Method for regulating oxidative stress of ischemic injury by GDF-15 based on AMPK signal channel

Abstract

The invention relates to the technical field of biological medicines, in particular to a method for regulating oxidative stress of ischemic injury by GDF-15 based on an AMPK (adenosine monophosphate kinase) signal pathway, which comprises the following steps: culturing AC16 myocardial cells, treating primary myocardial cells, and carrying out hypoxia experiment and function and molecule detection; carrying out quantitative analysis on fluorescence signals according to the obtained fluorescence image, and determining expression modes of GDF-15 and HI F-1alpha in the MI heart tissue; detecting the protein level of the GDF-15 in the MI heart tissue, verifying the expression mode of the GDF-15, or adjusting dyeing parameters; carrying out statistical analysis on the detection parameters, and determining a specific parameter group with significant differences among the parameters; determining a GDF-15 regulation pathway, and judging a production mode of ROS in AC16 myocardial cells and a phosphorylation state of an AMPK signal pathway; and carrying out exogenous drug delivery immunofluorescence analysis, and judging the expression relationship between the GDF-15 and the HI F-1alpha. According to the invention, a new insight is provided for the cardiac protection effect of the GDF-15 and the potential of the GDF-15 as a myocardial infarction treatment target by illuminating the molecular mechanism of the GDF-15.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway. Background Art

[0002] Ischemic heart disease is the leading cause of death worldwide, with acute myocardial infarction (AMI) being the leading cause of death. With advances in medicine and improved access to healthcare, more and more AMI patients are now eligible for revascularization treatments such as PCI, thrombolysis, and coronary artery bypass grafting (CABG), which have significantly saved lives and improved outcomes. However, two major challenges plaguing clinical treatment remain unresolved: First, the short golden period for treatment after MI, with only a minority of patients able to complete revascularization within 2 hours. Second, ischemia-reperfusion injury (I / R) inevitably triggers cardiomyocyte death and widespread inflammation as blood supply is restored, leading to further myocardial damage, also known as reperfusion injury. Due to the complex regulatory mechanisms of myocardial infarction and reperfusion, no effective therapeutic intervention has yet been developed. Therefore, further elucidating the regulatory mechanisms of myocardial I / R and identifying key regulators of reperfusion injury for drug development are of great importance.

[0003] Chinese Patent Publication No.: CN115478105A discloses an application of SIRT4 in the treatment of liver ischemia, particularly in the use as a drug target for screening or preparing drugs for treating liver ischemia caused by liver ischemia-reperfusion injury. The drug contains a SIRT4 agonist, SIRT4 gene or SIRT4 protein, which regulates the expression of SIRT4 to alleviate liver ischemia-reperfusion injury. SIRT4 gene knockout mice, hepatocyte-specific SIRT4 high-expression mice and wild-type C57 mice are used as experimental subjects to study the role of SIRT4 gene in liver ischemia-reperfusion injury model. Therefore, the application of SIRT4 in the treatment of liver ischemia has the following problems:

[0004] Despite advances in reperfusion therapy to restore blood flow in hepatic ischemia, the complex pathophysiological mechanisms underlying the onset of myocardial infarction and subsequent cardiac remodeling remain poorly understood;

[0005] In the prior art, growth differentiation factor-15 (GDF-15), as a member of the transforming growth factor β superfamily, plays a role in a variety of physiological and pathological processes, including inflammation and cancer. However, its role in myocardial infarction remains unclear. Summary of the Invention

[0006] To this end, the present invention provides a method for regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway, so as to overcome the problem in the prior art that GDF-15, as a stress response cytokine, has been involved in cardiac pathological and physiological processes, but its exact role in myocardial infarction is still controversial.

[0007] To achieve the above objectives, the present invention provides a method for regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway, comprising:

[0008] Animals underwent ischemia-reperfusion surgery to create a myocardial ischemia-reperfusion model. MI heart tissues were obtained before ischemia and at different time points after reperfusion for culture of AC16 cardiomyocytes and treatment of primary cardiomyocytes.

[0009] AC16 cardiomyocytes and primary cardiomyocytes were subjected to hypoxia experiments and several functional and molecular assays, including obtaining fluorescent images of MI heart tissue;

[0010] The fluorescence signals of the fluorescence images were analyzed, and the expression patterns of GDF-15 and HIF-1α in MI heart tissues were determined based on the mean fluorescence intensity of MI;

[0011] According to the expression pattern of GDF-15, whether the protein level of GDF-15 is increased in MI heart tissues is tested, the expression pattern of GDF-15 is confirmed, or the staining parameters of western blot analysis of AC16 cardiomyocytes and primary cardiomyocytes are adjusted;

[0012] After confirming the expression pattern of GDF-15, statistical analysis of the test parameters obtained from several functional and molecular tests was performed to determine the mean differences between samples. The regulatory pathways of GDF-15 were determined based on the specific parameter groups with significant differences between the test parameters.

[0013] Determine a difference evaluation value based on the mean difference, and judge the ROS generation pattern in AC16 cardiomyocytes based on the fluorescence intensity difference between the mean fluorescence intensities of ROS under different treatment conditions;

[0014] The phosphorylation status of the AMPK signaling pathway in AC16 cardiomyocytes and primary cardiomyocytes was determined based on the criteria for phosphorylation characteristics, the rationality of the ROS generation pattern was verified, and the determined difference evaluation value was adjusted;

[0015] Exogenous GDF-15 administration and immunofluorescence analysis were performed on MI heart tissues, and the expression relationship between GDF-15 and HIF-1α was obtained based on the difference evaluation value analysis;

[0016] The animals include C57BL / 6 normal mice and GDF15 gene knockout mice.

[0017] Furthermore, the process of determining the expression pattern of GDF-15 and HIF-1α in MI cardiac tissues included:

[0018] The mean fluorescence intensity was obtained by measuring the sum of the fluorescence intensities of the immunofluorescence-stained region and dividing it by the area of the region, and the MI mean fluorescence intensities of GDF-15 and HIF-1α in the heart tissues of MI mice were compared with the control mean fluorescence intensities of GDF-15 and HIF-1α in the sham-operated control group;

[0019] According to the comparison results, it is determined whether GDF-15 and HIF-1α in MI heart tissue are in the first expression pattern or the second expression pattern.

[0020] Furthermore, the process of confirming the expression pattern of GDF-15 includes,

[0021] When GDF-15 expression was in the first expression pattern, the protein level of GDF-15 in the heart tissue of MI mice was examined, the average optical density of the bands in the band images was measured, and the average optical density of MI was compared with the average optical density of the control;

[0022] Based on the comparison results, determine whether the protein level of GDF-15 is increased, and calculate the absolute expression level of the target protein based on the standard curve.

[0023] Furthermore, if the average optical density of MI is greater than the average optical density of the control, it is determined that the protein level of GDF-15 is increased, which can confirm that GDF-15 is in the first expression mode. The absolute expression level of the target protein is calculated in combination with the standard curve;

[0024] If the MI mean optical density is less than or equal to the control mean optical density, it is judged that there is no increase in the protein level of GDF-15, and the staining parameters for western blot analysis of AC16 cardiomyocytes and primary cardiomyocytes are adjusted.

[0025] Furthermore, the process of determining the mean difference between samples includes,

[0026] Paired two-tailed t-test was performed on the detection parameters to determine the mean difference between the two samples.

[0027] If the absolute value of the calculated t-value is greater than the critical value, the null hypothesis is rejected and it is considered that the difference in the means of the two samples is beyond the normal range;

[0028] If the absolute value of the calculated t-value is less than or equal to the critical value, the null hypothesis is accepted and the mean difference between the two samples does not exceed the normal range.

[0029] Furthermore, the mitochondrial membrane potential in AC16 cardiomyocytes and primary cardiomyocytes was evaluated based on the detection parameters corresponding to the samples whose mean differences exceeded the normal range, and the changes in the membrane potential of AC16 cardiomyocytes were determined.

[0030] Furthermore, the process of determining the ROS production pattern in AC16 cardiomyocytes includes:

[0031] For AC16 cardiomyocytes after hypoxia experiment, the mean fluorescence intensity of ROS under different treatment conditions was measured by fluorescence imaging, and the fluorescence intensity difference between the mean fluorescence intensities was calculated;

[0032] The fluorescence intensity difference between the mean fluorescence intensity of AC16 cardiomyocytes after adding COCL2 and GDF-15 antibodies, or after adding COCL2 and GDF-15, was calculated;

[0033] The fluorescence intensity difference is compared with the first difference evaluation value, and it is determined whether the ROS in the AC16 cardiomyocytes is in the first generation mode or the second generation mode according to the comparison result.

[0034] Furthermore, the process of determining the phosphorylation status of the AMPK signaling pathway in AC16 cardiomyocytes and primary cardiomyocytes included:

[0035] When ROS was in the first generation mode, the average optical density of the bands in the band images of p-AMPK, AMPK, and β-actin in AC16 cardiomyocytes and primary cardiomyocytes under different treatment conditions was measured;

[0036] If the average optical density of the p-AMPK band under different treatment conditions meets the first and second judgment conditions, it is judged that the AMPK signaling pathway is in the first phosphorylation state, AMPK activation is positively correlated with GDF-15, and the ROS generation pattern is reasonable;

[0037] If the average optical density of the p-AMPK band under different treatment conditions does not meet the first judgment condition or the second judgment condition, it is judged that the AMPK signaling pathway is in the second phosphorylation state, the activation of AMPK is negatively correlated with GDF-15, the ROS generation pattern is unreasonable, and the first difference evaluation value is adjusted.

[0038] Furthermore, the first determination condition is that the average optical density of the p-AMPK band of AC16 cardiomyocytes after the addition of GDF-15 is greater than the average optical density of the p-AMPK band of AC16 cardiomyocytes after the addition of COCL2, and greater than the average optical density of the p-AMPK band of AC16 cardiomyocytes after the addition of normal saline;

[0039] The second determination condition is that the average optical density of the p-AMPK band of the primary cardiomyocytes after adding GDF-15 is greater than the average optical density of the p-AMPK band of the primary cardiomyocytes after adding COCL2, and the average optical density of the p-AMPK band of the primary cardiomyocytes after adding COCL2 is less than the average optical density of the p-AMPK band of the primary cardiomyocytes after adding normal saline;

[0040] Wherein, the primary cardiomyocytes are primary cardiomyocytes of GDF15 gene knockout mice.

[0041] Furthermore, the process of determining the expression relationship between GDF-15 and HIF-1α includes:

[0042] After exogenous GDF-15 administration, if the MI mean fluorescence intensity of HIF-1α in the MI cardiac tissue is less than or equal to the control mean fluorescence intensity, and the absolute value of the difference between the MI mean fluorescence intensity and the control mean fluorescence intensity is greater than the second difference evaluation value, then GDF-15 and HIF-1α are judged to be in a first expression relationship;

[0043] If the MI mean fluorescence intensity of HIF-1α in MI heart tissue was greater than the control mean fluorescence intensity, GDF-15 and HIF-1α were judged to be in the second expression relationship;

[0044] The functional and molecular assays include determination of intracellular ROS levels, determination of mitochondrial membrane potential, protein immunoblotting analysis, histological analysis, immunofluorescence analysis, and O2K analysis.

[0045] Compared with the existing technology, the beneficial effect of the present invention lies in that the pathophysiological process of myocardial infarction involves multiple complex interactions such as ischemia, reperfusion injury, oxidative stress, inflammation and subsequent fibrosis, which together lead to adverse cardiac remodeling and impaired cardiac function; this method uses multiple functional and molecular tests on AC16 cardiomyocytes and primary cardiomyocytes to clarify how GDF-15 affects cardiac remodeling, mitochondrial function and the molecular mechanism of oxidative stress regulation after ischemic injury, providing new insights into the cardioprotective effect of GDF-15 and its potential as a therapeutic target for myocardial infarction.

[0046] Furthermore, this method fluorescently stains GDF-15 stress response protein and HIF-1α to obtain fluorescent images, calculates the average fluorescence intensity by summing the fluorescence intensities of the immunofluorescence staining areas in the fluorescent images, compares the average fluorescence intensities of the fluorescence images of MI heart tissue and sham-operated heart tissue, and determines the expression patterns of GDF-15 and HIF-1α, that is, the changes in GDF-15 and HIF-1α, thereby quantitatively analyzing the fluorescent signals; immunofluorescence staining shows that the expression of GDF-15 and HIF-1α is significantly increased in the infarct site, indicating that GDF-15 plays a key role in mediating cardiac remodeling after myocardial infarction.

[0047] Furthermore, immunofluorescence is a semi-quantitative analysis, and the average fluorescence intensity can only semi-quantitatively characterize the expression of specific proteins. This method uses protein immunoblotting analysis to examine the protein level of GDF-15 in the heart tissue of MI mice. By measuring the average optical density of the bands in the band image and comparing the average optical density of the fluorescence images of MI heart tissue and sham-operated heart tissue, it is determined whether there is an increase in the protein level of GDF-15, thereby confirming the increase in GDF-15 at the protein level and confirming that GDF-15 expression is in the first expression mode, thereby improving the accuracy of indicating that GDF-15 plays a key role in mediating cardiac remodeling after myocardial infarction.

[0048] Furthermore, the data obtained after functional and molecular testing of AC16 cardiomyocytes and primary cardiomyocytes were expressed as mean ± standard error r (SEM). The statistical comparison between the two groups of data was performed using an unpaired two-tailed t-test to determine whether the mean difference between the two independent samples was significant. This method performs an unpaired two-tailed t-test on multiple data obtained from functional and molecular testing to determine which corresponding parameters have significant differences, thereby avoiding the misjudgment that is easy to cause by manual judgment, reducing comparison errors, and increasing the accuracy of data analysis.

[0049] Furthermore, to elucidate the molecular mechanisms of GDF-15-mediated cardiac regulation, RNA sequencing analysis was performed. GO and KEGG pathway enrichment analysis compared differentially expressed genes in wild-type and GDF15-knockout heart tissues, revealing distinct metabolic profiles between wild-type and GDF15-knockout mice. To further investigate the metabolic effects of GDF-15, mitochondrial membrane potential was assessed in AC16 cardiomyocytes and primary cardiomyocytes under different treatment conditions. Immunofluorescence analysis revealed significant changes in the membrane potential of AC16 cells in different treatment groups, and similar effects were observed in primary cardiomyocytes. High-resolution respirometry analysis indicated that GDF-15 affected key parameters of mitochondrial function in AC16 cells, including basal respiration, volumetric respiration, ATP production, and non-mitochondrial respiration, indicating that GDF-15 deficiency impairs mitochondrial function and energy metabolism under hypoxic stress. This method demonstrated that GDF-15 maintains mitochondrial function and cellular energy metabolism, thereby protecting cardiomyocytes from hypoxic damage. After GDF-15 supplementation under hypoxic conditions, the restoration of mitochondrial membrane potential, cellular oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) emphasized the direct regulatory effect of GDF-15 on mitochondrial function.

[0050] Furthermore, this method measured ROS by fluorescence imaging. Under different treatment conditions, the ROS production patterns in AC16 cardiomyocytes were significantly different. p-AMPK (phosphorylated AMPK) is the activated form of AMPK (AMP-activated protein kinase). This method examined the AMPK signaling pathway by detecting the phosphorylation status of the AMPK signaling pathway to determine how GDF-15 regulates ROS. It was found that GDF-15 regulated the phosphorylation status of AMPK in AC16 cells and primary cardiomyocytes, indicating that GDF-15 regulates ROS production through the AMPK signaling pathway. GDF-15 inhibits hypoxia-induced ROS generation through AMPK signaling, showing the effect of GDF-15 on cellular oxidative stress. The AMPK signaling pathway is known to regulate energy metabolism and antioxidant responses. This method not only elucidates the molecular mechanism by which GDF-15 protects the heart, but also highlights its potential as a therapeutic target for myocardial infarction.

[0051] Furthermore, to investigate the therapeutic potential of GDF-15 in myocardial infarction, recombinant GDF-15 was injected into mice with myocardial infarction; histological analysis showed that GDF-15 treatment significantly reduced cardiac inflammation and fibrosis, consistent with the histological findings. Echocardiographic assessment indicated that cardiac function was improved in mice with myocardial infarction treated with GDF-15, and immunofluorescence analysis showed that GDF-15 administration significantly reduced HIF-1α expression in the infarcted myocardium, suggesting that GDF-15 may partially exert its cardioprotective effects by regulating the hypoxia response pathway. At the same time, GDF-15 significantly reduced inflammatory cell infiltration and collagen deposition in the heart after myocardial infarction, thereby alleviating adverse cardiac remodeling, suggesting that GDF-15 may exert its protective effects at least partially by regulating the immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of a method for regulating oxidative stress in ischemic injury by GDF-15 based on the AMPK signaling pathway according to an embodiment of the present invention;

[0053] Figure 2 This is an example code diagram of using Python to perform LSD testing in an embodiment of the present invention;

[0054] Figure 3 Schematic diagram of GDF-15 and HIF-1α fluorescence staining and protein immunoblotting analysis of MI cardiomyocytes in an embodiment of the present invention;

[0055] Figure 4 Schematic diagram of ROS fluorescence staining and AMPK signaling pathway immunoblotting analysis in AC16 cardiomyocytes in an embodiment of the present invention;

[0056] Figure 5 This is a fluorescence staining image showing that GDF15 treatment leads to decreased HIF-1α expression in MI mice in an example of the present invention. DETAILED DESCRIPTION

[0057] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0058] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0059] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside", and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0060] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0061] See also Figure 1-Figure 5 As shown, Figure 1 Schematic diagram of a method for regulating oxidative stress in ischemic injury by GDF-15 based on the AMPK signaling pathway according to an embodiment of the present invention; Figure 2 This is an example code diagram of using Python to perform LSD testing in an embodiment of the present invention; Figure 3 Schematic diagram of GDF-15 and HIF-1α fluorescence staining and protein immunoblotting analysis of MI cardiomyocytes in an embodiment of the present invention; Figure 4 Schematic diagram of ROS fluorescence staining and AMPK signaling pathway immunoblotting analysis in AC16 cardiomyocytes in an embodiment of the present invention; Figure 5 This is a fluorescence staining image showing that GDF15 treatment leads to decreased HIF-1α expression in MI mice in an example of the present invention.

[0062] A method for regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway, comprising:

[0063] Step S1, performing ischemia-reperfusion surgery on animals to create a myocardial ischemia-reperfusion model, culturing AC16 cardiomyocytes and treating primary cardiomyocytes;

[0064] The process of culturing AC16 cardiomyocytes and processing primary cardiomyocytes includes:

[0065] Step S101, AC16 cardiomyocytes are cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a humidity-controlled environment containing 5% carbon dioxide;

[0066] Step S102, primary cardiomyocytes are isolated from 0-3 day old Sprague-Dawley mice, the isolated ventricles are placed in pre-chilled HBSS, and cut into 1-3 mm fragments;

[0067] Step S103, the tissue fragments were enzymatically hydrolyzed with a mixture of enzyme 1 and enzyme 2 (20:1) at 37°C in 5% carbon dioxide for 30 minutes;

[0068] Step S104, after washing with HBSS, the cells were pre-seeded into a 15 cm culture dish in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured at 37°C for 90 minutes to remove non-cardiac myocytes;

[0069] Step S105 , collecting non-adherent primary cardiomyocytes and seeding them into a NEST confocal culture dish.

[0070] The enzyme 1 is collagenase, and the enzyme 2 is trypsin; the primary cardiomyocytes include primary cardiomyocytes of a sham-operated control group that did not undergo ischemia-reperfusion surgery and hypoxia experiment, and primary cardiomyocytes of an experimental group that underwent ischemia-reperfusion surgery.

[0071] The process of performing ischemia-reperfusion surgery on animals to create a myocardial ischemia-reperfusion model includes:

[0072] Step S111, 8-10 week old male C57BL / 6 normal mice or GDF15 knockout (GDF15KO) mice were subjected to ischemia-reperfusion surgery, and recombinant GDF-15 (rGDF-15) or normal saline was implanted during the surgery at a dose of 12 μg / day;

[0073] Step S112, creating a myocardial ischemia-reperfusion model by ligating the left anterior descending coronary artery (LAD), anesthetizing with 2% isoflurane, intubating, shaving the chest area, making an incision between the fourth and fifth intercostal spaces to reach the chest cavity, and securing it with a chest retractor;

[0074] Step S113, using a long 0-4 Prolene suture as a spacer, and suturing the LAD with a 0-7 Prolene suture, with a suture spacing of 2 mm, the electrocardiogram showing blanching of the ischemic area and ST segment elevation indicates successful ligation;

[0075] Step S114, after the chest cavity is closed, the mouse is extubated and placed on a heating pad for 90 minutes of ischemia, after which the chest cavity is reopened and the ligature is removed. The previously pale ischemic area of the heart turns red, indicating that cardiac reperfusion is ongoing;

[0076] In step S115, the chest cavity is closed to allow the mouse to recover. During this process, the ventilator parameters are maintained at an inspiration / expiration ratio of 1:1, a respiratory rate of 90-100 times / minute, and a tidal volume of 1-2 mL.

[0077] In this example, animals that did not survive the ischemic surgery were not included in the analysis.

[0078] Step S2, mouse hearts were collected before ischemia (0-1 hour) and at different time points after reperfusion, namely, 0 hour, 2 hours, 6 hours, 12 hours, 1 day, 3 days, and 7 days; a hypoxia experiment was performed, AC16 cardiomyocytes and primary cardiomyocytes were treated with or without 250 μM cobalt chloride, and 100 ng / ml recombinant GDF-15 was added;

[0079] In this example, for the hypoxia experiment, AC16 cardiomyocytes and primary cardiomyocytes from some mouse hearts were treated with or without 250 μM cobalt chloride, and 100 ng / ml recombinant GDF-15 was added.

[0080] The collected mouse hearts and partial mouse hearts include normal mouse hearts and mouse hearts with myocardial infarction (MI), and the tissue sections of the mouse hearts with myocardial infarction (MI) are MI heart tissues.

[0081] Step S3, performing functional and molecular tests on AC16 cardiomyocytes and primary cardiomyocytes, wherein the tests include measuring intracellular ROS levels, measuring mitochondrial membrane potential, protein immunoblotting analysis, histological analysis, immunofluorescence analysis, and O2K analysis;

[0082] Step S31, the process of determining the intracellular ROS level includes:

[0083] Intracellular ROS levels were assessed using a ROS detection kit. DCFH-DA and Hoechst 33342 were diluted in cell-free culture medium and incubated with AC16 cardiomyocytes or primary cardiomyocytes at 37°C in the dark for 15 min, and fluorescence images were captured by fluorescence microscopy.

[0084] Step S32, the process of measuring mitochondrial membrane potential includes:

[0085] Mitochondrial membrane potential was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit. AC16 cardiomyocytes or primary cardiomyocytes were incubated with the JC-1 reagent at 37°C for 20 minutes, and then fluorescence images were acquired using a fluorescence microscope.

[0086] Step S33, the process of protein immunoblotting analysis includes,

[0087] Cellular proteins were extracted using RIPA buffer supplemented with phosphatase and protease inhibitors, and protein concentrations were determined using a BCA protein assay kit;

[0088] Equal amounts of protein were separated by 8–12.5% SDS-PAGE and transferred to nitrocellulose membranes, which were blocked with TBS containing 0.1% Tween20 and 5% skim milk for 1 h at room temperature;

[0089] After incubation with the primary antibody at 4°C overnight, the membrane was washed three times with TBST for 5 minutes each time. Protein detection was performed using a horseradish peroxidase-labeled secondary antibody to obtain band images of GDF-15 protein levels in the heart tissue of MI mice.

[0090] Step S34, the process of histological analysis includes:

[0091] Mouse heart tissue was fixed in 10% neutral buffered formaldehyde overnight, dehydrated through ethanol gradients, and embedded in paraffin;

[0092] Tissue sections were prepared using a microtome and stained with hematoxylin, eosin, and Masson's trichrome according to standard procedures;

[0093] In this embodiment, the ethanol gradient is 100%, 95%, and 85%.

[0094] Step S35, the process of immunofluorescence analysis includes:

[0095] The tissue sections were deparaffinized at 70°C, and the deparaffinized tissue sections were rehydrated through a xylene and ethanol gradient;

[0096] After washing with PBS, the tissue sections were treated with 3% hydrogen peroxide for 8 minutes and antigen retrieval was performed using citric acid buffer;

[0097] Tissue sections were permeabilized with 0.3% TritonX-100 and blocked with 3% BSA for 1 h;

[0098] Primary antibodies against HIF-1α or GDF-15 were incubated overnight at 4°C, followed by incubation with fluorescein-conjugated goat anti-mouse IgG secondary antibodies at 37°C for 1 hour;

[0099] Cell nuclei were counterstained with DAPI for 5 min at room temperature;

[0100] Immunofluorescence analysis was performed to obtain fluorescent images of tissue sections.

[0101] Step S36, the O2K analysis process includes:

[0102] The oxygen concentration was set to 200 nmol O / ml in each chamber, and data were recorded every 2 seconds using DatLab 5 software;

[0103] AC16 cardiomyocytes were collected by trypsinization, and MiR05 respiration medium and 1 × 10 6 AC16 cardiomyocytes;

[0104] Oligomycin (5 mM / chamber), CCCP (0.5 μM / chamber), rotenone (0.5 μM / chamber) and antimycin A (2.5 μM / chamber) were added sequentially to evaluate different respiratory states of AC16 cardiomyocytes.

[0105] Specifically, the pathophysiological process of myocardial infarction involves a variety of complex interactions, including ischemia, reperfusion injury, oxidative stress, inflammation, and subsequent fibrosis, which together lead to adverse cardiac remodeling and impaired cardiac function. This method, through multiple functional and molecular assays of AC16 cardiomyocytes and primary cardiomyocytes, elucidates the molecular mechanisms of how GDF-15 affects cardiac remodeling, mitochondrial function, and oxidative stress regulation after ischemic injury, providing new insights into the cardioprotective effects of GDF-15 and its potential as a therapeutic target for myocardial infarction.

[0106] Step S4, performing image analysis on the fluorescence signal of the fluorescence image obtained in step S3, and determining the expression pattern of GDF-15 and HIF-1α in the MI heart tissue according to the MI mean fluorescence intensity;

[0107] The MI heart tissue is the heart tissue of the mouse heart with myocardial infarction, and the sham operation control group is the heart tissue of the normal mouse heart;

[0108] The fluorescence images of MI (mouse) heart tissue were compared with those of sham-operated control group. The fluorescence signal was quantitatively analyzed by measuring the mean fluorescence intensity by dividing the total fluorescence intensity (Integrated Density) of the immunofluorescence staining area by the area of the area.

[0109] If the MI mean fluorescence intensity of GDF-15 is greater than the control mean fluorescence intensity, GDF-15 expression in the MI heart tissue is judged to be in the first expression pattern;

[0110] If the MI mean fluorescence intensity of GDF-15 is less than or equal to the control mean fluorescence intensity, GDF-15 expression in the MI heart tissue is judged to be in the second expression pattern;

[0111] If the MI mean fluorescence intensity of HIF-1α was greater than the control mean fluorescence intensity, HIF-1α expression in the MI heart tissue was judged to be in the first expression pattern;

[0112] If the MI mean fluorescence intensity of HIF-1α is less than or equal to the control mean fluorescence intensity, it is determined that the HIF-1α expression in the MI heart tissue is in the second expression pattern.

[0113] In this example, GDF-15 and HIF-1α are expressed in the first expression pattern.

[0114] Specifically, this method fluorescently stains GDF-15 stress response protein and HIF-1α to obtain fluorescent images, calculates the average fluorescence intensity by summing the fluorescence intensities of the immunofluorescence-stained areas in the fluorescent images, and compares the average fluorescence intensities of the fluorescent images of MI heart tissue and sham-operated heart tissue to determine the expression patterns of GDF-15 and HIF-1α, that is, the changes in GDF-15 and HIF-1α, thereby quantitatively analyzing the fluorescent signals; immunofluorescence staining shows that the expression of GDF-15 and HIF-1α is significantly increased in the infarct site, indicating that GDF-15 plays a key role in mediating cardiac remodeling after myocardial infarction.

[0115] Step S5, testing whether the protein level of GDF-15 in the MI heart tissue is increased based on the expression pattern of GDF-15, confirming the expression pattern of GDF-15, or adjusting the staining parameters of the western blot analysis of AC16 cardiomyocytes and primary cardiomyocytes;

[0116] Wherein, the staining parameters include antibody concentration, incubation time and incubation temperature.

[0117] When GDF-15 expression is in the first expression mode, the protein level of GDF-15 in MI heart tissue is tested, the average optical density (OD value) of the bands in the band image is measured, and the absolute expression amount of the target protein is calculated based on the standard curve;

[0118] If the average optical density of MI is greater than that of the control, it is judged that the protein level of GDF-15 is increased, which can confirm that GDF-15 is in the first expression mode. The absolute expression level of the target protein is calculated based on the standard curve;

[0119] If the mean optical density of MI was less than or equal to the mean optical density of control, it was determined that there was no increase in the protein level of GDF-15, and the staining parameters for western blotting analysis of AC16 cardiomyocytes and primary cardiomyocytes were adjusted;

[0120] Specifically, adjusting the staining parameters is to increase the antibody concentration and incubation time in the staining parameters, thereby increasing the signal intensity of the bands in the band image, and the signal intensity is the clarity.

[0121] The target protein is GDF-15, and the step of calculating the absolute expression level of the target protein includes:

[0122] Step S51, using the determined protein concentration as the standard concentration as the horizontal axis and the optical density value as the vertical axis, a standard curve is drawn by linear regression analysis;

[0123] Step S52, measuring the optical density value of the target protein band from the experimental sample, mapping the optical density value of the target protein to the standard curve, and finding the corresponding concentration value;

[0124] Step S53, calculating the absolute expression level of the target protein based on the concentration of the target protein and the sample volume;

[0125] Target protein concentration = optical density value / standard curve slope + standard curve intercept;

[0126] Absolute expression level = target protein concentration × sample volume;

[0127] Specifically, immunofluorescence is a semi-quantitative analysis, and the average fluorescence intensity can only semi-quantitatively characterize the expression of specific proteins. This method uses protein immunoblotting analysis to examine the protein level of GDF-15 in the heart tissue of MI mice. By measuring the average optical density of the bands in the band image and comparing the average optical density of the fluorescence images of MI heart tissue and sham-operated heart tissue, it is determined whether there is an increase in the protein level of GDF-15, thereby confirming the increase in GDF-15 at the protein level and confirming that GDF-15 expression is in the first expression mode, thereby improving the accuracy of indicating that GDF-15 plays a key role in mediating cardiac remodeling after myocardial infarction.

[0128] Step S6, after confirming the expression pattern of GDF-15, statistical analysis is performed on the detection parameters obtained from several functional and molecular tests to determine the mean differences between samples and to identify specific parameter groups based on the significant differences between the detection parameters;

[0129] A paired two-tailed t-test was performed on the detection parameters to determine whether the mean difference between the two samples was significant.

[0130] Step S61, proposing hypotheses, the null hypothesis that the two samples come from populations with the same mean and the alternative hypothesis that the two samples come from populations with different means;

[0131] Step S62, calculate the t value and degrees of freedom. For each pair of groups, calculate their mean difference (i.e., the difference between the group means). Use the standard deviation of each group and the sample size to calculate the standard error of the mean difference. The t value is equal to the mean difference divided by the standard error. The t value calculation formula is:

[0132]

[0133] The degree of freedom calculation formula is, df = n1 + n2 - 2;

[0134] in, and are the means of the two samples, and are the variances of the two samples, n1 and n2 are the sample sizes of the two samples.

[0135] Step S63: Find the critical value in the t distribution table based on the degrees of freedom and significance level (usually α = 0.05).

[0136] If the absolute value of the calculated t value is greater than the critical value, the null hypothesis is rejected, and it is considered that the mean difference between the two samples is significant and the mean difference is beyond the normal range;

[0137] If the absolute value of the calculated t-value is less than or equal to the critical value, the null hypothesis is accepted, and it is believed that there is no significant difference in the means of the two samples and the mean difference does not exceed the normal range.

[0138] In this example, the specific parameter group with significant differences among the different treatment groups was the membrane potential of AC16 cardiomyocytes and primary cardiomyocytes.

[0139] Specifically, the data obtained after functional and molecular testing of AC16 cardiomyocytes and primary cardiomyocytes were expressed as mean ± standard error r (SEM). The statistical comparison between the two groups of data was performed using an unpaired two-tailed t-test to determine whether the mean difference between the two independent samples was significant. This method performs an unpaired two-tailed t-test on multiple data obtained from functional and molecular testing to determine which corresponding parameters have significant differences, thereby avoiding misjudgment that is easy to cause by manual judgment, reducing comparison errors, and increasing the accuracy of data analysis.

[0140] Step S7, determining the pathway regulated by GDF-15 based on the determined specific parameter group, determining the ROS production pattern in AC16 cardiomyocytes, and determining the phosphorylation status of the AMPK signaling pathway in AC16 cardiomyocytes and primary cardiomyocytes;

[0141] In this example, the mitochondrial membrane potential in AC16 cardiomyocytes and primary cardiomyocytes was evaluated under different treatment conditions. Immunofluorescence analysis showed that the membrane potential of AC16 cardiomyocytes changed significantly in the different treatment groups.

[0142] Specifically, RNA sequencing analysis was performed to elucidate the molecular mechanisms of GDF-15-mediated cardiac regulation. GO and KEGG pathway enrichment analyses were performed to compare differentially expressed genes in wild-type and GDF15 knockout heart tissue, revealing distinct metabolic profiles between wild-type and GDF15 knockout mice. To further investigate the metabolic effects of GDF-15, mitochondrial membrane potential was assessed in AC16 cardiomyocytes and primary cardiomyocytes under different treatment conditions. Immunofluorescence analysis revealed significant changes in the membrane potential of AC16 cells in different treatment groups, and similar effects were observed in primary cardiomyocytes. High-resolution respirometry analysis indicated that GDF-15 affected key parameters of mitochondrial function in AC16 cells, including basal respiration, volumetric respiration, ATP production, and non-mitochondrial respiration, indicating that GDF-15 deficiency impairs mitochondrial function and energy metabolism under hypoxic stress. This method demonstrated that GDF-15 maintains mitochondrial function and cellular energy metabolism, thereby protecting cardiomyocytes from hypoxic damage. After GDF-15 supplementation under hypoxic conditions, the restoration of mitochondrial membrane potential, cellular oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) emphasized the direct regulatory effect of GDF-15 on mitochondrial function.

[0143] For AC16 cardiomyocytes after hypoxia experiment, the average fluorescence intensity of ROS under different treatment conditions was measured by fluorescence imaging.

[0144] The different treatment conditions include adding COCL2 and GDF-15 antibodies to AC16 cardiomyocytes, or adding COCL2 and GDF-15 to AC16 cardiomyocytes. The different treatment conditions also include adding COCL2 to AC16 cardiomyocytes, or adding physiological saline;

[0145] The fluorescence intensity difference between the mean fluorescence intensity of AC16 cardiomyocytes after adding COCL2 and GDF-15 antibodies, or after adding COCL2 and GDF-15, was calculated;

[0146] If the fluorescence intensity difference is greater than the first difference evaluation value, it is determined that the ROS in the AC16 cardiomyocytes is in the first generation mode;

[0147] If the fluorescence intensity difference is less than or equal to the first difference evaluation value, it is determined that the ROS in the AC16 cardiomyocytes is in the second generation mode;

[0148] The first difference evaluation value is the product of the difference between the absolute value of the t value and the critical value and the ratio of the critical value calculated based on the membrane potential parameter group of AC16 cardiomyocytes and primary cardiomyocytes, and the average fluorescence intensity of ROS after adding COCL2 to AC16 cardiomyocytes;

[0149] In implementation, when a larger or smaller conventional evaluation value is used to judge the ROS production mode in several AC16 cardiomyocytes compared to the first difference evaluation value, the proportion of AC16 cardiomyocytes in which ROS is determined to be in the second production mode is higher, and the error in judging the ROS production mode is greater than 2.9%, while the error in judging the ROS production mode using the first difference evaluation value is less than 1%.

[0150] In this example, ROS in AC16 cardiomyocytes is in the first generation mode. Compared with the addition of GDF-15 antibody to AC16 cardiomyocytes, the ROS content is significantly reduced after the addition of GDF-15 to AC16 cardiomyocytes.

[0151] When ROS was in the first generation mode, the average optical density of the bands in the band images of p-AMPK, AMPK, and β-actin in AC16 cardiomyocytes and primary cardiomyocytes under different treatment conditions was measured.

[0152] The different treatment conditions of the primary cardiomyocytes are the same as those of the AC16 cardiomyocytes;

[0153] If the average optical density of the p-AMPK band under different treatment conditions meets the first and second judgment conditions, it is judged that the AMPK signaling pathway is in the first phosphorylation state, AMPK activation is positively correlated with GDF-15, and the ROS generation pattern is reasonable;

[0154] Furthermore, GDF-15 regulates ROS production through the AMPK signaling pathway, and GDF-15 inhibits hypoxia-induced ROS generation through AMPK signaling;

[0155] If the average optical density of the p-AMPK band under different treatment conditions does not meet the first judgment condition or the second judgment condition, it is judged that the AMPK signaling pathway is in the second phosphorylation state, the activation of AMPK is negatively correlated with GDF-15, and the ROS generation pattern is unreasonable, and the first difference evaluation value is adjusted;

[0156] During implementation, when the ROS generation pattern is unreasonable, the first difference evaluation value is increased according to the ratio of the average fluorescence intensities of the two, and the judgment standard for determining the ROS generation pattern is adjusted;

[0157] Specifically, the first determination condition is that the average optical density of the p-AMPK band of AC16 cardiomyocytes after adding GDF-15 is greater than the average optical density of the p-AMPK band of AC16 cardiomyocytes after adding COCL2, and greater than the average optical density of the p-AMPK band of AC16 cardiomyocytes after adding normal saline;

[0158] The second judgment condition is that the average optical density of the p-AMPK band of primary cardiomyocytes after adding GDF-15 is greater than the average optical density of the p-AMPK band of primary cardiomyocytes after adding COCL2, and the average optical density of the p-AMPK band of primary cardiomyocytes after adding COCL2 is less than the average optical density of the p-AMPK band of primary cardiomyocytes after adding normal saline;

[0159] Wherein, the primary cardiomyocytes are primary cardiomyocytes of GDF15 gene knockout mice;

[0160] In this example, the average optical density of the p-AMPK band under different treatment conditions meets the first and second determination conditions, the AMPK signaling pathway is in the first phosphorylation state, and the activation of AMPK is positively correlated with GDF-15.

[0161] Specifically, this method measures ROS through fluorescence imaging. Under different treatment conditions, the ROS production patterns in AC16 cardiomyocytes are significantly different. p-AMPK (phosphorylated AMPK) is the activated form of AMPK (AMP-activated protein kinase). This method examined the AMPK signaling pathway by detecting the phosphorylation status of the AMPK signaling pathway to determine how GDF-15 regulates ROS. It was found that GDF-15 regulated the phosphorylation status of AMPK in AC16 cells and primary cardiomyocytes, indicating that GDF-15 regulates ROS production through the AMPK signaling pathway. GDF-15 inhibits hypoxia-induced ROS generation through AMPK signaling, showing the effect of GDF-15 on cellular oxidative stress. The AMPK signaling pathway is known to regulate energy metabolism and antioxidant responses. This method not only clarifies the molecular mechanism by which GDF-15 protects the heart, but also highlights its potential as a therapeutic target for myocardial infarction.

[0162] Step S8, administering exogenous GDF-15 to MI mice and performing immunofluorescence analysis to determine the expression relationship between GDF-15 and HIF-1α;

[0163] The MI mice include C57BL / 6 normal mice and GDF15 gene knockout mice, and the normal mice serve as the control group;

[0164] After exogenous GDF-15 administration, if the MI mean fluorescence intensity of HIF-1α in the MI heart tissue is less than the control mean fluorescence intensity, and the absolute value of the difference between the MI mean fluorescence intensity and the control mean fluorescence intensity is greater than the second difference evaluation value, then GDF-15 and HIF-1α are judged to be in a first expression relationship;

[0165] The second difference evaluation value is the product of the difference between the absolute value of the t value and the critical value and the ratio of the critical value calculated according to the membrane potential parameter group of AC16 cardiomyocytes and primary cardiomyocytes, and the mean fluorescence intensity of the control;

[0166] If the MI mean fluorescence intensity of HIF-1α in MI heart tissue is greater than or equal to the control mean fluorescence intensity, GDF-15 and HIF-1α are judged to be in the second expression relationship;

[0167] In this example, immunofluorescence analysis showed that administration of GDF-15 significantly reduced the expression of HIF-1α in the infarcted myocardium, and GDF-15 and HIF-1α were in a primary expression relationship.

[0168] Specifically, to investigate the therapeutic potential of GDF-15 in myocardial infarction, recombinant GDF-15 was injected into mice with myocardial infarction; histological analysis showed that GDF-15 treatment significantly reduced cardiac inflammation and fibrosis, consistent with the histological findings. Echocardiographic assessment indicated that cardiac function was improved in mice with myocardial infarction treated with GDF-15, and immunofluorescence analysis showed that GDF-15 administration significantly reduced HIF-1α expression in the infarcted myocardium, suggesting that GDF-15 may partially exert its cardioprotective effects by regulating the hypoxia response pathway. At the same time, GDF-15 significantly reduced inflammatory cell infiltration and collagen deposition in the heart after myocardial infarction, thereby alleviating adverse cardiac remodeling, suggesting that GDF-15 may exert its protective effects at least partially by regulating the immune response.

[0169] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

[0170] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for regulating oxidative stress in ischemic injury by GDF-15 based on the AMPK signaling pathway, characterized in that: include: Animals underwent ischemia-reperfusion surgery to create a myocardial ischemia-reperfusion model. MI heart tissues were obtained before ischemia and at different time points after reperfusion for culture of AC16 cardiomyocytes and treatment of primary cardiomyocytes. AC16 cardiomyocytes and primary cardiomyocytes were subjected to hypoxia experiments and several functional and molecular assays, including obtaining fluorescent images of MI heart tissue; The fluorescence signals of the fluorescence images were analyzed, and the expression patterns of GDF-15 and HIF-1α in MI heart tissues were determined based on the mean fluorescence intensity of MI; According to the expression pattern of GDF-15, whether the protein level of GDF-15 is increased in MI heart tissues is tested, the expression pattern of GDF-15 is confirmed, or the staining parameters of western blot analysis of AC16 cardiomyocytes and primary cardiomyocytes are adjusted; After confirming the expression pattern of GDF-15, statistical analysis of the test parameters obtained from several functional and molecular tests was performed to determine the mean differences between samples. The regulatory pathways of GDF-15 were determined based on the specific parameter groups with significant differences between the test parameters. Determine a difference evaluation value based on the mean difference, and judge the ROS generation pattern in AC16 cardiomyocytes based on the fluorescence intensity difference between the mean fluorescence intensities of ROS under different treatment conditions; The phosphorylation status of the AMPK signaling pathway in AC16 cardiomyocytes and primary cardiomyocytes was determined based on the phosphorylation signature criteria, the rationality of the ROS generation pattern was verified, and the determined difference evaluation value was adjusted. Exogenous GDF-15 administration and immunofluorescence analysis were performed on MI heart tissues, and the expression relationship between GDF-15 and HIF-1α was obtained based on the difference evaluation value analysis; The animals include C57BL / 6 normal mice and GDF15 gene knockout mice.

2. The method of regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 1, characterized in that: The process of determining the expression pattern of GDF-15 and HIF-1α in MI cardiac tissue included: The mean fluorescence intensity was obtained by measuring the sum of the fluorescence intensities of the immunofluorescence-stained region and dividing it by the area of the region, and the MI mean fluorescence intensities of GDF-15 and HIF-1α in the heart tissues of MI mice were compared with the control mean fluorescence intensities of GDF-15 and HIF-1α in the sham-operated control group; According to the comparison results, it is determined whether GDF-15 and HIF-1α in MI heart tissue are in the first expression pattern or the second expression pattern.

3. The method of regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 2, characterized in that: The process of confirming the expression pattern of GDF-15 included: When GDF-15 expression was in the first expression pattern, the protein level of GDF-15 in the heart tissue of MI mice was examined, the average optical density of the bands in the band images was measured, and the average optical density of MI was compared with the average optical density of the control; Based on the comparison results, determine whether the protein level of GDF-15 is increased, and calculate the absolute expression level of the target protein based on the standard curve.

4. The method of regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 3, characterized in that: If the average optical density of MI is greater than that of the control, it is judged that the protein level of GDF-15 is increased, which can confirm that GDF-15 is in the first expression mode. The absolute expression level of the target protein is calculated based on the standard curve; If the MI mean optical density is less than or equal to the control mean optical density, it is judged that there is no increase in the protein level of GDF-15, and the staining parameters for western blot analysis of AC16 cardiomyocytes and primary cardiomyocytes are adjusted.

5. The method of regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 1, characterized in that: The process of determining the mean difference between samples involves, Paired two-tailed t-test was performed on the detection parameters to determine the mean difference between the two samples. If the absolute value of the calculated t-value is greater than the critical value, the null hypothesis is rejected and it is considered that the difference in the means of the two samples is beyond the normal range; If the absolute value of the calculated t-value is less than or equal to the critical value, the null hypothesis is accepted and the mean difference between the two samples does not exceed the normal range.

6. The method for regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 5, characterized in that: The mitochondrial membrane potential in AC16 cardiomyocytes and primary cardiomyocytes was evaluated based on the detection parameters corresponding to the samples whose mean differences exceeded the normal range, and the changes in the membrane potential of AC16 cardiomyocytes were determined.

7. The method of regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 1, characterized in that: The process of determining the ROS production pattern in AC16 cardiomyocytes includes: For AC16 cardiomyocytes after hypoxia experiment, the mean fluorescence intensity of ROS under different treatment conditions was measured by fluorescence imaging, and the fluorescence intensity difference between the mean fluorescence intensities was calculated; The fluorescence intensity difference between the mean fluorescence intensity of AC16 cardiomyocytes after adding COCL2 and GDF-15 antibodies, or after adding COCL2 and GDF-15, was calculated; The fluorescence intensity difference is compared with the first difference evaluation value, and it is determined whether the ROS in the AC16 cardiomyocytes is in the first generation mode or the second generation mode according to the comparison result.

8. The method for regulating oxidative stress in ischemic injury by GDF-15 based on the AMPK signaling pathway according to claim 7, characterized in that: The process of determining the phosphorylation status of the AMPK signaling pathway in AC16 cardiomyocytes and primary cardiomyocytes includes: When ROS were in the first generation mode, the average optical density of the bands in the band images of p-AMPK, AMPK, and β-actin in AC16 cardiomyocytes and primary cardiomyocytes under different treatment conditions was measured; If the average optical density of the p-AMPK band under different treatment conditions meets the first and second judgment conditions, it is judged that the AMPK signaling pathway is in the first phosphorylation state, AMPK activation is positively correlated with GDF-15, and the ROS generation pattern is reasonable; If the average optical density of the p-AMPK band under different treatment conditions does not meet the first judgment condition or the second judgment condition, it is judged that the AMPK signaling pathway is in the second phosphorylation state, the activation of AMPK is negatively correlated with GDF-15, the ROS generation pattern is unreasonable, and the first difference evaluation value is adjusted.

9. The method for regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 8, characterized in that: The first determination condition is that the average optical density of the p-AMPK band of AC16 cardiomyocytes after adding GDF-15 is greater than the average optical density of the p-AMPK band of AC16 cardiomyocytes after adding COCL2, and greater than the average optical density of the p-AMPK band of AC16 cardiomyocytes after adding normal saline; The second determination condition is that the average optical density of the p-AMPK band of the primary cardiomyocytes after adding GDF-15 is greater than the average optical density of the p-AMPK band of the primary cardiomyocytes after adding COCL2, and the average optical density of the p-AMPK band of the primary cardiomyocytes after adding COCL2 is less than the average optical density of the p-AMPK band of the primary cardiomyocytes after adding normal saline; Wherein, the primary cardiomyocytes are primary cardiomyocytes of GDF15 gene knockout mice.

10. The method of regulating ischemic injury oxidative stress by GDF-15 based on the AMPK signaling pathway according to claim 1, characterized in that: The process of determining the expression relationship between GDF-15 and HIF-1α includes: After exogenous GDF-15 administration, if the MI mean fluorescence intensity of HIF-1α in the MI cardiac tissue is less than or equal to the control mean fluorescence intensity, and the absolute value of the difference between the MI mean fluorescence intensity and the control mean fluorescence intensity is greater than the second difference evaluation value, then GDF-15 and HIF-1α are judged to be in a first expression relationship; If the MI mean fluorescence intensity of HIF-1α in MI heart tissue was greater than the control mean fluorescence intensity, GDF-15 and HIF-1α were judged to be in the second expression relationship; The functional and molecular assays include determination of intracellular ROS levels, determination of mitochondrial membrane potential, protein immunoblotting analysis, histological analysis, immunofluorescence analysis, and O2K analysis.

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