A method of improving hypoxia within an atherosclerotic hemorrhagic plaque and inhibiting ferroptosis

Abstract

The application discloses a method for improving hypoxia in atherosclerotic hemorrhagic plaque and inhibiting ferroptosis, and belongs to the technical field of molecular biology. The application discloses a method for improving hypoxia in atherosclerotic hemorrhagic plaque and inhibiting ferroptosis, and discloses, for the first time, that low-frequency low-intensity ultrasound has a double protection function of simultaneously improving hypoxia and inhibiting ferroptosis, breaks through the limitation that existing sonodynamic therapy researches are mostly concentrated in a single path, and provides a more comprehensive solution for the complex pathological environment of a hemorrhagic plaque.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a method for improving hypoxia within atherosclerotic hemorrhagic plaques and inhibiting ferroptosis. Background Technology

[0002] Intraplaque hemorrhage (IPH) is a key pathological step leading to a sharp increase in plaque vulnerability and triggering acute cardiovascular and cerebrovascular events. Following IPH, the degradation of hemoglobin released from erythrocyte lysis produces a large amount of free iron, causing iron overload in macrophages within the plaque. Simultaneously, the hemorrhage and accompanying thrombus formation disrupt the microcirculation within the plaque, exacerbating hypoxia in the core area. This hypoxic microenvironment further worsens cellular metabolism and inflammation.

[0003] It is noteworthy that hypoxia and iron overload synergistically drive ferroptosis in macrophages within hemorrhagic plaques. Ferroptosis is an iron-dependent, programmed cell death process characterized by systemic peroxidation of the cell membrane due to the accumulation of lipid reactive oxygen species. Within hemorrhagic plaques, macrophage ferroptosis releases large amounts of damage-associated molecular patterns (DAMPs) and pro-inflammatory factors, dramatically amplifying local inflammation and directly disrupting the integrity of the fibrous cap through the secretion of matrix metalloproteinases, making it one of the key drivers of plaque rupture. Currently, there is a lack of effective clinical interventions that can simultaneously target both hypoxia and ferroptosis. Summary of the Invention

[0004] This invention addresses the lack of effective means in the prior art to simultaneously intervene in the two key aspects of "hypoxia" and "ferroptosis" by providing a method to improve hypoxia within atherosclerotic hemorrhagic plaques and inhibit ferroptosis.

[0005] One of the objectives of this invention is to provide a method for improving hypoxia within atherosclerotic hemorrhagic plaques and inhibiting ferroptosis. The method includes the following steps: administering a sonosensitive agent to macrophages to induce an absorption and transformation reaction; and after the reaction is complete, subjecting the cells to irradiation treatment using low-frequency, low-intensity ultrasound.

[0006] Furthermore, the sound-sensing agent is an organic molecular sound-sensing agent, an inorganic nano-sound-sensing agent, or an organic-inorganic hybrid nano-sound-sensing agent.

[0007] Furthermore, the sound-sensitizing agent is administered via intravenous injection or in vitro incubation.

[0008] Furthermore, when the acoustic sensitizer is administered via intravenous injection, the irradiation treatment conditions are: an ultrasonic frequency of 0.5-1.5 MHz and an ultrasonic intensity of 0.4 W / cm². 2 The irradiation time is 15 minutes.

[0009] Furthermore, when the acoustic sensitizer is administered via in vitro incubation, the irradiation treatment conditions are: an ultrasonic frequency of 0.5-1.5 MHz and an ultrasonic intensity of 0.2 W / cm². 2 The irradiation time is 5 minutes.

[0010] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a method to improve hypoxia and inhibit ferroptosis in atherosclerotic hemorrhagic plaques, and for the first time reveals that low-frequency low-intensity ultrasound has a dual protective function of simultaneously improving hypoxia and inhibiting ferroptosis, breaking through the limitation of existing sonodynamic therapy research focusing on a single pathway, and providing a more comprehensive solution for the complex pathological environment of hemorrhagic plaques.

[0011] This invention establishes a model for the transformation from an in vitro cell model (hypoxia combined with iron overload macrophage model) to an in vivo animal model (ApoE). - / - The complete chain of evidence for the mouse hemorrhagic plaque is logically rigorous and highly persuasive. Specifically, in vitro experiments confirmed that very low intensity ultrasound therapy (SDT) significantly reduced the hypoxia of iron-overloaded macrophages (decreased Pimonidazole fluorescence intensity), effectively inhibited hypoxia-induced lipid peroxidation (significantly decreased Liperfluo fluorescence intensity, P<0.05), and reduced the expression of ferroptosis markers (decreased 4-HNE protein expression and decreased MDA content). In vivo animal experiments further demonstrated that the hypoxia-positive staining area in the carotid artery plaque of mice in the SDT treatment group was significantly smaller than that in the control group (P<0.05), the plaque area / total vessel area ratio was smaller and the lumen area was correspondingly larger (P<0.05), collagen fiber content was significantly increased (P<0.05), iron deposition particles in the plaque were significantly reduced (P<0.05), and 4-HNE protein expression level was significantly reduced (P<0.05), and MDA content was significantly decreased (P<0.05).

[0012] In summary, this invention provides a novel mechanism for improving hypoxia and inhibiting ferroptosis within atherosclerotic hemorrhagic plaques, effectively stabilizing the plaques and achieving the expected technical effects, thus providing a new strategy for the treatment of atherosclerotic hemorrhagic plaques. Attached Figure Description

[0013] Figure 1 Figure A shows the results of SDT-induced iron overload reduction in macrophage hypoxia detection; Figure B is an immunofluorescence staining detection graph, and Figure B is a quantitative statistical graph. Figure 2 Figure A shows the results of SDT detection of lipid peroxidation levels in hypoxia-induced iron-overloaded macrophages; Figure B is an immunofluorescence staining detection graph, and Figure B is a quantitative statistical graph of fluorescence intensity. Figure 3Figure A shows the effect of SDT on 4-HNE and MDA in hypoxia-induced iron-overloaded macrophages; A is a Western blot analysis, and B and C are quantitative statistical graphs. Figure 4 SDT treatment for ApoE - / - Assessment of hypoxia in mouse hemorrhagic plaques; A is the result of immunohistochemical staining, and B is a quantitative statistical chart of the positive area. Figure 5 SDT treatment for ApoE - / - Assessment of the morphology and composition of hemorrhagic plaques in mice; A is the result of H&E and Masson staining, B is the statistical chart of plaque area, C is the statistical chart of lumen area, and D is the statistical chart of collagen content quantification. Figure 6 SDT treatment for ApoE - / - Assessment of iron deposition in hemorrhagic plaques in mice; A is the result of Prussian blue staining, and B is a quantitative statistical chart of positive area. Figure 7 SDT treatment for ApoE - / - Image of 4-HNE content assessment in mouse hemorrhagic plaques; A is immunohistochemical staining of 4-HNE in mouse plaques, B is quantitative statistical graph of positive area, C is protein expression results detected by Western Blot, and D is quantitative statistical graph of protein expression. Figure 8 SDT treatment for ApoE - / - Statistical chart of MDA content detection in mouse hemorrhagic plaque tissue homogenate. Detailed Implementation

[0014] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0016] In the following embodiments, the method for improving hypoxia in atherosclerotic hemorrhagic plaques and inhibiting ferroptosis provided by the present invention will be referred to as SDT.

[0017] Example 1: A method for improving hypoxia within atherosclerotic hemorrhagic plaques and inhibiting ferroptosis 1. Inhibitory effect of SDT on ferroptosis in macrophages under hypoxia combined with iron overload in vitro (1) Establishment of cell model: Primary bone marrow macrophages were extracted from C57BL / 6 mice (purchased from Nanjing Qingzilan Technology Co., Ltd.). To simulate the pathological state within hemorrhagic plaques, the cells were first treated with a medium containing 100 μmol / L ferric ammonium citrate (FAC) (the medium consisted of commercially available RPMI 1640 culture medium) for 12-24 hours to induce the formation of iron-overloaded macrophages. Subsequently, the cells were divided into the following two groups; Hypoxia model group: The induced iron-overloaded macrophages were transferred to a three-gas incubator (37°C, 5% CO2, 1% O2, 94% N2) and cultured for 12-24 hours to simulate severe hypoxia.

[0018] Hypoxia + SDT treatment group: The induced iron-overloaded macrophages were first incubated with 0.2 μM sonosensitive agent DVDMS for 4 hours in the dark, and then placed in a 1% O2 environment to simulate severe hypoxia, and immediately treated with SDT (a method provided by the present invention to improve hypoxia in atherosclerotic hemorrhagic plaques and inhibit ferroptosis).

[0019] The SDT treatment steps are as follows: the cell culture dish is placed under an ultrasound probe with degassed water as the coupling medium, and low-frequency, low-intensity ultrasound is used for irradiation treatment; the irradiation treatment parameters are set as follows: ultrasound frequency 1.0 MHz, intensity 0.2 W / cm², irradiation time 5 minutes; this intensity is defined as "extremely low intensity".

[0020] (2) Detection and Results: Immunofluorescence staining was performed on the intracellular hypoxia marker pimonidazole (purchased from Hypoxyprobe) in the hypoxia model group and the hypoxia + SDT treatment group. The experimental procedure was as follows: After incubation, cells were washed three times with PBS, followed by fixation with 4% paraformaldehyde at room temperature for 15 min. After fixation, cells were washed again with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 10 min. Cells were blocked with 5% BSA at room temperature for 1 h, followed by incubation with anti-pimonidazole monoclonal antibody (Hypoxyprobe kit, included) overnight at 4°C. The next day, cells were washed three times with PBS for 5 min each time, and fluorescently labeled secondary antibody was added, followed by incubation at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI, and finally, slides were mounted with anti-fluorescence quenching mounting medium and imaged and analyzed under a fluorescence microscope.

[0021] The results are as follows Figure 1 As shown in section A, compared with the hypoxia group, the fluorescence intensity in cells of the hypoxia + SDT group was weakened. Semi-quantitative detection using a fluorescence microplate reader revealed, as... Figure 1 As shown in Part B, compared with the hypoxia group, the fluorescence intensity in the hypoxia + SDT group was reduced (P<0.05). These results indicate that very low-intensity SDT can alleviate hypoxia in iron-overloaded macrophages.

[0022] The lipid peroxidation level was detected in the hypoxia model group and the hypoxia + SDT treatment group using the Liperfluo fluorescent probe (purchased from DOJINDO). The experimental steps are as follows: BMDM was cultured in different 96-well plates and fluorescent culture dishes. A 100 μmol / L FAC culture medium was prepared, and the FAC working solution was added to the 96-well plates containing BMDM for incubation. The plates were cultured in a cell hypoxia incubator at 37°C (1% O2, 5% CO2, 94% N2) for 16 hours. The plates were then washed with sterile PBS buffer, and an appropriate concentration of Liperfluo working solution was added. The plates were cultured at 37°C for 30 minutes. After removing the supernatant, the plates were washed twice with serum-free culture medium. Cell nuclei were stained with Hoeschst 33342 for 10 minutes in the fluorescent culture dishes, followed by three washes with sterile PBS buffer, each for 2 minutes. Cells were observed under a fluorescence microscope, and images were acquired using a fluorescence microscope. The microplate reader was set to excitation wavelength 488 nm and emission wavelength 525 nm, and the fluorescence intensity was measured by placing the 96-well plate in the microplate reader.

[0023] The results are as follows Figure 2 As shown, the hypoxia model group exhibited strong green fluorescence, indicating intense lipid peroxidation; while the fluorescence intensity in the hypoxia + SDT group was significantly reduced (P<0.05). This result clearly demonstrates that SDT effectively inhibits hypoxia-induced lipid peroxidation.

[0024] Ferraphobia markers were detected in the hypoxia model group and the hypoxia + SDT treatment group by Western blot. The experimental steps are as follows: 1) Protein sample preparation: After treatment, BMDM was removed, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS buffer. Lysis buffer containing protease and phosphatase inhibitors was prepared according to the ratio. 60 μL of lysis buffer was added to the culture dish. Cells were scraped with a cell scraper and collected into pre-cooled 1.5 mL EP tubes. The EP tubes were shaken 3 times (10 minutes apart, 30 seconds each time) to ensure complete lysis. Then, the cells were centrifuged at 12000 rpm for 30 minutes at 4°C. The supernatant was collected and the volume was recorded. The protein concentration was determined using a BCA kit. 5× protein loading buffer was added to 1 / 4 of the sample volume. The sample was heated in a metal bath at 100°C for 10 minutes. The obtained protein sample was stored at -80°C. 2) Electrophoresis: Prepare the PAGE gel according to the molecular weight of the target protein; prepare the protein sample and calculate the loading volume, add the electrophoresis buffer and load the sample; use 60V for the stacking gel, and switch to 120V after entering the separating gel. Stop electrophoresis when the sample is 0.5cm from the bottom edge of the separating gel. 3) Transfer: Cut a PVDF membrane of appropriate size with a pore size of 0.45µm or 0.2µm, activate it by soaking it in methanol for 30 seconds, and place it in the transfer holder in the following order: sponge, filter paper, PVDF membrane, gel, filter paper, sponge. Place the membrane in the transfer tank, set the current to 300mA, and the transfer time depends on the molecular weight of the target protein. 4) Blocking and Incubation: After transfer, place the PVDF membrane in skim milk powder prepared with 5% PBST and block at room temperature for 90 minutes. After washing with PBST, place the membrane in the primary antibody working solution and incubate overnight at 4°C on a shaker. The next day, remove the membrane and warm it for 10 minutes, then wash it three times with PBST (10 minutes each time). Prepare the corresponding species-specific HRP-labeled secondary antibody, incubate on a shaker for 1.5 hours, and wash three times with PBST (10 minutes each time). 5) Color Development and Analysis: Prepare the ultrasensitive ECL color development solution (reagent A: reagent B = 1:1), add it evenly to the PVDF membrane, and perform color development and imaging in the ChemiDoc™ MP imaging system. Use ImageLab or ImageJ software for data analysis.

[0025] The results are as follows Figure 3 As shown in section AB, compared with the hypoxia group, the expression of 4-HNE protein in cells of the hypoxia + SDT group was reduced (P<0.01).

[0026] MDA content in the hypoxia model group and the hypoxia + SDT treatment group was detected using an MDA detection kit (purchased from Beyotime Biotechnology Co., Ltd.). The experimental steps were as follows: After treatment, cells were collected, and 0.1 mL of lysis buffer was used per million cells. After lysis, the cells were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was pipetted for subsequent measurements. The tissue protein concentration was measured using a BCA protein concentration assay kit. 0.1 mL of PBS buffer was added to the centrifuge tube as a blank control, and 0.1 mL of standards at different concentrations were added to create a standard curve. Then, 0.1 mL of sample was added for measurement. Subsequently, 0.2 mL of MDA detection working solution was added, mixed well, heated in a 100°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 1000 rpm for 10 minutes at room temperature. 0.2 mL of supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm using a microplate reader to calculate the intracellular MDA content.

[0027] The results are as follows Figure 3 As shown in section C, compared with the normoxic group, the hypoxia + SDT group showed increased intracellular MDA content. These results indicate that very low-intensity SDT reduces hypoxia-induced lipid peroxidation in iron-overloaded macrophages.

[0028] Therefore, the method (SDT) provided by this invention for improving hypoxia and inhibiting ferroptosis in atherosclerotic hemorrhagic plaques can effectively reduce lipid peroxidation of iron-overloaded macrophages under hypoxic conditions, inhibit ferroptosis, and improve cell viability in vitro.

[0029] 2. SDT reduces ApoE - / - In mice, plaque hemorrhage and hypoxia inhibited ferroptosis and stabilized the plaque. (1) Animal model and grouping: 8-week-old male ApoE - / - Mice (purchased from Nanjing Qingzilan Technology Co., Ltd.) were fed a high-fat diet and a right carotid artery plaque hemorrhage model was induced by surgery. At the end of the 10th week after modeling, the mice were randomly divided into the following two groups. Control group (Ctrl group): No treatment received.

[0030] SDT treatment group (SDT group): Received SDT treatment.

[0031] (2) SDT treatment plan: In the SDT group, model mice were injected with DVDMS (4 mg / kg) via the tail vein, anesthetized and fixed after 4 hours of protection from light; the right carotid artery area was exposed, and after applying coupling gel, local irradiation was performed using the SDT therapeutic probe; irradiation parameters: intensity 0.4 W / cm², frequency 1.0 MHz, time 15 minutes, once every other day, for a total of 3 times; the control group was given an equal volume of physiological saline.

[0032] Sample collection and comprehensive analysis: Samples were collected the day after the last treatment to perform multi-indicator testing on the carotid artery plaques.

[0033] Immunohistochemical staining with Pimonidazole (hypoxia probe) was used to assess plaque hypoxia in the Ctrl and SDT groups. The experimental procedure was as follows: A 30 mg / mL Pimonidazole sterile saline solution was prepared and injected intravenously into mice at a dose of 60 mg / kg 90 minutes before euthanasia. Paraffin sections were then prepared and baked at 62°C for 2 hours. The sections were dewaxed sequentially with xylene I and xylene II for 10 minutes each, followed by a gradient of ethanol dewaxing: 100% ethanol I and 100% ethanol II for 5 minutes each, then 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, and 75% ethanol for 5 minutes each, and then soaked in distilled water for 5 minutes. The sections were then blocked with 3% H2O2 in the dark for 15 minutes, rinsed with distilled water for 5 minutes, and then microwaved with citrate antigen retrieval solution (4 minutes on medium heat, 5 minutes on high heat, and 2 minutes on medium heat). After natural cooling, the sections were rinsed three times with PBS (5 minutes each time), blocked with goat serum for 30 minutes, dried, and then 1% H2O2 was added. The primary antibody prepared with BSA was incubated overnight at 4°C in a humidified chamber. The next day, the slide was warmed for 10 minutes, washed three times with PBS (5 minutes each time), and then incubated with HRP-labeled secondary antibody at room temperature for 1 hour. After washing three times with PBS, DAB staining was performed, followed by hematoxylin counterstaining for 50 seconds and rinsing with tap water for 5 minutes. Gradient ethanol dehydration was then performed: 75% ethanol and 80% ethanol for 30 seconds each, followed by 90% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II for 5 minutes each, and then cleared with xylene I and xylene II for 5 minutes each. Finally, the slide was mounted with neutral resin, dried at 60°C, and images were acquired using an inverted white light microscope. Data analysis was performed using IPP software.

[0034] The results are as follows Figure 4 As shown, the positive staining area in the plaques of the SDT group was significantly less than that of the Ctrl group (P<0.05), demonstrating that SDT improved plaque hypoxia.

[0035] The morphology and composition of plaques in the Ctrl and SDT groups were assessed using H&E staining (kit purchased from Beijing Solarbio Science & Technology Co., Ltd.) and Masson staining (kit purchased from Beijing Solarbio Science & Technology Co., Ltd.), respectively. The experimental procedures are as follows: HE staining procedure: Paraffin tissue sections were baked at 62℃ for 2 hours, then dewaxed sequentially with xylene I and xylene II for 10 minutes each, followed by gradient ethanol hydration (5 minutes each with 100% ethanol I, 100% ethanol II, 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, and 75% ethanol), and soaked in distilled water for 5 minutes; then hematoxylin staining for 1 minute, hydrochloric acid alcohol differentiation for 10 seconds, rinsed with tap water, eosin staining for 5 minutes, and rinsed with tap water; then rapid dehydration (30 seconds each with 75% ethanol and 80% ethanol), followed by sequential ethanol hydration (5 minutes each with 90% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II), and clearing with xylene I and xylene II for 5 minutes each; finally, the sections were mounted with neutral resin, dried at 60℃, and images were acquired using an inverted white light microscope. Data statistical analysis was performed using IPP software.

[0036] Masson staining procedure: After baking paraffin tissue sections at 62℃ for 2 hours, dewaxing and hydration were completed according to the HE staining procedure. Hematoxylin staining was performed for 5 minutes, followed by rinsing with distilled water, inversion with bluing solution, and rinsing with distilled water again. Ponceau S staining was performed for 5 minutes, followed by rinsing with weak acid working solution (2L distilled water + 5ml glacial acetic acid). 1% phosphomolybdic acid was added for 1 minute, followed by aniline blue staining for 40 seconds and soaking in weak acid working solution for 1 minute. Subsequently, rapid dehydration with 95% ethanol was performed, followed by soaking in 100% ethanol I and II for 5 minutes each, and clearing with xylene I and II for 5 minutes each. Finally, the sections were mounted with neutral resin, dried at 60℃, and images were acquired using an inverted white light microscope. Data analysis was performed using IPP software.

[0037] The results are as follows Figure 5 As shown, H&E staining results indicated that the plaque area / total vessel area ratio was significantly reduced in the SDT group, while the lumen area ratio was correspondingly increased (P<0.05). Masson staining results showed that the collagen fiber content (blue) in the plaque of the SDT group was significantly higher than that in the Ctrl group (P<0.05), suggesting that the plaque was more stable.

[0038] Iron content in plaques in the Ctrl and SDT groups was assessed using Prussian blue staining (kit purchased from Beijing Solarbio Science & Technology Co., Ltd.). The experimental procedure was as follows: Paraffin-embedded tissue sections were baked at 62℃ for 2 hours, then dewaxed and hydrated according to the HE staining procedure. Solutions A and B were mixed in a 1:1 ratio to prepare the iron staining working solution. The sections were soaked for 8 minutes, rinsed with distilled water for 1 minute, stained with nuclear solid red solution for 5 minutes, and rinsed with distilled water for 30 seconds. Subsequently, rapid dehydration was performed with 75% ethanol and 80% ethanol, followed by dehydration with 90% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II for 3 minutes each, and cleared with xylene I and II for 5 minutes each. Finally, the sections were mounted with neutral resin, dried at 60℃, and images were acquired using an inverted white light microscope. Data analysis was performed using IPP software.

[0039] The results are as follows Figure 6 As shown, the Prussian blue staining results indicated that the blue iron deposits in the SDT group were significantly fewer than those in the Ctrl group (P<0.05), suggesting that SDT promoted the removal of iron from the plaques.

[0040] Plaque ferroptosis was assessed in the Ctrl and SDT groups using 4-HNE immunohistochemistry and Western blotting, respectively. The 4-HNE immunohistochemistry procedure was the same as the Pimonidazole (hypoxia probe) immunohistochemistry staining procedure described above. Arterial tissue protein extraction: Carotid artery plaque tissue was weighed and ground, then lysed with a lysis buffer prepared from 990 μL RIPA lysis buffer and 10 μL PMSF (100 μL per tube). The mixture was thoroughly mixed on an ice plate, centrifuged at 12000 rpm for 20 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA kit. The remaining supernatant was added to 5×SDS loading buffer, denatured by heating in a 100°C metal bath for 10 minutes, centrifuged at 1000 rpm for 1 minute, and then stored at -80°C. Subsequent electrophoresis and membrane transfer procedures were performed as described above.

[0041] The results are as follows Figure 7-8 As shown, both immunohistochemistry and Western blotting of 4-HNE revealed that the expression level of 4-HNE protein in the plaques of the SDT group was significantly lower than that of the Ctrl group (P<0.05). MDA analysis of tissue homogenates showed that the MDA content in the SDT group was significantly lower than that in the Ctrl group (P<0.05).

[0042] In summary, the present invention provides a method (SDT) for improving hypoxia and inhibiting ferroptosis in atherosclerotic hemorrhagic plaques. By inhibiting the ferroptosis of iron-overloaded macrophages induced by hypoxia in hemorrhagic plaques, the present invention can effectively stabilize plaques and achieve the expected technical effects.

[0043] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A method for improving hypoxia within atherosclerotic hemorrhagic plaques and inhibiting ferroptosis, characterized in that, The method includes the following steps: administering a sonosensitive agent to macrophages to induce an absorption and transformation reaction, and after the reaction is complete, subjecting them to irradiation treatment using low-frequency, low-intensity ultrasound.

2. The method according to claim 1, characterized in that, The sound-sensing agent is an organic molecular sound-sensing agent, an inorganic nano sound-sensing agent, or an organic-inorganic hybrid nano sound-sensing agent.

3. The method according to claim 1, characterized in that, The sound-sensitive agent is administered via intravenous injection or in vitro incubation.

4. The method according to claim 3, characterized in that, When the acoustic sensitizer is administered intravenously, the irradiation treatment conditions are: ultrasonic frequency of 0.5-1.5 MHz and ultrasonic intensity of 0.4 W / cm². 2 The irradiation time is 15 minutes.

5. The method according to claim 3, characterized in that, When the acoustic sensitizer is administered via in vitro incubation, the irradiation treatment conditions are: ultrasonic frequency of 0.5-1.5 MHz and ultrasonic intensity of 0.2 W / cm². 2 The irradiation time is 5 minutes.

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