Use of alox5 gene in preparation of doxorubicin cardiomyopathy drugs

Abstract

The present application relates to the use of Alox5 gene in the preparation of doxorubicin cardiomyopathy drugs, belonging to the technical field of biological medicine. The drug is used for one or more selected from the following: 1) prevention and / or treatment of doxorubicin myocardial injury; 2) prevention and / or treatment of doxorubicin-induced heart failure; 3) prevention and / or treatment of doxorubicin-induced arrhythmia; 4) prevention and / or treatment of doxorubicin-induced dilated cardiomyopathy; the drug contains a substance with inhibitory effect on Alox5 gene expression. It is found that knocking down Alox5 gene can significantly reduce doxorubicin-induced myocardial injury, and overexpression of Alox5 gene can significantly aggravate doxorubicin-induced myocardial injury, therefore, Alox5 gene can be used as a new target for treating doxorubicin cardiomyopathy, and more doxorubicin cardiomyopathy treatment drugs can be developed to provide more selectivity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the pharmaceutical use of the Alox5 gene, particularly the use of the Alox5 gene or its active fragment in the preparation of drugs for the prevention or treatment of doxorubicin cardiomyopathy. Background Technology

[0002] Doxorubicin (DOX) belongs to the anthracycline class of drugs and is one of the most effective chemotherapy drugs for treating various tumors such as leukemia, breast cancer, lung cancer, and lymphoma. Unfortunately, its application is limited due to its cardiotoxicity. Currently, the pathogenesis of doxorubicin-induced cardiomyopathy includes: inhibition of DNA, RNA, and protein synthesis, increased production of reactive oxygen species, impaired energy metabolism, mitochondrial dysfunction, cardiomyocyte apoptosis, interstitial fibrosis, autophagy dysregulation, and disturbance of intracellular calcium homeostasis. Several antioxidants, such as vitamin E1(2), N-acetylcysteine, resveratrol, and 7-monohydroxyethyl rutin, have been used to alleviate doxorubicin cardiomyopathy. However, due to the limited efficacy of these drugs in treating doxorubicin cardiomyopathy, the results remain far from satisfactory.

[0003] Alox5 is a key enzyme in the biosynthesis of chemical inducers and vasoactive leukotrienes, catalyzing the production of reactive oxygen species (ROS). Alox5 is known to participate in various physiological and pathological processes. Studies have shown its application in diseases such as hepatitis, pancreatic cancer, asthma, Alzheimer's disease, and lymphedema. More importantly, research has been conducted on its application in pulmonary hypertension, atherosclerosis, ischemic heart disease, septic heart disease, pressure-induced heart failure, and diabetic cardiomyopathy. However, no reports have been found linking Alox5 to doxorubicin-induced cardiomyopathy. Summary of the Invention

[0004] Through extensive experimental research, the applicant discovered that the expression of Alox5 gene mRNA and protein was significantly upregulated in the myocardial tissue of mice with doxorubicin-induced myocardial injury, resulting in a significant decrease in left ventricular diastolic and systolic function and a significant increase in myocardial injury markers. Both in vivo and in vitro, knockdown of the Alox5 gene significantly reduced doxorubicin-induced myocardial injury, while overexpression of the Alox5 gene significantly aggravated doxorubicin-toxic myocardial injury. Based on this, the present invention provides the use of the Alox5 gene in the preparation of doxorubicin-induced cardiomyopathy drugs.

[0005] In a first aspect, the present invention provides the use of the Alox5 gene in the preparation of a drug, said drug being used in one or more of the following:

[0006] 1) Prevention and / or treatment of doxorubicin-induced myocardial injury;

[0007] 2) Prevention and / or treatment of doxorubicin-induced heart failure;

[0008] 3) Prevention and / or treatment of doxorubicin-induced arrhythmias;

[0009] 4) Prevention and / or treatment of doxorubicin-induced dilated cardiomyopathy;

[0010] The drug contains a substance that inhibits the expression of the Alox5 gene.

[0011] Based on the above technical solution, the substance that inhibits Alox5 gene expression is a nucleic acid, a nucleic acid construct, adenovirus, protein, or small molecule compound, which can reduce the levels of Alox5 gene mRNA and Alox5 protein in the myocardial tissue of mice with doxorubicin-induced myocardial injury.

[0012] Based on the above technical solution, the substance that inhibits Alox5 gene expression is an Alox5-targeting siRNA, the sequence of which is shown in SEQ ID NO:1.

[0013] Secondly, the present invention provides the use of Alox5-targeting siRNA in the preparation of a drug, wherein the sequence of the Alox5-targeting siRNA is shown in SEQ ID NO:1, and it is used to inhibit the expression of Alox5 protein by the Alox5 gene; the drug is used in one or more of the following:

[0014] 1) Prevention and / or treatment of doxorubicin-induced myocardial injury;

[0015] 2) Prevention and / or treatment of doxorubicin-induced heart failure;

[0016] 3) Prevention and / or treatment of doxorubicin-induced arrhythmias;

[0017] 4) Prevention and / or treatment of doxorubicin-induced dilated cardiomyopathy.

[0018] In a preferred embodiment of the above technical solution, the drug uses siRNA targeting Alox5 as the sole active ingredient.

[0019] In another preferred embodiment of the above technical solution, the drug comprises a combination of siRNA targeting Alox5 and other active ingredients, wherein the other active ingredients are used to treat one or more of the following:

[0020] 1) Prevention and / or treatment of doxorubicin-induced myocardial injury;

[0021] 2) Prevention and / or treatment of doxorubicin-induced heart failure;

[0022] 3) Prevention and / or treatment of doxorubicin-induced arrhythmias;

[0023] 4) Prevention and / or treatment of doxorubicin-induced dilated cardiomyopathy;

[0024] The combination of the Alox5-targeting siRNA and other active ingredients is selected from any of the following forms:

[0025] (i) The siRNA targeting Alox5 and other active ingredients are formulated separately;

[0026] (ii) Formulate a compound preparation by combining Alox5-targeting siRNA with other active ingredients.

[0027] Based on the above technical solution, the drug also includes an optional pharmaceutically acceptable carrier or excipient.

[0028] Thirdly, the present invention provides the use of a substance capable of inhibiting the activity of Alox5 protein or its active fragment in the preparation of a medicament, said medicament being used in one or more of the following:

[0029] 1) Prevention and / or treatment of doxorubicin-induced myocardial injury;

[0030] 2) Prevention and / or treatment of doxorubicin-induced heart failure;

[0031] 3) Prevention and / or treatment of doxorubicin-induced arrhythmias;

[0032] 4) Prevention and / or treatment of doxorubicin-induced dilated cardiomyopathy.

[0033] Fourthly, the present invention provides the use of reagents capable of detecting the expression level of Alox5 protein or its active fragments in the preparation of a kit, said kit being used for one or more of the following:

[0034] 1) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced myocardial injury;

[0035] 2) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis for doxorubicin-induced heart failure;

[0036] 3) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis for doxorubicin-induced arrhythmias;

[0037] 4) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced dilated cardiomyopathy.

[0038] Fifthly, the present invention provides a kit containing reagents for detecting the expression level of Alox5 protein or its active fragment, said kit being selected from one or more of the following:

[0039] 1) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced myocardial injury;

[0040] 2) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis for doxorubicin-induced heart failure;

[0041] 3) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis for doxorubicin-induced arrhythmias;

[0042] 4) Diagnosis, risk assessment, and / or evaluation of treatment efficacy and prognosis of doxorubicin-induced dilated cardiomyopathy.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0044] This invention provides a new target for the treatment of doxorubicin cardiomyopathy, which is expected to lead to the development of more drugs for the treatment of doxorubicin cardiomyopathy, thus providing more drug options for the treatment of doxorubicin cardiomyopathy. Attached Figure Description

[0045] In the following figures, &, $, # and * indicate P < 0.05:

[0046] Figure 1 The Alox5 gene is involved in doxorubicin cardiomyopathy in in vivo experiments. Figure A shows the grouping of mice and the establishment of doxorubicin cardiomyopathy models in C57BL / 6J mice in each group. Figure B shows the level of Alox5 mRNA in mice evaluated by quantitative real-time PCR. Group C shows the protein expression level of Alox5 in mice evaluated by Western blot. Group D shows the subcellular localization and fluorescence intensity of Alox5 in cardiomyocytes evaluated by immunofluorescence. Group E shows the expression level of Alox5 evaluated by immunohistochemistry. Figures F and G show the expression levels of LTB4 in serum and cardiac homogenate.

[0047] Figure 2 The Alox5 gene is involved in doxorubicin-induced cardiomyopathy in in vitro experiments. Figure A shows the grouping of primary cardiomyocytes and the establishment of a primary cardiomyocyte doxorubicin-induced cardiomyopathy injury model. Figure B evaluates the cell viability levels of each group. Figure C uses quantitative real-time PCR to evaluate the mRNA level of the Alox5 gene. Figure D uses Western blot to evaluate the protein expression level of the Alox5 gene. Figure E uses immunofluorescence to assess the subcellular localization of the Alox5 gene in primary cardiomyocytes. Figure F uses Western blot to evaluate the subcellular localization of the Alox5 gene in primary cardiomyocytes.

[0048] Figure 3The study investigated how overexpression of Alox5 in vivo exacerbated myocardial injury and worsened cardiac function in doxorubicin-induced cardiomyopathy. Figure A shows the survival analysis curves of mice in each group. Figure B shows the changes in body weight of mice in each group. Figure C shows the ratio of heart weight to tibia length of mice in each group. Figures DE show the size of cardiomyocytes in mice in each group assessed by HE staining. Figures F and J show the LVEF, LVFS, LVEDd, and LVEDs of mice in each group assessed by ultrasound. Figures K, M, N, and O show the hemodynamic parameters of each group, including pressure-volume curves, cardiac output (CO), rate of maximum pressure (+dP / dt max), and rate of minimum pressure decay (-dP / dt min). Figure PR shows the ELISA results for myocardial injury markers CK-MB, CTNT, and LDH in mice in each group.

[0049] Figure 4 The in vitro overexpression of Alox5 exacerbates cardiomyocyte damage in doxorubicin-induced cardiomyopathy. Figure A shows the grouping of primary cardiomyocytes and the establishment of the primary cardiomyocyte doxorubicin-induced cardiomyopathy damage model. Figures B and C show the evidence of Alox5 overexpression by quantitative real-time PCR and Western blot. Figure D evaluates the cell viability level of each group. Figure E shows the LDH release assay to evaluate the LDH level of each group.

[0050] Figure 5 This study investigated how knocking down Alox5 in vivo alleviated myocardial injury and cardiac function deterioration in doxorubicin-induced cardiomyopathy. Figure A shows the survival analysis curves of mice in each group. Figure B shows the changes in body weight of mice in each group. Figure C shows the ratio of heart weight to tibia length of mice in each group. Figures DE show the size of myocardial cells in mice in each group, assessed by HE staining. Figure FJ shows the LVEF, LVFS, LVEDd, and LVEDs of mice in each group, assessed by ultrasound. Figure KN shows the hemodynamic parameters of each group, including pressure-volume curves, cardiac output (CO), rate of maximum pressure (+dP / dt max), and rate of minimum pressure decay (-dP / dt min). Figure OQ shows the myocardial injury markers CK-MB, CTNT, and LDH in mice in each group, assessed by ELISA.

[0051] Figure 6 The in vitro knockdown of Alox5 alleviates cardiomyocyte damage in doxorubicin cardiomyopathy. Figure A: Indicates the establishment of an in vitro Alox5 knockdown model in doxorubicin cardiomyopathy. Figures B and C: Validation of Alox5 knockdown by quantitative real-time PCR and Western blot. Figure D: Evaluation of cell viability in each group. Figure E: Evaluation of LDH levels in each group by LDH release assay.

[0052] Figure 7Silencing Alox5 improves ferroptosis in vivo. Figure AC: Transcriptomic comparison of the DOX group and the Ko-Alox5+DOX group. Figure D: Quantitative real-time PCR evaluation of the mRNA expression levels of PTGS2, GPX4, TFR, FPN, FTH, and SLC7A11 in mice. Figure EI: Western blot comparison of the protein expression levels of Ferritin, FPN, GPX4, and TFR in each mouse group. Figure JK: Assessment of MDA expression levels in mouse serum and heart tissue homogenates. Figure LM: Assessment of non-hemeiron expression levels in mouse serum and heart tissue homogenates. Figure N: Assessment of NAPDH expression levels in mouse heart tissue homogenates. Figure O: Assessment of GSH expression levels in mouse heart tissue homogenates. Figure PQ: Immunohistochemistry assessment of 4-HNE and GPX4 expression levels. Figure R: DHE staining assessment of ROS levels in each mouse tissue group.

[0053] Figure 8 Silencing Alox5 improves ferroptosis in vitro. Figure A: Evaluation of MDA expression level in primary cardiomyocytes of each group. Figure B: Evaluation of NAPDH expression level in primary cardiomyocytes of each group. Figure C: Evaluation of GSH expression level in primary cardiomyocytes of each group. Figures DE: Evaluation of ROS expression level in primary cardiomyocytes of each group using DCFH-DA. Figure F: Evaluation of mRNA expression levels of PTGS2, GPX4, TFR, FPN, FTH, and SLC7A11 in mice using quantitative real-time PCR. Figure GK: Comparison of protein expression levels of Ferritin, FPN, GPX4, and TFR in mice of each group using Western blot.

[0054] Figure 9 Silencing Alox5 improves inflammation in vivo. Figure A: Quantitative real-time PCR evaluates the mRNA expression levels of TNF-α, IL-1β, IL-6, HMGB1, and MCP-1 in mice. Figures B and C: Western blot compares the protein expression levels of TNF-α, IL-1β, and IL-6 in different mouse groups. Figure F and H: ELISA assay assesses the expression levels of TNF-α, IL-1β, and IL-6 in mouse serum. Figure IL: Immunohistochemistry assesses the expression levels of TNF-α, HMGB1, CD45, and CD68.

[0055] Figure 10 Silencing Alox5 improves inflammation in vitro. Figure A: Quantitative real-time PCR evaluates the mRNA expression levels of TNF-α, IL-1β, IL-6, HMGB1, and MCP-1 in mice. Figures B and C: Western blot compares the protein expression levels of TNF-α, IL-1β, and IL-6 in different mouse groups. Detailed Implementation

[0056] This invention constructs a mouse model of doxorubicin-induced cardiomyopathy and utilizes (5-LOX) - / - Functional validation was performed on mice with Alox5 gene overexpression, confirming that Alox5 gene expression was upregulated in the cardiac tissue of mice with doxorubicin-induced cardiomyopathy and in primary cardiomyocytes of rats. Knockdown of the Alox5 gene, both in vivo and in vitro, improved cardiac function in the doxorubicin-induced cardiomyopathy model, reducing cardiomyocyte ferroptosis, inflammation, and reactive oxygen species levels. Conversely, Alox5 gene overexpression, both in vivo and in vitro, exacerbated the damage caused by doxorubicin-induced cardiomyopathy. The Alox5 gene could be used to prevent or treat doxorubicin-induced myocardial injury and related diseases, providing new evidence for the prevention, diagnosis, and treatment of doxorubicin-induced myocardial injury in clinical medicine. Further research revealed increased ferroptosis pathway proteins in the myocardial tissue of mice with doxorubicin-induced myocardial injury, while knockdown of the Alox5 gene reduced the expression of ferroptosis pathway proteins and inflammatory proteins following doxorubicin-induced myocardial injury. These results indicate that the Alox5 gene can be used to prevent or treat doxorubicin-induced myocardial injury and related diseases by reducing ferroptosis.

[0057] Based on the above findings, this invention provides for the first time the use of the Alox5 gene in the preparation of a drug, wherein the drug is selected from one or more of the following:

[0058] 1) Prevention and / or treatment of doxorubicin-induced myocardial injury;

[0059] 2) Prevention and / or treatment of doxorubicin-induced heart failure;

[0060] 3) Prevention and / or treatment of doxorubicin-induced arrhythmias;

[0061] 4) Prevention and / or treatment of doxorubicin-induced dilated cardiomyopathy;

[0062] The drug contains a substance that inhibits the expression of the Alox5 gene.

[0063] In this invention, the specific sequence of the Alox5 gene can be found in the NCBI Reference Sequence: Location: Chr6:116387038-116438139bp, -strand Genetic Position: Chr6, 53.79cM.

[0064] In this invention, the term "doxorubicin myocardial injury" has the meaning known in the art, referring to left ventricular dysfunction that occurs after the use of doxorubicin.

[0065] In this invention, the term "prevention and / or treatment of doxorubicin myocardial injury" refers to inhibiting or slowing the occurrence of doxorubicin myocardial injury and related diseases.

[0066] In this invention, the term "active fragment of the Alox5 gene" refers to a fragment that has the function of the Alox5 protein. It can be a part of the Alox5 protein or a fragment obtained by deleting, adding or substituting the amino acid sequence of the Alox5 protein. The method for preparing or obtaining the active fragment of the Alox5 protein is known in the art. For example, the active fragment is a fragment containing the part of the Alox5 protein that binds to a ligand or receptor, or a fragment that retains the function of the Alox5 protein after the deletion, addition or substitution of amino acids.

[0067] In this invention, using the detection of the expression level of Alox5 protein or its active fragment for prediction and / or evaluation means that when the expression level of Alox5 gene or its active fragment in blood, tissue or cells is lower than the reference value, it is possible to predict the occurrence of doxorubicin myocardial injury and related diseases, or to evaluate its treatment effect or prognosis.

[0068] In this invention, the expression level of Alox5 protein or its active fragment can be detected by methods known in the art, such as amplifying Alox5 mRNA by polymerase chain reaction and performing a quantitative reaction, or detecting the expression level of Alox5 protein by Western blot.

[0069] In this invention, the substance that has an inhibitory effect on Alox5 gene expression is known in the art, such as siRNA that targets Alox5, which can knock down the expression level of Alox5 gene.

[0070] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Unless otherwise specified, the following embodiments are all conventional methods.

[0072] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0073] In the following examples, the sequence of the Alox5 gene-targeting Alox5 siRNA (Si-Alox5) is shown in SEQ ID NO:1: 5′-GCATGACTTTGCTGACTTT-3′; the control sequence is shown in SEQ ID NO:2: 5′-GCTGCACAGAGTTGCCTAA-3′.

[0074] All experimental data in this invention are percentages. Chi-square tests were used to compare two samples, and Graphpad 8.0 software was used for data analysis. P < 0.05 was considered statistically significant.

[0075] Example 1

[0076] (1) Establishment of a C57BL / 6J mouse model of doxorubicin-induced myocardial injury

[0077] Forty 11-week-old male C57BL / 6J mice were randomly divided into four groups using a randomized table: NS group, DOX+1day group, DOX+3day group, and DOX+8day group, with 10 mice in each group. The DOX+1day group, DOX+3day group, and DOX+8day group received intraperitoneal injections of doxorubicin at a dose of 15 mg / kg for 1, 3, and 8 days, respectively, while the NS group received an equal volume of physiological saline via intraperitoneal injection.

[0078] (2) Detection of Alox5 gene mRNA expression in myocardial tissue by real-time quantitative PCR

[0079] Add 1 mL of Trizol to the left ventricle (LV) tissue, let stand for 10 min, extract with chloroform, add chloroform at a ratio of 200 μL / mL of Trizol, let stand at room temperature for 5 min, centrifuge at 12000 rpm at 4°C for 30 min, the supernatant after centrifugation is RNA, transfer to a new EP tube, add 500 μL of isopropanol, let stand at room temperature for 10 min, centrifuge at 12000 rpm at 4°C for 10 min, discard the supernatant, wash with 75% ethanol, centrifuge, dry at room temperature for 10 min, dissolve in DEPC water, and determine the RNA concentration and purity using a spectrophotometer, store in a -80°C freezer.

[0080] RT-PCR detection: The mRNA expression levels of apoptosis and oxidative stress-related factors were detected according to the LightCycler 480SYBR Green I Master instructions, with GADPH used as an internal control.

[0081] The results showed that, compared with the NS group, the expression level of Alox5 gene mRNA increased significantly with increasing doxorubicin stimulation days. Figure 1 B.

[0082] (3) Western blot detection of Alox5 protein expression in myocardial tissue

[0083] Proteins extracted from cardiac tissue were assessed using the BCA protein assay kit. Left ventricular (LV) tissue was added to a protein lysis buffer at a concentration of 10 mg / 100 μL (1 mL of protein lysis buffer contained 10 μL of 1 mmol / L benzyl sulfonyl fluoride, 50 μL of 1 mmol / L sodium chloride, 10 μL of 1 mmol / L sodium vanadate, 100 μL of Roche phosphatase inhibitor, 100 μL of complete lysis buffer, and 730 μL of RIFA strong lysis buffer). Cells from each group were collected and placed in 1.5 mL EP tubes. LV tissue was then lysed 15-20 times using an ultrasonic lysis device (5 kHz) and centrifuged at 12000 rpm for 30 min at 4 °C. Protein concentration was quantified using the BCA method. After denaturation at 72 °C, the proteins were stored at -80 °C. Protein electrophoresis was performed using a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were transferred to a PVDF membrane, blocked for 2 h, and then incubated overnight at 4 °C with primary antibody. The following day, after washing with TBST buffer, the membrane was incubated with secondary antibody for 1.5 hours and then developed using a chemical scanning membrane analyzer. Protein bands were analyzed using ImageLab software, with GADPH as an internal control.

[0084] The results showed that, compared with the NS group, the expression level of Alox5 protein increased significantly with increasing doxorubicin stimulation days. Figure 1 C.

[0085] (4) Immunofluorescence detection of subcellular localization and expression level of Alox5 in doxorubicin cardiomyopathy

[0086] Place the paraffin sections on a slide holder with the same orientation and bake them in a 55°C oven for 30 minutes. Simultaneously, place the dewaxing solution container in the 55°C oven. Place the paraffin sections, along with the slide holder, into the dewaxing solution container, then remove them from the oven and allow them to cool to room temperature. After 5 minutes, remove the sections and immerse them in the room-temperature dewaxing solution container. Add the paraffin sections in the container in the following order: dewaxing solution, dewaxing solution, anhydrous ethanol, anhydrous ethanol, and another anhydrous ethanol (or alternatively, a gradient concentration of ethanol), for 5 minutes per container. Rinse the sections with running water for 5 minutes. In a pressure cooker, add the antigen retrieval solution and preheat on high heat. Once the retrieval solution boils, place the sections in the pressure cooker, ensuring complete tissue immersion. Cover the cooker, close the pressure valve, and continue heating on high heat. Once the pressure valve begins to release steam, reduce the heat to medium and start timing for 2 minutes. After timing, remove the pressure cooker from the heat source and allow it to depressurize naturally. Then, transfer the pressure cooker to cold water for slow cooling. Once the retrieval solution has cooled to room temperature, wash the sections three times with PBS buffer, 1 minute each time. Draw circles around the slide with an immunohistochemical pen, wash with PBS for 5 min x 3 times, block with sheep serum for 30 min, discard the serum (do not wash), incubate overnight at 37°C in a humidified chamber, warm to room temperature for 1 h (to prevent slide drop), wash with PBS for 5 min x 3 times, incubate with secondary antibody at 37°C in a humidified chamber for 1 h (protect from light), wash with PBS for 5 min x 3 times, counterstain the nuclei with DAPI for 10 min, rinse twice with PBS, dehydrate and clear, and mount with neutral resin.

[0087] The results showed that, compared with the NS group, the doxorubicin group had a higher fluorescence intensity of the Alox5 gene and it was localized in the cell nucleus. Figure 1 D.

[0088] (5) Immunohistochemical detection of Alox5 gene expression level in doxorubicin cardiomyopathy

[0089] Place the paraffin sections on a slide rack with the same orientation and bake them in a 55°C oven for 30 minutes. Simultaneously, place one tank of dewaxing solution into the 55°C oven. Place the paraffin sections, along with the slide rack, into the dewaxing solution tank, then remove them from the oven and place them at room temperature. After 5 minutes, remove the sections and immerse them in the room-temperature dewaxing solution tank. Add the paraffin sections in the following order: dewaxing solution, dewaxing solution, anhydrous ethanol, anhydrous ethanol, and anhydrous ethanol (or alternatively, a gradient concentration of ethanol), for 5 minutes per tank. Rinse the sections with running water for 5 minutes. In a pressure cooker, add antigen retrieval solution and preheat on high heat. Once the retrieval solution boils, place the sections in the pressure cooker, ensuring complete tissue immersion. Cover the cooker, close the pressure valve, and continue heating on high heat. Once the pressure relief valve begins to release steam, reduce the heat to medium and start timing for 2 minutes. After timing, remove the cooker from the heat source and allow it to depressurize naturally before transferring it to cold water for slow cooling. After the retrieval solution cooled to room temperature, wash three times with PBS buffer, 1 min each time. Wash with 3% hydrogen peroxide for 20 min, then rinse with PBS, block with 8% sheep serum for 40 min, incubate with primary antibody at 4°C overnight, warm to room temperature for 30 min the next day, rinse with PBS for 5 min each time (3 times), incubate with secondary antibody for 1 h, rinse with PBS for 5 min each time (3 times), perform DAB staining, rinse with PBS for 5 min, counterstain with hematoxylin, dehydrate and clear, and mount with neutral resin.

[0090] The results showed that, compared with the NS group, the doxorubicin group had a higher Alox5 gene expression level. Figure 1 E.

[0091] (6) Expression of LTB4 in serum and cardiac homogenate

[0092] The expression of LTB4 in serum and homogenate of mice in the NS group, DOX+1 day group, DOX+3 day group, and DOX+8 day group was detected by ELISA. The results showed that Alox5 enzyme activity was significantly increased in doxorubicin-induced cardiomyopathy. Figure 1 FG.

[0093] Example 2: Alox5 involvement in doxorubicin-induced myocardial injury

[0094] (1) Detection of neonatal rat cardiomyocyte (NRVM) activity using CCK-8 assay

[0095] 100 μL of cell suspension was added to each well of a 96-well plate and cultured for 24 h. Six replicates were set up for each group. Different drugs were administered for 24 h according to the groups of PBS, 0.25 μmol / L DOX, 0.5 μmol / L DOX and 1 μmol / L DOX. Then, 10 μL of LCK-8 solution was added to each well and the culture plate was incubated in an incubator for 4 h. The absorbance of each group of cells at 450 nm was measured using a microplate reader.

[0096] The results showed that cell viability decreased with increasing doxorubicin concentration. Figure 2 B.

[0097] (2) Detection of Alox5 gene mRNA expression in primary cardiomyocytes by real-time PCR

[0098] ① RNA extraction: Add 1 mL of Trizol to a 6-well plate inoculated with cells, let stand for 10 min, extract with chloroform, add chloroform at a ratio of 200 μL / mL Trizol, let stand at room temperature for 5 min, centrifuge at 12000 rpm at 4℃ for 15 min, the supernatant is RNA, transfer to a new EP tube, add 500 μL of isopropanol, let stand at room temperature for 10 min, centrifuge at 12000 rpm at 4℃ for 10 min, discard the supernatant, wash with 75% ethanol, centrifuge, dry at room temperature for 10 min, dissolve in DEPC water, and determine the RNA concentration and purity using a spectrophotometer, store at -80℃. ② RT-PCR detection: Detect the mRNA expression levels of apoptosis and oxidative stress-related factors according to the LightCycler 480SYBR Green I Master instructions, using GADPH as an internal control.

[0099] The results showed that the expression level of Alox5 gene mRNA increased significantly with increasing doxorubicin concentration. Figure 2 C.

[0100] (3) Western blot detection of Alox5 protein expression in primary cardiomyocytes

[0101] ① Protein Extraction and Quantification: Nuclear protein was calculated using a nuclear and cytoplasmic protein extraction kit. Protein concentrations were standardized before all Western blotting. Cells were seeded in six-well plates according to the following groups: PBS group, 0.25 μmol / L DOX, 0.5 μmol / L DOX, and 1 μmol / L DOX group. After 24 h of culture, 30 mL of protein lysis buffer (1 mL of protein lysis buffer contains 10 μL of 1 mmol / L benzyl sulfonyl fluoride, 50 μL of 1 mmol / L sodium chloride, 10 μL of 1 mmol / L sodium vanadate, 100 μL of Roche phosphatase inhibitor, 100 μL of complete, and 730 μL of RIFA strong lysis buffer) was added to each well. Cells from each group were collected and transferred to 1.5 mL EP tubes. Cells were then lysed 15-20 times using an ultrasonic lysate (5 kHz) and centrifuged at 12000 rpm for 15 min at 4 °C. Protein concentration was quantified using the BCA method. After denaturation at 72°C, the protein was stored at -80°C. ② Western blotting procedure: Protein electrophoresis was performed using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were transferred to a PVDF membrane, blocked for 2 hours, and then incubated overnight at 4°C with primary antibody. The next day, the membrane was washed with TBST buffer, incubated with secondary antibody for 1.5 hours, and developed using a chemical scanning membrane analyzer. Protein bands were analyzed using ImageLab software, with GADPH as an internal control.

[0102] The results showed that the expression level of Alox5 gene protein increased significantly with increasing doxorubicin concentration; compared with the NS group, the expression level of Alox5 gene nuclear protein was also increased (see [reference needed]). Figure 2 D、 Figure 2 F.

[0103] (4) Detection of subcellular localization of Alox5 gene in NRVM cells using cell fluorescence technology

[0104] Wash the cell-covered slides in the culture plate three times with PBS for 3 minutes each time; fix the slides with 4% paraformaldehyde for 15 minutes, then wash the slides three times with PBS for 3 minutes each time; add 0.5% Triton... X-100 (prepared with PBS) was used for permeation at room temperature for 20 min. The slides were then washed three times with PBS for 3 min each time. The PBS was blotted dry with absorbent paper. Normal goat serum was added to the slides, and the slides were blocked at room temperature for 30 min. The blocking solution was then blotted off with absorbent paper, but the slides were not washed. Sufficient diluted primary antibody was added to each slide, and the slides were placed in a humidified chamber and incubated overnight at 4°C. The next day, fluorescent secondary antibody was added: the slides were washed three times with PBST for 3 min each time. The excess liquid on the slides was blotted dry with absorbent paper, and the diluted fluorescent secondary antibody was added. The slides were incubated in a humidified chamber at 20-37°C for 1 h. The slides were then washed three times with PBST for 3 min each time. Note: From the time the fluorescent secondary antibody was added, all subsequent steps should be performed in a dark place as much as possible. Counterstaining of the nucleus: DAPI was added and incubated in the dark for 5 min to stain the nucleus. The excess DAPI was washed off with PBST for 5 min × 4 times. The liquid on the slides was blotted dry with absorbent paper, and the slides were mounted with mounting solution containing anti-fluorescence quencher. The images were then observed and acquired under a fluorescence microscope.

[0105] The results showed that, compared with the PBS group, the doxorubicin group exhibited higher fluorescence intensity in primary cardiomyocytes, with the subcellular localization being the cell nucleus. Figure 2 E.

[0106] Example 3: Alox5 overexpression accelerates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy.

[0107] (1) Establishing an adriamycin-induced myocardial injury model in rAAV9-Alox5 mice

[0108] Two weeks ago, the experimental group received a tail vein injection of 200 μl of normal saline containing ≥5*10 11 Recombinant rAAV9-Alox5-CMV was administered via vg. The control group was injected with an equal volume of physiological saline. Ten male 11-week-old rAAV9-Alox5 mice and their littermate control mice (rAAV9-GFP) were randomly divided into a control group and an experimental group, totaling four groups: rAAV9-GFP, rAAV9-Alox5, rAAV9-GFP+DOX, and rAAV9-Alox5+DOX, with 10 mice in each group. An doxorubicin-induced myocardial injury model was established according to the method described in Example 1.

[0109] (2) Weighing the small animals and measuring the heart weight and tibia length of the mice

[0110] The results showed that compared with the rAAV9-GFP+DOX group, the rAAV9-Alox5+DOX group mice had decreased body weight, heart weight, and HW / TL, suggesting that Alox5 overexpression accelerates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy. Figure 3 BC.

[0111] (3) Small animal ultrasound evaluation of mouse cardiac diastolic and systolic function and hemodynamic assessment of mouse cardiac function

[0112] Mice were anesthetized with isoflurane, and echocardiography was performed using a MyLab 30CV system. Left ventricular (LV) morphology was measured over five consecutive cardiac cycles, including left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameter (LVEDd), left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF). Hemodynamics were measured using cardiac catheterization as previously described. Hemodynamic parameters such as cardiac output (CO), maximum pressure incidence (dP / dt max), and minimum pressure decay rate (dP / dt min) were examined.

[0113] The results showed that compared with the rAAV9-GFP+DOX group mice, the rAAV9-Alox5+DOX group mice had decreased LVEF, LVFS, CO, (dP / dt max), and (dP / dt min), while their LVEDd and LVEDs were increased. This suggests that Alox5 overexpression accelerates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy. Figure 3 FK, Figure 3 MO. (4) Expression of serum myocardial injury markers CK-MB, LDH and cTNT.

[0114] The operation method is described in Example 1.

[0115] The results showed that compared with the rAAV9-GFP+DOX group mice, the expression of myocardial injury markers CK-MB, LDH, and cTNT was significantly increased in the rAAV9-Alox5+DOX group mice. Figure 3 PR studies have demonstrated that Alox5 overexpression accelerates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy.

[0116] (5) HE staining to evaluate the gross morphology of the heart

[0117] Procedure: Place paraffin sections on a 55℃ baking machine and bake for 1 hour. Then, sequentially dewax the sections by immersing them in xylene for 10 minutes, followed by xylene for 10 minutes, xylene for 10 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and 50% ethanol for 5 minutes. Next, stain the paraffin sections with hematoxylin for 0.5-1 minute, rinse with tap water, differentiate with 1% hydrochloric acid ethanol for a few seconds, rinse with tap water, then blue with 1% ammonia solution for 1 minute, rinse with running water for a few seconds, stain with eosin for a few seconds, and rinse with running water. Finally, sequentially immerse the paraffin sections in 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and xylene for 5 minutes to achieve transparency. Remove the sections from the xylene solution and mount them with neutral resin.

[0118] The results showed that, compared with the rAAV9-GFP+DOX group, the cardiomyocytes in the rAAV9-Alox5+DOX group were significantly smaller. Figure 3 DE indicates that Alox5 overexpression accelerates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy.

[0119] Example 4: Alox5 overexpression accelerates doxorubicin-induced cardiomyocyte damage

[0120] (1) Construction of primary cardiomyocytes with Alox5 overexpression

[0121] Newborn rat cardiomyocytes (NRVMs) were infected with the adenovirus particle Ad-Alox5 encoding Alox5 at an MOI of 50 for 12 hours, designated as the Ad-Alox5+DOX group; as a control, newborn rat cardiomyocytes (NRVMs) were infected with the empty virus Ad-GFP at an MOI of 50 for 12 hours, designated as the Ad-GFP+DOX group; a total of 4 groups were designated as Ad-GFP, Ad-Alox5, Ad-GFP+DOX, and Ad-Alox5+DOX.

[0122] (2) Detection of NRVM cell viability using CCK-8 assay

[0123] The method is as described in 1.

[0124] The results show: See Figure 4 D. Compared with the Ad-GFP+DOX group, the Ad-Alox5+DOX group had lower cell viability, indicating that Alox5 overexpression accelerates doxorubicin-induced cardiomyocyte damage.

[0125] (3) Lactate dehydrogenase (LDH) release experiment

[0126] LDH release into the supernatant was considered an indicator of cytotoxicity. Following the specified treatment, culture supernatants were collected using a commercially available kit to detect LDH release, following the manufacturer's instructions. The absorbance of the samples was measured at 490 nm and 600 nm using a spectrophotometer.

[0127] The results show: See Figure 4 E. Compared with the Ad-GFP+DOX group, the Ad-Alox5+DOX group had a higher LDH ratio, indicating that Alox5 overexpression accelerates doxorubicin-induced cardiomyocyte damage.

[0128] (4) Detection of Alox5 gene mRNA expression in primary cardiomyocytes by real-time PCR

[0129] Operating method: See Example 1.

[0130] The results show: See Figure 4 C. Compared with the Ad-GFP+DOX group, the Alox5 gene mRNA expression level was significantly increased in the Ad-Alox5+DOX group.

[0131] (5) Western blot detection of Alox5 protein expression in primary cardiomyocytes

[0132] Operating method: See Example 1.

[0133] The results show: See Figure 4 B. Compared with the Ad-GFP+DOX group, the Alox5 gene protein expression level was significantly increased in the Ad-Alox5+DOX group.

[0134] Example 5: Knockdown of Alox5 prevents myocardial damage and improves cardiac function deterioration in doxorubicin cardiomyopathy

[0135] (1) Establishment of an adriamycin-induced myocardial injury model in Ko-Alox5 mice

[0136] Ko-Alox5 mice were constructed by knocking out the gene sequence shown in the NCBI Reference Sequence: Location: Chr6:116387038-116438139bp, -strand Genetic Position: Chr6, 53.79cM. Male 11-week-old Ko-Alox5 mice and their littermate control mice were divided into a DOX group and an experimental group (Ko-Alox5+DOX group), for a total of 4 groups, with 10 mice in each group. An doxorubicin-induced myocardial injury model was established according to the method described in Example 1.

[0137] (2) Weighing the small animals and measuring the heart weight and tibia length of the mice

[0138] The results showed that compared with the DOX group mice, the Ko-Alox5+DOX group mice had decreased body weight, heart weight, and HW / TL, suggesting that Alox5 overexpression accelerates myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy. Figure 3 BC.

[0139] (3) Small animal super evaluation of mouse cardiac diastolic and systolic function and hemodynamic assessment of mouse cardiac function are described in Example 3.

[0140] The results showed that, compared with the DOX group mice, the Ko-Alox5+DOX group mice had higher LVEF, LVFS, CO, (dP / dtmax), and (dP / dtmin) values, and lower LVEDd and LVEDs values. Alox5 knockdown improved myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy. Figure 5 FN.

[0141] (4) Expression of serum myocardial injury markers CK-MB, LDH and cTNT

[0142] The expression of myocardial injury markers CK-MB, LDH, and cTNT in the serum of Ko-Alox5+DOX group and DOX group mice was detected by ELISA to determine the degree of myocardial injury.

[0143] The results showed that, compared with the DOX group, the Ko-Alox5+DOX group had significantly lower expression of myocardial injury markers CK-MB, LDH, and cTNT. Figure 5 OQ demonstrates that lowering Alox5 can improve myocardial damage and worsening cardiac function in doxorubicin cardiomyopathy.

[0144] (5) HE staining to evaluate the gross morphology of the heart

[0145] See Example 3 for instructions.

[0146] The results showed that, compared with the DOX group, the Ko-Alox5+DOX group had larger cardiomyocytes, demonstrating that Alox5 reduction can improve myocardial damage and cardiac function deterioration in doxorubicin cardiomyopathy. Figure 5 DE.

[0147] Example 6: Alox5 knockdown alleviates doxorubicin-induced cardiomyocyte damage

[0148] (1) Construction of primary cardiomyocytes with Alox5 knockdown

[0149] To knock down Alox5 in primary cardiomyocytes, cells were transfected with Alox5-targeting siRNA (abbreviated as Si-Alox5, sequence as shown in SEQ ID NO:1: 5′-GCAAGAGACCTCATGTTT-3′) for 8 h, designated as the Si-Alox5+DOX group; cells were transfected with scRNA (sequence as shown in SEQ ID NO:2: 5′-GCTGCACAGAGTTGCCTAA-3′) for 8 h, designated as the DOX group.

[0150] (2) Detection of NRVM cell viability using CCK-8 assay

[0151] The method is as described in Example 2.

[0152] The results show: See Figure 6 D. Compared with the DOX group, the Si-Alox5+DOX group had lower cell activity, indicating that Alox5 knockdown alleviated doxorubicin-induced cardiomyocyte damage.

[0153] (3) LDH release experiment

[0154] The method is as described in Example 4.

[0155] The results show: See Figure 6 E. Compared with the DOX group, the Si-Alox5+DOX group had a lower LDH ratio, indicating that Alox5 knockdown alleviated cardiomyocyte damage caused by doxorubicin.

[0156] (4) Detection of Alox5 gene mRNA expression in primary cardiomyocytes by real-time PCR

[0157] The method is as described in Example 2.

[0158] The results show: See Figure 6 C. Compared with the DOX group, the expression level of Alox5 gene mRNA in the Si-Alox5+DOX group was significantly reduced.

[0159] (5) Western blot detection of Alox5 protein expression in primary cardiomyocytes

[0160] The method is as described in Example 2.

[0161] The results show: See Figure 6 B. Compared with the DOX group, the expression level of Alox5 gene protein in the Si-Alox5+DOX group was significantly reduced.

[0162] Example 7: Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis

[0163] (1) The mRNA expression of PTGS2, GPX4, SLC7A11, TFR, FPN and FTH in cardiac tissue was detected by real-time PCR. There were a total of 6 groups: NS group, DOX group, Ko-Alox5 group, Ko-Alox5+DOX group, DOX+Fer-1 group and DOX+DXZ group.

[0164] The operation method is described in Example 1.

[0165] The results showed that compared with the DOX group, the mRNA expression levels of PTGS2, TFR, and FTH were significantly decreased in the Ko-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group, while the expression levels of GPX4, SLC7A11, and FPN were significantly decreased. Figure 7 D,Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0166] (2) Western blot analysis of the expression levels of GPX4, TFR, FPN, and Ferritin proteins in cardiac tissue.

[0167] The operation method is described in Example 1.

[0168] The results showed that, compared with the DOX group, the expression levels of TFR and Ferritin proteins were significantly decreased in the Ko-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group, while the expression levels of GPX4 and FPN were significantly decreased. Figure 7 EI, Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0169] (3) Expression levels of MDA / non-hen in serum and cardiac homogenate

[0170] For operating instructions, please refer to the MDA / non-hene operating kit.

[0171] The results showed that, compared with the DOX group, the expression of MDA / non-hen in serum and cardiac homogenate was significantly decreased in the Ko-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group. Figure 7 JM, Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0172] (4) Expression levels of GSH and NAPDH in cardiac homogenate

[0173] For operating instructions, please refer to the GSH and NAPDH kit instructions.

[0174] The results showed that compared with the DOX group, the expression levels of NAPDH in cardiac homogenates from the Ko-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group were significantly decreased, while the expression levels of GSH were significantly upregulated. Figure 7 NO, Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0175] (5) Immunohistochemistry

[0176] The operation method is described in Example 1.

[0177] The results showed that compared with the DOX group, the expression level of GPX4 was significantly increased in the Ko-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group, while the expression level of 4-HNE was significantly decreased. Figure 7 PQ, Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0178] (6) DHE staining

[0179] For operating instructions, please refer to the DHE kit instructions.

[0180] The results showed that, compared with the DOX group, the levels of ROS in cardiac tissue were significantly decreased in the Ko-Alox5+DOX group, the DOX+Fer-1 group, and the DOX+DXZ group. Figure 7 R,Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0181] (7) Omics

[0182] Procedure: Total RNA was isolated from heart tissues of DOX+Ko-Alox5 and DOX mice using TRIzol reagent and the RNeasy mini kit (QIAGEN). RNA quality was assessed using a 2100 bioanalyzer, demonstrating high RNA purity and integrity. RNA-seq libraries were prepared according to the NEB Next μLtra II Directive RNA Library Prep Kit (England Biolabs) and sequenced using an Illumina HiSeq 2500 System (60 bp single read). Library preparation and sequencing were performed by the Genomics Department of CNIC. FastQ sequencing files were then processed and analyzed using the network tool RNA-seq. Differentially expressed genes between groups were determined using Limma's moderate t-test, adjusted by Benjamini-Hochberg (P < 0.1). Genome enrichment analysis was performed using KEGG. Only significant enrichment with a Benjamini-Hochberg adjusted P value less than 0.01 was considered. For heatmaps, the frontier gene set from the selected enrichment pathways is extracted, annotated with their respective TPM values, then converted to raw Z-scores and imported into GraphPadPrism 8.0 (GraphPad Software, USA) for heatmap generation.

[0183] The results showed that, compared with the DOX group, the Ko-Alox5+DOX group had significant differences in ferroptosis and inflammatory pathways. Figure 7 AC.

[0184] Example 8: Alox5 knockdown improves doxorubicin cardiomyopathy by preventing ferroptosis

[0185] (1) Quantitative real-time PCR was used to detect the mRNA expression of PTGS2, GPX4, SLC7A11, TFR, FPN, and FTH in primary cardiomyocytes. There were a total of 6 groups: PBS group, DOX group, si-Alox5 group, si-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group.

[0186] The operation method is described in Example 2.

[0187] The results showed that compared with the DOX group, the mRNA expression levels of PTGS2, TFR, and FTH were significantly reduced in the Si-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group, while the expression levels of GPX4, SLC7A11, and FPN were significantly reduced. Figure 8 F,Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0188] (2) The procedure for detecting the expression levels of GPX4, TFR, FPN and Ferritin proteins in primary cardiomyocytes by Western blot is described in Example 2.

[0189] The results showed that, compared with the DOX group, the expression levels of TFR and Ferritin proteins were significantly decreased in the Si-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group, while the expression levels of GPX4 and FPN were significantly decreased. Figure 8 GK, Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0190] (3) Expression levels of GSH, NAPDH and MDA in primary cardiomyocyte homogenate proteins.

[0191] For operating instructions, please refer to the GSH, NAPDH, and MDA kits.

[0192] The results showed that compared with the DOX group, the expression levels of MDA and NAPDH were significantly decreased in the Si-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group, while the expression level of GSH was significantly upregulated. Figure 8 AC. (4) Expression level of ROS in primary cardiomyocytes.

[0193] The procedure involves diluting DCFH-DA with serum-free culture medium at a volume ratio of 1:1000 to achieve a final concentration of 10 μmol / L. After removing the cell culture medium, add an appropriate volume of the diluted DCFH-DA and incubate for 3 hours. Then, wash three times with PBS to remove as much unbound probe as possible. Finally, photograph the sample using an inverted microscope.

[0194] The results showed that, compared with the DOX group, the ROS levels in the Si-Alox5+DOX group, DOX+Fer-1 group, and DOX+DXZ group were significantly reduced. Figure 8 DE,Alox5 knockdown alleviates doxorubicin cardiomyopathy by preventing ferroptosis.

[0195] Example 9: Alox5 knockdown improves inflammation in doxorubicin cardiomyopathy

[0196] (1) Detection of mRNA expression of TNF-α, IL-6, IL-1β, HMGB1 and MCP-1 in cardiac tissue by quantitative real-time PCR.

[0197] The operation is described in Example 1.

[0198] The results showed that compared with the DOX group, the mRNA expression levels of TNF-α, IL-6, IL-1β, HMGB1, and MCP-1 were significantly reduced in the Ko-Alox5+DOX group. Figure 9A indicates that Alox5 knockdown can improve inflammation in doxorubicin cardiomyopathy.

[0199] (2) Western blot was used to detect the expression levels of TNF-α, IL-6 and IL-1β proteins in cardiac tissue.

[0200] The operation is described in Example 1.

[0201] The results showed that, compared with the DOX group, the protein expression levels of TNF-α, IL-6, and IL-1β were significantly reduced in the Ko-Alox5+DOX group. Figure 9 BE indicates that Alox5 knockdown can improve inflammation in doxorubicin cardiomyopathy. (3) Expression of TNF-α, IL-6, and IL-1β in serum and cardiac homogenate.

[0202] The operation is described in Example 1.

[0203] The results showed that, compared with the DOX group, the serum protein expression levels of TNF-α, IL-6, and IL-1β were significantly reduced in the Ko-Alox5+DOX group. Figure 9 FH indicates that Alox5 knockdown can improve inflammation in doxorubicin cardiomyopathy.

[0204] (4) Immunohistochemistry

[0205] The operation is described in Example 1.

[0206] The results showed that compared with the DOX group, the expression levels of TNF-α, HMGB(1)CD45, and CD68 in the Ko-Alox5+DOX group were significantly reduced (see 9I-L), indicating that Alox5 knockdown can improve inflammation in doxorubicin cardiomyopathy.

[0207] Example 10: Alox5 knockdown improves inflammation in doxorubicin cardiomyopathy

[0208] (1) Detection of mRNA expression of TNF-α, IL-6, IL-1β, HMGB1 and MCP-1 in primary cardiomyocytes by quantitative real-time PCR.

[0209] See Example 2 for instructions.

[0210] The results showed that, compared with the DOX group, the mRNA expression levels of TNF-α, IL-6, IL-1β, HMGB1, and MCP-1 were significantly reduced in the Si-Alox5+DOX group. Figure 10 A indicates that Alox5 knockdown can improve inflammation in doxorubicin cardiomyopathy.

[0211] (2) Western blot was used to detect the expression levels of TNF-α, IL-6 and IL-1β proteins in primary cardiomyocytes.

[0212] See Example 2 for instructions.

[0213] The results showed that, compared with the DOX group, the expression levels of TNF-α, IL-6, and IL-1β proteins in the Si-Alox5+DOX group were significantly reduced. Figure 10 BE indicates that Alox5 knockdown can improve inflammation in doxorubicin cardiomyopathy.

[0214] As demonstrated by the above examples, the Alox5 gene of this invention has an ameliorative effect on a mouse model of doxorubicin-induced cardiomyopathy. Cardiac function was assessed using echocardiography and hemodynamics; reactive oxygen species levels, ferroptosis, and inflammation-related markers were detected using histological staining in mice and primary cardiomyocytes; and molecular biology was used to detect ferroptosis and inflammation-related protein and mRNA levels in mice and primary cardiomyocytes. These findings indicate that the Alox5 gene can improve doxorubicin-induced cardiomyopathy. In conclusion, Alox5 is found to be applicable as a drug for the prevention and treatment of doxorubicin-induced cardiomyopathy and its complications.

Claims

1. The use of Alox5-targeting siRNA in the preparation of a drug for treating doxorubicin-induced myocardial injury, characterized in that: The sequence of the Alox5-targeting siRNA is shown in SEQ ID NO:1, and it is used to inhibit the expression of Alox5 protein by the Alox5 gene.

2. The use according to claim 1, characterized in that: The drug uses siRNA that targets Alox5 as its sole active ingredient.

3. The use according to claim 1, characterized in that: The drug comprises a combination of siRNA targeting Alox5 and other active ingredients, which are used to treat doxorubicin-induced myocardial injury; The combination of the Alox5-targeting siRNA and other active ingredients is selected from any of the following forms: (i) Formulate the Alox5-targeting siRNA and other active ingredients separately; (ii) Formulate a compound preparation by combining Alox5-targeting siRNA with other active ingredients.

4. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 2, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

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