A physical method of modulating ferroptosis in cells or tissues ex vivo

Abstract

This invention belongs to the field of biomedicine and cell protection technology, and provides a method for regulating ferroptosis in isolated cells or tissues by applying a magnetic field. The method includes: applying a magnetic field to isolated cells or tissues during treatment to reduce the level of ferroptosis; the treatment process refers to a process that increases the level of ferroptosis in isolated cells or tissues. This invention, by applying a magnetic field during cell or tissue treatment, can reduce the level of ferroptosis in cells or tissues compared to a control condition without a magnetic field, accompanied by improvements in various related biological indicators. This invention utilizes a magnetic field as a regulatory means, eliminating the need for exogenous chemical inhibitors, thereby avoiding potential toxicity and residue risks, and possessing higher safety and application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and cell protection technology, specifically relating to a method for regulating ferroptosis in isolated cells or tissues by applying a magnetic field, and particularly relating to its application in inhibiting ferroptosis and improving cell or tissue stability during cryopreservation. Background Technology

[0002] During in vitro processing of cells and tissues, especially under low-temperature conditions, cells are often subjected to multiple damaging factors such as oxidative stress and energy metabolism disorders, leading to various forms of programmed cell death and severely affecting cell viability and tissue functional stability. Recent studies have shown that in addition to traditional cell death mechanisms such as necrosis, apoptosis, and autophagy, several new programmed cell death patterns exist. Among them, ferroptosis is a form of cell death characterized by iron-dependent lipid peroxidation accumulation. Its typical biochemical characteristics include: depletion of intracellular glutathione (GSH), decreased glutathione peroxidase 4 (GPX4) activity, reduced lipid peroxide scavenging capacity, and decreased iron ion concentration. 2+ Excessive reactive oxygen species (ROS) are generated during the oxidative reactions involved, leading to membrane lipid peroxidation damage and ultimately cell death. Under low-temperature treatment conditions, the aforementioned ferroptosis-related processes are particularly pronounced and are considered one of the important mechanisms leading to cell damage and tissue dysfunction.

[0003] Current technologies typically intervene in ferroptosis by adding chemical inhibitors (such as Ferrostatin-1). However, these methods have drawbacks including potential toxicity, residue risks, and limitations in clinical application. Therefore, there is an urgent need to develop a physical modulation method that does not require the addition of exogenous chemicals to effectively intervene in ferroptosis. Summary of the Invention

[0004] To address the potential toxicity, residual risks, and limited clinical applications associated with existing technologies that involve adding chemical inhibitors to intervene in the ferroptosis process, this invention provides a physical method for regulating ferroptosis in isolated cells or tissues.

[0005] This invention is achieved through the following technical solution: A physical method for regulating ferroptosis in isolated cells or tissues includes: applying a magnetic field to isolated cells or tissues during the treatment of isolated cells or tissues to reduce the level of ferroptosis in isolated cells or tissues; the treatment process refers to a process that increases the level of ferroptosis in isolated cells or tissues.

[0006] Preferably, the magnetic induction intensity of the magnetic field is 10~500 μT.

[0007] Furthermore, the magnetic induction intensity of the magnetic field is 50~200 μT, more preferably 100 μT.

[0008] Preferably, the magnetic field is an alternating magnetic field, a constant magnetic field, a pulsed magnetic field, or an oscillating magnetic field.

[0009] Furthermore, the magnetic field is an alternating magnetic field with a frequency of 1~100 Hz, preferably 5~50 Hz, and more preferably 10 Hz.

[0010] Preferably, the isolated cells or tissues are cells or tissues derived from mammals.

[0011] Furthermore, the isolated cells are isolated small intestinal epithelial cells, and the isolated tissue is isolated small intestinal tissue or a segment of small intestine.

[0012] Furthermore, the small intestinal epithelial cells are small intestinal crypt epithelial cells, such as IEC-6 cells or primary small intestinal epithelial cells.

[0013] Preferably, the treatment process is cryopreservation, that is, applying a magnetic field during the cryopreservation of isolated cells or tissues, thereby improving the preservation effect of isolated cells or tissues by inhibiting ferroptosis.

[0014] Furthermore, the temperature for cryopreservation is -8 to -12°C, more preferably -10°C.

[0015] Furthermore, the cryopreservation time for isolated cells is 1–24 hours, and the cryopreservation time for isolated tissues is 24–72 hours.

[0016] Preferably, the reduction in ferroptosis levels in isolated cells or tissues is characterized by at least one of the following indicators: (a) Intracellular Fe 2+ The content decreased; (b) Reduced levels of reactive oxygen species; (c) Decreased lipid peroxidation levels; (d) The decrease in mitochondrial membrane potential was inhibited; (e) Elevated glutathione levels or an elevated GSH / GSSG ratio; (f) Increased expression or activity of GPX4.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention has found that applying a magnetic field during the treatment of cells or tissues can reduce ferroptosis levels in cells or tissues compared to control conditions without a magnetic field, accompanied by improvements in various related biological indicators. Specifically, at least one of the following effects can be achieved: (1) Improved antioxidant capacity: including but not limited to increased intracellular reduced glutathione (GSH) levels, increased GSH / GSSG ratio, and enhanced expression or activity of glutathione peroxidase 4 (GPX4); (2) Reduced lipid peroxidation levels: manifested as changes in lipid peroxidation probe detection signals and reduced content of lipid peroxidation end products (such as MDA); (3) Alleviation of ferroptosis-related phenotypes: including intracellular Fe 2+ The content decreased, the level of reactive oxygen species (ROS) decreased, and the damage to mitochondrial function was reduced; (4) the cell or tissue function was improved: including increased cell viability, improved energy metabolism and enhanced tissue structure stability. In order to further verify the correlation between the above effects and the ferroptosis pathway, the inventors conducted a comparative study by introducing different types of intervention conditions. The results showed that under specific conditions, the effect of the magnetic field and the ferroptosis regulation-related indicators showed a consistent trend, suggesting that the magnetic field may participate in the regulation of ferroptosis by affecting the cellular antioxidant system and lipid peroxidation process. Based on the experimental results, it can be considered that the method described in this invention can regulate the intracellular ferroptosis-related process through physical means, thereby reducing iron-dependent lipid peroxidation damage. This invention uses the magnetic field as a regulation means, without relying on exogenous chemical inhibitors, thereby avoiding potential toxicity and residual risks, and has higher safety and application potential. This invention not only reduces lipid peroxidation and oxidative stress at the cellular level, but also reduces toxic Fe². + This invention accumulates and maintains mitochondrial function, thereby effectively preserving the integrity of villous structures, mucosal barrier function, and digestive and absorptive functions at the tissue level, achieving comprehensive protection from cell viability to tissue function. This invention proposes a ferroptosis regulation strategy that differs from traditional chemical interventions, providing new research directions and technical means for related fields. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 In Experiment 1, after IEC-6 cells were treated at low temperature for 24 h, the following are the representative images of (a) F-actin cytoskeleton fluorescence staining and (b) average fluorescence intensity of the control group, low temperature group and magnetic field group.

[0020] Figure 2 The bar chart shows the quantitative results of IEC-6 cells after 4 h and 24 h of low-temperature treatment in Experiment 1, including (a) ATP content, (b) cell viability (CCK-8 assay), (c) intracellular ROS level, and (d) lipid peroxidation level. Lipid peroxidation level is expressed as the red / green fluorescence intensity ratio detected by the C11-BODIPY 581 / 591 probe.

[0021] Figure 3 In Experiment 1, after IEC-6 cells were treated at low temperature for 4 h, the results of flow cytometry using the JC-1 method were obtained. (a) The mitochondrial membrane potential was measured and (b) was quantitatively analyzed.

[0022] Figure 4 In Experiment 1, after IEC-6 cells were treated with low temperature for 4 h, intracellular Fe was detected using the RhoNox-6 probe. 2+ (a) Horizontal fluorescence representation image and (b) quantitative analysis of fluorescence intensity.

[0023] Figure 5 The bar chart shows the quantitative results of (a) GSH / GSSG ratio, (b) SOD activity, and (c) MDA content of IEC-6 cells after 4 h and 24 h of low-temperature treatment in Experiment 1.

[0024] Figure 6 In Experiment 1, after IEC-6 cells were treated at low temperature for 24 h, Western blot was used to detect the expression of GPX4 protein. (a) Representative bands and (b) Quantitative analysis of gray values ​​were performed.

[0025] Figure 7 In Experiment 2, after BSO intervention, the expression of GPX4 protein was detected by Western blot, and (a) a representative band image and (b) a quantitative analysis of gray values ​​were obtained.

[0026] Figure 8 The bar chart shows the quantitative results of (a) GSH / GSSG ratio and (b) cell viability (CCK-8 assay) after intervention with different inhibitors in Experiment 2.

[0027] Figure 9 The bar chart shows the quantitative results of (a) MDA content, (b) lipid peroxidation level (R / G ratio detected by C11-BODIPY 581 / 591), and (c) LDH activity in cell lysate after intervention with different inhibitors in Experiment 2.

[0028] Figure 10The bar chart shows the quantitative results of (a) cell viability (CCK-8 assay), (b) ATP content, (c) intracellular ROS level, (d) lipid peroxidation level (R / G ratio detected by C11-BODIPY 581 / 591), (e) MDA content and (f) SOD activity after Fer-1 intervention in Experiment 3.

[0029] Figure 11 In Experiment 3, after Fer-1 intervention, intracellular Fe²⁺ was detected using the RhoNox-6 probe. + (a) Horizontal fluorescence representation image and (b) quantitative analysis of fluorescence intensity.

[0030] Figure 12 The images shown are HE staining representations of small intestinal tissue after the addition of magnetic field and / or Fer-1 intervention in Experiment 4, along with quantitative analysis results of (a) Chiu's score, (b) tissue integrity, (c) cavity condition, and (d) submucosal integrity.

[0031] Figure 13 The bar chart shows the quantitative results of (a) ROS level, (b) MDA content, (c) SOD activity and (d) GSH / GSSG ratio in small intestinal tissue after the addition of magnetic field and / or Fer-1 intervention in Experiment 4.

[0032] Figure 14 The bar chart shows the quantitative results of (a) glucose absorption capacity of small intestinal tissue, (b) maltase activity, (c) lactase activity and (d) intestinal permeability after treatment with static cold preservation and deep supercooling (with or without Fer-1) in Experiment 5. Detailed Implementation

[0033] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0035] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not intended to limit the order of the method steps or define the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0036] Experiment 1: Magnetic field alleviates hypothermia-induced damage to rat small intestinal epithelial cells 1. Materials and Methods 1.1 Experimental Cells This experiment used the rat small intestinal crypt epithelial cell line IEC-6 (purchased from icell). Cells were cultured in DMEM complete medium containing: DMEM basal medium (containing 1.5 g / L NaHCO3), 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 0.01 mg / mL bovine insulin. Culture conditions were 37°C, 5% CO2, and saturated humidity (70%-80%).

[0037] 1.2 Drugs and Treatment IEC-6 cells were divided into three groups for treatment. The cryogenic treatment conditions were -10℃ for 4 hours or 24 hours, respectively. After treatment, all samples were uniformly replaced with DMEM complete medium and incubated at 37℃ for 4 hours, followed by various parameter measurements. The basal preservation solution was UW (University of Wisconsin) solution, supplemented with 0.2 mol / L 1,4-cyclohexanediol (1,4-CHD) and 5% polyethylene glycol (PEG 8000).

[0038] (1) Control group: Cells were cultured at 37°C without low temperature treatment or magnetic field.

[0039] (2) Low temperature group: Cells were treated in a -10℃ preservation solution for a specified time (4 h or 24 h) without applying a magnetic field during the treatment.

[0040] (3) Magnetic field group: Cells were treated in a -10℃ preservation solution for a specified time (4 h or 24 h), during which an alternating magnetic field (frequency 10 Hz, magnetic induction intensity 100 μT) was continuously applied.

[0041] 1.3 Cell morphology observation (F-actin staining) IEC-6 cells were seeded in six-well plates (seeding density approximately 1 × 10⁶ cells / well). 6 Cells / well). The cells were treated according to the grouping method described in 1.2. After treatment, the culture medium was discarded, and the cells were washed three times with phosphate-buffered saline (PBS). Subsequently, F-actin cytoskeleton staining was performed according to the kit instructions, and the cells were observed and photographed using a confocal laser scanning microscope.

[0042] like Figure 1 As shown, compared with the control group, the cytoskeleton integrity of the low-temperature group was significantly reduced, and the average fluorescence intensity decreased significantly, indicating that low-temperature treatment led to a decrease in cytoskeleton structure and cell integrity. The cytoskeleton integrity of the magnetic field group was significantly improved compared with the low-temperature group, and the average fluorescence intensity was also significantly increased, indicating that magnetic field treatment improved the problem of reduced cytoskeleton structure and cell integrity caused by low temperature.

[0043] 1.4 Cell viability and function assay (1) Cell viability (CCK-8 assay): IEC-6 cells were seeded in 96-well plates (approximately 5000 cells / well) and treated according to the grouping method described in 1.2. After treatment, the culture medium was discarded, and 100 μL of CCK-8 working solution (prepared by mixing DMEM complete culture medium and CCK-8 reagent at a volume ratio of 9:1) was added to each well. The cells were incubated at 37°C in the dark for 30 minutes. The absorbance (OD value) at a wavelength of 450 nm was measured using a microplate reader, and the OD value reflected cell viability.

[0044] (2) Cell energy status (ATP content detection): IEC-6 cells were seeded in 96-well plates (approximately 5,000 cells / well) and treated according to the grouping method described in 1.2. After treatment, the chemiluminescence signal of each well was detected according to the instructions of the Promega ATP detection kit. The luminescence value reflects the intracellular ATP content and characterizes the cell energy status.

[0045] like Figure 2 (a) and Figure 2 As shown in (b), compared to the control group, the cell viability and cell energy status of the low-temperature groups treated for 4 h and 24 h were significantly decreased. In contrast, the magnetic field group improved cell viability and cell energy status compared to the low-temperature group.

[0046] 1.5 Detection of intracellular reactive oxygen species (ROS) IEC-6 cells were seeded in 96-well plates (approximately 5000 cells / well) and treated according to the grouping method described in 1.2. After treatment, complete culture medium containing a final concentration of 10 μM DCFH-DA fluorescent probe was added, and the plates were incubated at 37°C in the dark for 1 hour. The cells were then washed three times with PBS to remove unbound probe. Fluorescence intensity (excitation wavelength 488 nm, emission wavelength 525 nm) was measured using a multi-functional microplate reader. The fluorescence intensity value was directly proportional to the intracellular ROS level.

[0047] like Figure 2 As shown in (c), the intracellular ROS levels in the low-temperature groups treated for 4 h and 24 h were significantly higher than those in the control group, while the intracellular ROS levels in the magnetic field group were significantly lower than those in the low-temperature group. This indicates that magnetic field treatment can reduce the increase in intracellular ROS levels caused by low temperature.

[0048] 1.6 Detection of lipid peroxidation level (C11-BODIPY) 581 / 591 (Probe method) IEC-6 cells were seeded in 96-well plates (approximately 5000 cells / well) and treated according to the grouping method described in 1.2. After treatment, a solution containing 2 μM C11-BODIPY was added. 581 / 591 The probe was incubated in complete culture medium at 37°C in the dark for 1 hour. After washing with PBS, the intensity of red fluorescence (excitation wavelength 581 nm, emission wavelength 591 nm) and green fluorescence (excitation wavelength 488 nm, emission wavelength 510 nm) was detected using a multi-functional microplate reader. The intensity ratio of red fluorescence to green fluorescence (R / G ratio) was calculated, which is expressed as LPO (R / G) in this invention. A higher ratio indicates a lower level of lipid peroxidation; a lower ratio indicates a higher level of lipid peroxidation.

[0049] like Figure 2 As shown in (d), compared with the control group, the lipid peroxidation level was increased in the low temperature group, while the magnetic field treatment reduced the lipid peroxidation level. This was reflected in the increased LPO (R / G ratio) in the C11-BODIPY 581 / 591 assay, indicating that the magnetic field treatment significantly improved the problem of increased lipid peroxidation level in IEC-6 cells caused by low temperature.

[0050] 1.7 Mitochondrial membrane potential detection (JC-1 method) IEC-6 cells were seeded in six-well plates (approximately 1 × 10⁻⁶ cells). 6Cells were grouped and treated according to the method described in 1.2 (cells / well). After treatment, cells were collected, washed with PBS, and JC-1 staining working solution was added. The cells were incubated at 37°C in the dark for 30 minutes. After incubation, the staining solution was discarded, and the cells were washed twice with JC-1 staining buffer. The cells were then digested and collected. The mean fluorescence intensity (MFI) of the PE channel was immediately detected by flow cytometry to evaluate the mitochondrial membrane potential level.

[0051] like Figure 3 As shown, compared with the control group, the average fluorescence intensity of the low temperature group decreased, indicating a decrease in mitochondrial membrane potential. The decrease in mitochondrial membrane potential is one of the phenotypes of ferroptosis-related damage. Magnetic field treatment significantly inhibited the decrease in mitochondrial membrane potential and maintained the mitochondrial membrane potential.

[0052] 1.8 Intracellular Fe 2+ Content detection IEC-6 cells were seeded in six-well plates (approximately 1 × 10⁻⁶ cells). 6 Cells / well) were treated according to the grouping method described in 1.2. After treatment, the cells were washed with PBS, and a solution containing a final concentration of 2 μM RhoNox-6 probe was added. The cells were incubated at 37°C in the dark for 30 minutes. The probe solution was discarded, and the cells were washed with PBS. Immediately afterward, the cells were observed and photographed using a fluorescence microscope. Fluorescence intensity and intracellular Fe... 2+ The level is directly proportional.

[0053] like Figure 4 As shown, compared with the control group, the fluorescence intensity of the low-temperature group was significantly increased, indicating that the intracellular Fe in the low-temperature group was higher. 2+ The levels of Fe were significantly increased, while the fluorescence intensity in the magnetic field group was significantly lower than that in the low temperature group, indicating that magnetic field treatment significantly reduced intracellular Fe levels. 2+ level.

[0054] 1.9 Detection of biochemical indicators related to oxidative stress and lipid peroxidation IEC-6 cells were seeded in six-well plates (approximately 1 × 10⁻⁶ cells). 6 Cells were processed according to the grouping method described in 1.2 (cells / well). After processing, the cells were washed with PBS, scraped, and collected into EP tubes. An appropriate amount of PBS was added, and the cells were sonicated and then centrifuged (e.g., 12000 g, 10 minutes, 4°C) to collect the supernatant. The protein concentration in the supernatant was determined using a BCA protein quantification kit. Subsequently, the contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the supernatant were detected according to the instructions of the respective kits, and the GSH / GSSG ratio, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity were calculated.

[0055] from Figure 5As can be seen from (a) to (c), compared with the control group, the low temperature group had a lower GSH / GSSG ratio, lower SOD activity, and higher MDA content. In contrast, the magnetic field group showed an increased GSH / GSSG ratio, increased SOD activity, and decreased MDA content after magnetic field treatment.

[0056] 1.10 Detection of GPX4 expression in cells IEC-6 cells were seeded in six-well plates (approximately 1 × 10⁻⁶ cells). 6 Cells / well were grouped and processed according to the method described in 1.2. After treatment, the cells were washed with PBS, and RIPA lysis buffer containing protease inhibitor (PMSF) was added. The cells were lysed on ice for 30 minutes. The lysate was collected, centrifuged at 12000 g for 15 minutes at 4°C, and the supernatant was collected for protein quantification using the BCA method. An equal volume of protein was separated by SDS-PAGE gel electrophoresis, and then the protein was transferred to a PVDF membrane. After blocking with blocking buffer at room temperature for 1 hour, GPX4 primary antibody (dilution ratio 1:5000) and internal control β-actin primary antibody (dilution ratio 1:5000) were added, and the membrane was incubated overnight at 4°C. The next day, the membrane was thoroughly washed with TBST buffer, and horseradish peroxidase (HRP)-labeled secondary antibody (dilution ratio 1:5000) was added. The membrane was incubated at room temperature for 1 hour. After washing again, the membrane was developed using enhanced chemiluminescence (ECL) buffer, and the protein bands were quantitatively analyzed by grayscale value using image analysis software.

[0057] like Figure 6 As shown, compared with the control group, the expression level of GPX4 protein in the low temperature group was significantly reduced, while the magnetic field treatment upregulated the expression level of GPX4 protein.

[0058] 1.11 Statistical Methods All experimental data are expressed as mean ± standard deviation (Mean ± SD). Data analysis was performed using GraphPad Prism 7.0 statistical software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0059] 2. Results In summary, compared with the conventional culture conditions at 37℃, IEC-6 cells exhibited multiple damage-related changes under -10℃ low-temperature treatment, including cytoskeleton disruption, decreased cell viability and ATP levels, and increased oxidative stress. This was further manifested as increased intracellular reactive oxygen species (ROS) levels, increased lipid peroxidation (reduced R / G ratio in C11-BODIPY assay), and decreased mitochondrial membrane potential, accompanied by increased Fe... 2+The changes included increased levels, decreased GSH / GSSG ratio, decreased SOD activity, increased MDA content, and decreased GPX4 protein expression levels.

[0060] After applying a magnetic field during the above-mentioned low-temperature treatment, the cell state was improved compared with the low-temperature group without a magnetic field, specifically as follows: (1) Improved cell structure stability: The degree of disorder in the cytoskeleton structure is reduced, and the integrity of cell morphology is improved; (2) Improved cell viability and energy metabolism: Cell viability and ATP levels were higher in the low-temperature group; (3) Decreased oxidative stress level: Decreased intracellular ROS level; (4) Decreased lipid peroxidation: The R / G ratio increased in C11-BODIPY assay, while the MDA content decreased; (5) Improved mitochondrial functional status: The degree of decline in mitochondrial membrane potential was reduced; (6) Easing of iron-related changes: intracellular Fe 2+ The level decreased; (7) Enhanced antioxidant capacity: The GSH / GSSG ratio and SOD activity are increased; (8) Changes in the expression of ferroptosis-related proteins: GPX4 protein expression level increased or remained unchanged.

[0061] The results indicate that applying a magnetic field during cryogenic treatment helps to improve various cell damage-related changes induced by cryogenicity, and shows a consistent regulatory trend across multiple biological indicators related to ferroptosis.

[0062] Therefore, it can be considered that the method described in this invention can reduce the level of cell ferroptosis under low temperature treatment conditions, and its effect may be related to the regulation of intracellular antioxidant state and lipid peroxidation process.

[0063] Experiment 2: Verification of the molecular mechanism of magnetic field protection 1. Materials and Methods 1.1 Experimental Cells This experiment used the rat small intestinal crypt epithelial cell line IEC-6 (purchased from icell). Cells were cultured in DMEM complete medium containing: DMEM basal medium (containing 1.5 g / L NaHCO3), 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 0.01 mg / mL bovine insulin. Culture conditions were 37°C, 5% CO2, and saturated humidity (70%-80%).

[0064] 1.2 Drugs and Treatment (1) Major inhibitors: 1) BSO (L-Buthionine-sulfoximine): Glutamine cysteine ​​synthase inhibitor, used to deplete intracellular glutathione (GSH), as a GSH synthesis inhibitor.

[0065] 2) RSL3 (Ras-selective lethal 3): GPX4 enzyme activity inhibitor.

[0066] 3) Fer-1 (Ferrostatin-1): A classic inhibitor of ferroptosis.

[0067] (2) Low temperature preservation solution: Same as Experiment 1, but add 0.2 mol / L 1,4-cyclohexanediol and 5% PEG8000 to the UW solution.

[0068] (3) Experimental grouping and treatment procedures: IEC-6 cells were treated according to the following groups. All groups involving low-temperature treatment were treated at -10℃ for 24 hours. After treatment, the cells were replaced with DMEM complete medium and cultured at 37℃ for 4 hours before detection. A magnetic field (if applicable) was applied continuously only during the -10℃ low-temperature treatment phase (frequency 10Hz, magnetic induction intensity 100 μT). The specific concentrations and timing of inhibitor use are as follows: 1) Control group: Routine culture at 37℃, without low temperature treatment, without adding inhibitors, and without applying a magnetic field.

[0069] 2) Low temperature group: -10℃ for 24 h, without adding inhibitors or applying magnetic field.

[0070] 3) Magnetic field group: treated at 10℃ for 24 h without adding inhibitors, and a magnetic field was applied.

[0071] 4) Low temperature + BSO group: BSO (250 nM) was added for pretreatment for 24 h during the conventional culture stage at 37℃, and then BSO (250 nM) was added again during the low temperature treatment stage at -10℃ without applying a magnetic field.

[0072] 5) Low temperature + RSL3 group: RSL3 (2 μM) was added during the -10℃ low temperature treatment stage, without applying a magnetic field.

[0073] 6) Magnetic field + BSO group: BSO (250 nM) was added for pretreatment for 24 h during the conventional culture stage at 37℃, followed by BSO (250 nM) added during the low temperature treatment stage at -10℃, and a magnetic field was applied.

[0074] 7) Magnetic field + RSL3 group: RSL3 (2 μM) was added during the -10℃ low temperature treatment stage, and a magnetic field was applied.

[0075] 8) Magnetic field + RSL3 + Fer-1 group: RSL3 (2 μM) and Fer-1 (2 μM) were added simultaneously during the -10℃ low temperature treatment stage, and a magnetic field was applied.

[0076] 1.3 GPX4 protein expression detection (Western blot) Cell seeding, treatment, and Western blotting procedures were the same as in Experiment 1. This experiment aimed to detect changes in GPX4 protein expression levels under different intervention groups, particularly the effect of BSO pretreatment on the upregulation of GPX4 by magnetic field.

[0077] like Figure 7 As shown, compared with the magnetic field group, the expression level of GPX4 protein decreased in the magnetic field + BSO group, indicating that BSO can weaken the effect of magnetic field on the upregulation / maintenance of GPX4 expression.

[0078] 1.4 Detection of the reduced glutathione / oxidized glutathione (GSH / GSSG) ratio Cell seeding, processing, and detection procedures were the same as in Experiment 1. This experiment aimed to investigate the effect of BSO intervention on the increase in the GSH / GSSG ratio by magnetic field.

[0079] The results are as follows Figure 8 As shown in (a), compared with the magnetic field group, the GSH / GSSG ratio decreased in the magnetic field + BSO group, indicating that BSO intervention can significantly weaken the effect of magnetic field on increasing the GSH / GSSG ratio.

[0080] 1.5 Cell viability assay (CCK-8) Cell seeding, treatment, and detection procedures were the same as in Experiment 1. After cell treatment, the culture medium was discarded, and 100 μL CCK-8 working solution was added. The cells were incubated at 37°C in the dark for 30 minutes. The absorbance (OD value) at 450 nm was measured using a microplate reader. This experiment aimed to evaluate the effects of BSO, RSL3, and Fer-1 intervention on the protective effect of magnetic field on cell viability.

[0081] The results are as follows Figure 8 As shown in (b), compared with the magnetic field group, the cell viability of the magnetic field + BSO group and the magnetic field + RSL3 group decreased, while the cell viability of the magnetic field + RSL3 + Fer-1 group increased compared with the magnetic field + RSL3 group. This indicates that magnetic field treatment can improve the cell viability decrease caused by low temperature, and BSO and RSL3 can inhibit the effect of magnetic field. The cell viability of the RSL3 + Fer-1 combined intervention group significantly recovered.

[0082] 1.6 LPO, MDA, and LDH Detection (1) Lipid peroxidation level (C11-BODIPY) 581 / 591R / G ratio): The detection method is the same as in Experiment 1. After cell treatment, add cells containing a final concentration of 2 μM C11-BODIPY. 581 / 591 The probe was incubated in complete culture medium at 37°C in the dark for 1 hour, washed with PBS, and detected using a multi-well microplate reader. This experiment focuses on the effect of RSL3 intervention on the reduction of lipid peroxidation by magnetic field.

[0083] (2) Malondialdehyde (MDA) content detection: The detection method is the same as in Experiment 1. After cell treatment, cells were scraped, collected, and sonicated. Then, the supernatant was collected by centrifugation, and the MDA content was detected according to the instructions of the MDA kit. This experiment focuses on the effect of RSL3 intervention on the reduction of MDA by magnetic field.

[0084] (3) Intracellular lactate dehydrogenase (LDH) activity assay: Cell pellets were collected, and the supernatant was taken after sonication. The LDH enzyme activity in the cell lysate was measured according to the LDH activity assay kit instructions. This indicator is used to characterize the maintenance of the activity of key enzymes in intracellular energy metabolism, rather than LDH released from cells into the culture medium (the latter is usually used to detect cell membrane integrity).

[0085] The results are as follows Figure 9 As shown in (a) to (c), comparing the low-temperature group and the magnetic field group, it can be seen that magnetic field treatment can significantly improve the increase of MDA and the decrease of LDH and LPO caused by low temperature. Comparing the magnetic field group with the magnetic field + BSO group and the magnetic field + RSL3 group, it can be seen that BSO and RSL3 interventions significantly weakened the effect of magnetic field. The cell state significantly improved after the combined intervention of RSL3 and Fer-1.

[0086] 1.7 Statistical Methods All experimental data are expressed as mean ± standard deviation (Mean ± SD). Data analysis was performed using GraphPad Prism 7.0 statistical software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0087] 2. Results This experiment systematically verified the relationship between magnetic field effects and ferroptosis-related processes by introducing different types of intervention conditions.

[0088] (1) Verification of upstream correlation: After adding glutathione synthesis inhibitor (BSO) during low temperature treatment, the intracellular GSH level decreased. Under this condition, the trend of increased GPX4 expression or activity induced by magnetic field treatment was weakened, and its effect on improving cell state was reduced, indicating that the magnetic field effect is closely related to intracellular glutathione-related metabolic processes.

[0089] (2) Validation of key nodes: After adding the GPX4 inhibitor (RSL3) to inhibit its activity, the effect of magnetic field treatment on lipid peroxidation level and cell viability was significantly weakened, indicating that GPX4-related processes play an important role in magnetic field regulation.

[0090] (3) Pathway association verification: When GPX4 activity was inhibited, the cell damage state was improved after the addition of the ferroptosis inhibitor (Fer-1). The trend of change was consistent with the results observed under magnetic field treatment, further indicating that the magnetic field effect is associated with the ferroptosis regulation pathway.

[0091] In summary, under different intervention conditions, multiple ferroptosis-related indicators showed a consistent trend, indicating that the magnetic field treatment method described in this invention can regulate ferroptosis-related processes. Its effects may involve antioxidant regulatory pathways represented by GSH / GPX4 and lipid peroxidation processes, but are not limited to the mechanisms mentioned above.

[0092] Experiment 3: Verification of the pathway overlap between magnetic field and the ferroptosis inhibitor Fer-1 1. Materials and Methods 1.1 Experimental Cells This experiment used the rat small intestinal crypt epithelial cell line IEC-6 (purchased from icell). Cells were cultured in DMEM complete medium containing: DMEM basal medium (containing 1.5 g / L NaHCO3), 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 0.01 mg / mL bovine insulin. Culture conditions were 37°C, 5% CO2, and saturated humidity (70%-80%).

[0093] 1.2 Drugs and Treatment Main reagent: Fer-1 (Ferrostatin-1), a classic inhibitor of ferroptosis.

[0094] IEC-6 cells were treated in five groups as described below. The cryopreservation conditions were -10°C for 4 hours or 24 hours. After treatment, all samples were uniformly replaced with DMEM complete medium and incubated at 37°C for 4 hours before analysis. A magnetic field (if applicable) was continuously applied only during the -10°C cryopreservation phase (frequency 10 Hz, magnetic induction intensity 100 μT). Fer-1 was used at a concentration of 2 μM and added only during the cryopreservation phase. The basal preservation solution was UW (University of Wisconsin) solution, supplemented with 0.2 mol / L 1,4-cyclohexanediol (1,4-CHD) and 5% polyethylene glycol (PEG 8000).

[0095] (1) Control group: conventional culture at 37℃, without Fer-1 and without magnetic field.

[0096] (2) Low temperature group: -10℃ treatment, no Fer-1 added, no magnetic field applied.

[0097] (3) Magnetic field group: -10℃ treatment, without adding Fer-1, and applying magnetic field.

[0098] (4) Low temperature + Fer-1 group: -10℃ treatment, Fer-1 (2 μM) added, no magnetic field applied.

[0099] (5) Magnetic field + Fer-1 group: -10℃ treatment, Fer-1 (2 μM) was added and a magnetic field was applied.

[0100] 1.3 Detection of cell viability and energy status (1) Cell viability (CCK-8 assay): Cells were seeded in 96-well plates and treated in groups (4 h and 24 h). The detection procedure was the same as in Experiment 1. After cell treatment, 100 μL of CCK-8 working solution was discarded, and the cells were incubated at 37°C in the dark for 30 minutes. The absorbance (OD value) at 450 nm was measured using an ELISA reader.

[0101] (2) Detection of intracellular ATP content: Cells were seeded in 96-well plates and treated in groups (4 h and 24 h). The chemiluminescence signal of each well was detected according to the instructions of the Promega ATP assay kit. The luminescence value reflects the intracellular ATP content and characterizes the cell energy state.

[0102] like Figure 10 As shown in (a) to (b), compared with the magnetic field group, there was no significant change in cell viability and ATP content in the magnetic field + Fer-1 group, and the magnetic field + Fer-1 group had similar effects to the magnetic field group.

[0103] 1.4 Detection of intracellular reactive oxygen species (ROS) Cells were seeded in 96-well plates and treated in groups (4 h and 24 h). Complete culture medium containing a final concentration of 10 μM DCFH-DA fluorescent probe was added, and the cells were incubated at 37°C in the dark for 1 h. Cells were then washed three times with PBS to remove unbound probe. Fluorescence intensity (excitation wavelength 488 nm, emission wavelength 525 nm) was measured using a multi-functional microplate reader. Fluorescence intensity values ​​were directly proportional to intracellular ROS levels.

[0104] like Figure 10 As shown in (c), the ROS content of the magnetic field + Fer-1 group did not change significantly compared to the magnetic field group, and the magnetic field + Fer-1 group had similar effects to the magnetic field group.

[0105] 1.5 Detection of lipid peroxidation level (C11-BODIPY) 581 / 591 LPO (R / G ratio) Cells were seeded in 96-well plates and treated in groups (4 h or 24 h). The detection method was the same as in Experiment 1. After cell treatment, cells containing a final concentration of 2 μM C11-BODIPY were added. 581 / 591 The probe was incubated in complete culture medium at 37°C in the dark for 1 hour, washed with PBS, and then detected using a multi-functional microplate reader.

[0106] like Figure 10 As shown in (d), the results of the magnetic field + Fer-1 group did not change significantly compared to the magnetic field group, and the results of the magnetic field + Fer-1 group were similar to those of the magnetic field group.

[0107] 1.6 Detection of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in cells Cells were seeded in six-well plates and treated in groups (4 h and 24 h). Cell lysate supernatant was collected and detected using the same method as in Experiment 1. After cell treatment, cells were scraped, collected, and sonicated. The supernatant was then centrifuged and the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected according to the instructions of the corresponding kits.

[0108] like Figure 10 As shown in (e) to (f), compared with the magnetic field group, the MDA and SOD contents of the magnetic field + Fer-1 group did not change significantly, and the magnetic field + Fer-1 group had similar effects to the magnetic field group.

[0109] 1.7 Detection of ferrous ion content in cells Cells were seeded in six-well plates, treated in groups (4 h), washed with PBS, and then incubated with a solution containing a final concentration of 2 μM RhoNox-6 probe at 37°C in the dark for 30 min. The probe solution was discarded, and after washing with PBS, the cells were immediately observed and photographed using a fluorescence microscope. Fluorescence intensity was correlated with intracellular Fe... 2+ The level is directly proportional.

[0110] like Figure 11 As shown, the magnetic field did not exhibit a further reduction in Fe in the presence of the Fer-1 inhibitor. 2+ The effects of the magnetic field + Fer-1 group and the magnetic field group are similar.

[0111] 1.8 Statistical Methods All experimental data are expressed as mean ± standard deviation (Mean ± SD). Data analysis was performed using GraphPad Prism 7.0 statistical software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0112] 2. Results Compared to the low-temperature group, the application of a magnetic field during the low-temperature treatment improved cell damage-related phenotypes, manifested as increased cell viability and energy status, decreased intracellular ROS levels, and decreased lipid peroxidation levels (increased R / G ratio in C11-BODIPY assay). Simultaneously, MDA content and Fe... 2+ The levels decreased. In the low-temperature + Fer-1 group following the addition of the ferroptosis inhibitor Fer-1, compared to the low-temperature group, cell damage-related indicators also showed an improving trend, manifested as decreased lipid peroxidation levels, and reduced ROS and Fe levels. 2+ The decrease in levels suggests that Fer-1 has an inhibitory effect on low-temperature-induced ferroptosis-related damage. Further comparison of the effects of magnetic field treatment, Fer-1 treatment, and a combination of both showed that Fer-1 significantly reduced ferroptosis-related parameters (including R / G ratio, ROS, Fe). 2+ In MDA and other studies, the combined magnetic field and Fer-1 treatment (magnetic field + Fer-1 group) did not show a significantly better enhancement trend than magnetic field alone (magnetic field group) or Fer-1 alone (low temperature + Fer-1 group). These results indicate that magnetic field treatment and Fer-1 treatment have similar trends in improving ferroptosis-related phenotypes, and their combined effect did not show a significant additive effect, suggesting that they may involve the same or partially overlapping pathways in regulating ferroptosis-related processes.

[0113] Therefore, it can be considered that the magnetic field treatment method described in this invention is related to the classical ferroptosis inhibition pathway, and its effect may involve the regulation of iron-dependent lipid peroxidation, but is not limited to the above mechanism.

[0114] Experiment 4: Magnetic Field's Effect on Hypothermia-Induced Small Intestinal Tissue Damage in Rats and its Reversal of Ferraphobia Inhibitor Effect 1. Materials and Methods 1.1 Obtaining experimental animals and small intestine This experiment used 5-7 week old SD rats. Rats were fasted for 8 hours prior to the experiment, but had free access to water. Rats were anesthetized by intraperitoneal injection of 10% sodium pentobarbital (0.6 mL / 200 g body weight). After anesthesia, the abdomen was opened through a midline incision to fully expose the abdominal organs. The proximal (towards the stomach) and distal small intestine were ligated sequentially, along with major vascular branches such as the renal artery and vein, lumbar artery and vein, portal vein, abdominal aorta, and splenic artery. Subsequently, histidine-tryptophan-ketoglutarate (HTK) solution (approximately 20 mL) was slowly perfused through the abdominal aorta, while the portal vein was incised as an outflow tract to thoroughly flush the mesenteric vascular bed until the liver and mesentery turned white. The jejunum was completely removed and immediately placed in pre-cooled phosphate-buffered saline (PBS). The intestinal lumen was gently flushed 2-3 times with pre-cooled PBS to remove contents, followed by flushing with PBS and UW solution sequentially until the intestinal lumen was clean. The prepared intestinal segments were placed in a modified cryopreservation solution (UW + 0.2 mol / L 1,4-cyclohexanediol + 5% PEG 8000) and kept on ice. To maintain the stability of the intestinal lumen structure and prevent mucosal collapse, 1% (w / v) of preheated liquid gelatin was slowly injected into the intestinal lumen until liquid flowed out from the other end of the intestinal segment. The two ends of the intestinal segment were then tied and sealed, and the segment was placed at 0-4°C for about 20 minutes to allow the gelatin to solidify.

[0115] 1.2 Drugs and Treatment Main reagent: Fer-1 (Ferrostatin-1), a specific inhibitor of ferroptosis.

[0116] Using isolated small intestinal tissue as the research subject, the following experimental groups were set up. The cryogenic treatment condition was -10℃, with treatment durations of 1 day (D1) or 3 days (D3). After treatment, all tissue samples were first placed on ice at 4℃ for 20 minutes to rewarm, then transferred to a 37℃ water bath to melt and remove the gelatin within the lumen, followed by the detection of various indicators. A magnetic field (if applicable) was continuously applied only during the -10℃ cryogenic treatment phase (frequency 10 Hz, magnetic induction intensity 100 μT). The concentration of Fer-1 used was 2 μM, added only to the preservation solution during the cryogenic treatment phase. The preservation solution was UW + 0.2 mol / L 1,4-CHD + 5% PEG 8000.

[0117] (1) Control group: Freshly obtained small intestine tissue without any low-temperature treatment.

[0118] (2) Low temperature group: -10℃ for 1 or 3 days, without adding Fer-1 to the preservation solution and without applying a magnetic field.

[0119] (3) Magnetic field group: -10℃ for 1 or 3 days, without adding Fer-1 to the preservation solution, and apply magnetic field.

[0120] (4) Low temperature + Fer-1 group: -10℃ for 1 or 3 days, Fer-1 (2 μM) was added to the preservation solution, and no magnetic field was applied.

[0121] (5) Magnetic field + Fer-1 group: -10℃ for 1 or 3 days, Fer-1 (2 μM) was added to the preservation solution and a magnetic field was applied.

[0122] 1.3 Organizational Structure Evaluation After treatment, small intestinal tissues from each group were collected, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The intestinal tissue structure was observed and evaluated under a light microscope, focusing on the morphology of intestinal villi, the integrity of intestinal epithelial cells, the submucosal structure, and the layering of the intestinal wall. The Chiu scoring system was used to quantitatively assess the degree of intestinal mucosal damage.

[0123] like Figure 12 HE staining results showed that, without magnetic field treatment, the low-temperature group exhibited significant damage to the small intestinal tissue structure, manifested as villus breakage, widening of the subepithelial space at the villus apex, intestinal wall edema, and villus shedding, accompanied by a significant reduction in intestinal lumen area. In contrast, after magnetic field intervention, the small intestinal tissue structure damage in the magnetic field group was significantly reduced, the degree of villus breakage decreased, the intestinal lumen area relatively increased, intestinal wall edema significantly improved, and the overall tissue structure was more intact. The low-temperature + Fer-1 group also showed significant changes in the integrity of the small intestinal tissue structure. Compared with the magnetic field group, the improvement effect of the magnetic field + Fer-1 group was not significant. This suggests that at the tissue level, magnetic fields improve tissue structure, and at the small intestinal tissue morphology and structure level, the inhibitor Fer-1 has a similar effect to magnetic field treatment.

[0124] 1.4 Detection of tissue oxidative stress and lipid peroxidation indicators Take appropriate amounts of small intestinal tissue from each group, add pre-chilled PBS or a specific lysis buffer, and homogenize on ice. Centrifuge the homogenate and collect the supernatant for the following assays: Protein quantification: The protein concentration of the supernatant of each sample was determined using the BCA protein quantification kit for standardized analysis of subsequent indicators.

[0125] (1) Reactive oxygen species (ROS) level: The level of ROS in small intestinal tissue was detected by fluorescent probe method. Approximately 50 mg of small intestinal tissue that had been stored for 1 day and thawed for 3 days was taken and added to pre-cooled phosphate buffer (PBS, 0.01 M, pH 7.4) at a ratio of 1:10 (w / v). The tissue was homogenized thoroughly in an ice-water bath using a tissue homogenizer. The homogenate was transferred to a centrifuge tube and centrifuged at 12000 × g for 10 min at 4°C. The supernatant was collected for later use. The ROS detection kit (Beyotime) was used for detection. The specific steps were as follows: 100 μL of tissue supernatant was added to a 96-well black microplate, and 100 μL of DCFH-DA working solution (final concentration 10 μM) was added to each well. The plate was gently vortexed to mix. Blank control wells (PBS + DCFH-DA) and positive control wells were also set up. The microplate was incubated in a cell culture incubator at 37°C in the dark for 30 min. Immediately after incubation, the fluorescence intensity (RFU) of each well was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0126] (2) Malondialdehyde (MDA) content: The MDA content in small intestinal tissue was detected using the thiobarbituric acid method. Approximately 50 mg of small intestinal tissue, after being stored for 1 day and thirteen days and then rewarmed, was added to pre-cooled phosphate-buffered saline (PBS, 0.01 M, pH 7.4) at a ratio of 1:10 (w / v) and homogenized thoroughly using a tissue homogenizer in an ice-water bath. The homogenate was transferred to centrifuge tubes and centrifuged at 12000 × g for 10 min at 4°C. The supernatant was collected for later use. The MDA detection kit (Beyotime) was used for detection. The specific steps were as follows: 100 μL of tissue supernatant was added to a centrifuge tube, followed by 200 μL of MDA detection working solution (containing thiobarbituric acid), and vortexed to mix. Standard wells and blank control wells were also prepared. The centrifuge tubes were heated in a 95°C water bath for 30 min, then cooled to room temperature in an ice bath. The reaction solution was transferred to a 96-well plate, and the absorbance of each well was measured at 532 nm using a microplate reader.

[0127] (3) Superoxide dismutase (SOD) activity: OD activity was measured using the WST-8 method with a total superoxide dismutase activity assay kit (Beyotime). An appropriate amount of tissue supernatant was added to a 96-well plate, along with the corresponding reaction reagents. After incubation at 37 °C for 30 min, the absorbance was measured at 450 nm using a microplate reader. The inhibition percentage was calculated using the formula: Inhibition percentage (unit) = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%.

[0128] like Figure 13Figures (a) to (c) show that the addition of a magnetic field can inhibit the accumulation of ROS and MDA, and increase SOD activity. Compared with the low-temperature group, the addition of Fer-1 (low-temperature + Fer-1 group) showed an improvement trend in all the above indicators; compared with the magnetic field group, the improvement effect of adding Fer-1 (magnetic field + Fer-1 group) was not significant. This suggests that at the tissue level, magnetic fields can inhibit oxidative stress, and in this respect, the inhibitor Fer-1 has a similar effect to magnetic field treatment.

[0129] 1.5 Detection of glutathione redox status in tissues Glutathione levels in small intestinal tissue were detected using the DTNB cyclic colorimetric method. The GSH / GSSG assay kit (Beyotime) was used according to the manufacturer's instructions. Small intestinal tissue, flash-frozen in liquid nitrogen, was ground into powder. An appropriate amount of protein removal reagent M solution was added to every 10 mg of tissue, and the mixture was thoroughly homogenized and incubated at 4 °C for 10 min. Subsequently, it was centrifuged at 10000 × g for 10 min, and the supernatant was collected for detection. For total GSH determination, GSH detection working solution was added to the sample, and after incubation at room temperature in the dark, NADPH solution was added. The absorbance change was measured at 412 nm, and the total GSH content was calculated based on the standard curve. For GSSG determination, a GSH scavenging reagent was first added to remove reduced GSH from the sample, and then the GSSG content was measured using the same method. Finally, the GSH / GSSG ratio was calculated based on the total GSH and GSSG contents.

[0130] like Figure 13 (d) shows that the addition of a magnetic field decreased the level of glutathione oxidation in the small intestine. Compared with the low-temperature group, the addition of Fer-1 (low-temperature + Fer-1 group) showed an improving trend in the accumulation of ROS. Compared with the magnetic field group, the improvement effect of adding Fer-1 (magnetic field + Fer-1 group) was not significant. This suggests that at the tissue level, magnetic fields can inhibit glutathione oxidation, and the inhibitor Fer-1 has a similar effect to magnetic field treatment in this respect.

[0131] 1.6 Statistical Methods All experimental data are expressed as mean ± standard deviation (Mean ± SD). Data analysis was performed using GraphPad Prism 7.0 statistical software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0132] 2. Results This experiment expanded the research system from the cellular level to the in vitro tissue level to evaluate the changes in ferroptosis-related processes and the effects of intervention under low-temperature treatment. The main results are as follows: 2.1 Changes in ferroptosis-related changes in small intestinal tissue under hypothermia treatment Compared with the fresh control, after treatment at -10℃ for 1 or 3 days, the small intestinal tissue showed morphological and structural damage, accompanied by changes in multiple ferroptosis-related indicators, including increased lipid peroxidation levels (decreased R / G ratio and increased MDA content in C11-BODIPY assay), decreased antioxidant capacity (decreased GSH / GSSG ratio and decreased SOD activity), and decreased GPX4 protein expression levels.

[0133] The synergistic changes in the above-mentioned indicators suggest that biological processes related to ferroptosis exist in the small intestinal tissue under low-temperature treatment conditions.

[0134] 2.2 Verification of the role of ferroptosis intervention at the tissue level To evaluate the impact of interventions on ferroptosis-related processes on tissue damage, chemical and physical methods were employed, including: (1) Add the ferroptosis inhibitor Fer-1 to the cryopreservation solution; (2) Apply a magnetic field during the low-temperature treatment process.

[0135] The results showed that under both treatment conditions, the degree of damage to the small intestine tissue was reduced, manifested as improved tissue structure integrity, reduced lipid peroxidation level (increased R / G ratio and decreased MDA content), enhanced antioxidant capacity (increased SOD activity and GSH / GSSG ratio), and increased or maintained GPX4 expression level.

[0136] 2.3 Pathway correlation analysis of different intervention methods To further analyze the relationship between different intervention methods, a comparison was made between magnetic field treatment, Fer-1 treatment, and a combination of both. The results showed that, across the various indicators tested, the magnetic field combined with Fer-1 treatment did not exhibit a significantly better enhancement trend than either magnetic field treatment alone or Fer-1 treatment alone.

[0137] The above results indicate that magnetic field treatment and Fer-1 treatment have similar trends in improving ferroptosis-related phenotypes, and their combined effect does not show a significant additive effect, suggesting that the two may involve the same or partially overlapping action pathways in regulating ferroptosis-related phenotypes.

[0138] In summary, at the tissue level, the magnetic field treatment method described in this invention can improve low-temperature induced tissue damage and shows a consistent regulatory trend in multiple ferroptosis-related indicators, indicating that its effect is related to ferroptosis-related processes, but is not limited to the above-mentioned mechanisms.

[0139] Experiment 5: Biofunctional Evaluation of Magnetic Field Protection Scheme – Comparison with Traditional Static Cryogenic Storage Method 1. Materials and Methods 1.1 Obtaining experimental animals and small intestine This experiment used 5-7 week old SD rats. Rats were fasted for 8 hours prior to the experiment, but had free access to water. Rats were anesthetized by intraperitoneal injection of 10% sodium pentobarbital (0.6 mL / 200 g body weight). After anesthesia, the abdomen was opened through a midline incision to fully expose the abdominal organs. The proximal (towards the stomach) and distal small intestine were ligated sequentially, along with major vascular branches such as the renal artery and vein, lumbar artery and vein, portal vein, abdominal aorta, and splenic artery. Subsequently, histidine-tryptophan-ketoglutarate (HTK) solution (approximately 20 mL) was slowly perfused through the abdominal aorta, while the portal vein was incised as an outflow tract to thoroughly flush the mesenteric vascular bed until the liver and mesentery turned white. The jejunum was completely removed and immediately placed in pre-cooled phosphate-buffered saline (PBS). The intestinal lumen was gently flushed 2-3 times with pre-cooled PBS to remove contents, followed by flushing with PBS and UW solution sequentially until the intestinal lumen was clean. The prepared intestinal segments were placed in a modified cryopreservation solution (UW + 0.2 mol / L 1,4-cyclohexanediol + 5% PEG 8000) and kept on ice. To maintain the stability of the intestinal lumen structure and prevent mucosal collapse, 1% (w / v) of preheated liquid gelatin was slowly injected into the intestinal lumen until liquid flowed out from the other end of the intestinal segment. The two ends of the intestinal segment were then tied and sealed, and the segment was placed at 0-4°C for about 20 minutes to allow the gelatin to solidify.

[0140] 1.2 Drugs and Treatment Main reagent: Fer-1 (Ferrostatin-1), a specific inhibitor of ferroptosis.

[0141] Using isolated small intestinal tissue as the research subject, the following experimental groups were set up. The treatment duration was 1 day or 3 days. After treatment, all samples were first warmed at 4℃ for 20 minutes, then transferred to a 37℃ water bath to remove gelatin, followed by various functional tests. The magnetic field was continuously applied only during the low-temperature treatment stage (frequency 10 Hz, magnetic induction intensity 100 μT). The concentration of Fer-1 used was 2 μM, which was added to the preservation solution only during the low-temperature treatment stage.

[0142] (1) Static cold preservation group: traditional control. Small intestinal tissue was stored at 4°C for 1 or 3 days. The preservation solution was a modified preservation solution, no magnetic field was applied, and no gelatin was injected into the intestinal lumen.

[0143] (2) Static cold preservation group + Fer-1 group: Based on the preservation at 4℃, Fer-1 (2 μM) was added to the preservation solution. No magnetic field was applied and no gelatin was injected into the intestinal lumen.

[0144] (3) Deep cryopreservation group: Small intestinal tissue was stored at -10°C for 1 or 3 days, with an alternating magnetic field (10 Hz, 100 μT) continuously applied during storage. Fer-1 was not added to the storage solution.

[0145] (4) Deep supercooling preservation + Fer-1 group: Deep supercooling + magnetic field + Fer-1 group, based on storage at -10℃ and application of magnetic field, Fer-1 (2 μM) was added to the storage solution.

[0146] 1.3 Assessment of intestinal absorption function (glucose absorption test) After treatment, an isolated intestinal segment perfusion system was constructed. Each group of intestinal segments was connected to the perfusion device and continuously perfused with Hanks' Balanced Salt Solution (HBSS, containing a certain concentration of glucose) at a constant flow rate and pressure. The effluent was collected after 2 hours of perfusion, and its glucose concentration was measured. The difference in glucose concentration between the perfused and effluent was calculated to assess the intestinal mucosa's ability to absorb glucose. Higher glucose absorption indicates better preservation of absorption function.

[0147] like Figure 14 Figure (a) shows that, compared with the static cryopreservation group, the deep cryopreservation group with added magnetic field had higher glucose uptake. Furthermore, the static cryopreservation group with added Fer-1 also showed some improvement compared with the static cryopreservation group without Fer-1, although this improvement was not significant, suggesting that Fer-1 also significantly improves small intestinal permeability at 4°C. However, adding Fer-1 to the deep cryopreservation group with added magnetic field (deep cryopreservation + Fer-1 group) did not show further improvement.

[0148] 1.4 Assessment of intestinal digestive function (disaccharidase activity detection) Small intestinal tissues from each group were collected, homogenized, and the supernatant was collected by centrifugation. Protein quantification was performed using the BCA method. Maltase and lactase activities in the tissue homogenate supernatant were measured using commercially available maltase and lactase activity assay kits, following the instructions. Higher enzyme activity indicates better preservation of digestive function.

[0149] like Figure 14 Figures (b) to (c) show that, compared with static cryopreservation, the deep cryopreservation group with added magnetic field had higher maltase and lactase activities, and the static cryopreservation group + Fer-1 group also showed significant improvement in maltase and lactase activities, suggesting that Fer-1 can also improve the digestive function of the small intestine at 4°C. However, there was no further improvement when Fer-1 was added to the deep cryopreservation group with added magnetic field (deep cryopreservation + Fer-1 group).

[0150] 1.5 Assessment of intestinal barrier permeability Small intestinal tissue was collected from each group, and the intestinal contents were washed three times with sterile, pre-cooled PBS. 50 μL of blue dextran 2000 (20 mg / mL) was pipetted into the small intestine of each group, and both ends were sealed with sterile cotton thread. The plate was then placed in a 6-well cell culture plate, and colorless DMEM medium (Solepro, China) containing 100 U / mL penicillin and 100 g / mL streptomycin was added to a final volume of 4 mL. The plate was incubated at 37°C with 5% (v / v) CO2 for 2 hours. Afterward, 100 μL of the culture medium was aspirated from the cell culture plate, and the absorbance at 610 nm was measured to assess the integrity of small intestinal permeability.

[0151] like Figure 14 As shown in Figure (d), the deep cryopreservation group with added magnetic field had lower absorbance compared to the static cryopreservation group, indicating better intestinal barrier permeability. Furthermore, the static cryopreservation group + Fer-1 showed significant improvement compared to the static cryopreservation group, suggesting that Fer-1 also significantly improves small intestinal permeability at 4°C. However, adding Fer-1 to the deep cryopreservation group with added magnetic field (deep cryopreservation + Fer-1 group) did not provide further improvement.

[0152] 1.6 Statistical Methods All experimental data are expressed as mean ± standard deviation (Mean ± SD). Data analysis was performed using GraphPad Prism 7.0 statistical software. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0153] 2. Results This experiment aimed to evaluate the role of the "inhibition of ferroptosis" strategy in cryopreservation by comparing the function of small intestinal tissue under different preservation conditions, and to verify the effectiveness of the proposed solution (-10℃ deep supercooling combined with a magnetic field) in achieving this strategy. The main results are as follows: (1) Inhibition of ferroptosis is associated with tissue protection Under conventional static cryopreservation conditions at 4°C, the addition of the ferroptosis-specific inhibitor Fer-1 improved the absorption, digestion, and barrier function of small intestinal tissue. This suggests that ferroptosis may be involved in the process of tissue dysfunction under cryopreservation conditions, and targeted intervention in ferroptosis can alleviate damage and has a potential role in tissue protection.

[0154] (2) The present invention provides protection against ferroptosis. Small intestinal tissue treated with a "-10℃ deep supercooling + magnetic field" approach showed improved performance across multiple functional indicators, demonstrating a higher protective effect compared to static cryopreservation at 4℃. Further experiments showed that adding Fer-1 to this approach did not significantly increase tissue function, suggesting that the proposed method can exert a protective effect similar to or superior to chemical inhibitors by physically intervening in ferroptosis-related processes.

[0155] (3) Physical intervention provides the possibility of preservation without chemical residues. The above results indicate that intervening in ferroptosis-related processes through physical means (magnetic fields with specific parameters) holds promise for improving cryopreservation without relying on exogenous chemical inhibitors. This provides feasible experimental evidence and technical reference for developing safe, efficient, and chemical-free novel organ preservation technologies.

[0156] In summary, the results of this experiment support the potential application of the "inhibition of ferroptosis" strategy in the preservation of the small intestine at low temperatures, and verify that the scheme of this invention, as a feasible physical intervention to achieve this strategy, can improve tissue function and alleviate low-temperature-related damage to some extent.

[0157] The above embodiments are used to illustrate the technical solution and effects of the present invention, and are not intended to limit the scope of protection of the present invention. The method described in this invention is applicable not only to small intestinal epithelial cells and small intestinal tissue, but also to epithelial cell types from other organs or tissues; the cell source can be any mammalian cell, including but not limited to human, mouse, and porcine cells.

[0158] The alternating magnetic field parameters described in this invention are exemplary embodiments. Those skilled in the art can make equivalent adjustments to the magnetic field type and parameters without departing from the principle of this invention. For example, alternating magnetic fields with different frequencies and magnetic induction intensity ranges can be used, and / or constant magnetic fields, pulsed magnetic fields, oscillating magnetic fields, etc. As long as improvements can be achieved on ferroptosis-related indicators (such as the GSH / GPX4 system, lipid peroxidation, etc.) under low-temperature treatment conditions, they should all fall within the protection scope of this invention.

[0159] Furthermore, the cell types, treatment durations, drug concentrations, and detection conditions used in the embodiments can be routinely adjusted and equivalently replaced according to actual needs, and should not be regarded as limitations on the scope of protection of this invention.

Claims

1. A physical method for regulating ferroptosis in isolated cells or tissues, characterized in that, include: Applying a magnetic field to isolated cells or tissues during the processing of isolated cells or tissues can reduce the level of ferroptosis in isolated cells or tissues. The treatment process refers to a process that increases the level of ferroptosis in isolated cells or tissues.

2. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 1, characterized in that, The magnetic field strength is 10~500 μT.

3. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 2, characterized in that, The magnetic field is an alternating magnetic field, a constant magnetic field, a pulsed magnetic field, or an oscillating magnetic field.

4. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 1, characterized in that, The magnetic field is an alternating magnetic field with a frequency of 5~50 Hz.

5. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 1, characterized in that, The isolated cells or tissues are of mammalian origin.

6. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 5, characterized in that, The isolated cells are isolated small intestinal epithelial cells, and the isolated tissue is isolated small intestinal tissue or a segment of small intestine.

7. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 1, characterized in that, The process involves low-temperature preservation.

8. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 7, characterized in that, The cryopreservation temperature is -8 to -12℃.

9. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 7, characterized in that, The cryopreservation time for isolated cells is 1 to 24 hours, and the cryopreservation time for isolated tissues is 24 to 72 hours.

10. The physical method for regulating ferroptosis in isolated cells or tissues according to claim 1, characterized in that, The reduction in ferroptosis levels in isolated cells or tissues is characterized by at least one of the following indicators: (a) Intracellular Fe 2+ The content decreased; (b) Reduced levels of reactive oxygen species; (c) Decreased lipid peroxidation levels; (d) The decrease in mitochondrial membrane potential was inhibited; (e) Elevated glutathione levels or an elevated GSH / GSSG ratio; (f) Increased expression or activity of GPX4.

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