the cell nuclei and incubate at room temperature in the dark for 15 minutes. After staining, wash the sample twice with PBS for 10 minutes each time in the dark.

[0045] e. Mounting and Image Acquisition: The prepared islet samples were mounted using DAPI mounting medium containing an anti-fluorescence quencher. Images were acquired using a laser confocal microscope, wherein:

[0046] The fluorescence signal of DAPI was excited and acquired using a 405 nm laser; The fluorescent secondary antibody signal corresponding to Insulin was excited and acquired using a 488 nm laser. The fluorescent secondary antibody signal corresponding to 4-HNE was excited and acquired using a 594 nm laser.

[0047] The expression level of 4-HNE was semi-quantitatively analyzed by analyzing the average fluorescence intensity of the 594 nm channel in the pancreatic islet region.

[0048] (3) Glucose-stimulated insulin secretion function test: The glucose-stimulated insulin secretion test (GSIS) was performed using the Ezassay kit. The function of the pancreas was assessed by measuring the amount of insulin secreted by the pancreas under stimulation with different concentrations of glucose. The specific steps included:

[0049] 3.1 Buffer solution preparation a. Preparation of insulin basal buffer (Krebs-Ringer Bicarbonate Buffer): Mix 12.5 mL of 20x Solution A stock solution with 12.5 mL of 20x Solution B stock solution, add ultrapure water, and bring the volume to 250 mL. Then, purify the solution with pure oxygen for 25 minutes. Adjust the pH to 7.5 using hydrochloric acid solution, and add 0.8 g of bovine serum albumin to bring the final concentration to 0.32% (w / v). This yields the insulin basal buffer.

[0050] b. Preparation of glucose-stimulated buffer solution: 3.3mM glucose buffer: Take 330μL of 1M glucose stock solution and 100μL of DMSO, and add the above insulin basal buffer to a final volume of 100mL.

[0051] 16.7mM glucose buffer: Take 1.67mL of 1M glucose stock solution and 100μL of DMSO, and add the above insulin basal buffer to a final volume of 100mL.

[0052] c. Preparation of acidic ethanol solution: Mix 35 mL of anhydrous ethanol, 750 μL of 12N hydrochloric acid and 14.25 mL of ultrapure water to prepare an acidic ethanol solution of 70% ethanol and 1.5% HCl, which is used for insulin extraction.

[0053] 3.2 Insulin secretion stimulation and sample collection a. Pre-stabilization: Transfer the islets to be tested to 3.3 mM glucose buffer and incubate at 37°C for 1 hour.

[0054] b. Basal secretion phase collection: Transfer the islets to fresh 3.3 mM glucose buffer and incubate at 37°C for 30 minutes. After incubation, collect the supernatant and record it as the basal secretion sample.

[0055] c. Collection of the stimulated secretion phase: The islets were transferred to 16.7 mM glucose buffer and incubated at 37°C for 1 hour. After incubation, 100 μL of the supernatant was collected and recorded as the stimulated secretion sample.

[0056] d. Intracellular insulin extraction: The islets stimulated in step c were collected into a 1.5 mL centrifuge tube, 1 mL of acidic ethanol solution was added, and the cells were sonicated to fully lyse the cells and release insulin. The resulting solution was recorded as the intracellular insulin sample.

[0057] 3.3 Sample Dilution and Detection All collected samples were diluted according to the following protocol and processed using the Ezassay High Sensitive Insulin Kit. The absorbance was measured at 450 nm to calculate the insulin concentration.

[0058] Basal secretion sample: Take 40 μL of sample, add 460 μL of 3.3 mM glucose buffer, and dilute 1:12.5.

[0059] Stimulating secretion sample: Take 10 μL of sample, add 490 μL of 16.7 mM glucose buffer, and dilute 1:50.

[0060] Intracellular insulin sample: First, take 5 μL of sample and add 495 μL of 16.7 mM glucose buffer to dilute 1:100; then take 100 μL of the diluted sample and add 900 μL of 16.7 mM glucose buffer to dilute 1:10, that is, the total dilution is 1:1000.

[0061] The 20x Solution A stock solution contains the following final concentrations of components: 118 mM NaCl, 5 mM KCl, 3 mM CaCl2, and 1 mM MgSO4.

[0062] The 20x Solution B stock solution contains the following final concentration components: 1 mM KH2PO4, 25 mM NaHCO3.

[0063] 1 M HEPES stock solution: Weigh 2.383 g HEPES and dissolve it in 10 mL of ultrapure water.

[0064] 1 M Glucose Stock Solution: Weigh 1.8016 g of glucose and dissolve it in 10 mL of ultrapure water.

[0065] II. Test Results 1. Combined treatment with deferoxamine (DFO) and deferoxone (DFP) on the effects of iron deficiency on cultured pancreatic islets in vitro. 2+ The cumulative effect The results are as follows Figure 1 As shown, compared with the 0-hour baseline group, the intracellular Fe²⁺ fluorescence intensity of pancreatic islet cells was significantly enhanced after 48 hours of conventional in vitro culture, indicating significant Fe²⁺ accumulation during culture. However, after the combined addition of DFO and DFP to the culture system, the Fe²⁺ fluorescence signal in the islets was significantly weakened compared with the 48-hour control group. This result indicates that the combined treatment with DFO and DFP can effectively chelate and remove abnormally accumulated Fe²⁺ in in vitro cultured islets. Since excessive Fe²⁺ is a key initiator of oxidative stress and ferroptosis, removing Fe²⁺ can reduce its cytotoxic effects on cells at the source, thus laying the foundation for maintaining cell survival and reducing mortality. Therefore, the combined treatment with DFO and DFP lays the foundation for improving islet survival by removing intracellular Fe²⁺.

[0066] 2. Effects of combined treatment with deferoxamine (DFO) and deferoxone (DFP) on the accumulation of 4-HNE, a lipid peroxidation product, in cultured pancreatic islets. As a key downstream event of Fe²⁺ toxicity, lipid peroxidation levels are a direct indicator for assessing the occurrence of ferroptosis and the degree of cell damage. For example... Figure 2 As shown, consistent with the Fe²⁺ accumulation trend, the fluorescence signal of 4-HNE, a lipid peroxidation product, was significantly enhanced in pancreatic islets cultured for 48 hours under normal conditions. This indicates that the accumulation of 4-HNE was significantly inhibited after combined treatment with DFO and DFP. This demonstrates that the combined treatment with DFO and DFP directly blocks the ferroptosis pathway by inhibiting lipid peroxidation. This result, together with the clearance of Fe²⁺, forms a complete chain of evidence, jointly confirming that this combined treatment regimen effectively blocks the Fe²⁺-dependent ferroptosis pathway. The reduction in lipid peroxidation directly implies a reduction in damage to cell membrane structure, which is strong evidence for maintaining cell structural integrity and preventing cell disintegration and death.

[0067] 3. Effects of combined treatment with deferoxamine (DFO) and deferoxone (DFP) on GSIS of insulin secretion in cultured pancreatic islets. The functional activity of the pancreatic islets is directly related to their survival status. For example... Figure 3As shown, conventional culture for 48 hours significantly weakens the core physiological function of pancreatic islets—the ability to stimulate insulin secretion. However, islets treated with a combination of DFO and DFP showed significantly improved GSIS function. Intact GSIS function depends on the healthy metabolic state of pancreatic β-cells, signal transduction pathways, and vesicle secretion mechanisms; the normal operation of these processes presupposes cellular structural integrity and survival. Therefore, the significant improvement in GSIS function strongly and indirectly demonstrates that the combined treatment of DFO and DFP effectively protects the overall activity of pancreatic islet cells, reduces functional cell loss due to ferroptosis, and functionally confirms its positive effect on improving the effective survival rate of islets.

[0068] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0069] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of deferoxamine and deferoxone in the preparation of biological agents to alleviate or prevent hypoxic damage in in vitro culture of pancreatic islets.

2. The application according to claim 1, characterized in that, The deferoxamine in the biological agent has a final concentration of 5 μM to 100 μM, and the deferoxone has a final concentration of 10 μM to 100 μM.

3. The application according to claim 2, characterized in that, The solvent for the biological agent is DMSO.

4. The application according to claim 3, characterized in that, The biological agent that alleviates or prevents hypoxic injury in in vitro pancreatic islet culture includes the following steps: After purification and culture, the islets are placed in a complete culture medium containing the aforementioned biological agent for further culture, thereby reducing islet hypoxia damage. The volume ratio of the biological agent to the complete culture medium is 2:2000-2100.

5. The application according to claim 4, characterized in that, The complete culture medium is based on RPMI-1640 medium, with 5.2mM to 5.5mM glucose, 1% to 2% penicillin-streptomycin antibiotics, and 8% to 10% fetal bovine serum added to every 500ml of the basic medium.

6. The application according to claim 5, characterized in that, The culture conditions are: 35℃~37℃, 5% CO2 culture for 48~50 hours, with deferoxamine and deferoxone supplemented every 23h~24 hours.

7. The application according to claim 4, characterized in that, The islet purification and culture steps are as follows: the islets are placed in the complete culture medium and incubated at 35℃~37℃ and 5% CO2 for 25~30 minutes, and the supernatant is discarded.

8. According to claim 1, the biological agent has the following functions: Reduce Fe 2+ The accumulation of 4-HNE, a lipid peroxidation product, reduces hypoxic damage to pancreatic islets during in vitro culture.

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