Application of NiMn-PB in the preparation of drugs for protecting abdominal aortic aneurysms

Abstract

This invention belongs to the field of biomedicine, specifically disclosing the application of NiMn-PB in the preparation of drugs to protect against abdominal aortic aneurysms. It reveals that NiMn-PB can significantly improve the degradation of the aortic elastic fiber layer induced by Ang II treatment, and can alleviate immune cell infiltration, inflammatory release, ferroptosis and angiogenesis in the aorta. Intervention with NiMn-PB after Ang II treatment can alleviate the occurrence and development of Ang II-induced abdominal aortic aneurysms, indicating that NiMn-PB can be used to prepare drugs to alleviate abdominal aortic aneurysms.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of NiMn-PB in the preparation of drugs for protecting against abdominal aortic aneurysms. Background Technology

[0002] Abdominal aortic aneurysm (AAA) is a highly lethal vascular disease characterized by progressive dilation of the abdominal aorta and degeneration of the arterial wall structure, with an extremely high mortality rate after rupture. Its onset is insidious, and its progression is driven by multiple risk factors (hypertension, hyperglycemia, smoking, aging, etc.) and local microenvironment dysregulation, making safe and effective drug intervention tools urgently needed in clinical practice.

[0003] Existing research has identified macrophage-mediated chronic inflammation as one of the core drivers of AAA development. The tumor site recruits large numbers of immune cells, including monocytes / macrophages, neutrophils, and T cells, releasing pro-inflammatory cytokines and chemokines such as IL-6, IL-1β, and TNF-α, activating pathways like NF-κB, and upregulating matrix metalloproteinases (MMP-2 / 9), accelerating the degradation of the extracellular matrix (collagen and elastic fibers). Simultaneously, vascular smooth muscle cells (VSMCs) undergo dedifferentiation, apoptosis, or phenotypic transformation, resulting in impaired vascular endothelial function and a significant decrease in arterial wall compliance and mechanical stability, thus creating a risk factor for persistent arterial dilation and rupture.

[0004] Ferroprelation is an iron-dependent, lipid peroxidation-regulated cell death process, essentially caused by iron metabolism disorders leading to the accumulation of reactive oxygen species (ROS) and lipid peroxidation. Inhibition of ferroprelation pathways (such as SLC7A11 / systemic Xc–, glutathione, and glutathione peroxidase GPX4) is crucial. AAA lesions commonly show elevated iron overload and lipid peroxidation, along with abnormal expression of key molecules such as GPX4 and ACSL4, which promote VSMC death and amplify inflammation, synergistically enhancing MMP-mediated matrix degradation, thereby exacerbating arterial wall degeneration and dilation.

[0005] Driven by inflammation and oxidative stress, endothelial cells interact with immune cells, upregulating pro-angiogenic factors such as VEGF and ANGPT2, as well as MMP-2 / 9, inducing the formation of new microvessels. These new blood vessels are highly permeable and prone to bleeding, further attracting inflammatory cell infiltration and the release of proteolytic enzymes, leading to elastic fiber rupture and medial structure destruction, significantly increasing tumor instability and the risk of rupture.

[0006] Currently, the main treatment and intervention methods for abdominal aortic aneurysms are surgical treatment, which involves replacing the diseased aortic segment through open-chest or open-abdomen surgery, or using minimally invasive vascular interventional techniques to place stents in the abdominal aortic aneurysm to reinforce the aneurysm wall and prevent further expansion and rupture. Although treatment with drugs such as antihypertensive drugs and beta-blockers can reduce the risk to the aorta, they cannot directly eliminate the abdominal aortic aneurysm. Some ferroptosis inhibitors (such as Ferrostatin-1 (Fer-1) and Liproxstatin-1) have shown the potential to alleviate inflammation and reduce aortic diameter dilation in animal models, but they generally have poor water solubility, weak tissue affinity, poor intrinsic stability, short half-life, dose-drug interaction risks, and the possibility of high doses inducing cell cycle arrest or apoptosis, which limits their clinical translation. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the application of NiMn-PB in the preparation of drugs to protect against abdominal aortic aneurysms. Through intervention with NiMn-PB nanomaterials, it is evident that there is no significant thickening of the arterial wall and a significant reduction in elastin degradation. Furthermore, it inhibits inflammatory responses, ferroptosis, and angiogenesis in aneurysm tissue, thereby achieving the effect of delaying the progression of abdominal aortic aneurysms.

[0008] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is the application of NiMn-PB as the sole active ingredient in the preparation of a drug for protecting against abdominal aortic aneurysms. The preparation method of NiMn-PB includes the following steps: S1. Dissolve 0.4 mmol K3[Fe(CN)6] and 1.5 g PVP in 20 mL of deionized water to obtain a clear solution A; S2. Separately dissolve 0.1 mmol MnCl2·4H2O, 0.1 mmol NiCl2·6H2O and 1.5 g PVP in 20 mL of deionized water to obtain clear solution B; S3. At room temperature, add solution B dropwise to solution A and stir continuously for 10 minutes. Then add 40 mL of 0.01 MHCl solution and mix thoroughly. Finally, react the mixture at 80°C for 20 hours. S4. After the reaction was completed, the product was collected by centrifugation and washed repeatedly with deionized water and ethanol. After vacuum freeze-drying at -40℃ for 48h, NiMn-PB nanomaterials were finally obtained.

[0009] The second technical solution provided by the present invention is an abdominal aortic aneurysm protection drug, comprising NiMn-PB nanomaterial as the sole active ingredient, and a medically acceptable carrier.

[0010] Compared with existing technologies, this invention reveals that the prepared NiMn-PB nanomaterials can effectively improve the dilation trend of the abdominal aorta in mice induced by Ang II treatment. After treatment with NiMn-PB nanomaterials, significant reductions in arterial wall thickening and elastin degradation were observed. Further studies found that NiMn-PB nanomaterial treatment reduced cellular inflammation levels, alleviated ferroptosis in vascular smooth muscle cells, and reduced angiogenesis, without producing cytotoxicity. These results indicate that NiMn-PB nanomaterials mainly delay the occurrence and development of abdominal aortic aneurysms in mice by inhibiting inflammatory responses, ferroptosis, and angiogenesis in the abdominal aortic tissue. Attached Figure Description

[0011] Figure 1 The characteristics of NiMn-PB nanomaterials are as follows: A. Surface morphology, particle size, and dispersion state of the material; B. Crystal structure and phase composition of the material; C. SOD inhibition rate of NiMn-PB nanomaterials; D. H2O2 scavenging rate of NiMn-PB nanomaterials.

[0012] Figure 2 To investigate the effect of NiMn-PB nanomaterials on inhibiting ferroptosis in mouse vascular smooth muscle cell lines: A. Schematic diagram of experimental design; B. Effect of different concentrations of NiMn-PB nanomaterials on cell viability; C. The effect of NiMn-PB nanomaterials treatment on alleviating RSL3-induced reactive oxygen species (ROS) levels in cells; D. The effect of NiMn-PB nanomaterials treatment on alleviating RSL3-induced lipid peroxidation levels in cells.

[0013] Figure 3 To assess the in vitro antioxidant and anti-inflammatory properties of NiMn-PB nanomaterials: A. Flow cytometry analysis of intracellular reactive oxygen species (ROS); B. The effect of NiMn-PB nanomaterial treatment on reducing intracellular ROS levels; C. The effect of NiMn-PB nanomaterial treatment on reducing macrophage expression of the inflammatory cytokine IL-6; D. The effect of NiMn-PB nanomaterial treatment on reducing macrophage expression of the inflammatory cytokine IL-1β; E. The effect of NiMn-PB nanomaterial treatment on reducing macrophage expression of the inflammatory cytokine TNF-α.

[0014] Figure 4Results of testing the effects of NiMn-PB nanomaterials on the treatment and relief of abdominal aortic aneurysm development in mice: A. Schematic diagram of animal experimental design; B. Morphological images of aortic tissue in mice under different treatment groups; C. Incidence of abdominal aortic aneurysm in mice under different treatment groups; D. Vascular ultrasound images of aorta in mice under different treatment groups; E. HE staining images of mouse aortic tissue; F. EVG staining images of mouse aortic tissue; G. Statistical graph of elastin degradation assessment scores in mouse aorta.

[0015] Figure 5 The effects of NiMn-PB nanomaterial treatment on inflammation, ferroptosis, and angiogenesis in mice: A. Immunofluorescence staining of mouse aortic tissue (left) and statistical chart of macrophage (F4 / 80 positive cells) percentage (right); B. Representative immunofluorescence staining of IL-1β (pro-inflammatory cytokine) in mouse aortic tissue (left) and statistical bar chart of IL-1β positive cell percentage (right); C. Representative immunofluorescence staining of GPX4 (glutathione peroxidase 4, a key regulator of ferroptosis) in mouse aortic tissue (left) and statistical bar chart of GPX4 positive cell percentage (right); D. Representative immunofluorescence staining of CD31 in mouse aortic tissue (left) and statistical bar chart of CD31 positive cell percentage (right).

[0016] Figure 6 Safety profile of NiMn-PB nanomaterials for therapeutic use in vivo: A. Statistical bar chart of mouse major organ index; B. Statistical bar chart of mouse serum alanine aminotransferase (ALT) activity; C. Statistical bar chart of mouse serum aspartate aminotransferase (AST) activity; D. Statistical bar chart of mouse serum urea level; E. Statistical bar chart of mouse serum creatinine level; F. Statistical bar chart of mouse peripheral blood red blood cell (RBC) count; G. Statistical bar chart of mouse peripheral blood white blood cell (WBC) count; H. Statistical bar chart of mouse peripheral blood hemoglobin (HGB) level; I. Statistical bar chart of mouse peripheral blood platelet (PLT) count; J. HE staining images of multiple mouse organs (heart, liver, spleen, lung, kidney). Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0018] Example 1: Preparation and Characterization of NiMn-PB Nanomaterials In NiMn-PB nanomaterials, PB refers to Prussian blue (PB), a classic metal-integrated nanozyme with multi-enzyme-like activities (such as peroxidase-like (POD) and catalase (CAT) activities). It can decompose excess H₂O₂, scavenge ROS, inhibit the release of inflammatory factors, and reduce inflammatory infiltration. It also exhibits excellent biocompatibility (already used for heavy metal / radioactive thallium detoxification). By introducing other metal ions into the PB framework, its structure and catalytic performance can be modulated, enabling synergistic intervention against pathological axes such as inflammation, oxidative stress, and ferroptosis, thus possessing the potential to become a candidate for AAA drugs. This embodiment utilizes NiMn to modify Prussian blue.

[0019] 0.4 mmol K3[Fe(CN)6] and 1.5 g PVP were dissolved in 20 mL of deionized water to obtain a clear solution A. Separately, 0.1 mmol MnCl2·4H2O, 0.1 mmol NiCl2·6H2O, and 1.5 g PVP were dissolved in 20 mL of deionized water to obtain a clear solution B. At room temperature, solution B was added dropwise to solution A, and the mixture was stirred continuously for 10 minutes. Then, 40 mL of 0.01 M HCl solution was added and thoroughly mixed. The mixture was then reacted at 80 °C for 20 hours. After the reaction was complete, the product was collected by centrifugation and repeatedly washed with deionized water and ethanol. After freeze-drying under vacuum at -40 °C for 48 hours, NiMn-PB nanomaterials were obtained. Figure 1 As shown in Figure A, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images reveal that the NiMn-PB nanomaterials are cubic in shape and well-dispersed. Elemental mapping confirms the uniform distribution of Fe, Ni, and Mn within the nanoparticles, indicating that the prepared nanomaterials are not a physical mixture. Figure 1 As shown in Figure B, X-ray diffraction (XRD) analysis confirmed the crystal structure and phase purity of the NiMn-PB nanomaterial. Its XRD pattern was consistent with the standard pattern of PB, and no other phases or impurities were detected, indicating that it is a single-phase structure. Simultaneously, this embodiment used a superoxide dismutase (SOD) assay kit and a catalase (CAT) assay kit to detect the scavenging of superoxide radicals (•O2) by the NiMn-PB nanomaterial. − The ability to decompose hydrogen peroxide (H2O2), such as Figure 1 C and Figure 1 As shown in D, the results indicate that NiMn-PB nanomaterials possess enzyme-mimicking antioxidant activity.

[0020] To verify that the NiMn-PB nanomaterials of this embodiment can be used to prepare drugs for protecting against abdominal aortic aneurysms, in vitro cell experiments as in Examples 2 and 3 and in vivo animal experiments as in Example 4 were conducted.

[0021] Example 2, In vitro cell experiment: NiMn-PB nanomaterials inhibit ferroptosis in vascular smooth muscle Inside the clean bench, MOVAS cells in the logarithmic growth phase (purchased from Beina Biotechnology Co., Ltd.) were prepared into a cell suspension and seeded into 96-well plates, with 3,000 cells per well. After inoculation, 24-well plates were incubated overnight at 37°C in a 5% CO2 incubator. The next day, the cells were divided into 6 groups: control group (containing DMSO, Sigma, not exceeding 0.1%), experimental group (containing 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL NiMn-PB and 500 nM RSL3 (purchased from MCE, catalog number: HY-100218A), and positive control group (containing 500 nM RSL3). After 24 hours of treatment, Hoechst 33342 (purchased from MCE, catalog number: HY-15630A) and PI (propidium iodide, purchased from MCE, catalog number: HY-D0815) were added to a final concentration of 1 μg / mL. The plates were incubated at 37°C in the dark for 15 minutes. The total cell count was analyzed using a high-content imaging system (Hoechst). 33342 positive cell count) and dead cells (PI positive cell count). For example... Figure 2 A- Figure 2 As shown in B, NiMn-PB nanomaterials significantly inhibited RSL3-induced ferroptosis in vascular smooth muscle cells, and the inhibitory effect increased with the concentration of NiMn-PB nanomaterials.

[0022] In a clean bench, MOVAS cell lines in logarithmic growth phase were prepared into a cell suspension and seeded into 24-well plates, maintaining a cell density of approximately 50%. The seeded 24-well plates were incubated overnight at 37°C with 5% CO2 until the cells adhered and achieved 70%–80% confluence. The cells were then divided into three groups and treated for 6 hours: a blank control group (DMSO group, not exceeding 0.1%), an experimental group (containing 100 μg / mL NiMn-PB and 500 nM RSL3), and a positive control group (containing 500 nM RSL3). After treatment, the complete culture medium in each well was aspirated, and the cells were gently washed twice with pre-warmed PBS buffer to remove residual serum. Add pre-warmed DCFH-DA working solution (final concentration 5 μM) or C11BODIPY (final concentration 5 μM) staining solution to each well. Incubate the 24-well plate at 37°C in a 5% CO2 incubator for 20–30 min in the dark, gently shaking the plate every 10 min. After incubation, discard the probe working solution from each well, wash with pre-warmed PBS buffer, and centrifuge. Repeat three times, 5 min each time, to remove free probes that have not entered the cells. Resuspend the cells in 0.2% (w / v) BSA solution containing DAPI (4',6-diamidinyl-2-phenylindole dye, purchased from MCE, catalog number: HY-D0814) to a final DAPI concentration of 1 μg / mL, and then perform flow cytometry analysis. Results are as follows: Figure 2 C- Figure 2 As shown in Figure D, treatment with NiMn-PB nanomaterials significantly inhibited ROS and lipid peroxidation levels induced by RSL3. These results indicate that NiMn-PB nanomaterials possess the biological function of inhibiting vascular smooth muscle.

[0023] Example 3, In vitro cell experiments: Effects of NiMn-PB nanomaterials on oxidative stress and inflammatory factor expression in RAW264.7 cells. In a clean bench, logarithmically growing RAW264.7 cells (purchased from ATCC) were prepared into a cell suspension and seeded into 24-well plates, maintaining a cell density of approximately 50%. The seeded 24-well plates were incubated overnight at 37°C in a 5% CO2 incubator until the cells adhered and achieved 70%–80% confluence. Cells were divided into three groups and treated for 6 hours: a blank control group (no treatment), an experimental group (treated with 100 μg / mL NiMn-PB and 500 nM lipopolysaccharide (LPS, purchased from MERCK Biotech), and a positive control group (treated with 500 nM LPS). After treatment, the complete culture medium in each well was discarded, and the cells were gently washed twice with pre-warmed PBS buffer to remove residual serum. Pre-warmed working solution containing 5 μM DCFH-DA (dissolved in PBS) was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 20-30 minutes in the dark. During incubation, the culture plate was gently shaken every 10 minutes. After incubation, the probe working solution in each well was discarded, and the cells were washed with pre-warmed PBS buffer and centrifuged three times for 5 minutes each time to remove free probes that had not entered the cells. The cells were resuspended in 0.2% (w / v) BSA solution containing 1 μg / mL DAPI, and then flow cytometry was performed. The results are shown below. Figure 3 A- Figure 3 As shown in B, NiMn-PB treatment significantly inhibited LPS-induced ROS levels in RAW264.7 cells.

[0024] Following the same treatment method described above, cells were divided into three groups: a blank control group (no treatment), an experimental group (treated with 100 μg / mL NiMn-PB and 500 nM lipopolysaccharide (LPS), and a positive control group (treated with 500 nM LPS). Complete culture medium was discarded from each well, and cells were gently washed twice with pre-warmed PBS buffer to remove residual serum. RNA was extracted from the cells in all three groups using a rapid RNA extraction kit, and reverse transcription was performed to convert it into mRNA. Subsequently, total RNA was extracted from the cells in all three groups using the rapid RNA extraction kit, and converted into cDNA using a reverse transcription kit. Using cDNA as a template, the mRNA expression levels of inflammatory factors such as IL-6, IL-1β, and TNF-α were detected using quantitative real-time PCR. Results are as follows: Figure 3 C- Figure 3As shown in Figure D, compared with the positive control group, the expression levels of the aforementioned inflammatory factors in LPS-induced RAW264.7 cells were significantly reduced in the NiMn-PB nanomaterial treatment group. These results indicate that NiMn-PB nanomaterials possess biological functions in inhibiting ROS and inflammatory factors.

[0025] Example 4: Animal in vivo experiment 1. Abdominal aortic aneurysm modeling experiment like Figure 4 As shown in A, ApoE of 6-8 weeks old is selected based on the specific pathogen-free (SPF) grade. - / - Twenty male C67BL / 6 mice (purchased from Guangdong Vital River Technology Co., Ltd.) were weighed and randomly divided into four groups of five mice each. After separation, the mice were housed in controlled-temperature environments (23℃-25℃) and under artificial lighting conditions (14 hours light, 10 hours darkness), respectively, fed standard pelleted feed, with free access to water and food. Mice were anesthetized with 0.67% (w / v) pentobarbital solution and fixed in a prone position on the operating table. The fur on the back of the neck was disinfected, and a transverse incision of approximately 1 cm was made to separate the subcutaneous tissue, creating a space. A micro-osmotic pump loaded with Ang II at a dosage concentration of 800 ng / kg / min was implanted subcutaneously in the back. The experiment was repeated at least twice; mice that died during the experiment were not included in the final statistics.

[0026] The skin incision was sutured and the surface disinfected. After the mouse regained consciousness, it was returned to its cage and continued routine feeding for 28 days to allow the abdominal aortic aneurysm to develop. Figure 4 As shown in A, starting the day after modeling, mice in each group were intraperitoneally injected with the drug every 4 days: the first group was treated with physiological saline and was designated the Ang II + Saline group; the second group was treated with 5 mg / kg NiMn-PB (with physiological saline as the solvent) and was designated the Ang II + NiMn-PB group; the third group did not undergo modeling, and the surgical procedure was performed normally without the implantation of a micro-osmotic pump subcutaneously in the back, and was designated the sham group. On days 14, 21, and 28 after Ang II modeling, the diameter of the abdominal aorta was measured by ultrasound, and the survival time was recorded.

[0027] like Figure 4 As shown in D: On day 28 after Ang II modeling, the diameter of the abdominal aorta in the Ang II group mice was significantly larger than that in the Ang II + saline group, while the diameter of the abdominal aorta in the Ang II + NiMn-PB group was significantly smaller, indicating that NiMn-PB nanomaterials can effectively alleviate the abdominal aortic dilation in mice caused by Ang II + saline treatment.

[0028] Mice were sacrificed and their tissues collected 28 days after Ang II modeling. Figure 4 As shown in Figure B, the Ang II + NiMn-PB nanomaterial treatment group significantly reduced the diameter of the abdominal aorta compared to the Ang II + Saline group and the NiMn-PB group, a result consistent with ultrasound findings. The incidence of abdominal aortic aneurysms in mice after modeling was also shown in Figure B. Figure 4 As shown in C, no abdominal aortic aneurysm was detected in mice in the sham group, the incidence of abdominal aortic aneurysm in mice in the Ang II + Saline group was 50%, and the incidence of abdominal aortic aneurysm in mice in the Ang II + NiMn-PB group was 16.6%, indicating that NiMn-PB nanomaterial treatment can significantly improve the incidence of Ang II-induced abdominal aortic aneurysm.

[0029] 2. HE and EVG staining of the abdominal aorta Mice were anesthetized with 0.67% (w / v) pentobarbital solution, placed on a foam board, and their limbs were fixed. The chest cavity was opened to expose the heart. A needle was inserted at the apex of the heart with the right hand, while the right atrial appendage was cut open with ophthalmic scissors with the left hand. Approximately 30-40 mL of PBS was injected at the injection site. Vascular perfusion was completed after the liver turned white and the outflowing fluid became clear. The abdominal cavity was opened, other organs were removed, and the entire aorta was dissected and immersed in 4% (w / v) paraformaldehyde (PFA). After 2 hours, it was photographed. The abdominal aortic aneurysm was fixed, embedded, sectioned, and stained with hematoxylin and eosin (HE) to observe cell infiltration in the mouse aorta. Figure 4 As shown in Figure E, compared to the untreated control group, the abdominal aorta of mice in the Ang II + saline group exhibited significant lesions, including luminal dilation, marked thickening of the aortic wall, and significant inflammatory cell infiltration in the media and adventitia. In contrast, the aortic wall of mice in the Ang II + NiMn-PB group showed significant improvement, and macrophages in the adventitia were not prominent. This indicates that NiMn-PB nanomaterials can effectively improve Ang II-induced abdominal aortic lesions in mice. The degree of degradation of elastic fibers and collagen fibers was observed using EVG staining. Elastic fiber layer degradation was classified into four grades (I-IV) from mild to severe: Grade I, damage to the elastic fiber layer and reduction of vascular smooth muscle cells are limited to one elastic fiber layer; Grade II, damage involves two or all layers, but is limited to 1 / 4 of the vessel; Grade III, damage involves all elastic fiber layers, but is limited to below 1 / 2 of the vessel; Grade IV, damage involves all elastic fiber layers and extends to above 3 / 4 of the vessel. Figure 4 F- Figure 4As shown in G, the elastic fiber layer of the abdominal aorta in mice in the PBS group was intact and rated as Grade I. In mice in the Ang II + saline group, 50% had Grade III lesions and 50% had Grade IV lesions. In mice in the Ang II + NiMn-PB group, 75% had Grade I vascular elastic fiber scores and 25% had Grade II scores. This demonstrates that NiMn-PB nanomaterial treatment can significantly improve Ang II-induced degradation of the elastic fiber layer of the abdominal aorta in mice.

[0030] 3. Immunofluorescence staining of the abdominal aorta To clarify how NiMn-PB nanomaterials improve aortic dilation in vivo, we performed immunofluorescence staining experiments on mouse aortas 28 days after Ang II treatment. First, OCT-embedded tissue blocks stored at -80°C were equilibrated to -20°C in a cryostat, cut into 7μm thick sections, attached to pre-cooled, non-detachable glass slides, and immediately placed at room temperature for 30 minutes to ensure firm attachment. Fixation was then performed: sections were immersed in freshly prepared 4% (w / v) paraformaldehyde (PFA) and fixed at room temperature for 15 minutes; after fixation, they were placed in a staining jar containing PBS, repeated three times to completely remove the fixative. Permeabilization was then performed: permeabilization was performed using PBS containing 0.25% Triton X-100 at room temperature for 10 minutes, followed by washing three times with PBS. To reduce non-specific binding, blocking was performed: sufficient blocking solution (2% (w / v) BSA) was added to the tissue area to ensure complete coverage, and the tissue was blocked in a humidified chamber at room temperature for 1 hour. After sealing, carefully blot away the blocking solution around the slide with absorbent paper, but keep the tissue moist. Then perform primary antibody incubation: Prepare the primary antibody working solution using PBS containing 1% BSA, with the following antibody and dilution ratios: anti-F4 / 80 (1:100), anti-IL-1β (1:100), anti-GPX4 (1:100), anti-CD31 (1:50). Ensure the liquid covers the tissue and is free of air bubbles. Place the slide flat in a humidified chamber and incubate overnight (16-18 hours) at 4°C. The next day, bring the humidified chamber to room temperature and perform washing: Immerse the slide in a staining jar containing PBS, let it stand for 5 minutes, repeat three times to thoroughly remove unbound primary antibody. After washing, dry the surrounding area and perform secondary antibody incubation: Add fluorescently labeled secondary antibody working solution prepared with antibody dilution buffer (strictly protected from light), and incubate in a humidified chamber at room temperature for 1 hour. After incubation, wash three more times with PBS in the dark, 5 minutes each time. Next, nuclear staining and mounting were performed: DAPI staining solution (final concentration 1 μg / mL) was added to cover the tissue, and incubated at room temperature in the dark for 5 minutes. Then, the tissue was washed twice with PBS in the dark, 5 minutes each time. Finally, excess liquid was carefully blotted off the slide with absorbent paper (but the tissue should not be allowed to dry completely). An appropriate amount of anti-fluorescence quenching mounting medium was placed in the center of the tissue, and a coverslip was gently placed on top, pressing down slowly from one side to avoid air bubbles. The coverslip was sealed around its edges with clear nail polish. After it was completely dry, it could be observed and images acquired under a fluorescence microscope or confocal microscope. Figure 5 A- Figure 5 As shown in B, by F4 / 80 and IL-1β staining, we found that F4 / 80 in the aorta after Ang II treatment... + The proportion of macrophages and IL-1β expression were significantly increased, while treatment with NiMn-PB nanomaterials significantly reduced the F4 / 80 ratio in the aorta after Ang II treatment. +Cell proportion and IL-1β expression level suggest that NiMn-PB nanomaterials can effectively reduce Ang II-mediated inflammatory cell infiltration and inflammation levels in the aorta. (As...) Figure 5 As shown in C, GPX4 staining significantly reduced the expression level of GPX4 (antiferroptosis protein) in the aorta after Ang II treatment, while NiMn-PB nanomaterial treatment significantly increased the GPX4 level in the aorta after Ang II treatment, suggesting that NiMn-PB nanomaterials can effectively reduce Ang II-mediated ferroptosis in the aorta. Figure 5 The D-staining assay showed that, through CD31 staining, we found CD31 levels in the aorta after Ang II treatment. + Increased number of angiogenic cells; treatment with NiMn-PB nanomaterials significantly reduced CD31. + The cell count suggests that NiMn-PB can effectively reduce Ang II-mediated angiogenesis in the aorta.

[0031] 4. Biosafety assessment of NiMn nanomaterials After weighing the model mice, they were euthanized, and whole blood was collected by enucleation of the eyeballs. One unit of whole blood was immediately injected into an EDTA anticoagulant tube and gently mixed for subsequent analysis of white blood cell, red blood cell, platelet, and hemoglobin counts using a fully automated hematology analyzer. Figure 6 F- Figure 6 As shown in Figure I, the nanomaterial treatment did not significantly affect the number of blood cells. Another sample of whole blood was injected into a non-anticoagulant tube, allowed to coagulate at room temperature, and then centrifuged at 3000 rpm for 15 minutes to obtain the supernatant serum. This serum was aliquoted and stored at -80℃ for use in an automated biochemical analyzer to detect biochemical indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen. Results Figure 6 B- Figure 6 The E value indicates that nanomaterial treatment did not affect blood biochemical parameters. Subsequently, the mice were perfused with PBS until the major organs and abdominal aorta turned white. The heart, liver, spleen, lungs, and both kidneys were completely removed. After rinsing with pre-cooled PBS and drying with filter paper, the wet weight was measured on a precision electronic balance, and the organ index (organ wet weight mg / mouse body weight g) was calculated. Figure 6 As shown in A, nanomaterial treatment had no significant effect on the major organ indices. All organs were then fixed in sufficient tissue fixative (4% (w / v) PFA) for 24-48 hours, followed by standard procedures for embedding, sectioning, and hematoxylin-eosin (HE) staining. Finally, the histological structure, morphology, and pathological changes of each organ were observed and evaluated under an optical microscope. Figure 6As shown in Figure J, after treatment with the nanomaterial, no significant changes were observed in the tissue structure and morphology of the major organs of mice, including the heart, liver, spleen, lungs, and kidneys. These results indicate that the NiMn-PB nanomaterial possesses good biocompatibility.

[0032] In summary, NiMn-PB nanomaterials can alleviate Ang II-induced abdominal aortic aneurysm by reducing cellular inflammation, ferroptosis, and angiogenesis, indicating that NiMn-PB nanomaterials can be used as the sole active ingredient in the preparation of drugs to alleviate abdominal aortic aneurysm.

[0033] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The application of NiMn-PB as the sole active ingredient in the preparation of drugs for protecting against abdominal aortic aneurysms, characterized in that, The preparation method of NiMn-PB includes the following steps: S1. Dissolve 0.4 mmol K3[Fe(CN)6] and 1.5 g PVP in 20 mL of deionized water to obtain a clear solution A; S2. Separately dissolve 0.1 mmol MnCl2·4H2O, 0.1 mmol NiCl2·6H2O and 1.5 g PVP in 20 mL of deionized water to obtain clear solution B; S3. At room temperature, add solution B dropwise to solution A and stir continuously for 10 minutes. Then add 40 mL of 0.01 M HCl solution and mix thoroughly. Finally, react the mixture at 80°C for 20 hours. S4. After the reaction was completed, the product was collected by centrifugation and washed repeatedly with deionized water and ethanol. After being freeze-dried in vacuum at -40℃ for 48h, NiMn-PB nanomaterials were finally obtained. The abdominal aortic aneurysm protective drug exerts its protective effect by inhibiting inflammatory responses, ferroptosis, and angiogenesis in the abdominal aortic tissue.

2. A drug for protecting against abdominal aortic aneurysms, characterized in that, The drug includes the NiMn-PB nanomaterial as the sole active ingredient as described in claim 1, and a medically acceptable carrier; wherein the drug treats or alleviates abdominal aortic aneurysm by inhibiting inflammatory responses, ferroptosis, and angiogenesis in abdominal aortic tissue.

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