Application of S-adenosylmethionine or salt thereof in preparation of medicine for preventing and treating ATO cardiotoxicity

Abstract

The invention relates to application of S-adenosylmethionine or salt thereof in preparation of a medicine for preventing and treating ATO cardiotoxicity, and belongs to the field of biological medicine. In order to solve the problem that a general ferroptosis inhibitor cannot prevent and treat ATO cardiotoxicity from the source, the invention provides application of S-adenosylmethionine or salt thereof in preparation of drugs for preventing and treating ATO cardiotoxicity. The invention discloses a key molecular pathway of cardiac toxicity caused by ATO: ATO methylation metabolism exhausts endogenous methyl donors of myocardial cells, causes methylation imbalance of histone H3K9me3, and causes abnormal high expression of ferroptosis key protein HO-1 and mitochondrial iron overload. On the basis, the invention creatively provides a targeted intervention strategy of exogenous supplementation of S-adenosylmethionine, methylation imbalance and ferroptosis pathway activation are blocked from the upstream, root prevention and treatment of ATO cardiotoxicity are realized, and cardiac insufficiency is improved. The targeted intervention strategy is good in safety and has the advantages of clinical transformation and long-term application.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of S-adenosylmethionine or its salt in the preparation of drugs for the prevention and treatment of ATO cardiotoxicity. Background Technology

[0002] Arsenic trioxide (ATO) is a first-line treatment for acute promyelocytic leukemia (APL), significantly improving complete remission and long-term survival rates, and holds an irreplaceable position in clinical treatment. However, ATO treatment is prone to causing serious cardiotoxicity such as QT interval prolongation, ventricular arrhythmias, and even sudden cardiac death, which greatly limits its clinical application, often leading to treatment interruption or even endangering patients' lives, becoming a key bottleneck restricting the full realization of ATO's clinical value.

[0003] Existing research confirms that ferroptosis, as a novel cell death mechanism driven by iron-dependent lipid peroxidation, is a core pathway mediating ATO myocardial injury. It can be directly activated by ATO and act as an upstream node, amplifying secondary damage such as oxidative stress and inflammation, forming a multi-pathway synergistic damage network. Therefore, targeting ferroptosis has become a key direction for preventing and controlling ATO cardiotoxicity.

[0004] However, currently used ferroptosis inhibitors, such as Fer-1, Lip-1, and DFO, are all designed based on a universal ferroptosis model and only target downstream effects of ferroptosis, such as directly clearing lipid peroxides or chelating free iron. This is a passive blockade of damage; they cannot inhibit the initiation of ferroptosis at its root and are difficult to effectively reverse or block severe myocardial damage caused by ATO. Long-term use may interfere with normal cellular iron metabolism and pose potential safety risks.

[0005] Therefore, elucidating the core role and regulatory mechanism of ferroptosis in ATO-induced cardiotoxicity and developing targeted interventions to fundamentally regulate the ATO-specific ferroptosis pathway are key challenges that urgently need to be addressed. This is of great significance for ensuring the safety of ATO in clinical applications and expanding its clinical value. Summary of the Invention

[0006] To address the problem that existing general-purpose ferroptosis inhibitors cannot fundamentally prevent and treat ATO cardiotoxicity, this invention provides the application of S-adenosylmethionine or its salt in the preparation of drugs for preventing and treating ATO cardiotoxicity.

[0007] The technical solution of the present invention:

[0008] Application of S-adenosylmethionine or its salts in the preparation of drugs for the prevention and treatment of ATO cardiotoxicity.

[0009] Furthermore, the salt is a pharmaceutically acceptable salt of S-adenosylmethionine, selected from one or more of hydrochloride, sulfate, phosphate, citrate, maleate, fumarate, tartrate, or succinate.

[0010] Furthermore, the drug for preventing ATO cardiotoxicity is prepared with S-adenosylmethionine or its pharmaceutically acceptable salt as the core active ingredient, supplemented with pharmaceutically acceptable carriers, excipients or excipients.

[0011] Furthermore, the dosage form of the drug for preventing ATO cardiotoxicity is an oral preparation or an injectable preparation; the oral preparation is selected from tablets, capsules, granules, suspensions or oral solutions, and the injectable preparation is selected from injection solutions, lyophilized powder injections or infusion solutions.

[0012] Furthermore, the purity of S-adenosylmethionine or its pharmaceutically acceptable salt in the injectable formulation is not less than 98%.

[0013] Furthermore, the drug for preventing ATO cardiotoxicity has at least one of the following uses:

[0014] (1) Reduces iron overload in cardiomyocytes caused by ATO and reduces ferroptosis in cardiomyocytes;

[0015] (2) Improve heart failure caused by ATO.

[0016] The beneficial effects of this invention are:

[0017] This invention reveals for the first time the key molecular pathway of ATO-induced cardiotoxicity—ATO triggers the depletion of endogenous methyl donors in cardiomyocytes via arsine methyltransferase, leading to an imbalance in histone H3K9me3 methylation modification, ultimately triggering abnormally high expression of the key ferroptosis protein HO-1, initiating a pathological chain of mitochondrial iron overload. This provides a novel target for the development of targeted drugs against ATO-induced cardiotoxicity. Based on the discovery of this pathway and target, this invention innovatively proposes a targeted intervention strategy of exogenously supplementing the methyl donor S-adenosylmethionine (SAM). By directly supplementing the methyl donor, it blocks the upstream activation of methylation imbalance and ferroptosis pathways, achieving precise prevention and treatment of ATO-induced cardiotoxicity at its root cause, significantly improving the accuracy and effectiveness of ferroptosis control. This invention inhibits upstream nodes of ferroptosis, blocking the initiation of cardiomyocyte ferroptosis, and simultaneously blocking its amplification effect on secondary damage pathways such as oxidative stress and inflammation, achieving overall inhibition of multi-pathway synergistic damage.

[0018] This invention, through animal experiments, demonstrates that exogenous supplementation with S-adenosylmethionine (SAM) significantly improves ATO-induced cardiac failure in mice, overcoming the limitation of existing universal ferroptosis inhibitors that only alleviate local damage, and achieving an integrated prevention and treatment effect of "damage blocking and functional repair." This invention works by regulating endogenous epigenetic metabolic pathways, reducing interference with normal cellular iron metabolism; simultaneously, SAM, as a natural intracellular methyl donor, precisely repairs the depletion of the methyl donor pool after supplementation, possessing both targeted efficacy and clinical translational feasibility, providing a novel solution for improving the safety of ATO clinical medication. Attached Figure Description

[0019] Figure 1 This is a graph showing the GSEA enrichment analysis of the porphyrin metabolic pathway in cardiomyocytes after ATO treatment in Example 1.

[0020] Figure 2 This is a heatmap of differential gene expression in cardiomyocytes between the ATO group and the control group in Example 1;

[0021] Figure 3 This is a volcano diagram of differentially expressed genes in cardiomyocytes of the ATO group and the control group in Example 1;

[0022] Figure 4 This is a comparison of HO-1 protein expression levels in cardiomyocytes of the ATO group and the control group in Example 1. A is a Western blot result, and B is a quantitative statistical graph.

[0023] Figure 5 This is an immunofluorescence image showing the co-localization of HO-1 protein and mitochondria in cardiomyocytes of the ATO group and the control group in Example 1.

[0024] Figure 6 The image shows a comparison of the protein expression levels of HO-1, GPX4, FTH-1, FTL-1, and GAPDH in four groups of cardiomyocytes in Example 1. A is a Western blot result, B is a quantitative statistical graph of HO-1, C is a quantitative statistical graph of GPX4, D is a quantitative statistical graph of FTH-1, and E is a quantitative statistical graph of FTL-1.

[0025] Figure 7 This is a comparison chart of JC-1 staining results of four groups of cardiomyocytes in Example 1. A is a JC-1 staining chart, and B is a quantitative statistical chart.

[0026] Figure 8 The diagram shows the screening and analysis of differentially expressed genes related to histone modifying enzymes in Example 2. A is the Venn diagram of the intersection of histone methyltransferases and differentially expressed genes in the transcriptome, and B is the volcano bubble diagram of differentially expressed histone modifying enzymes.

[0027] Figure 9This is a comparison of the expression levels of H3K9me1, H3K9me2, and H3K9me3 proteins in cardiomyocytes of the ATO group and the control group in Example 2.

[0028] Figure 10 This is a comparison of the expression levels of H3K9me3, H3, SUV39, HO-1, FTH-1, FTL-1, GPX4, and GAPDH proteins in three groups of cardiomyocytes in Example 2. A is the Western blot result, B is the quantitative statistical graph of SUV39, C is the quantitative statistical graph of H3K9me3, D is the quantitative statistical graph of HO-1, E is the quantitative statistical graph of FTH, F is the quantitative statistical graph of FTL, and G is the quantitative statistical graph of GPX4.

[0029] Figure 11 This is a comparison of the JC-1 staining results of three groups of cardiomyocytes in Example 2. A is the JC-1 staining image, and B is the quantitative statistical graph.

[0030] Figure 12 This is a comparison chart of the survival rates of the three groups of mice in Example 3;

[0031] Figure 13 The images show photographs of the heart morphology of the three groups of mice in Example 3 and HE-stained longitudinal sections of the mouse heart. A is a photograph of the heart morphology, and B is an HE-stained image.

[0032] Figure 14 This is a comparison chart of the heart weight / body weight ratio of the three groups of mice in Example 3;

[0033] Figure 15 This is a comparison of WGA staining results of heart sections from three groups of mice in Example 3. A is a WGA staining image, and B is a statistical diagram of the cross-sectional area of ​​myocardial cells.

[0034] Figure 16 The images show a comparison of echocardiographic results of three groups of mice in Example 3. A is an echocardiogram, B is a comparison of left ventricular ejection fraction, and C is a comparison of left ventricular shortening fraction.

[0035] Figure 17 The images show a comparison of Masson staining results for heart sections from three groups of mice in Example 3. A is a Masson staining image, and B is a comparison of collagen levels in heart sections.

[0036] Figure 18 The image shows a comparison of the 4HNE immunohistochemical results of heart slices from three groups of mice in Example 3. A is the 4HNE immunohistochemical result image, and B is the 4HNE immunohistochemical statistical graph.

[0037] Figure 19The image shows a comparison of the HO-1 immunohistochemical results of heart slices from three groups of mice in Example 3. A is the HO-1 immunohistochemical result image, and B is the HO-1 immunohistochemical statistical graph.

[0038] Figure 20 The image shows a comparison of Prussian blue staining results for three groups of mouse heart sections in Example 3. A is the Prussian blue staining image, and B is a statistical chart of relative iron deposition area. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0040] Example 1

[0041] This embodiment reveals the core pathological mechanism by which ATO upregulation of HO-1 induces cardiomyocyte ferroptosis through in vitro cell experiments.

[0042] HO-1 (heme oxygenase-1) is a core protein in the porphyrin metabolic pathway, primarily catalyzing the degradation of heme to generate free iron ions, biliverdin, and carbon monoxide. Under normal conditions, HO-1 is expressed at low levels, maintaining only basic iron metabolism regulation and intracellular iron homeostasis. However, when HO-1 is abnormally overexpressed, it leads to iron accumulation and exacerbates oxidative stress, thereby inducing ferroptosis.

[0043] To investigate the effect of ATO on HO-1 expression in cardiomyocytes, this study used primary rat cardiomyocytes as the research subject, and the cells were grown to a size of 5 × 10⁻⁶. 6 The cells were divided into two groups:

[0044] (1) Control group: The cells were treated with a replacement solution;

[0045] (2) ATO group: treated with 10 μM ATO for 24 hours.

[0046] (a) Transcriptomics testing

[0047] RNA was extracted from two groups of cells for transcriptomics analysis to obtain expression levels of all genes in both groups. Pathway enrichment analysis was performed on the RNA-seq data using GSEA software to obtain... Figure 1 The pathway enrichment analysis diagram is shown; differentially expressed genes between the two groups are screened, and generated... Figure 2The differential gene expression heatmap shown is as follows: Figure 3 The volcano diagram shown.

[0048] Figure 1 GSEA analysis results showed that ATO significantly upregulated the porphyrin metabolic pathway. Figure 2 and Figure 3 The expression level of the Hmox1 gene (heme oxygenase 1 gene), which encodes HO-1, increased after ATO treatment, indicating that ATO significantly upregulated HO-1 expression. This suggests that ATO increases iron ion release by upregulating HO-1, leading to ferroptosis in cardiomyocytes.

[0049] (ii) Western blot detection

[0050] Two groups of cellular proteins were extracted and Western blotted using standard methods to detect HO-1 protein expression levels. Figure 4 As shown in the comparison figure, the HO-1 protein expression level in the ATO group was significantly increased compared with the control group.

[0051] (III) Immunofluorescence detection

[0052] Primary rat cardiomyocytes were seeded onto confocal culture dishes and cultured until 60-80% confluence. They were divided into a control group (cells were treated with a different medium) and an ATO group (treated with 10 μM ATO for 24 hours). Mito-tracker staining, cell fixation, permeabilization, blocking, HO-1 antibody incubation, nuclear DAPI staining, and mounting imaging were performed in both groups. Figure 5 The image shows the co-localization immunofluorescence image of HO-1 protein and mitochondria. Green fluorescence represents the location of the HO-1 protein, red fluorescence represents the location of the mitochondrial probe Mito-Tracker, and blue fluorescence (DAPI) represents the location of the cell nucleus. Figure 5 As shown, compared with the control group, ATO significantly increased HO-1 expression. The green fluorescence of HO-1 in the ATO group highly overlapped with the red fluorescence of mitochondria, indicating that after ATO treatment, HO-1 protein was mainly located in mitochondria. At the same time, compared with the control group, ATO significantly reduced the red fluorescence intensity of mitochondria, indicating that it caused mitochondrial dysfunction.

[0053] To further verify the role of HO-1 in ATO-induced cardiomyocyte ferroptosis, this study used si-HO-1 transfection to rat primary cardiomyocytes. The effect of HO-1 downregulation on ATO-induced cardiomyocyte ferroptosis was investigated by knocking down HO-1 expression. The specific experimental method is as follows:

[0054] Rat primary cardiomyocytes were extracted and cultured until the cells grew to 5 × 10⁶. 6 The cells were divided into four groups:

[0055] (1) Control group: The cells were treated with a different medium;

[0056] (2) ATO group: treated with 10 μATO for 24 hours;

[0057] (3) ATO + si-HO-1 group: transfected with si-HO-1, 24h later, 10μM ATO was administered;

[0058] (4) ATO + si-NC group: transfected with si-NC, 24h later, 10μM ATO was administered.

[0059] siRNA transfection of primary rat cardiomyocytes was performed using jetPRIME as the transfection reagent according to conventional methods. The sense strand sequence of si-HO-1 siRNA is shown in SEQ ID No:1, and the antisense strand sequence is shown in SEQ ID No:2. Specifically, the sense strand sequence is 5'-CCACCAAGUUCAAACAGCUTT-3'; the antisense strand sequence is 5'-AGCUGUUUGAACUUGGUGGTT-3'.

[0060] To comply with WIPO ST.26 standards, uracil in RNA is represented by T in the nucleotide sequence shown in SEQ ID NO:1, where T replaces U at positions 9, 10, and 19 at the 5' end, and uracil in RNA is represented by T in the nucleotide sequence shown in SEQ ID NO:2, where T replaces U at positions 4, 6, 7, 8, 13, 14, and 17 at the 5' end.

[0061] The negative control si-NC is a non-targeted sequence that has no homology with known genes in the rat genome and does not produce a gene knockout effect. It is used only as a negative control for transfection and experimentation.

[0062] (a) Western blot detection

[0063] Four groups of cellular proteins were extracted and their expression levels of HO-1, GPX4 (glutathione peroxidase 4), FTH-1 (ferritin heavy chain 1), FTL-1 (ferritin light chain 1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were detected by Western blot using standard methods. Figure 6 The comparison diagram shows that, compared with the control group, ATO treatment alone increased HO-1 expression in cardiomyocytes, while significantly decreasing the core inhibitor of ferroptosis, GPX4, indicating the initiation of ferroptosis. The iron storage protein FTH / FTL was significantly increased, indicating that cells reduce free iron by increasing iron storage, which is a compensatory response to ferroptosis.

[0064] After knocking down HO-1, HO-1 expression was significantly downregulated, GPX4 was higher than in the ATO group, and FTH / FTL was lower than in the ATO group, indicating that the level of ferroptosis inhibitory factor was restored and the compensatory iron storage response was weakened. This suggests that knocking down HO-1 alleviated the ferroptosis stress in cardiomyocytes caused by ATO, implying that HO-1 is a promoting factor for ATO-induced ferroptosis in cardiomyocytes.

[0065] (ii) JC-1 staining detection

[0066] Four groups of cells were stained with a commercial JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbonyl cyanide iodide) staining kit and observed under a fluorescence microscope. Figure 7 The staining results shown indicate that, compared with the control group, the ATO group showed a significant increase in green fluorescence, indicating that ATO significantly reduced mitochondrial membrane potential and caused severe damage, consistent with the characteristics of ferroptosis in mitochondria. However, after knocking down HO-1, red fluorescence was restored and green fluorescence was reduced, indicating that the degree of mitochondrial membrane potential decline was alleviated and mitochondrial damage was reduced.

[0067] The results of this embodiment show that ATO upregulates HO-1, induces mitochondrial iron overload, and leads to cardiomyocyte ferroptosis. Knocking down HO-1 can reverse ATO-induced HO-1 upregulation, iron deposition, ferroptosis, and mitochondrial dysfunction in cardiomyocytes. This indicates that HO-1 is a key factor promoting mitochondrial damage during ATO-induced cardiomyocyte ferroptosis.

[0068] Example 2

[0069] This embodiment elucidates, through in vitro experiments, the regulatory mechanism by which ATO metabolism consumes endogenous SAM, leading to an imbalance in the methylation modification of histone H3K9me3 (trimethylation of lysine at position 9 of histone H3), and upregulating HO-1 to cause ferroptosis in cardiomyocytes.

[0070] SAM (S-adenosylmethionine), as the only methyl donor in the cell, not only participates in arsenic methylation but also extensively regulates various epigenetic modification processes, including DNA methylation, RNA methylation, and histone methylation. Arsenic methylation metabolism depletes SAM, leading to the depletion of the intracellular methyl donor pool, which in turn causes an imbalance in epigenetic modifications, affecting chromatin structure and transcriptional regulation. H3K9me3 is an epigenetic marker for transcriptional silencing. When SAM levels decrease, H3K9me3 modification of HO-1 is weakened, and HO-1 transcription is activated; when SAM levels increase, H3K9me3 modification of HO-1 is enhanced, and HO-1 transcription is inhibited.

[0071] This embodiment uses SAM and ATO to co-treat cardiomyocytes to investigate and verify the core role of H3K9me3-mediated epigenetic regulation in ATO-induced HO-1 expression and myocardial ferroptosis, while also clarifying the rescue function of SAM as a methyl donor. Specifically, primary rat cardiomyocytes were extracted and cultured until they reached a growth rate of 5 × 10⁶ cells / year. 6 The cells were divided into three groups:

[0072] (1) Control group: The cells were treated with a change of medium.

[0073] (2) ATO group: treated with 10 μM ATO for 24 h.

[0074] (3) ATO+SAM group: After pretreatment with 5μM SAM for 2h, it was treated with 10μM ATO for 24h.

[0075] (a) Transcriptomics testing

[0076] RNA was extracted from two groups of cells for transcriptomics analysis. The differentially expressed genes in the transcriptome were then intersected with histone modifying enzymes from a public database to obtain... Figure 8 The intersection Venn diagram and volcano bubble diagram shown indicate that five modifying enzymes changed, with SUV39 (H3K9 methyltransferase) being downregulated most significantly.

[0077] (ii) Western blot detection

[0078] Western blot was used to detect the protein expression levels of H3K9me1 (monomethylation of histone H3 at lysine 9), H3K9me2 (dimethylation of histone H3 at lysine 9), and H3K9me3 in primary cardiomyocytes of rats in the control and ATO groups. Figure 9 As shown in the comparison figure, compared with the control group, the expression level of H3K9me3 protein in the ATO group was significantly downregulated, indicating that ATO can specifically inhibit the modification of histone H3K9me3 in cardiomyocytes. Combined with the experimental results of ATO-induced high expression of HO-1 in Example 1, it suggests that ATO relieves its transcriptional repression of the Hmox1 gene by downregulating the epigenetic regulation of H3K9me3 modification, thereby upregulating the expression of HO-1.

[0079] To investigate whether H3K9me3 binds to HO-1 in cardiomyocytes, this study retrieved Chip-seq sequencing results of H3K9me3 in mouse cardiomyocytes from the Cistrome DB (Chromatin Regulationome Database). The results showed that H3K9me3 and HO-1 significantly bind in mouse cardiomyocytes.

[0080] Western blot was used to detect the expression levels of H3K9me3, H3 (histone H3), SUV39, HO-1, FTH-1, FTL-1, GPX4, and GAPDH proteins in each group of cells. Figure 10 As shown in the comparison figure, compared with the control group, ATO treatment significantly downregulated the expression of H3K9me3, SUV39 and GPX4, while increasing the expression of HO-1, FTH-1 and FTL-1 proteins. SAM supplementation reversed the ATO-induced downregulation of H3K9me3, high expression of HO-1, compensatory increase of ferritin and decrease of GPX4 in cardiomyocytes, suggesting that SAM can block the ATO-induced epigenetic imbalance, iron overload and ferroptosis process from upstream.

[0081] (III) JC-1 staining detection

[0082] JC-1 staining was used to detect changes in mitochondrial membrane potential in three groups of cells. Figure 11 As shown in the comparison figure, compared with the control group, ATO significantly reduced mitochondrial membrane potential, and SAM can reverse the decrease in mitochondrial membrane potential and mitochondrial dysfunction caused by ATO.

[0083] This embodiment experimentally demonstrates that exogenous methyl donor supplementation can restore H3K9me3 modification levels and inhibit HO-1 expression. This result validates the epigenetic regulatory pathway of ATO-induced myocardial ferroptosis: ATO consumes endogenous methyl donors in cardiomyocytes, leading to depletion of their reserves, thereby reducing H3K9me3 modification, relieving the inhibition of HO-1, and ultimately upregulating HO-1 expression, triggering mitochondrial iron overload and myocardial ferroptosis. Clarifying this mechanism provides direct experimental evidence for identifying key molecules in the "methyl donor-H3K9me3-HO-1" pathway and lays a core target foundation for subsequent drug development targeting ATO cardiotoxicity.

[0084] Example 3

[0085] This embodiment verifies, through in vivo experiments, the fundamental preventive and therapeutic effects of exogenous SAM supplementation on ATO-induced heart failure and myocardial ferroptosis in mice.

[0086] To verify whether exogenous SAM supplementation can improve ATO-induced myocardial ferroptosis in vivo, this example divided C57BL / 6 mice into three groups:

[0087] (1) Control group: C57BL / 6 mice, intraperitoneally injected with physiological saline.

[0088] (2) ATO group: C57BL / 6 mice were intraperitoneally injected with 5 mg / kg / d ATO injection solution.

[0089] (3) ATO+SAM group: C57BL / 6 mice were intraperitoneally injected with 25 mg / kg / d SAM injection and 5 mg / kg / d ATO injection.

[0090] Mice were administered the drug continuously for 28 days, and sacrificed on day 29. At the same time, the cardiac function of mice in each group was tested.

[0091] (a) Lifespan

[0092] Record the survival rate of mice in each group and obtain Figure 12 The survival curve comparison chart shows that, compared with the control group, the survival rate of the ATO group continued to decline over time, and the survival rate decreased significantly at 30 days. Although the survival rate of the ATO+SAM group was lower than that of the control group, it was significantly higher than that of the ATO group, and the survival status was closer to that of the control group. This indicates that SAM supplementation can improve the survival rate of mice after ATO treatment and reduce the risk of death, verifying the in vivo protective effect of SAM against ATO toxicity.

[0093] (II) Gross morphology and histopathological sections of the heart

[0094] Hearts were harvested from each group of mice and histopathological sections were stained with hematoxylin and eosin (HE) to obtain... Figure 13 The heart morphology and staining images shown indicate that, compared with the control group, the ATO group showed significant cardiac enlargement and widened intercellular spaces, suggesting edema and significant tissue damage. In contrast, the ATO+SAM group showed significantly reduced cardiac enlargement and significantly improved myocardial cell arrangement and structure. This suggests that SAM supplementation can effectively improve the cardiac pathological changes induced by ATO, restore the normal morphology and tissue structure of the heart, and further corroborate the protective effect of SAM against ATO cardiotoxicity.

[0095] (iii) Heart weight

[0096] Weigh the hearts and bodies of the mice in each group to obtain... Figure 14 The comparison chart of heart weight / body weight ratio shows that, compared with the control group, the ATO group had increased heart weight, while the SAM group had significantly reduced heart weight, essentially returning to a level close to the normal level of the Ctrl group. This indicates that SAM supplementation can effectively alleviate ATO-induced cardiac weight gain.

[0097] (iv) WGA staining detection

[0098] Heart tissue sections from each group of mice were subjected to routine WGA staining to obtain... Figure 15As shown in the comparison figures, compared with the control group, the cardiomyocytes in the ATO group mice were significantly larger in volume and had coarser cell outlines, indicating cardiomyocyte hypertrophy. The cardiomyocytes in the ATO+SAM group were significantly smaller than those in the ATO group, essentially returning to a normal morphology close to that of the Ctrl group. This indicates that SAM supplementation can reduce the cross-sectional area of ​​cardiomyocytes after ATO treatment, maintaining it at a near-normal level, demonstrating the ameliorative effect of SAM on ATO-induced structural damage to cardiomyocytes, and indicating that SAM rescues ATO-induced myocardial hypertrophy.

[0099] (v) Echocardiographic examination

[0100] Before euthanizing the mice in week 4 of the experiment, echocardiography was performed on the mice in each group to obtain... Figure 16 As shown in the comparison chart, compared with the control group, the ATO group exhibited significantly reduced amplitude of myocardial motion waveform fluctuations, smoother signal intensity, and significantly decreased left ventricular ejection fraction and left ventricular short-axis shortening rate, indicating weakened cardiac contractility. In the ATO+SAM group, after SAM supplementation, the amplitude of myocardial motion waveforms recovered significantly compared to the ATO group, and both left ventricular ejection fraction and left ventricular short-axis shortening rate increased substantially. This demonstrates that SAM supplementation can improve ATO-induced cardiac contractile dysfunction, restoring cardiac function indicators to near-normal levels.

[0101] (vi) Masson staining

[0102] Heart tissue sections from each group of mice were subjected to routine Masson staining to obtain... Figure 17 The comparison figures show that, compared with the control group, the ATO group had increased myocardial collagen levels, suggesting that ATO treatment induced significant myocardial fibrosis. In the ATO+SAM group, after SAM supplementation, the collagen fiber area was significantly reduced compared to the ATO group, essentially returning to a state close to the normal state of the Ctrl group. This indicates that SAM supplementation can reduce collagen fiber deposition in myocardial tissue after ATO treatment and improve the degree of myocardial fibrosis.

[0103] (vii) Immunohistochemical detection

[0104] Myocardial tissue was collected from mice in each group, and routine immunohistochemical detection was performed on lipid peroxidation markers 4HNE (4-hydroxynonenal) and HO-1 to obtain... Figure 18 and Figure 19 The comparison chart shows the detection results of 4HNE and HO-1.

[0105] Figure 18 A and Figure 18 B showed that, compared with the control group, the expression of 4HNE in myocardial tissue was increased in the ATO group, indicating that the oxidative damage to myocardial cells was aggravated. The expression of 4HNE in the SAM+ATO group was significantly reduced, which indicates that SAM supplementation can alleviate the damage to myocardial cells caused by oxidative stress induced by ATO.

[0106] Figure 19 A and Figure 19 B showed that, compared with the control group, HO-1 expression was increased in the myocardial tissue of mice in the ATO group, while HO-1 expression was decreased in the myocardial tissue of mice in the SAM+ATO group compared with the ATO group. This indicates that SAM supplementation can downregulate HO-1 expression, thereby blocking the initiation of the ferroptosis pathway upstream.

[0107] (viii) Prussian blue staining

[0108] Myocardial tissue from each group of mice was routinely stained with Prussian blue to obtain... Figure 20 The comparison figures show that, compared with the control group, the ATO group exhibited increased iron deposition in myocardial tissue, indicating that ATO induces myocardial iron overload. The iron deposition area in the ATO+SAM group was significantly reduced compared to the ATO group, falling back to levels close to those of the Ctrl group. This demonstrates that SAM supplementation can reduce ATO-induced myocardial iron overload, an effect directly corresponding to the inhibition of the ferroptosis pathway, further enriching the evidence chain regarding the mechanism by which SAM improves ATO cardiotoxicity.

[0109] The experimental results of this embodiment demonstrate that exogenous SAM supplementation can effectively improve ATO-induced heart failure through multiple pathways. In terms of overall survival, it significantly reduced the mortality rate of ATO-treated mice. Regarding cardiac morphology and structure, it alleviated cardiac enlargement, improved pathological changes such as disordered cardiomyocyte arrangement and widened interstitial spaces, restored heart weight and heart weight / body weight ratio to near-normal levels, and alleviated cardiomyocyte hypertrophy. In terms of cardiac function indicators, echocardiographic examination showed that it restored myocardial motion amplitude, increased left ventricular ejection fraction and short-axis shortening, and enhanced cardiac contractile function. Simultaneously, it reduced myocardial collagen fiber deposition to improve fibrosis, decreased the expression of the lipid peroxidation product 4HNE to alleviate oxidative stress damage, downregulated HO-1 expression and reduced iron ion deposition in myocardial tissue, playing a role in inhibiting key aspects of the ferroptosis pathway and comprehensively reversing the cardiotoxic effects of ATO.

Claims

1. Application of S-adenosylmethionine or its salts in the preparation of drugs for the prevention and treatment of ATO cardiotoxicity.

2. The application according to claim 1, characterized in that, The salt is a pharmaceutically acceptable salt of S-adenosylmethionine, selected from one or more of hydrochloride, sulfate, phosphate, citrate, maleate, fumarate, tartrate, or succinate.

3. The application according to claim 2, characterized in that, The aforementioned drug for preventing ATO cardiotoxicity is prepared with S-adenosylmethionine or its pharmaceutically acceptable salt as the core active ingredient, supplemented with pharmaceutically acceptable carriers, excipients or excipients.

4. The application according to claim 3, characterized in that, The dosage form of the drug for preventing ATO cardiotoxicity is an oral preparation or an injectable preparation; the oral preparation is selected from tablets, capsules, granules, suspensions or oral solutions, and the injectable preparation is selected from injection solutions, lyophilized powder injections or infusion solutions.

5. The application according to claim 4, characterized in that, The purity of S-adenosylmethionine or its pharmaceutically acceptable salt in the injectable formulation is not less than 98%.

6. The application according to claim 5, characterized in that, The drug for preventing and treating ATO cardiotoxicity has at least one of the following uses: (1) Reduces iron overload in cardiomyocytes caused by ATO and reduces ferroptosis in cardiomyocytes; (2) Improve heart failure caused by ATO.

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