Use of a net inhibitor for the preparation of a medicament for the prevention and / or treatment of an IBMIR response in a pancreatic islet transplantation

Abstract

The application discloses application of a NETs inhibitor in preparation of a medicine for preventing and / or treating an IBMIR reaction after islet transplantation. The application is based on a clinicopathological finding that NETs is related to islet function after islet transplantation, and further verified through experiments that a NETs inhibitor such as DNase I and knockout of a PAD4 gene can significantly inhibit IBMIR occurring in an early stage after islet transplantation, prevent formation of an ischemic focus, and significantly improve sugar metabolism function after islet transplantation. Meanwhile, the application also proves that NETs promotes the IBMIR reaction after islet transplantation by activating NF-kappa B, MAPK and NLRP3 inflammatory pathways, and the application effectively inhibits activation of the above pathways and expression of TF and CitH3 by blocking the NETs. The technical scheme of the application has important significance for islet transplantation treatment of diabetic patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of NETs inhibitors in the preparation of medicaments for the prevention and / or treatment of islet transplantation IBMIR response. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by persistently high blood sugar levels. Its core pathological mechanism lies in abnormal pancreatic function: when the pancreatic beta cells responsible for secreting insulin are destroyed by the autoimmune system, the body cannot produce sufficient insulin, resulting in type 1 diabetes; when the body's cells cannot respond normally to insulin and develop insulin resistance, it is type 2 diabetes. Insulin, a hormone secreted by the pancreas, plays a crucial role in regulating the entry of glucose from the blood into cells to provide energy for the body. Once this regulatory system is imbalanced, blood sugar levels remain elevated, triggering a series of health problems.

[0003] An integrated treatment strategy for diabetes encompasses multiple levels, including basic interventions, drug regulation, device-assisted therapy, and cell replacement therapy. Lifestyle interventions such as diet, exercise, and sleep management, along with self-monitoring of blood glucose, form the cornerstone of all treatment plans. For type 2 diabetes, metformin monotherapy is the first-line treatment. If blood glucose control is inadequate, it can be combined with oral hypoglycemic agents of different mechanisms, such as SGLT-2 inhibitors or GLP-1 receptor agonists, or insulin therapy can be initiated. Type 1 diabetes, due to an absolute deficiency of endogenous insulin, requires insulin replacement therapy. Closed-loop insulin infusion systems (artificial pancreas) can achieve automated linkage between real-time blood glucose monitoring and insulin infusion, significantly improving blood glucose control. For fragile type 1 diabetes patients with hypoglycemia perception impairment or recurrent severe hypoglycemia, islet transplantation can achieve functional cure by restoring endogenous insulin secretion, and approximately 70% of recipients can be weaned off insulin injections long-term.

[0004] Islet transplantation, as an effective treatment to replace insulin secretion function, has made significant progress in clinical application in recent years. However, immediate blood-mediated inflammatory response (IBMIR) occurs after portal vein islet transplantation, leading to an early loss of up to 50%-70% of transplanted islets, severely limiting clinical efficacy. Anti-inflammatory drugs such as TNF-α inhibitors (e.g., etanercept) and IL-1 receptor antagonists (e.g., anaerobiculin) can alleviate IBMIR to some extent, but due to the complexity of the IBMIR response mechanism, the effect of single drug intervention remains limited, and a high level of islet cell loss still exists in the early post-transplantation period. This indicates that the occurrence of IBMIR is not caused by a single factor, but is the result of multiple mechanisms working together.

[0005] NETs are complexes released by neutrophils, consisting of a chromatin backbone and attached with various granule proteins, including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (CitH3). The physiological function of NETs is to physically capture and efficiently kill pathogens such as bacteria and fungi, preventing the spread of infection. They can induce immune dysregulation in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.

[0006] Therefore, in order to further reduce the initial damage after islet transplantation, it is urgent to explore the specific mechanisms of the immediate blood-mediated inflammatory response that occurs early after portal vein islet transplantation. By further elucidating the mechanism of platelet-neutrophil interaction and developing new intervention strategies, it is hoped that early damage to the transplanted islets can be reduced, thereby improving the survival rate and efficacy of transplantation. Summary of the Invention

[0007] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of NETs inhibitors in the preparation of medicaments for the prevention and / or treatment of IBMIR response in islet transplantation.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides the use of NETs inhibitors in the preparation of products that improve glycemic regulation after pancreatic islet transplantation.

[0009] In some embodiments of the present invention, the improvement of blood glucose regulation function after islet transplantation includes improving blood glucose homeostasis maintenance function and / or glucose tolerance after islet transplantation; the glucose tolerance is the body's ability to regulate ingested or infused glucose.

[0010] In some embodiments of the present invention, the NETs inhibitors include at least one of PAD4 inhibitors, NADPH oxidase inhibitors, neutrophil elastase inhibitors, and DNA degrading enzymes.

[0011] In some embodiments of the present invention, the DNA-degrading enzyme includes DNase I.

[0012] In some embodiments of the present invention, the NETs inhibitor further includes a platelet-neutrophil aggregate inhibitor; the platelet-neutrophil aggregate inhibitor includes at least one of a P-selectin inhibitor, a PSGL-1 inhibitor, and a GPIbα inhibitor.

[0013] In some embodiments of the present invention, the P-selectin inhibitor includes an anti-CD62P antibody.

[0014] In some embodiments of the present invention, the islet transplantation includes at least one of normothermic mechanical perfusion of the liver combined with islet transplantation and portal vein islet transplantation.

[0015] In some embodiments of the present invention, the NETs inhibitor includes use before and / or after islet transplantation.

[0016] A second aspect of the invention provides the use of NETs inhibitors in the preparation of medicaments for the prevention and / or treatment of ischemic injury following islet transplantation.

[0017] A third aspect of the invention provides the use of NETs inhibitors in the preparation of medicaments for the prevention and / or treatment of blood-mediated inflammatory responses immediately following islet transplantation.

[0018] In some embodiments of the present invention, the prevention and / or treatment of immediate blood-mediated inflammatory response after islet transplantation includes achieving prevention and / or treatment of immediate blood-mediated inflammatory response after islet transplantation by regulating NLRP3 expression, inhibiting activation of the NF-κB inflammatory pathway after islet transplantation, and inhibiting activation of the MAPK pathway after islet transplantation.

[0019] A fourth aspect of the present invention provides a product for improving islet function after islet transplantation, the product comprising a therapeutically effective amount of a NETs inhibitor.

[0020] In some embodiments of the present invention, the pancreatic function includes a blood glucose regulation function.

[0021] In some embodiments of the present invention, the product comprises a drug or a drug composition.

[0022] The beneficial effects of this invention are as follows: This invention discloses the application of NETs inhibitors in the preparation of drugs for preventing and / or treating islet transplantation-induced IBMIR. Based on clinical pathological findings regarding the correlation between NETs and islet function after islet transplantation, this invention further experimentally verifies that NETs inhibitors, such as DNase I, can significantly inhibit early IBMIR after islet transplantation, prevent the formation of ischemic foci, and significantly improve glucose metabolism after islet transplantation. Furthermore, the experimental results of this invention demonstrate that DNase I has a more significant effect on improving glucose metabolism after islet transplantation compared to other NETs inhibitors. Simultaneously, this invention also demonstrates that NETs promote IBMIR after islet transplantation by activating the NF-κB, MAPK, and NLRP3 inflammatory pathways. This invention effectively inhibits the activation of these pathways and the expression of TF and CitH3 by blocking NETs. The technical solution of this invention has significant implications for the treatment of islet transplantation in diabetic patients. Attached Figure Description

[0023] Figure 1 The images show the observation results of isolated and purified C57 mouse islets. A shows mouse islets under a light microscope; B shows the FDA / PI staining of mouse islets.

[0024] Figure 2 The graph shows the results of drug-induced NETs blocking the activity of pancreatic islet cells. A shows the results of immunofluorescence detection of the activity of pancreatic islet cells cultured in vitro for 24 hours in different groups; B shows the results of quantitative analysis of FDA / PI staining fluorescence intensity in different groups.

[0025] Figure 3 The image shows the results of detecting NETs in the liver of mice 2 hours after islet transplantation using drugs and PAD4 gene knockout. A shows the observation results of liver ischemic lesions in each group of mice 2 hours after islet transplantation; B shows the detection results of hematoxylin-eosin staining and NETs marker immunofluorescence in liver tissue 2 hours after surgery. In the hematoxylin-eosin staining image, the red circle represents the islets, and the scale bar is 100 μm.

[0026] Figure 4 The figure shows the results of detecting serum MPO-DNA levels 2 hours after mouse islet transplantation by using drugs and PAD4 gene knockout.

[0027] Figure 5 The figures show the results of blood glucose and glucose tolerance tests after islet transplantation in mice. A is a graph showing the blood glucose change within 60 days after islet transplantation; B is an analysis of the proportion of mice with normal blood glucose after islet transplantation; C is a graph showing the results of the intraperitoneal glucose tolerance test after islet transplantation; and D is a bar chart showing the AUC values ​​of the intraperitoneal glucose tolerance test results after islet transplantation.

[0028] Figure 6 The images show the serum detection results 2 hours after mouse islet transplantation. A shows the serum TNF-α detection results; B shows the serum IL-1β detection results; C shows the serum IL-6 detection results; D shows the serum C-peptide detection results; and E shows the serum TAT detection results.

[0029] Figure 7 The images show the results of peripheral blood platelet activation in mice 2 hours after islet transplantation. A is the FSC-H / SSC-H gating chart; B is the FITC-CD41 / SSC-H gating chart; C is the result chart for the WT group; D is the result chart for the control group; E is the result chart for PAD4. - / - The results of the test are shown in Figure 1; F is the results of the test in the anti-CD62P group; G is the histogram of PE-CD62P detection; H is the bar chart of the percentage of CD62P positive platelets.

[0030] Figure 8 The following graphs show the platelet-neutrophil aggregate (PNA) levels in mice 2 hours after islet transplantation. A is a gating chart of FSC-A / SSC-A; B is a gating chart of APC-CD45 / SSC-A; C is a gating chart of CD11b / Ly6G; D is the result graph for the WT group; E is the result graph for anti-CD62P; F is the result graph for the control group; G is the result graph for PAD4. - / - The detection results for the group are shown in Figure 1; H is the histogram of PCD41 detection; I is the histogram of CD41. + With LY6G + Cell percentage bar chart.

[0031] Figure 9 Image showing the results of NET formation in the liver of mice 2 hours after islet transplantation following anti-CD62P treatment. Scale bar = 100 μm.

[0032] Figure 10 The graph shows the results of KEGG analysis of differentially expressed genes in the transcriptome after islet transplantation in WT and PAD4 knockout mice. A is a scatter plot of enrichment factors; B is a bar chart of KEGG enrichment analysis.

[0033] Figure 11 GSEA proteomic analysis of pancreatic islet transplantation in WT and PAD4 knockout mice.

[0034] Figure 12 The images show the Western blot (WB) results of liver NF-κB inflammatory pathway detection after mouse islet transplantation. A shows the detection results of PAD4, p-IκB, and IκB; B shows the bar chart of relative expression levels of PAD4; C shows the bar chart of relative expression levels of p-IκB and IκB; D shows the detection results of p-p65 and p65; E shows the bar chart of relative expression levels of p-p65 and p65.

[0035] Figure 13 The images show the Western blot (WB) results of the MPAK pathway in the liver of mice after pancreatic islet transplantation. A shows the results of ACTIN and TF detection; B shows the bar chart of TF expression relative to ACTIN; C shows the bar chart of p-JNK expression relative to JNK; D shows the results of p-JNK and JNK detection; E shows the results of p-ERK, ERK, and CitH3 detection; F shows the bar chart of CitH3 relative expression; and G shows the bar chart of p-ERK relative to ERK expression.

[0036] Figure 14 The image shows the Western blot (WB) results of NLRP3 expression levels in the liver of mice after islet transplantation. A shows the NLRP3 detection results; B shows the bar chart of relative NLRP3 expression. Detailed Implementation

[0037] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art. Statistical analysis of the embodiments was performed using R language software (version 4.0.2) and GraphPad Prism 8. Experimental results are expressed as mean ± standard deviation or median (interquartile range). For normally distributed data, a t-test was used for comparisons between two independent samples; for non-normally distributed data, a nonparametric test was used. Correlation analysis was performed using the Spearman correlation test. A p-value less than 0.05 was considered statistically significant. P <0.05, P <0.01, P <0.001. The calculation of islet equivalents in the examples is shown in Table 1 below.

[0038] Table 1. Islet Equivalent Table

[0039] Multiply the number of prepared islets by the corresponding coefficient, and finally add up the equivalents under each coefficient to get the total equivalent of islets used.

[0040] Example 1 This embodiment provides an assessment of the effects of NETs on pancreatic islet cells. DNase I and neutrophil elastase inhibitor sivelestat can be used as NET blocking drugs to inhibit NET formation or clear NETs. This embodiment uses DNase I and sivelestat for testing, and the specific experimental steps are as follows.

[0041] Male SPF-grade C57BL / 6 mice, aged 8-12 weeks and weighing 20-25g, were selected from the Laboratory Animal Center of Sun Yat-sen University. All mice were housed in the specific pathogen-free (SPF) grade animal facility at Sun Yat-sen University, with free access to food and water, and a 12-hour diurnal cycle. All animal experimental procedures in this study were approved by the Animal Ethics Committee of Sun Yat-sen University and strictly adhered to the national guidelines for laboratory animal ethics and welfare.

[0042] The steps for islet isolation, extraction, and culture are as follows: (1) Select C57BL / 6 mice weighing 20-25g and fast them for 4 hours before the operation. After anesthesia with 1% (w / v) sodium pentobarbital injected intraperitoneally, the heart was punctured and blood was released.

[0043] (2) After disinfecting the skin with 75% alcohol, cut open the abdomen to expose the common bile duct and duodenal papilla, and use vascular clamps to close the common bile duct and duodenal papilla.

[0044] (3) Using a stereomicroscope, perform retrograde common bile duct puncture and cannulation with a 5mL syringe and a 30G needle. After confirming that the puncture needle has entered the common bile duct, inject 2.5mL of type V collagenase (1mg / mL, Sigma-Aldrich) retrogradely.

[0045] (4) After the V-type collagenase is infused, the well-expanded pancreas is removed and placed into a 50mL centrifuge tube. V-type collagenase is added to make the total volume of liquid in the centrifuge tube 7mL. Then the centrifuge tube is placed in a water bath at 37±0.2℃ and allowed to stand for digestion for 9 to 9.5 minutes until the pancreatic islet tissue is loose.

[0046] (5) After digestion, remove the centrifuge tube, shake the centrifuge tube appropriately to loosen the pancreatic islet tissue to a muddy state, and then quickly add 35 mL of pre-cooled (4℃) Hanks solution to stop digestion. Centrifuge at 2000 r / min for 6 seconds.

[0047] (6) After gently discarding the supernatant, add 35 mL of 4℃ Hanks solution to wash the tissue again, and centrifuge at 2000r / min for 6 seconds.

[0048] (7) Discard the supernatant again and add the precipitate to 5 mL Histopaque ®Resuspend the cells in -1119 nucleated and granulocyte separation medium (Sigma-Aldrich), mix thoroughly, and slowly add 2 mL of Histopaque. ® Add 1077 lymphocyte separation medium (Sigma-Aldrich), then slowly add 2 mL of Hanks' solution. After establishing the density gradient, centrifuge at 2000 rpm for 5 minutes, followed by slow increasing and decreasing the flow rate.

[0049] (8) After centrifugation, pour the supernatant containing islets into another sterile 15 mL centrifuge tube, wash three times with Hanks' solution, transfer the precipitate to a black glass culture dish, and rinse with Hanks' solution. Manually select islets using a 200 μL pipette under a stereomicroscope.

[0050] (9) Manually selected high-purity islets were added to RPMI-1640 medium (containing 10% (v / v) fetal bovine serum (Gibco) and 1% (v / v) penicillin-streptomycin) and cultured in a 37°C, 5% CO2 incubator. Islet equivalent (IEQ) and purity were calculated. Islet activity was detected by FDA / PI staining. Both FDA and PI reagents were from Sigma-Aldrich. FDA and PI dyes were added to the islets according to the reagent instructions. Staining time was 15 to 30 minutes. After staining, cells were observed using a fluorescence microscope. Live cells emitted green fluorescence, while dead cells showed red fluorescence.

[0051] The preparation steps for NETs are as follows: First, isolate mouse bone marrow neutrophils: (1) The mice were euthanized by overdose of anesthesia, their tibia and femur were separated, the muscle tissue around the tibia and femur was removed, and the mice were disinfected by soaking in 75% alcohol.

[0052] (2) After rinsing with pre-cooled PBS three times, cut off both ends of the bone marrow to expose the bone marrow cavity. Use a 10mL syringe to draw pre-cooled RPMI-1640 medium (Gibco) to rinse the bone marrow cavity until it is filtered through a 70μm nylon sieve. Collect the filtered rinsing solution into a 50mL centrifuge tube and centrifuge at 3000rpm for 4min.

[0053] (3) Discard the supernatant and resuspend the bone marrow cells in 3 mL of RPMI-1640 medium solution.

[0054] (4) Take another sterile 15mL centrifuge tube, first add 3mL of 78% (v / v) Percoll solution (Beijing Solarbio Science & Technology Co., Ltd.), then slowly add 3mL of 65% (v / v) Percoll solution (Beijing Solarbio Science & Technology Co., Ltd.), and finally slowly add the 3mL of bone marrow cells from step (3). Centrifuge at 800×g at room temperature for 35 minutes without braking.

[0055] (5) After centrifugation, the loosely packed cell layer between the 65% and 78% (v / v) Percoll solution layers in the centrifuge tube is the neutrophil layer. Carefully aspirate this cell layer, add 3 mL of PBS buffer, mix well, and centrifuge at 4°C and 1000 rpm for 3 minutes.

[0056] (6) Discard the supernatant and wash twice with 3 mL of PBS buffer.

[0057] (7) Add 1 mL of red blood cell lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), mix well and react for 1 minute to lyse the red blood cells.

[0058] (8) Add PBS buffer to stop the lysis, centrifuge at 4°C and 1000 rpm for 3 minutes.

[0059] (9) Discard the supernatant, wash twice with 3 mL PBS buffer, discard the supernatant again, and the precipitate is neutrophils. Resuspend the cells in RPMI-1640 medium (containing 10% (v / v) fetal bovine serum and 1% (v / v) streptomycin / penicillin).

[0060] The mouse bone marrow neutrophils obtained above were then used for NET induction and extraction, and the specific steps are as follows: (1) Freshly extracted neutrophils were seeded into 6-well cell culture plates at a cell density of 1×10⁻⁶ cells / well. 7 Neutrophils were stimulated with 200 nmol / L phorbol 12-myristate 13-acetate (PMA) per well for 4 hours.

[0061] (2) After 4 hours, gently aspirate the supernatant, add 2 mL of pre-cooled sterile PBS buffer and gently wash twice. Collect the washing solution into a sterile 15 mL centrifuge tube and centrifuge at 450 × g for 10 minutes at 4°C.

[0062] (3) After centrifugation, the precipitate consists of residual neutrophils. The supernatant is collected into a sterile EP tube.

[0063] (4) The supernatant EP tube was placed in an EP tube centrifuge and centrifuged at 20000×g, 4℃ for 20 minutes. The precipitate is NETs.

[0064] (5) Add 2 mL of pre-cooled PBS buffer to resuspend the precipitate. Quantify the extracted NETs using the Histone Elisa kit (Shanghai Maclean Biotechnology Co., Ltd.). Store the remaining NETs at -80℃ for subsequent experiments.

[0065] The isolated islets were transferred to 6-well ultra-low adsorption plates at a density of 100 IEQ per well. 2 mL of RPMI-1640 medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin was added to each well. The plates were then divided into four groups according to the following treatment: Blank control group: The blank control group received no treatment; NETs + Islets Group (NETs Group): NETs were added to the islets to a final concentration of 200 μg / mL; DNase I + NETs + Islets Group (DNase + NETs Group): DNase I and NETs at a final concentration of 0.02 mg / mL and NETs at a final concentration of 200 μg / mL were added to the islets; Civelestat sodium + NETs + islets group: Civelestat sodium with a final concentration of 20 μg / mL and NETs with a final concentration of 200 μg / mL were added to the islets.

[0066] Each culture group was placed in a 37°C cell culture incubator and cultured for 24 hours. After 24 hours, FDA and PI reagents were added for activity detection.

[0067] Experimental results of islet isolation and extraction are as follows: Figure 1 As shown, Figure 1 As shown in Figure A, this embodiment successfully isolated morphologically intact and sufficient pancreatic islets, with an extracted islet purity exceeding 95%. Furthermore, FDA / PI staining results of the isolated islets showed islet viability greater than 90%, indicating that the isolated islets exhibited good cell viability. Figure 1 (B)

[0068] The results of the in vitro culture experiment of pancreatic islets and NETs are as follows: Figure 2 As shown in Figure A, the activity of islets significantly decreased after the addition of NETs to isolated islets, the islet structure became loose, the cell boundaries became unclear, the capsule became incomplete, and the morphology was obviously damaged; while in the DNase I and Sivelestat drug treatment groups, the PI / FDA fluorescence intensity ratio was significantly lower than that in the NETs group ( Figure 2 In the study (B), islet activity was significantly improved, islet morphology remained intact, and the capsule showed no obvious damage. These experimental results indicate that NETs have a significant impact on islet activity, while DNase I and Sivelestat can effectively prevent NETs from damaging the islets.

[0069] Example 2 This embodiment provides the use of NETs blocking drugs and an animal model of NET deficiency to verify the relationship between NETs and islet transplantation prognosis after islet transplantation. This embodiment uses NETs blocking drugs DNase I, PAD4 inhibitor Cl-amidine, and Sivelestat for testing. Islet isolation and extraction are performed in the same steps as in Example 1, as detailed below.

[0070] Male SPF-grade C57BL / 6 mice, aged 8-12 weeks and weighing 20-25g, were selected. The animals were obtained from the Laboratory Animal Center of Sun Yat-sen University. All mice were housed in a specific pathogen-free (SPF) level animal facility at Sun Yat-sen University, with free access to food and water, and a 12-hour diurnal cycle. All animal experimental procedures in this study were approved by the Animal Ethics Committee of Sun Yat-sen University and strictly adhered to the national guidelines for laboratory animal ethics and welfare.

[0071] The specific steps for establishing a type 1 diabetes model using streptozotocin are as follows: Select healthy male C57 mice approximately 10 weeks old, weighing 20-25g, and fast them overnight while allowing free access to water. Weigh them and administer 1% (w / v) streptozotocin (STZ) intraperitoneally at a dose of 200mg / kg, according to the drug dosage and mouse weight ratio. Starting on the 4th day after the intraperitoneal injection of STZ, blood is collected from the tail vein and random blood glucose is measured using a glucometer. Mice with two consecutive random blood glucose levels greater than 16.8 mmol / L are considered to have successfully established a type 1 diabetes model and are selected as islet transplant recipients.

[0072] The animals were grouped as follows, with 8 animals in each group. The islet isolation and extraction were performed in the same manner as in Example 1: Control group: C57 mice were injected with the same volume of physiological saline after portal vein puncture; Islet transplantation group (WT group): C57 mice underwent portal vein puncture followed by islet transplantation, with each mouse receiving 300 IEQ islets. DNAse I+ Islet Transplantation Group (DNAse I Group): C57 mice underwent portal vein puncture followed by islet transplantation. Each mouse received 300 IEQ islets. DNase I was injected intraperitoneally 30 minutes before islet transplantation in C57 mice. The injection dose was 2.5 mg / kg based on the mouse's body weight. Sivelestat+Islet Transplantation Group (Sivelestat Group): C57 mice underwent portal vein puncture followed by islet transplantation, with 300 IEQ islets transplanted per mouse; C57 mice were injected intraperitoneally with cerevesta sodium 30 minutes before islet transplantation, at a dose of 100 mg / kg based on mouse body weight, and were injected intraperitoneally again one day after surgery at a dose of 100 mg / kg of cerevesta sodium. Cl-amidine + Islet Transplantation Group (Cl-amidine Group): C57 mice underwent portal vein puncture followed by islet transplantation, with 300 IEQ islets transplanted per mouse; C57 mice were injected intraperitoneally with Cl-amidine 30 minutes before islet transplantation, at a dose of 100 mg / kg based on mouse body weight; and 100 mg / kg Cl-amidine was injected intraperitoneally again one day after islet transplantation. Two hours after islet transplantation, serum and liver tissue samples were collected from each group of mice to observe the condition of the transplanted livers and to detect the levels of MPO-DNA in the liver and serum. The steps for MPO-DNA detection are as follows: The apoptosis detection ELISA kit (Roche, Switzerland) and human myeloperoxidase (MPO) antibody (Bio-Rad, USA) were used according to the instructions. The steps are as follows: (1) Dilute the MPO antibody to 5 μg / mL and add it to each well of an ELISA 96-well plate in a volume of 100 μL. Incubate overnight at 4°C.

[0073] (2) Discard the MPO antibody and gently wash the 96-well plate twice with PBS buffer. After washing, add 100 μL of 1% (v / v) bovine serum albumin (BSA) to each well and block at room temperature for 1 hour.

[0074] (3) Wash three times with PBS buffer, and prepare a 100 μL test sample system by mixing 40 μL of serially diluted standard or serum sample with 60 μL of anti-DNA antibody. Add 100 μL of the test sample system to each well and incubate at room temperature for 2 hours.

[0075] (4) After washing three times with PBS buffer, add peroxidase substrate and incubate at 37°C for 1 hour.

[0076] (5) After incubation, wash the sample three times with PBS, add TMB substrate for color development, and then add 2N sulfuric acid stop solution. Use an ELISA reader to detect the absorbance of the sample at a wavelength of 405 nm.

[0077] Immunofluorescence co-localization detection of myeloperoxidase (MPO), citrullinated histone (CitH3), and insulin (NETs) and hematoxylin-eosin staining (HE staining) were performed on liver tissue specimens. The specific steps of tissue immunofluorescence staining are as follows: (1) Take liver tissue samples and quickly place them in 4% paraformaldehyde solution for fixation for about 24 hours.

[0078] (2) The fixed tissue was rinsed with physiological saline to remove residual fixative. Then, it was dehydrated by conventional methods, and was successively immersed in 70%, 80%, 90%, 95% and 100% alcohol concentrations, with the alcohol changed each time, and treated for about 1 hour in each concentration.

[0079] (3) After dehydration, clear the tissue sample with xylene, soaking it 2-3 times for about 15 minutes each time. Next, immerse the tissue sample in melted paraffin liquid for 4-6 hours until the tissue is fully permeated with paraffin. After the paraffin has permeated the tissue, embed the tissue in the paraffin and cut it into sections about 4-5 micrometers thick.

[0080] (4) Heat the prepared tissue paraffin sections in a microwave oven for 3 minutes to dewax them, and then soak them in different concentrations of ethanol (100%-90%-80%-70%-50%-30%) for 4 minutes each time.

[0081] (5) Heat the antigen retrieval solution (Shanghai Beyotime Biotechnology Co., Ltd.) to 96-98°C and maintain for 15 minutes. After the slides have cooled, add the blocking solution (Shanghai Beyotime Biotechnology Co., Ltd.) and incubate in the dark for 1 hour.

[0082] (6) Add diluted primary antibody solution to the slides and incubate overnight at 4°C. After incubation, wash the slides three times with PBS buffer for 5 minutes each time. The primary antibodies used were Anti-MPO antibody (AF3667, R&D Company, USA), Anti-Histone H3 (citrulline R2+R8+R17) antibody (Abcam, UK), and insulin antibody (Abcam, UK).

[0083] (7) Add diluted fluorescently labeled secondary antibody to the slides and incubate for 1 hour. Wash the slides three times with PBS buffer for 5 minutes each time. The secondary antibodies used were goat anti-rabbit IgG (Alexa Flour 488, Abcam, UK), donkey anti-goat IgG (Alexa Flour 467, Abcam, UK) and donkey anti-guinea pig IgG (AF555, Abcam, UK).

[0084] (8) Add DAPI staining agent to the slides and stain for 5 minutes. After staining, wash the slides three times with PBS buffer. Finally, add anti-fluorescence quenching mounting medium to mount the slides and observe the fluorescence under a fluorescence microscope. Use ImageJ software to perform quantitative analysis of the fluorescence intensity of the images.

[0085] The steps for HE staining are as follows: (1) Take liver tissue samples and quickly place them in 4% paraformaldehyde solution for fixation for about 24 hours.

[0086] (2) The fixed tissue was rinsed with physiological saline to remove residual fixative. Then, it was dehydrated by conventional methods, and was successively immersed in 70%, 80%, 90%, 95% and 100% alcohol concentrations, with the alcohol changed each time, and treated for about 1 hour in each concentration.

[0087] (3) After dehydration, clear the tissue sample with xylene, soaking it 2-3 times for about 15 minutes each time. Next, immerse the tissue sample in melted paraffin liquid for 4-6 hours until the tissue is fully permeated with paraffin. After the paraffin has permeated the tissue, embed the tissue in the paraffin and cut it into sections about 4-5 micrometers thick, placing them on a glass slide.

[0088] (4) Place the slices on a 60°C heating plate for pre-baking for 30 minutes to ensure the slices are fixed. Use xylene to remove the paraffin by treating twice with xylene for about 10 minutes each time. After removing the paraffin, treat the slices in 100%, 95%, 80%, and 70% alcohol solutions in sequence, soaking for 3-5 minutes at each step.

[0089] (5) Rinse the sections with distilled water. Soak the sections in hematoxylin solution for 5-10 minutes, then rinse with tap water until the color no longer appears in the water.

[0090] (6) Immerse the slides in eosin solution for 2-3 minutes, then rinse with tap water and dehydrate with alcohol. Finally, clear the slides in xylene and mount them with mounting glue. After the specimens are prepared, observe the liver tissue structure and inflammatory cell infiltration under a microscope for pathological analysis.

[0091] The levels of inflammatory factors, C-peptide, and thrombin-antithrombin complex (TAT) in serum within 2 hours post-islet transplantation were measured using ELISA kits following the instructions. The following kits (all purchased from Wuhan Sanying Biotechnology Co., Ltd.) were used: Mouse TNF-α ELISA kit, Mouse IL-1β ELISA kit, Mouse IL-6 ELISA kit, Mouse C-peptide ELISA kit, and Mouse TAT ELISA kit. PAD4 was also used. - / -C57 mice (Wuhan Youdu Biotechnology Co., Ltd.) were used as a control, along with PAD4 mice. - / - C57 mice were treated the same as those in the islet transplantation group.

[0092] Blood glucose levels in all mice were randomly measured every two days via tail vein blood collection for 60 days post-surgery using blood glucose test strips and a blood glucose meter. Simultaneously, 20 days post-islet transplantation, intraperitoneal glucose tolerance tests were performed on all groups of mice, with normal mice serving as controls (Normal group). The specific procedures were as follows: Mice were fasted for 12 hours before the experiment but allowed free access to water. A glucose solution with a concentration of 200 mg / mL was prepared in advance using physiological saline and glucose. First, fasting basal blood glucose was measured via tail vein blood collection, and the measured value was defined as the 0-minute blood glucose level. A 1 mL syringe was used to inject glucose solution intraperitoneally, with a glucose dose of 2 g / kg based on mouse body weight. Blood glucose was collected via tail vein blood collection at 15, 30, 45, 60, and 90 minutes after injection, and blood glucose levels were recorded using a blood glucose meter. Blood glucose values ​​at different time points were plotted as curves, and the area under the curve (AUC) for each group of mice was calculated to assess and compare the glucose tolerance levels of mice after islet transplantation.

[0093] Results of each group of islet transplantation experiments are as follows Figure 3 and 4 As shown, the WT group had significantly more ischemic lesions on the liver surface after islet transplantation than the other four groups. Figure 3 (A) indicates a strong coagulation and thrombosis response. Meanwhile, Figure 3 The results showed that the WT group mice had significant inflammatory infiltration of the pancreatic islets, unclear staining of the islet nuclei, and damaged islet morphology. In contrast, the inflammatory infiltration of the islets was significantly reduced by DNAse I, Cl-amidine, and Sivelestat, and the islet morphology was more intact. Figure 3 The immunofluorescence results of the liver in the WT group showed that the fluorescence intensity of the liver NETs marker proteins MPO and Cith3 after pancreatic islet transplantation was significantly higher than that in the other four groups, indicating that the level of NETs in the liver after WT was significantly higher than that in other groups.

[0094] Figure 4 The MPO-DNA detection results showed that the serum NETs marker MPO-DNA level in the WT group was significantly increased 2 hours after surgery, significantly higher than that in the other four groups. These results indicate that NETs levels in the liver and serum increased after portal vein islet transplantation in wild-type diabetic mice, and ischemic foci appeared in the liver. Treatment with DNAse I, Cl-amidine, and Sivelestat effectively reduced NETs levels in the liver and serum of mice after portal vein islet transplantation, preventing the occurrence of ischemic foci in the liver.

[0095] Random blood glucose test results as follows Figure 5 As shown, the results indicated that postoperative blood glucose control in the DNAse I, Cl-amidine, and Sivelestat groups was significantly better than that in the WT group. Figure 5 (A) Only one mouse in the WT group achieved normal blood glucose levels, all mice in the DNase group recovered to normal blood glucose levels, and the Cl-amidine group and PAD4 group... - / - Seven mice in each group recovered to normal blood glucose levels, and six mice in the Sivelestat group recovered to normal blood glucose levels. Figure 5 (B). The glucose tolerance test results showed that the glucose tolerance of the WT group mice was significantly worse than that of the other four groups ( Figure 5 (C and D) indicate that blocking NETs significantly improved glucose metabolism after islet transplantation. These results suggest that blocking NETs through drugs or gene knockout can effectively improve the efficacy of mouse islet transplantation, with the DNAse I group showing a significantly better effect on improving the efficacy of mouse islet transplantation than other groups.

[0096] ELISA kit test results are as follows: Figure 6 As shown, compared with the WT group mice, the levels of major inflammatory factors such as TNF-α, IL-1β, and IL-6, as well as C-peptide and TAT in the serum of mice in each treatment group were significantly decreased after NETs blockade. Figure 6 (Middle AE). TNF-α, IL-1β, and IL-6 reflected the degree of inflammation in the IBMIR response, TAT levels reflected the degree of coagulation system activation in the IBMIR response, and elevated early C-peptide levels reflected the degree of islet damage. These results indicate that NETs play an important role in the IBMIR response after mouse islet transplantation. Blocking NETs can significantly reduce the intensity of the post-transplant inflammatory response, alleviate coagulation system activation, and effectively slow down islet damage.

[0097] Example 3 This embodiment uses animal experiments to further verify the relevant mechanisms of NETs after islet transplantation. The specific experimental steps are as follows.

[0098] To further verify platelet activation after islet transplantation, mice were divided into four groups for islet transplantation: WT group, Control group, anti-CD62P group, and PAD4 gene knockout group. The PAD4 gene knockout group received PAD4 gene knockout. - / - The experiment was conducted using C57 mice (Wuhan Youdu Biotechnology Co., Ltd.), and the islet isolation and extraction were performed in accordance with the steps in Example 1.

[0099] Control group: C57 mice were injected with the same volume of physiological saline after portal vein puncture; Islet transplantation group (WT group): C57 mice underwent portal vein puncture followed by islet transplantation, with each mouse receiving 300 IEQ islets. PAD4 gene knockout group: PAD4 - / - C57 mice underwent portal vein puncture followed by islet transplantation, with 300 IEQ islets transplanted into each mouse. The anti-CD62P group: C57 mice underwent portal vein puncture followed by islet transplantation, with each mouse receiving 300 IEQ islets. Mice were injected intraperitoneally with 20 μg of anti-CD62P antibody (Abcam) 30 minutes before surgery and 1 day after islet transplantation.

[0100] Two hours after transplantation, the platelet activation and PNA production in the peripheral blood of mice were detected by flow cytometry. The specific experimental steps are as follows.

[0101] This experiment used CD41 as a surface marker for mouse platelets and CD62P as a marker of platelet activation. Platelet activation was analyzed by flow cytometry. Both the CD41 and CD62P antibodies used were from BD Biosciences (USA). The specific experimental procedures are as follows: (1) Antibody preparation: FITC-labeled CD41 antibody: used to label platelets; PE-labeled CD62P antibody: used to label activated platelets.

[0102] (2) Flow cytometry tube coding: Aspirate 200 μL of pre-separated and processed platelets into a flow cytometry tube. Avoid vigorous shaking or blowing during the process to prevent platelet aggregation. The tubes are labeled as follows: blank tube, CD41 labeled tube, CD62P labeled tube, and experimental tube.

[0103] (3) Antibody incubation: Add 2 μL of the corresponding antibody to each flow cytometer tube labeled with the antibody and reagent, gently shake the bottom of the tube to ensure that the sample and antibody are thoroughly mixed, and incubate each tube at room temperature in the dark for 15 minutes. After incubation, add 500 μL of pre-cooled 1% paraformaldehyde to each tube for fixation.

[0104] (4) Platelet activation detection: Flow cytometry was used for detection. First, a single-stain tube was used for compensation, and the compensation was adjusted to ensure accurate differentiation of different fluorescence signals. Platelets were labeled using the FITC channel of CD41 and platelet activation was analyzed using the PE channel of CD62P. Data from at least 10,000 platelets were collected.

[0105] Meanwhile, using CD41 as a surface marker for mouse platelets and LY6G and CD11b as markers for neutrophils, PNA can be detected by detecting neutrophils expressing CD41.

[0106] (1) Antibody and reagent preparation: FITC-labeled CD41 antibody (BD Biosciences, USA): used for labeling platelets; APC-labeled CD45 antibody (BD Biosciences, USA): used for labeling leukocytes; PE-CY7-labeled LY6G antibody (BD Biosciences, USA) and AF700-labeled CD11b antibody (BD Biosciences, USA): used for labeling neutrophils; FITC Mouse IgG1 isotype control antibody (BD Biosciences, USA): used as an isotype control antibody for CD41. Other reagents: wash-free flow cytometry erythrocyte lysis buffer (containing fixative, Beijing Solarbio Science & Technology Co., Ltd.): used for lysing and fixing erythrocytes; mouse Fc receptor blocker (CD16 / CD32, BD Biosciences, USA): used to reduce nonspecific binding.

[0107] (2) Sample collection: After anesthetizing C57 mice, whole blood samples were collected via ocular sampling. Citrate anticoagulant tubes were used for blood collection to avoid shaking the blood during collection and processing, which could prevent spontaneous platelet activation. The collected blood samples should be immediately incubated with flow cytometry antibodies and tested to reduce spontaneous platelet activation.

[0108] (3) Flow cytometry tube coding: Prepare flow cytometry test tubes, add 100 μL of blood sample to each tube and label them as follows: blank tube, CD41 labeled tube, CD45 labeled tube, LY6G labeled tube, CD11b labeled tube, CD41+FITC-IgG1 isotype control tube, and experimental tube.

[0109] (4) Flow cytometry antibody incubation: To avoid platelet activation, centrifugation and resuspending of cells were avoided throughout the mouse PNA flow cytometry assay. First, 100 μL of whole blood was added to each flow cytometry tube, along with 1 μL of Fc receptor blocker, and incubated at room temperature for 10 minutes to reduce non-specific binding. Subsequently, according to the different tube numbers, 1 μL of anti-CD45 flow cytometry antibody, 1 μL of anti-LY6G flow cytometry antibody, 1 μL of anti-CD11b flow cytometry antibody, 1 μL of anti-CD41 flow cytometry antibody, and IgG1 positive control were added, and incubated in the dark for 15 minutes. After incubation, 500 μL of wash-free flow cytometry erythrocyte lysis buffer (containing fixative) was added to each tube, and erythrocytes were lysed at room temperature for 10 minutes.

[0110] (5) PNA detection and analysis: The prepared flow cytometry samples were analyzed by flow cytometry. First, the white blood cell population was screened using SSC and FSC signals. Neutrophil population was then screened based on the positive expression of CD45, LY6G, and CD11b. Within the neutrophil population, PNA was determined by screening for CD41-positive cells, i.e., CD41... + Neutrophils. Collect data on at least 20,000 cell counts to ensure statistical reliability of the data.

[0111] The results of the platelet activation assay are as follows: Figure 7 As shown in the AH, the level of CD62P-positive platelets in the WT group mice after transplantation was significantly higher than that in the Control group, the anti-CD62P group, and the PAD4 gene knockout group, indicating that platelets were significantly activated after islet transplantation. However, the platelet activation level in mice was significantly reduced after administration of anti-CD62P antibody and PAD4 gene knockout, with no significant difference from the control group.

[0112] Results of peripheral blood PNA production detection as follows Figure 8 As shown in the AI, PNA significantly increased in the WT group after transplantation, reaching 63% ( Figure 8 (D). Postoperative PNA levels in the Control group, anti-CD62P group, and PAD4 gene knockout group were significantly lower than those in the WT group ( Figure 8 Among the groups treated with CD62P antibody, the postoperative PNA level was the lowest (2.13%). These results indicate that platelet activation after islet transplantation promotes PNA production, PAD4 gene knockout significantly reduces PNA levels, while anti-CD62P treatment significantly inhibits PNA production.

[0113] Example 4 This embodiment provides further verification of the association between anti-CD62P treatment and the generation of NETs after islet transplantation, as detailed below.

[0114] Two hours after pancreatic islet transplantation, livers from mice in the WT group and anti-CD62P group of Example 3 were subjected to immunofluorescence detection and HE staining, following the same steps as in Example 2.

[0115] Experimental results are as follows Figure 9 As shown, the expression of MPO and Cith3 in the liver of mice treated with anti-CD62P was significantly lower than that in wild-type mice two hours after islet transplantation. Furthermore, anti-CD62P treatment significantly reduced inflammatory infiltration around the islets, and the islets remained morphologically intact. These results indicate that anti-CD62P treatment significantly reduced the formation of NETs in the liver after islet transplantation in mice.

[0116] Example 5 This embodiment provides the mechanism by which PAD4 knockout affects the expression levels of key molecules in inflammation-related pathways after liver islet transplantation. Details are as follows.

[0117] Two groups of mice (WT group and PAD4 gene knockout group) from Example 3, with three mice in each group, were used. Liver tissue was harvested two hours after transplantation for transcriptomic and proteomic sequencing. The sequencing was performed by Hangzhou Lianchuan Biotechnology Co., Ltd. This example further included GSEA enrichment analysis of liver proteomic sequencing after islet transplantation in wild-type C57 mice and PAD4 gene knockout mice.

[0118] Sequencing results as follows Figure 10 and 11 As shown, Figure 10 Analysis revealed significant differences in NETs-related pathways, complement-related pathways, and coagulation-related pathways between PAD4 knockout mice and WT mice. Furthermore, in inflammation-related pathways, differentially expressed genes were enriched in the MAPK and NF-κB signaling pathways, both of which play crucial roles in the inflammatory response after islet transplantation. Figure 11 The analysis showed that the MAPK signaling pathway, chemokine signaling pathway, NF-κB signaling pathway, neutrophil extracellular trap formation pathway, and leukocyte transendothelial migration pathway were significantly enriched in the WT group after islet transplantation, indicating that these pathways were activated in the WT group. In contrast, the enrichment fractions of these pathways were significantly reduced in PAD4 gene knockout mice, suggesting that PAD4 gene knockout inhibited the activation of these pathways. These results suggest that NETs may exacerbate the inflammatory response after islet transplantation by participating in the activation of MAPK and NF-κB inflammatory pathways, thereby increasing islet inflammatory damage.

[0119] Example 6 This embodiment further verifies the effect of PAD4 gene knockout on the MAPK and NF-κB inflammatory pathways through specific experimental steps.

[0120] C57 mice and PAD4 - / - C57 mice were used to establish a diabetes model, following the same steps as in Example 2. They were then grouped and treated as follows. Pancreatic islet extraction from the mice was performed as in Example 1.

[0121] Control group: C57 mice were used and only an equal volume of physiological saline was infused during islet transplantation; WT group: C57 mice underwent portal vein puncture followed by islet transplantation, with 400 IEQ islets transplanted per mouse; PAD4 gene knockout islet transplantation group (PAD4) - / - Group): PAD4 - / - C57 mice underwent portal vein puncture followed by islet transplantation, with 400 IEQ islets transplanted into each mouse; Two hours after surgery, liver tissue from three groups of mice was collected for Western blot analysis to detect NF-κB pathway-related proteins. The specific steps are as follows.

[0122] (1) Mice were euthanized after being anesthetized, and liver tissue was quickly harvested and rinsed three times in pre-cooled (4℃) PBS phosphate buffer to remove residual blood. The tissue samples were flash-frozen in liquid nitrogen and then transferred to an ultra-low temperature freezer at -80℃ for storage. Before the experiment, the tissue samples were thawed in an ice box, and pre-cooled RIPA lysis buffer (Guangzhou Boluteng Biotechnology Co., Ltd.) was added. The samples were homogenized intermittently in an ice bath at 15000 rpm using a tissue homogenizer (10 seconds homogenization / 30 seconds interval, repeated 3 times). After centrifugation at 12000×g for 15 minutes at 4℃, the supernatant was collected and quantified using a BCA protein quantification kit (Thermo Fisher). The absorbance was measured at 562 nm using an ELISA reader, and the protein concentration was calculated using a bovine serum albumin (BSA) standard curve. All samples were adjusted to a uniform concentration (2 μg / μL), and after adding 5×SDS-PAGE loading buffer (containing β-mercaptoethanol) (Guangzhou Boluteng Biotechnology Co., Ltd.), they were denatured in a metal bath at 95℃ for 5 min, aliquoted and stored at -20℃ for later use.

[0123] (2) SDS-PAGE gel preparation: SDS-PAGE gel was prepared using a rapid PAGE gel preparation kit (Shanghai Yamei Biomedical Technology Co., Ltd.).

[0124] (3) Sample loading and electrophoresis: Fix the gel in the electrophoresis tank, add 1× electrophoresis buffer (Guangzhou Boluteng Biotechnology Co., Ltd.), and remove the comb. Load 40 μg of protein into each well, and add 2 μL of pre-stained protein marker to both wells. Adjust the initial electrophoresis voltage to 100 V, and after the sample enters the separating gel, adjust it to 120 V. Electrophore until the bromophenol blue reaches the bottom of the gel.

[0125] (4) Transfer: Cut the PVDF membrane and then immerse it in methanol for 30 seconds to activate it. Assemble the transfer clamp in a "sandwich" structure (sequence: negative electrode - filter paper - gel - membrane - filter paper - positive electrode). The transfer parameters are 250mA constant current transfer, and the transfer time is adjusted according to the molecular weight of the target protein (usually 90-120 minutes). Keep the temperature low during the transfer in an ice bath.

[0126] (5) Blocking and antibody incubation: After transfer, the PVDF membrane was blocked with 5% BSA or skim milk at room temperature for 2 hours. The primary antibody (diluted according to the instructions) was incubated overnight at 4°C, and the membrane was washed 3 times with TBST (15 minutes each time). Then, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour, and the membrane was washed 3 times with TBST. The antibodies used included CitH3 antibody (Abcam), PAD4 antibody (Abcam), GADPH antibody (CST), ACTIN antibody (CST), TF antibody (Abcam), p-IκB antibody (CST), IκB antibody (CST), p65 antibody (Abcam), p-p65 antibody (Abcam), ERK1 / 2 antibody (Abcam), p-ERK1 / 2 antibody (Abcam), JNK1 / 2 / 3 antibody (Abcam), p-JNK1 / 2 / 3 antibody (Abcam) and NLRP3 antibody (Abcam). The secondary antibody used was HRP-labeled secondary antibody (CST, USA). (6) Development and Quantification: The membrane was uniformly covered with ECL chemiluminescence reagent (Guangzhou Boluteng Biotechnology Co., Ltd.), and the band development signal was acquired using a gel imaging system. After saving the developed images, the gray values ​​of the bands were analyzed using ImageJ software, and semi-quantitative analysis was performed using the target protein / internal reference ratio. For phosphorylated proteins, the phosphorylated protein / total protein ratio was used for analysis.

[0127] Experimental results are as follows Figure 12-14 As shown, Figure 12 The results showed that, compared with the WT group, the PAD4 gene knockout group mice had significantly lower PAD4 protein expression levels, and the expression levels of p-IκB and p-p65 proteins were also significantly decreased. These results indicate that blocking NETs through PAD4 gene knockout can inhibit the activation of the liver NF-κB inflammatory pathway after islet transplantation.

[0128] Figure 13 The results showed that CitH3 protein expression was significantly increased in the WT group mice after transplantation, while CitH3 protein expression was significantly decreased in the PAD4 gene knockout group compared to the WT group. Simultaneously, p-JNK and p-ERK protein expression were significantly increased in the WT group, while PAD4 gene knockout reduced p-JNK and p-ERK protein expression. Furthermore, TF expression was significantly increased in the WT group mice compared to the control group post-transplantation, while TF expression was significantly decreased after PAD4 gene knockout, suggesting a decreased IBMIR response level after PAD4 knockout. These results indicate that NET production increases and the MAPK pathway is activated after islet transplantation, while PAD4 gene knockout inhibits MAPK pathway activation, reduces NETs, ​​and lowers IBMIR levels.

[0129] Figure 14 The results showed that the NLRP3 protein expression level in the WT group was significantly higher than that in the Control group after transplantation, while the NLRP3 protein expression level in the PAD4 gene knockout group was significantly lower than that in the WT group. These results suggest that the NLRP3 pathway plays an important role in the inflammatory response after islet transplantation, and that PAD4 gene knockout may alleviate the postoperative inflammatory response by regulating NLRP3 expression.

[0130] This study, using a PAD4 gene knockout mouse islet transplantation model and combining transcriptome sequencing and Western blotting, revealed the molecular mechanism by which NETs exacerbate IBMIR after islet transplantation by activating the NF-κB and MAPK inflammatory pathways. It also confirmed that PAD4 knockout significantly reduces the activation levels of downstream inflammatory signaling pathways by inhibiting NET production, providing new theoretical basis for improving early inflammatory damage after islet transplantation. Western blot results further confirmed that the levels of key phosphorylated proteins such as p-IκB, p-p65, p-JNK, and p-ERK were significantly lower in the PAD4 knockout group compared to the wild-type group. This suggests that NETs may activate Toll-like receptors (TLRs) or directly induce oxidative stress by releasing pro-inflammatory components such as histones and myeloperoxidase (MPO), triggering the NF-κB nuclear translocation and MAPK phosphorylation cascade. Meanwhile, this study found that the NLRP3 protein expression level in the WT group mice was significantly higher than that in the control group, while the PAD4 gene knockout group showed lower NLRP3 expression, suggesting that NLRP3 plays an important role in NETs-mediated inflammatory response after islet transplantation. PAD4 knockout blocking NETs may alleviate the inflammatory response after islet transplantation by regulating NLRP3 expression.

[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of NETs inhibitors in the preparation of products that improve glycemic regulation after pancreatic islet transplantation.

2. The application according to claim 1, characterized in that, The improvement of blood glucose regulation function after islet transplantation includes improving blood glucose homeostasis and / or glucose tolerance after islet transplantation.

3. The application according to claim 1, characterized in that, The NETs inhibitors include at least one of PAD4 inhibitors, NADPH oxidase inhibitors, neutrophil elastase inhibitors, and DNA degrading enzymes.

4. The application according to claim 3, characterized in that, The DNA-degrading enzymes include DNase I.

5. The application according to claim 1, characterized in that, The NETs inhibitor further includes platelet-neutrophil aggregate inhibitors; the platelet-neutrophil aggregate inhibitors include at least one of P-selectin inhibitors, PSGL-1 inhibitors, and GPIbα inhibitors; preferably, the P-selectin inhibitors include anti-CD62P antibodies.

6. The application according to claim 1, characterized in that, The islet transplantation includes at least one of normothermic mechanical perfusion of the liver combined with islet transplantation and portal vein islet transplantation.

7. The use of NET inhibitors in the preparation of drugs for the prevention and / or treatment of ischemic injury after islet transplantation.

8. The use of NET inhibitors in the preparation of drugs for the prevention and / or treatment of blood-mediated inflammatory responses immediately following islet transplantation.

9. A product for improving islet function after islet transplantation, characterized in that, The product includes a therapeutically effective amount of a NETs inhibitor.

10. The product according to claim 9, characterized in that, The pancreatic function includes blood glucose regulation.

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