Use of slc1a4 inhibitors for the preparation of a medicament for the prevention, alleviation and / or treatment of diabetic cardiomyopathy

Abstract

The application discloses application of SLC1A4 inhibitors in preparation of medicines for preventing, alleviating and / or treating diabetic cardiomyopathy, and belongs to the technical field of biological medicines.The application discloses a new function of SLC1A4 in diabetic cardiomyopathy for the first time, and proves that SLC1A4 can be used as a drug target for preventing and treating diabetic cardiomyopathy.The application has extremely high clinical conversion value and commercial development prospect, and provides a brand-new strategy for treating diabetic cardiomyopathy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of SLC1A4 inhibitors in the preparation of drugs for the prevention, relief and / or treatment of diabetic cardiomyopathy. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by elevated blood sugar levels that can damage multiple organ systems, including the liver, kidneys, and heart. Diabetic cardiomyopathy (DCM) refers to impaired systolic and / or diastolic function of the myocardium in the presence of diabetes (regardless of the presence of other risk factors and diseases). Epidemiological data show that approximately 25%–30% of diabetic patients eventually develop DCM, and its incidence and progression are showing a trend of increasing in tandem with the global obesity epidemic.

[0003] The pathogenesis of diabetic muscular dystrophy (DCM) is highly complex. Besides metabolic disorders involving glucose, lipids, and amino acids, and insulin resistance, pathological processes such as myocardial inflammation cascade, interstitial fibrosis and remodeling, and mitochondrial energy metabolism disorders collectively constitute a vicious cycle of "metabolism-structure-function," exacerbating the progression of DCM. Clinically, simply controlling blood glucose or blood lipids is insufficient to effectively curb the incidence of cardiovascular events. Therefore, in-depth research into the pathogenesis of DCM will provide new ideas and solutions for its clinical prevention and treatment.

[0004] In recent years, ferroptosis, as a novel regulatory necrosis mechanism, has been extensively studied and its crucial role in the progression of diabetic cardiomyopathy has been confirmed. Unlike classic apoptosis, ferroptosis is characterized by iron metabolism disorder, inducing cell death through lipid peroxidation of long-chain polyunsaturated fatty acids in cell membrane phospholipids catalyzed by the Fenton reaction. In a high-glucose microenvironment, increased reactive oxygen species, inhibited glutathione peroxidase 4 (GPX4) activity, and reduced glutathione (GSH) synthesis in cardiomyocytes lead to decreased cellular antioxidant capacity, disruption of membrane structural integrity, and ultimately, the induction of cardiomyocyte ferroptosis, exacerbating heart failure. Notably, amino acid metabolism plays a vital role in regulating ferroptosis. Among them, serine, as a core regulatory amino acid of one-carbon metabolism, plays a crucial role in regulating cardiomyocyte energy homeostasis, oxidative stress levels, and cell survival. Specifically, serine can promote the production of NADPH and glutathione (GSH) by participating in the folate cycle and transsulfurization pathway, thereby enhancing cellular antioxidant capacity and exerting an anti-ferroptosis effect. Clinical evidence shows that serum serine levels are decreased in patients with diabetes and heart failure; metabolomics analysis further indicates that increased cardiac serine efflux is associated with worsening heart failure. Therefore, elucidating the bioavailability of serine and the key transport mechanisms affecting transmembrane transport efficiency may be a potential key to regulating the ferroptosis sensitivity of cardiomyocytes.

[0005] SLC1A4, a member of solute carrier family 1 (SLC1A4), is a neutral amino acid transporter responsible for mediating the transmembrane transport of neutral amino acids such as serine, alanine, cysteine, and threonine. Our previous research has shown that cyclophosphamide-induced premature ovarian failure is closely associated with elevated SLC1A4 expression. Furthermore, studies have reported that SLC1A4 dysfunction is associated with neurological disorders and acute myeloid leukemia. In the cardiovascular system, microarray data analysis (RNA-seq) has shown that SLC1A4 and SLC1A5 are key genes in dilated and hypertrophic cardiomyopathy and are closely related to ferroptosis. Meanwhile, its family member SLC1A5 has been shown to play a crucial role in heart disease through glutamine transport. However, the role of SLC1A4 in heart disease, particularly dilated cardiomyopathy (DCM), has not yet been reported. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides the application of SLC1A4 inhibitors in the preparation of drugs for the prevention, mitigation, and / or treatment of diabetic cardiomyopathy (DCM). This invention reveals for the first time a novel function of SLC1A4 in DCM and confirms its potential as a drug target for the prevention and treatment of DCM. This invention has extremely high clinical translational value and commercial development prospects, providing a completely new strategy for the treatment of diabetic cardiomyopathy.

[0007] To achieve the above objectives, the present invention provides, in one aspect, the use of SLC1A4 inhibitors in the preparation of medicaments for the prevention, relief and / or treatment of diabetic cardiomyopathy, wherein the SLC1A4 inhibitors exert their preventive, relief and / or therapeutic effects by inhibiting myocardial ferroptosis.

[0008] Furthermore, the drug is a substance capable of downregulating the mRNA expression level and protein expression level of SLC1A4, and / or inhibiting the transport activity and biological function of SLC1A4.

[0009] Furthermore, the SLC1A4 inhibitor is selected from at least one of SLC1A4 gene silencing agents and SLC1A4 protein activity inhibitors.

[0010] Furthermore, the SLC1A4 gene silencing agent is selected from at least one of siRNA, shRNA, miRNA, and antisense oligonucleotides; the SLC1A4 protein activity inhibitor is selected from at least one of specific neutralizing antibodies and small molecule compounds.

[0011] Furthermore, the inhibition of cardiomyocyte ferroptosis includes at least one of the following: reducing intracellular lipid peroxidation levels, reducing intracellular ferrous ion content, upregulating GPX4 protein expression levels, downregulating NCOA4 protein expression levels, and upregulating the content of the antioxidant core molecule GSH.

[0012] Furthermore, the drug also includes pharmaceutically acceptable carriers, excipients, or diluents.

[0013] Furthermore, the dosage form of the drug is selected from at least one of injections, tablets, capsules, granules, and aerosols.

[0014] A second aspect of the present invention provides a kit for detecting diabetic cardiomyopathy, comprising a reagent for detecting the expression level of SLC1A4 protein in a biological sample.

[0015] Furthermore, the biological sample is selected from at least one of serum, plasma, peripheral blood mononuclear cells, and myocardial tissue biopsy samples; the reagent for detecting the expression level of SLC1A4 protein includes an antibody that specifically binds to SLC1A4 protein.

[0016] A third aspect of the present invention provides a method for screening candidate drugs for treating diabetic cardiomyopathy, comprising the following steps: (1) Apply the candidate drug to cardiomyocytes expressing SLC1A4 or to a non-human animal model of diabetic cardiomyopathy; (2) Detect the expression level or activity of SLC1A4 in the cardiomyocytes or non-human animal models; (3) If the candidate drug can significantly downregulate the expression level of SLC1A4 or inhibit its activity, then the candidate drug is a potential drug for the treatment of diabetic cardiomyopathy; (4) Test the inhibitory effect of the candidate drug on myocardial cell ferroptosis. If it can inhibit myocardial cell ferroptosis, it will be further confirmed as a potential drug for the treatment of diabetic cardiomyopathy.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: 1. First discovery of a novel target: This invention reveals for the first time the function and mechanism of action of SLC1A4 in diabetic cardiomyopathy (DCM), breaking through the limitations of existing research on SLC1A4 (there are currently no reports on its role in DCM), and confirming that SLC1A4 is abnormally highly expressed in in vivo and in vitro DCM models and is closely related to the occurrence and development of DCM, providing a novel and previously unreported molecular target for the prevention and treatment of DCM.

[0018] 2. Advantages of cell-specific targeting: By constructing a cardiomyocyte-specific SLC1A4 gene knockout mouse model, this invention precisely locates the role of SLC1A4 in cardiomyocytes of diabetic mice without affecting the weight, blood glucose, and blood lipid levels of diabetic mice. This avoids the metabolic side effects that may be caused by systemic intervention, providing an important theoretical basis for the future development of cardiomyocyte-targeted drug delivery systems and is expected to achieve precision treatment.

[0019] 3. Significant preventive and therapeutic effects: In a mouse model of diabetic cardiomyopathy, cardiomyocyte-specific knockout of SLC1A4 significantly improved cardiac function inhibition, reduced cardiac hypertrophy and myocardial fibrosis, suggesting that targeting SLC1A4 has strong potential for the prevention and treatment of diabetic cardiomyopathy.

[0020] 4. Broad Application Prospects: Various drug formulations can be developed targeting this site, including small molecule inhibitors of SLC1A4, RNA interference drugs targeting SLC1A4 (such as siRNA and shRNA), and targeted formulations capable of delivering these inhibitors to cardiomyocytes (such as cardiomyocyte-targeted adeno-associated viruses or nanodelivery systems). This invention has extremely high clinical translational value and commercial development prospects, providing a novel strategy for the treatment of diabetic cardiomyopathy. Attached Figure Description

[0021] Figure 1 This is an illustration of the abnormally elevated SLC1A4 expression in cardiomyocytes of diabetic mice in one embodiment of the present invention; (AB) Representative images (left) and quantitative analysis (right) of SLC1A4 and SLC1A5 protein expression in the hearts of type 2 diabetic mice (A) and type 1 diabetic mice (B) detected by Western blot; (C) Representative images and quantitative analysis of SLC1A4 protein expression in cardiomyocytes after HG+PA treatment detected by Western blot; (D) RT-qPCR detection of SLC1A4 and SLC1A5 mRNA expression levels in H9c2 cells after HG+PA treatment. *p<0.05, **p<0.01; ns, no significant difference, n=3.

[0022] Figure 2 This is a graph showing the results of blood glucose and lipid levels in diabetic mice after specific knockout of SLC1A4 in cardiomyocytes in Example 2 of one embodiment of the present invention; (A) the weight gain curve of each group of mice was measured regularly; (B) the fasting blood glucose change curve of each group of mice was measured regularly; (C) the percentage curve of insulin tolerance measured by ITT test one week before sample collection (left graph) and the quantitative analysis of the area under the curve (AUC) (right graph) of the curve (left graph) of the curve ...

[0023] Figure 3This is a diagram showing the results of cardiomyocyte-specific knockout of SLC1A4 in Example 3 of one embodiment of the present invention, which alleviated diabetes-induced cardiac dysfunction; (A) Representative images of echocardiography analysis of mice in each group. M Mode represents ultrasound images acquired in M ​​mode, PW represents pulse wave Doppler echocardiography, and Tissue represents tissue Doppler echocardiography; (B) Quantitative analysis of left ventricular ejection fraction (LVEF%); (C) Quantitative analysis of left ventricular fractional shortening (LVFS%); (D) Quantitative analysis of the ratio of early to late peak diastolic velocity of mitral valve blood flow (E / A); (E) Quantitative analysis of the ratio of early to late diastolic motion velocity of mitral valve annulus (E' / A'); (F) Quantitative analysis of the ratio of early diastolic velocity of mitral valve blood flow to early diastolic motion velocity of mitral valve annulus (E / e'). *p<0.05, **p<0.01, ****p<0.0001; ns, no significant difference, n=6-8.

[0024] Figure 4 This is an illustration of the results of cardiomyocyte-specific knockout of SLC1A4 in Example 3 of one embodiment of the present invention, which alleviated diabetes-induced ventricular remodeling; (A) Representative images of histopathological (H&E staining and Masson staining) and apoptosis staining of heart tissue sections from each group of mice, H&E: Scale bar = 100 µm; Masson: Scale bar = 100 µm; Tunel: Scale bar = 20 µm; (B) Quantitative analysis of the proportion of Tunel-positive cells in apoptosis staining of the heart tissues of each group of mice; (C) Detection of lactate dehydrogenase (LDH) activity in the heart tissues of each group of mice using the corresponding kits. ***p<0.001, ****p<0.0001; ns, no significant difference, n=6-8.

[0025] Figure 5This is a diagram showing the results of Example 4 in one embodiment of the present invention, where cardiomyocyte-specific knockout of SLC1A4 promotes the expression of the anti-ferroptosis core molecule GPX4 and reduces the expression of the ferritin autophagy carrier NCOA4; (A) Representative images and quantitative analysis of SLC1A4, GPX4, and NCOA4 protein expression in mouse hearts of each group detected by Western blot; (B) Representative images and quantitative analysis of ferrous ion (Fe2+) levels in mouse heart tissues of each group detected by Prussian blue staining, scale bar = 100 µm; (C) Ferrous ion (Fe2+) content in mouse heart tissues of each group detected by appropriate kits; (D) Level of malondialdehyde (MDA), a lipid peroxidation product, in mouse heart tissues of each group detected by appropriate kits; (E) Level of glutathione (GSH) in mouse heart tissues of each group detected by appropriate kits. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, no significant difference, n=6-8. Detailed Implementation

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0029] Experimental materials: 1. Cells: The H9c2 rat cardiomyocyte line was selected for in vitro experiments. The cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (100 U / mL penicillin and 100 µg / mL streptomycin) in a cell culture incubator at 37°C.

[0030] 2. Animals and Feeding: This experiment used SLC1A4 conditional knockout mice (SLC1A4).flox / flox ) and SLC1A4 cardiomyocyte-specific knockout mice (SLC1A4 Mlc-KO Mice, all of strain C57BL / 6, including SLC1A4. flox / flox Mice were purchased from Cyagen Biosciences (Suzhou, China), USA. Mlc (Mlc-2v) Cre mice were provided by the Experimental Animal Center of the Institute of Translational Medicine, Nanchang University. SLC1A4 mice were used to... flox / flox Mouse was crossed with Mlc (Mlc-2v) Cre mice to obtain cardiomyocyte-specific SLC1A4 gene knockout (SLC1A4). Mlc-KO Mice. All mice used were male mice aged 6-8 weeks. All experimental animals were housed in the SPF-grade animal facility of the Laboratory Animal Center of the Institute of Translational Medicine, Nanchang University, in standard cages with a 12-hour light / dark cycle. All animal experiments were conducted in strict accordance with the institutional guidelines (Agreement No.: NCULAE-20240426099) established by the Laboratory Animal Welfare and Ethics Committee of Nanchang University.

[0031] Example 1: SLC1A4 expression is abnormally elevated in cardiomyocytes of diabetic mice. 1. Establishment of a type 1 diabetes model: Six- to eight-week-old mice were intraperitoneally injected with STZ at a dose of 45 mg / kg / day for five consecutive days. Fasting blood glucose (FBG) was measured one week after administration. A type 1 diabetes model was considered successfully established if two consecutive fasting blood glucose levels were significantly higher than 11.1 mM. The mice were then fed normally for three months to induce a type 1 diabetic cardiomyopathy model.

[0032] 2. Establishment of a Type 2 Diabetes Model: A type 2 diabetic cardiomyopathy (DCM) model was established using 6-8 week old mice fed a high-fat diet (HFD) combined with a low-dose STZ. After 2 months of feeding with the high-fat diet (Research Diets, D12492; control diet: D12450J), intraperitoneal glucose tolerance test (IPGTT) and insulin resistance test (ITT) were performed. Mice exhibiting insulin resistance and glucose resistance were used for subsequent model establishment. Selected mice were fasted for 6 hours and then intraperitoneally injected with STZ at a dose of 45 mg / kg / day (3 times consecutively). Mice with a fasting blood glucose (FBG) ≥ 11.1 mmol / L (more than 2 times consecutively) one week later were considered to have diabetes. The mice were then fed the high-fat diet for another 4 months, with blood glucose and body weight measured every two weeks to induce the type 2 diabetic cardiomyopathy model.

[0033] 3. Cell Model Construction: To simulate the damage to the mouse heart caused by diabetes, we induced H9c2 rat cardiomyocytes with high glucose (HG, 33.3 mM) combined with palmitic acid (PA, 0.3 mM) to construct an in vitro cell lipotoxicity damage model. At the same time, we administered mannitol (MO) to exclude the effects of high osmotic stress on cells.

[0034] 4. Sample collection and detection: Mouse heart tissue and induced cardiomyocytes were collected, and the expression of SLC1A4 and its family protein SLC1A5 was detected by Western blot and RT-qPCR.

[0035] The results showed that (STZ: type 1 diabetic model mouse group; HFD+STZ: type 2 diabetic model mouse group; control: control mouse group; NG: normal control group; HG+PA: cytotoxic injury model group; MO: intervention group): the expression level of serine transporter SLC1A4 in the heart of diabetic mice was significantly higher than that in the control group, while the expression of its family member SLC1A5 was not significantly different between the hearts of diabetic mice and control mice. Figure 1 In vitro HG+PA-induced cardiomyocytes also showed significantly increased gene and protein expression of SLC1A4. Figure 1 (CD). The above results suggest that abnormally elevated SLC1A4 expression has significant specificity in diabetes-induced myocardial injury.

[0036] Example 2: Myocardial-specific knockout of SLC1A4 has no direct effect on systemic blood glucose and lipid changes caused by diabetes. 1. Gene knockout mouse construction: SLC1A4 cardiomyocyte-specific knockout mice (SLC1A4) were constructed using the FLOX-p system. Mlc-KO Then, a mouse model of type 2 diabetic cardiomyopathy was constructed using the method described above.

[0037] 2. During modeling and after sample collection, the mice's weight gain, blood glucose and lipid levels, and systemic glucose homeostasis regulation capacity as reflected by the glucose tolerance test (GTT) and insulin tolerance test (ITT) were measured.

[0038] 2.1 Testing of four lipid markers (TC, TG, LDL-C, and HDL-C): 1) Sample preparation: Blood was collected from the orbital cavity, and after standing at room temperature for 4-5 hours, the supernatant serum was collected by centrifugation at 3000 rpm for 15 minutes. 2) Detection Procedure: The lipid profile four-item test kit from Nanjing Jiancheng Bioengineering Institute was used, following the microplate method. Blank solution, standard or test sample (2.5 μL), and working solution (250 μL) were added to each well sequentially. The samples were incubated at 37°C in the dark for 10 min. The OD values ​​of each indicator were measured using a microplate reader at the specified wavelengths. The levels of TC, TG, HDL-C, and LDL-C in the sample were calculated based on the standard curve. HDL-C and LDL-C were detected using a two-reagent method; please refer to the kit instructions for specific steps.

[0039] 2.2 The detection steps for GTT and ITT are as follows: 1) GTT: Mice were fasted for 16 h before the experiment and injected intraperitoneally with glucose solution (1.5 g / kg). Blood was collected from the tail vein at 15, 30, 60, 90 and 120 minutes after injection to measure blood glucose levels. 2) ITT: Fasting for 4 hours before the experiment, insulin was injected intraperitoneally at a dose of 0.75-1.0 U / kg, and tail vein blood glucose levels were measured at 15, 30, 60, 90 and 120 minutes after injection.

[0040] The results showed that the fasting blood glucose level and body weight of mice in the DM group (model group) were significantly higher than those in the control group (control group). Figure 2 The presence of AB in the middle of the spectrum suggests that the diabetes model was successfully constructed. However, compared with SLC1A4... flox / flox Compared to mice, SLC1A4 Mlc-KO Mouse weight gain ( Figure 2 (A) Blood glucose level ( Figure 2 (B) Glucose homeostasis regulation capacity ( Figure 2 CD) and blood lipid levels ( Figure 2 There were no significant differences in either the mean or mean (E). These results suggest that cardiomyocyte-specific knockout of SLC1A4 does not affect blood glucose or lipid levels in diabetic mice.

[0041] Example 3: Cardiac cell-specific knockout of SLC1A4 alleviates diabetes-induced cardiac dysfunction and ventricular remodeling 1. After the above model was constructed, the abnormal systolic and diastolic functions of the mouse heart were assessed by echocardiography. The specific detection steps are as follows: This study used the VisualSonics Vevo3100 system from the Transgenic Animal Center of the Institute of Translational Medicine, Nanchang University, to perform echocardiographic examination of mouse hearts. First, the mice were anesthetized in an anesthesia box and then quickly transferred to the ultrasound imaging platform. 1.0% to 2.0% isoflurane was administered via a nasal anesthesia device for continuous anesthesia. To reduce the potential interference of heart rate fluctuations on ultrasound detection, the anesthesia dosage was fine-tuned to maintain a stable heart rate of 400 to 450 beats per minute. Next, hair was removed from the chest region of the mice using depilatory cream, and images were acquired using an ultrasound probe with the assistance of a coupling gel. For analysis, all raw images were imported into Vevo LAB offline analysis software (version 5.10.0, FUJIFILM VisualSonics). Overall structural changes of the heart were assessed using both long-axis and short-axis views, and the optimal view for calculating cardiac function indices was selected. The long-axis view was used to observe the systole and diastole of the left and right ventricles, while the short-axis view was more helpful in observing ventricular wall thickness and myocardial motility. Simultaneously, images acquired in the short-axis view were used for M-mode echocardiography analysis. Furthermore, to further assess cardiac diastolic function, four-chamber views taken at the apex were used to analyze changes in diastolic function, and pulse-wave Doppler and tissue Doppler techniques were employed to analyze cardiac diastolic parameters.

[0042] The results showed that diabetic model mice in the control group exhibited significant cardiac dysfunction, manifested as decreased LVEF, LVFS, E / A ratio, and E' / A' ratio, and increased E / e' ratio, while SLC1A4... Mlc-KO All of the above indicators in diabetic mice (DM group) were significantly improved. Figure 3 These results suggest that knocking out SLC1A4 in cardiomyocytes can effectively improve diabetic-induced cardiac dysfunction in mice.

[0043] 2. The structure, fibrosis, and apoptosis of mouse heart tissue were detected by H&E staining, Masson trichrome staining, and TUNEL staining; LDH, a cardiac injury marker, was detected in mouse serum.

[0044] 2.1 H&E staining procedure: 1) Tissue fixation: Fresh heart tissue collected from mice was quickly placed in an embedding cassette and then fixed in 4% paraformaldehyde (PFA) at 4°C for 48 h. 2) Dehydration, clearing and wax impregnation: After washing with running water for 5 min, the product is dehydrated sequentially by 70% ethanol (overnight), 80% ethanol (1 h), 90% ethanol (1 h), and 100% ethanol (two passes, 1 h each); then cleared by equal volume mixture of xylene and anhydrous ethanol, xylene I, and xylene II for 30 min each; finally, it is embedded in a 60℃ embedding machine by sequentially impregnating with soft wax (1-2 h) and hard wax (two passes, 2 h each); 3) The heart tissue was sectioned (5 μm) and stored at room temperature; 4) Dewaxing and hydration: The paraffin slices were placed in a 60℃ oven for 3 hours to facilitate subsequent dewaxing. Then, they were immersed in xylene for dewaxing (5 minutes each time, twice in total). Next, they were placed in ethanol of different concentrations (100%, 95%, 80%, 75%) for hydration treatment. 5) Hematoxylin staining and differentiation: The sections were stained in hematoxylin staining solution for 5 min; then they were differentiated using 1% hydrochloric acid alcohol and treated with blueing solution to make the cell nuclei appear deep blue. 6) Eosin counterstaining and dehydration: After rinsing with distilled water, immerse the sections in eosin staining solution for 3 min to make the cytoplasm pink; then dehydrate with gradient ethanol (75%, 100%) and clear with xylene. 7) Mount the slide with neutral resin and observe it under a microscope.

[0045] 2.2 Masson's trichrome staining procedure: 1) The steps before hematoxylin staining and differentiation are the same as those for H&E staining; 2) Muscle fiber staining: Add Ponceau S staining solution and immerse at room temperature for 3-5 minutes; 3) Prepare the weak acid working solution in advance according to the ratio of distilled water: weak acid solution = 2:1, and wash for 30 seconds by adding the weak acid working solution dropwise; 4) Pour off the excess liquid, add phosphomolybdic acid solution for 1-2 minutes; then add weak acid working solution to wash for 30 seconds; 5) Collagen fiber staining: Add aniline blue staining solution and stain for 1-2 minutes; 6) Dehydration and clearing: After staining, rinse the sections with a weak acid working solution for the final rinse; first dehydrate the sections quickly in 95% ethanol (2-3 s), then dehydrate them twice with anhydrous ethanol (5-10 s each time), and finally clear them twice with xylene (5 min each time). 7) Mount the slide with neutral resin and observe it under a microscope.

[0046] 2.3 Tunel staining procedure: 1) Tissue embedding: PFA-fixed mouse heart tissue was subjected to gradient dehydration and sucrose precipitation treatment with 30% and 15% sucrose solutions, then embedded with OGT embedding agent and quickly frozen at -80℃. 2) The embedded tissue was fixed on a cryostat and trimmed and sectioned to a thickness of 5 μm; 3) TdT enzyme labeling: Add TdT enzyme reaction solution (Equilibration Buffer, biotin-11-dUTP, TdTEnzyme, mix well), incubate at 37°C in the dark for 60 min, and wash with PBS; 4) FITC labeling: Add Streptavidin-FITC working solution (Streptavidin-FITC:Labeling Buffer = 1:9), incubate at 37°C in the dark for 30 min, and wash with PBS; 5) Counterstaining and mounting: Add DAPI staining solution, counterstain cell nuclei at room temperature in the dark for 10 min, wash away excess staining solution, add anti-fluorescence quenching mounting medium, mount, and observe and photograph under a fluorescence microscope.

[0047] 2.4 LDH Detection: Mouse serum was diluted 10-fold with physiological saline and placed on ice for testing. Following the instructions of the LDH detection kit from Nanjing Jiancheng Biotechnology Institute, the sample to be tested, matrix buffer, coenzyme I, etc., were added sequentially, mixed well, and incubated. The absorbance at 450 nm was then measured using an ELISA reader.

[0048] The results showed that the myocardial tissue of diabetic model mice (DM group) exhibited significant collagen deposition, aggravated fibrosis, and an increased proportion of apoptotic cells. Cardiac cell-specific knockout of SLC1A4 significantly improved these histopathological indicators. Figure 4 In summary, cardiomyocyte-specific knockout of SLC1A4 exerts a significant cardioprotective effect in the pathological environment of diabetes by improving cardiac systolic and diastolic function and inhibiting myocardial hypertrophy, fibrosis, and apoptosis.

[0049] Example 4: Cardiac cell-specific knockout of SLC1A4 inhibits diabetic-induced cardiac ferroptosis in mice 1. Proteins were extracted from mouse heart tissue by grinding, and the expression levels of ferroptosis-related markers GPX4 and NCOA4 were then detected by Western blot.

[0050] The results showed that, compared with the control group, cardiomyocyte-specific knockout of SLC1A4 (DM+Mlc-KO) promoted the expression of the anti-ferroptosis core molecule GPX4 and reduced the expression of the ferritin autophagy carrier NCOA4. Figure 5 (A)

[0051] 2. Changes in ferrous ion content in mouse heart tissue were detected using a commercially available kit.

[0052] 2.1 Prussian blue staining (ferrous ion specific, DAB enhanced): 1) This kit was provided by Solarbio (Beijing, China). Staining was performed according to the instructions. 2) Cut the paraffin sections into 3-7µm sections, then perform routine dewaxing and rehydration; 3) Prepare Perls staining working solution at a 1:1 ratio, add it to the slide until it completely covers the tissue, and incubate in a humidified chamber at 37°C for 20 min; 4) Add incubation solution, incubate at 37°C in the dark for 20 min, and rinse 3 times with PBS; 5) Add DAB colorimetric solution (C1:C2:PBS=1:1:18), incubate at 37°C in the dark for 20 min, and rinse 3 times with PBS; 6) Counterstain with hematoxylin for 3 min, then rinse with distilled water for 10 min; 7) Dehydrate with graded ethanol, clear with xylene, mount with neutral resin, and acquire images under an optical microscope; 8) Staining results: ferrous iron (brownish-yellow to brownish-red); cell nucleus (light blue); cytoplasm (light brown or colorless).

[0053] 2.2 Detection of ferrous ion content: 1) Sample processing: Collect cells or myocardial tissue, add lysis buffer and sonicate, centrifuge at 4℃ and 10000 g for 10 min, and collect the supernatant for later use; 2) Detection procedure: Dilute the sample 5 times, add the sample to the test tube, standard tube and blank tube, mix well and let stand at 37℃ for 10 min; add 100 μL of chloroform to the test tube, mix well and let stand at room temperature for 10 min; measure the absorbance at 593 nm wavelength with the microplate reader, and calculate the ferrous ion concentration according to the standard curve.

[0054] The results showed that, compared with the control group, SLC1A4 mice had significantly higher levels of SLC1A4. Mlc-KO The content of ferrous ions in the heart of diabetic (DM) mice was reduced. Figure 5 (Chinese BC).

[0055] 3. Determination of MDA content in mouse heart tissue: 1) Sample collection: Weigh the myocardial tissue sample, add extraction buffer at a mass-to-volume ratio of 1:9 for homogenization, centrifuge at 4℃ and 12000 rpm for 10 min, and collect the supernatant for later use. 2) Prepare the reaction system according to the kit instructions, and heat the reaction mixture at 95°C or in a boiling water bath for 30-60 min; 3) After the reaction solution is cooled to room temperature, it is centrifuged at 3000 rpm for 10 min. The supernatant is then transferred to a 96-well plate, and the absorbance at 532 nm is measured using a microplate reader.

[0056] The results showed that cardiomyocyte-specific knockout of SLC1A4 (DM+Mlc-KO) reduced MDA levels in the mouse heart. Figure 5 (D).

[0057] 4. Determination of GSH content in mouse heart tissue: 1) Sample processing: Weigh the myocardial tissue sample, add extraction buffer at a mass-to-volume ratio of 1:9, homogenize, centrifuge at 3500 rpm for 10 min at 4℃, and collect the supernatant for later use. 2) Colorimetric reaction: Add samples to blank wells (100 μL of reagent 1), standard wells (100 μL of GSH standard), and test wells (100 μL of supernatant). Add 25 μL of reagent 3 and 100 μL of reagent 2 to each well, mix well, and incubate at room temperature in the dark for 5 min. 3) The absorbance was measured at a wavelength of 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the GSH content was calculated based on the standard curve.

[0058] The results showed that diabetes leads to a decrease in GSH levels in the heart of mice (DM+flox), while knocking out SLC1A4 (DM+Mlc-KO) can restore GSH levels. Figure 5 (E).

[0059] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. The use of SLC1A4 inhibitors in the preparation of drugs for the prevention, relief, and / or treatment of diabetic cardiomyopathy, characterized in that, The SLC1A4 inhibitor exerts preventive, alleviating, and / or therapeutic effects by inhibiting myocardial ferroptosis.

2. The application according to claim 1, characterized in that, The drug is a substance that can downregulate the mRNA expression level and protein expression level of SLC1A4, and / or inhibit the transport activity and biological function of SLC1A4.

3. The application according to claim 2, characterized in that, The SLC1A4 inhibitor is selected from at least one of SLC1A4 gene silencing agents and SLC1A4 protein activity inhibitors.

4. The application according to claim 3, characterized in that, The SLC1A4 gene silencing agent is selected from at least one of siRNA, shRNA, miRNA, and antisense oligonucleotides; the SLC1A4 protein activity inhibitor is selected from at least one of specific neutralizing antibodies and small molecule compounds.

5. The application according to claim 1, characterized in that, The inhibition of cardiomyocyte ferroptosis includes at least one of the following: reducing intracellular lipid peroxidation levels, reducing intracellular ferrous ion content, upregulating GPX4 protein expression levels, downregulating NCOA4 protein expression levels, and upregulating the content of the antioxidant molecule GSH.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers, excipients, or diluents.

7. The application according to claim 1, characterized in that, The dosage form of the drug is selected from at least one of injections, tablets, capsules, granules, and aerosols.

8. A kit for detecting diabetic cardiomyopathy, characterized in that, This includes reagents for detecting the expression level of SLC1A4 protein in biological samples.

9. The reagent kit according to claim 8, characterized in that, The biological sample is selected from at least one of serum, plasma, peripheral blood mononuclear cells, and myocardial tissue biopsy samples; the reagent for detecting the expression level of SLC1A4 protein includes an antibody that specifically binds to SLC1A4 protein.

10. A method for screening candidate drugs for treating diabetic cardiomyopathy, characterized in that, Includes the following steps: (1) Apply the candidate drug to cardiomyocytes expressing SLC1A4 or to a non-human animal model of diabetic cardiomyopathy; (2) Detect the expression level or activity of SLC1A4 in the cardiomyocytes or non-human animal models; (3) If the candidate drug can significantly downregulate the expression level of SLC1A4 or inhibit its activity, then the candidate drug is a potential drug for the treatment of diabetic cardiomyopathy; (4) Test the inhibitory effect of the candidate drug on myocardial cell ferroptosis. If it can inhibit myocardial cell ferroptosis, it will be further confirmed as a potential drug for the treatment of diabetic cardiomyopathy.

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