Use of flvcr2 protein inhibitors in the preparation of a medicament for treating diabetic cardiomyopathy

Abstract

The application of FLVCR2 protein inhibitor in the preparation of a drug for treating diabetic cardiomyopathy belongs to the technical field of biological medicine.The application of FLVCR2 protein inhibitor in the preparation of a drug for treating diabetic cardiomyopathy is provided to solve the problem that there is no efficient and low-side-effect method for treating diabetes and its complications in the prior art, the amino acid sequence of the FLVCR2 protein is shown in SEQ ID NO.1, the expression of the FLVCR2 protein is inhibited, the uptake of abnormal hematin by myocardial cells is reduced, and the purposes of reducing myocardial cell death and improving heart failure symptoms are achieved.The application of FLVCR2 protein inhibitor in the preparation of a drug for treating diabetic cardiomyopathy is provided, the expression of the FLVCR2 protein is inhibited, the pyroptosis of myocardial cells is relieved, myocardial damage is improved, a new method for treating diabetic cardiomyopathy is provided, and the application has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of FLVCR2 protein inhibitors in the preparation of drugs for treating diabetic cardiomyopathy. Background Technology

[0002] Diabetes mellitus is a chronic disease centered on metabolic and endocrine disorders. Early stages are often asymptomatic, but as it progresses to the symptomatic stage, it manifests as the classic "three highs and one low" (polydipsia, polyphagia, polyuria, and weight loss) along with symptoms such as excessive thirst, hunger, fatigue, and obesity. Type 2 diabetes accounts for over 90% of cases, with its core pathological mechanism being insulin resistance accompanied by relative insulin insufficiency. While patients do not depend on exogenous insulin for survival, they require it to control hyperglycemia. It is noteworthy that insulin resistance and hyperinsulinemia are not only characteristics of type 1 diabetes but also a common pathological basis for diseases such as obesity, hypertension, arteriosclerosis, dyslipidemia, hyperuricemia, hyperleptinemia, and microalbuminuria. These interconnected syndromes are now collectively referred to as "metabolic syndrome" (replacing the old terms "insulin resistance syndrome" or "Syndrome X"), highlighting the central role of glucose and lipid metabolism disorders in multi-system diseases.

[0003] Studies have shown that compared to non-diabetic individuals, diabetic patients have a 2 to 2.5 times higher risk of developing cardiovascular disease, with heart failure being the most common cardiovascular complication and a leading cause of premature death. Even with good management of cardiovascular risk factors, the risk of heart failure in diabetic patients remains 2 to 5 times higher. The concept of "diabetic cardiomyopathy (DCM)" was first proposed in 1972, defining it as a specific cardiomyopathy independent of hypertension and coronary artery disease, associated with a high incidence and mortality rate of heart failure. A 2024 expert consensus statement published by the European Heart Failure Association and the European Society of Cardiology defines diabetic cardiomyopathy as heart failure occurring in the presence of diabetes, regardless of the presence of other risk factors and diseases. DCM has been proven to be a leading cause of death in diabetic patients.

[0004] Type 2 diabetes is closely related to non-alcoholic fatty liver disease (NASH). Nearly one-third of patients with NASH already have diabetes or fasting hyperglycemia at the time of diagnosis. The incidence of fatty liver in patients with type 2 diabetes is as high as 21-78%, while the incidence is lower in patients with type 1 diabetes. Diabetic patients have a seven-fold increased risk of developing liver fibrosis; therefore, diabetes is an independent predictor of severe liver fibrosis in NASH. Studies have shown that patients with advanced NASH often experience heart failure, but the underlying mechanisms remain unclear.

[0005] Heme is an iron porphyrin compound composed of four pyrrole subunits forming a cyclic group with a ferrous ion at its center. Heme is primarily synthesized in immature erythrocytes in the bone marrow, with the liver being the main site of heme metabolism. Heme is an important non-protein component of hemoglobin; one globin molecule and four heme molecules are linked by four polypeptide chains to form Hb. The ferrous ion in the center of each heme group can bind oxygen, making Hb oxyhemoglobin. Heme is not only a cofactor for Hb but also a cofactor for myoglobin, cytochromes, and peroxidases, playing important roles in oxygen binding, electron transport, and signal transduction. Free heme is a hemolytic product, a metabolic product of erythrocyte rupture. Free heme is highly hydrophobic, easily permeates cell membranes, and acts as an oxidant with direct cytotoxic effects.

[0006] Although existing studies have found that free heme has hepatotoxicity, the relationship between heme levels and myocardial tissue, and whether free heme can serve as a key factor in the treatment of diabetic cardiomyopathy, remains unclear. Therefore, those skilled in the art are eager to develop a highly effective drug with low side effects for diabetes and its complications by targeting heme metabolism and uptake pathways. Summary of the Invention

[0007] This invention addresses the lack of efficient and low-side-effect treatment methods for diabetes and its complications in the prior art by providing the application of FLVCR2 protein inhibitors in the preparation of drugs for treating diabetic cardiomyopathy.

[0008] One of the objectives of this invention is to provide the application of FLVCR2 protein inhibitors in the preparation of drugs for treating diabetic cardiomyopathy.

[0009] In a preferred embodiment of the present invention, the amino acid sequence of the FLVCR2 protein is shown in SEQ ID NO.1.

[0010] In a preferred embodiment of the invention, the application is achieved by inhibiting the expression of FLVCR2 protein, thereby reducing the uptake of heme in the heart.

[0011] In a preferred embodiment of the invention, the drug comprises an FLVCR2 protein inhibitor and pharmaceutically acceptable excipients.

[0012] In a preferred embodiment of the invention, the pharmaceutically acceptable excipient is selected from one or more of dispersants, diluents, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, or flavoring agents.

[0013] Beneficial effects of the present invention: The present invention provides the application of FLVCR2 protein inhibitor in the preparation of drugs for treating diabetic cardiomyopathy. The amino acid sequence of the FLVCR2 protein is shown in SEQ ID NO.1. By inhibiting the expression of FLVCR2 protein, the uptake of heme in the heart is reduced, thereby achieving the purpose of reducing myocardial cell death and improving heart failure symptoms.

[0014] The results showed that (1) in the diabetic cardiomyopathy mouse model, liver dysfunction led to disordered heme metabolism, which in turn caused an abnormal increase in peripheral free heme; (2) FLVCR2 protein is located on the surface of cardiomyocytes, and its expression level was significantly increased in the diabetic cardiomyopathy model; (3) Excessive free heme was taken up by FLVCR2 protein on the surface of cardiomyocytes, and heme combined with high sugar and high fat could further aggravate the damage to cardiomyocytes; (4) by gene knockdown technology, FLVCR2 protein was intervened, which showed that the ability of cardiomyocytes to take up heme was significantly reduced, and the damage to cardiomyocytes could be alleviated.

[0015] This invention provides the application of an FLVCR2 protein inhibitor in the preparation of drugs for treating diabetic cardiomyopathy. By inhibiting the expression of FLVCR2 protein, it alleviates cardiomyocyte pyroptosis and improves myocardial damage, providing a new method for treating diabetic cardiomyopathy and possessing significant clinical application value. Attached Figure Description

[0016] Figure 1 Figure A shows the correlation graphs of serum heme levels in different groups of patients; Figure B shows the statistical graph of left ventricular ejection fraction (LVEF); Figures C and D show the correlation analysis results of serum heme with BNP, ALT, AST, DBIL, and GGT levels, respectively. Wherein, Serum Heme is serum heme, LVEF is left ventricular ejection fraction, BNP is brain-type natriuretic peptide, ALT is alanine aminotransferase (ALT), AST is aspartate aminotransferase (AST), DBIL is direct bilirubin, and GGT is... -Glutamine transferase;

[0017] Figure 2Figures show the results of different groups of diabetic mouse models; A is a bar chart of blood glucose detection; B is a line chart of glucose tolerance test; C is an image of liver tissue appearance; D is a Masson staining result of liver tissue; E is a graph of triglyceride content in liver tissue; F is a graph of ALT (alanine aminotransferase) content in liver tissue; G is a graph of AST (aspartate aminotransferase) content in liver tissue; H is a graph of DBIL (direct bilirubin) content in liver tissue; I is a graph of ALB (albumin) content in liver tissue; J is a Masson staining result of heart tissue; K is a graph of ANP mRNA expression in heart tissue; L is a graph of BNP mRNA expression in heart tissue; where Blood glucose refers to blood glucose, Glucose tolerance test refers to glucose tolerance test, Hepatic refers to liver, and Relative Expression refers to expression level.

[0018] Figure 3 Figure 1 shows the detection results of diabetic mouse models in different treatment groups; A is the serum heme detection figure; B is the liver tissue heme detection figure; C is the serum heme-binding protein detection figure; D is the heart tissue heme detection figure; where Serum Heme is serum heme, Hepatic Heme is liver heme, Serum hemopexin is serum heme-binding protein, and Cardiac Heme is cardiac heme.

[0019] Figure 4 Figure A shows the results of HPX and LRP1 detection in diabetic mouse models under different treatment groups; Figure B shows the results of HPX and LRP1 protein expression level detection; Figure C shows the statistical graph of LRP1 protein expression level; Figure C shows the statistical graph of HPX protein expression level.

[0020] Figure 5 Figure A shows the results of HO-1 and HO-2 detection in diabetic mouse models under different treatment groups; Figure B shows the results of HO-1 and HO-2 protein expression level detection; Figure C shows the statistical graph of HO-1 protein expression level; Figure C shows the statistical graph of HO-2 protein expression level.

[0021] Figure 6 Cell distribution map for FLVCR2 detection by immunofluorescence;

[0022] Figure 7 The graph shows the statistical results of heme uptake in different treatment groups; ZnPP is a heme analog, and si-FLVCR2 represents the FLVCR2 gene expression level knocked down using small interference technology.

[0023] Figure 8Image showing the results of cardiomyocyte death in different treatment groups detected by immunofluorescence; Calcein-AM labeled live cells; PI labeled dead cells; Pal represents palmitate; HG represents high glucose; Heme represents heme.

[0024] Figure 9 The image shows the results of myocardial cell death in different treatment groups detected by immunofluorescence; Calcein-AM labeled live cells, PI labeled dead cells, Pal was palmitate, HG was high glucose, and Heme was heme.

[0025] Figure 10 The image shows the results of myocardial cell death in different treatment groups detected by immunofluorescence. Calcein-AM labeled live cells, PI labeled dead cells, Pal was palmitate, HG was high glucose, Heme was heme, MCC950 was a pyroptosis inhibitor, Nec-1 was a necroptosis inhibitor, and PBS was phosphate buffer.

[0026] Figure 11 The image shows the results of Western blotting of cardiomyocyte pyroptosis levels in different treatment groups; GSDMD is a key regulatory protein of pyroptosis, Caspase-1 is a marker of inflammasome activation, and β-actin is an internal reference protein.

[0027] Figure 12 Figure 1 shows the results of Western Blot analysis of pyroptosis levels in cardiomyocytes from different treatment groups: si-FLVCR2 represents the gene-level knockdown of FLVCR2 protein expression using small interference technology; heme represents heme; GSDME represents a key protein in the non-classical pyroptosis pathway; and IL-18 represents the interleukin-18 inflammatory factor. Detailed Implementation

[0028] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0030] The patients involved in the following examples are all inpatients from the Department of Cardiology at the First Affiliated Hospital of Harbin Medical University.

[0031] Example 1: Application of FLVCR2 protein inhibitors in the preparation of drugs for treating diabetic cardiomyopathy

[0032] 1. Collection of clinical samples:

[0033] Twenty-five patients with diabetic cardiomyopathy and abnormal liver function were selected as the experimental group, and 25 patients without the above-mentioned disease were selected as the control group (non-diabetic cardiomyopathy patients with normal liver function). Peripheral blood samples were collected from the patients in the above groups, centrifuged, and serum was collected. The heme level was detected using the Sigma-Aldrich heme assay kit. Routine blood tests were also performed on the above patients. Based on the test results, the correlation between serum heme and BNP, ALT, AST, DBIL, and GGT levels was analyzed.

[0034] BNP (B-type natriuretic peptide) is a polypeptide hormone mainly secreted by ventricular myocytes. It plays an important role in monitoring cardiac function and in the diagnosis and treatment of cardiovascular diseases. As an important biomarker of heart failure, its level is positively correlated with the severity of heart failure. Changes in ALT, AST, DBIL, and GGT levels are mainly used for clinical assessment of liver function, as the liver is one of the main organs for heme metabolism.

[0035] like Figure 1 As shown in Part A, the serum heme level in patients with diabetic cardiomyopathy and abnormal liver function (experimental group) was significantly higher than that in the control group (non-diabetic cardiomyopathy patients with normal liver function).

[0036] like Figure 1 As shown in Part B, serum heme levels in the experimental group were negatively correlated with cardiac ejection fraction, meaning that higher heme levels correlated with lower ejection fraction. Conversely, serum heme levels in the experimental group were positively correlated with BNP, ALT, AST, DBIL, and GGT levels. This indicates that impaired liver function may lead to elevated serum heme levels, and that heme levels are correlated with the severity of heart failure.

[0037] 2. Establishment of animal models:

[0038] Eight-week-old db / + mice (18-20 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with equal numbers of males and females, were selected as the normal control group; db / db mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with equal numbers of males and females, were used to establish a type 2 diabetes animal model. The animals were divided into a normal control group and a db / db mouse group (type 2 diabetes group). After 20 weeks of rearing, both groups of mice were sacrificed, and serum heme levels and related biochemical indicators were measured in the mice.

[0039] (1) Myocardial and liver tissues were fixed with 4% paraformaldehyde, and structural changes in myocardium and liver were detected by hematoxylin-eosin (HE) staining. The specific operation is as follows:

[0040] a. Take tissue sections from the heart and liver of the mice to be tested, and soak them in xylene, anhydrous ethanol, 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, 75% ethanol and distilled water for 5 minutes each.

[0041] b. Place the above tissue sections into a staining box containing hematoxylin staining solution for 2.5 minutes, then quickly rinse with distilled water for 10 minutes; then stain the sections in eosin staining solution for 1.5 minutes.

[0042] c. Soak the treated sections sequentially in 95% alcohol, anhydrous ethanol, and xylene for 5 minutes each; remove the sections from the xylene and mount them with neutral resin; observe the tissue staining under a microscope and take pictures for storage; analyze the images using ImageJ.

[0043] (2) Masson staining was used to observe changes in liver and myocardial fibrosis. The specific procedure is as follows:

[0044] a. Take tissue sections from the heart and liver of the mice to be tested, and soak them sequentially in xylene, 100% anhydrous ethanol, 95% anhydrous ethanol, 90% anhydrous ethanol, 85% anhydrous ethanol, 80% anhydrous ethanol, 75% anhydrous ethanol, and distilled water for 5 minutes each. Wipe away any residual liquid around the tissues with filter paper, immerse the sections in Weiger's iron hematoxylin staining solution for 8 minutes, and rinse the sections with running tap water for 1.5 minutes. Wipe away any residual liquid around the tissues with filter paper, cover the tissues with acidic ethanol differentiation solution for 10 seconds, and rinse the sections with running tap water for 5 minutes. Wipe away any residual liquid around the tissues with filter paper, add Masson's blue solution to cover the tissues for bluening for 2 minutes, rinse the sections with running tap water for 5 minutes, and rinse with distilled water for 1 minute. Wipe away any residual liquid around the tissues with filter paper, add Ponceau S and acid fuchsin solution for staining for 10 minutes, and rinse the sections with running tap water for 1 minute.

[0045] b. Prepare a weak acid working solution with a volume ratio of distilled water to weak acid solution of 2:1. Rinse the sections treated in step a with the weak acid working solution for 1.5 minutes. Wipe away any residual liquid around the tissue with filter paper. Incubate the tissue with a 1% phosphomolybdic acid aqueous solution for 2 minutes. Rinse the sections with the weak acid working solution for 1 minute. Wipe away any residual liquid around the tissue with filter paper. Add aniline blue staining solution and stain for 2 minutes. Rinse the sections with the weak acid working solution for 1 minute. Wipe away any residual liquid around the tissue with filter paper. Add 95% ethanol for 4-6 seconds, then add anhydrous ethanol for 10 seconds. Immerse the sections in xylene three times for 2 minutes each time. Mount with neutral resin. Observe the tissue staining under a microscope and take pictures for storage. Analyze the images using Image J.

[0046] (3) The changes in heme levels in liver and heart tissues were detected using a heme assay kit (Sigma, MAK316). Proteins were extracted from liver and heart tissues (specific procedures are as follows). The extracted proteins were quantified using the Bradford method. The specific steps for determining the heme concentration in the protein extract are as follows:

[0047] a. will ddH2O and The heme calibrator was added to a 96-well plate, and heme was added to each blank well and calibration reaction well. ddH2O, diluted heme calibration is equivalent to ;

[0048] b. The sample to be tested is added into the wells of the well plate, and the sample is added to each well. Mix the heme reagent (Heme Reagant), incubate at room temperature for 5 minutes, and measure the absorbance at 400 nm using a microplate reader.

[0049] (4) Western Blot was used to detect changes in liver and heme metabolic pathway related indicators. Tissue proteins were extracted for Western Blot experiments. The steps were as follows: The entire protein extraction process was performed on ice. The myocardial and liver tissues of the mice to be tested were repeatedly ground with a tissue grinding rod until each tissue piece was approximately 1 mm. 3 (Tissue); Add protease lysis buffer and PMSF and incubate on ice for about 20 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant, and obtain the protein from the mouse myocardium and liver tissue to be tested. Use BSA as the standard and Bradford method to quantify the protein in the supernatant.

[0050] a.SDS-PAGE

[0051] Gel preparation and electrophoresis apparatus assembly: After aligning and clamping the glass plates, add water to check for leaks, and then prepare gels of the corresponding concentration according to the molecular weight of the target protein; slowly pour the separating gel along one side of the glass plate, and then pour anhydrous ethanol to flatten the liquid surface. This process requires a slow speed to prevent the separating gel from being deformed. Time for 15 minutes. When there is a clear refractive line between the ethanol and the gel, it means that the gel has fully solidified. Discard the ethanol and blot dry with filter paper. Then slowly add the prepared upper gel and insert the comb. Let it stand for 30 minutes. Remove the glass plates and place them in the electrophoresis tank. Add the electrophoresis solution and slowly remove the comb.

[0052] Sample loading and electrophoresis: Add samples to the lanes on both sides of the edge. Marker: The protein sample loading volume is based on the volume calculated using the BCA method. Upper gel 80V, lower gel 120V. Stop electrophoresis when bromophenol blue reaches the bottom of the gel.

[0053] b. Transfer and sealing

[0054] Prepare four layers of filter paper and a PVDF membrane. Cut the PVDF membrane and filter paper to the appropriate size according to the size of the glue block. Pre-cool the transfer solution and filter paper. Soak the PVDF membrane in formaldehyde for 1 minute for activation. Place the membrane on the transfer apparatus in the order of filter paper + PVDF membrane + glue + filter paper, and perform the transfer at a constant current of 200 mA on ice. After the transfer is complete, seal with 5% skim milk powder at room temperature for 1 hour.

[0055] c. Primary and secondary antibody immune responses

[0056] The sealed PVDF membrane was cut according to the molecular weight of the target protein and placed in an incubator containing the primary antibody pre-diluted with TBS-T according to the instructions. The membrane was incubated overnight at 4°C. The next day, the membrane was washed 6 times with TBS-T for 5 minutes each time. The secondary antibody was diluted similarly, and the membrane was incubated at room temperature with gentle shaking for 1 hour, followed by 6 washes with TBS-T for 5 minutes each time.

[0057] d.ELC color development

[0058] Remove the strip from the TBS-T solution, absorb excess water, prepare color development solutions A and B at a 1:1 volume ratio, and then evenly cover the membrane for color development.

[0059] like Figure 2 As shown in section AB, the fasting blood glucose levels of the db / db group mice were significantly higher than those of the db / + group mice, indicating impaired glucose tolerance. This suggests that the db / db group mice can serve as a typical animal model of type 2 diabetes. Figure 2 As shown in the CD section, the livers of db / db group mice were enlarged, lighter in color, and softer in texture; after hematoxylin-eosin (HE) staining, obvious fatty degeneration was observed in liver sections, manifested as numerous lipid droplets within hepatocytes, including macrovesicular and microvesicular fatty degeneration, and ballooning degeneration of hepatocytes was also observed. Figure 2 As shown in the EI section, the db / db group mice exhibited not only fatty liver but also significant liver function impairment; specifically, compared to the db / + group mice, the db / db group mice showed significantly elevated levels of triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and direct bilirubin. Figure 2 As shown in section J, Masson staining of the heart tissue revealed significant fibrosis in the hearts of the db / db group mice. Figure 2 As shown in the KL section, the expression results of ANP and BNP mRNA in cardiac tissue indicate that, compared with the db / + group mice, the db / db group mice exhibited impaired cardiac function, heart failure, and diabetic cardiomyopathy. Therefore, the db / db group mice can serve as a mouse model of type 2 diabetes, exhibiting severe hepatic steatosis and decreased cardiac function.

[0060] like Figure 3 As shown, the heme level in the peripheral serum of db / db group mice was significantly higher than that of db / + group mice, and the content of serum heme-binding protein in db / db group mice was significantly higher than that in db / + group mice; the heme levels in the liver and heart tissues of both db / db and db / + groups mice were elevated to varying degrees. Therefore, the serum, liver, and heart heme levels of db / db group mice were significantly higher than those of the control group (db / + group mice).

[0061] HPX and LRP1 are known heme transport proteins that assist in the transport of heme on the surface and inside hepatocytes. Therefore, this invention detected the expression levels of HPX and LRP1 proteins in the liver tissue of db / db group mice. The results are as follows: Figure 4 As shown, the expression levels of HPX and LRP1 proteins in the liver tissue of db / db group mice were significantly higher than those in the control group (db / + group mice).

[0062] This invention also detected the key enzymes HO-1 (heme oxygenase-1) and HO-2 (heme oxygenase-2) in hepatocytes that metabolize heme, and the results are as follows. Figure 5 As shown, HO-2 is a constitutively expressed enzyme. Unlike HO-1 (heme oxygenase-1), HO-2 expression is relatively stable and does not depend on external stimuli or stress responses. Experiments revealed no significant changes in HO-2 expression in the liver tissue of the db / db group mice compared to the db / + group, while HO-1 levels were significantly elevated. This indicates that abnormal liver function in the db / db group mice led to an imbalance in heme homeostasis.

[0063] 3. Establishment of cell model:

[0064] High-fat treatment of cardiomyocytes: Using 1-3 day old mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), the apical portion of the mouse heart was minced in DMEM solution and digested five times. After centrifugation, the supernatant was discarded, and the sedimented cells were collected and placed in culture dishes. The dishes were incubated at 37°C in a CO2 incubator for 2 hours to induce differential adhesion. 5'-bromodeoxyuridine was then added to inhibit the culture of non-cardiomyocyte cells. palmitate and A type 2 diabetic cardiomyocyte model was established by culturing the cells in glucose for 48 hours, simulating the high-glucose or high-fat environment in db / db mice; simultaneously, exogenous heme was added. Simulate the high heme environment in db / db mice.

[0065] (1) In order to determine the pathway of heme transfer into cardiomyocytes in the heart, this invention anchors the FLVCR2 protein based on proteomics results, detects the cellular distribution of FLVCR2 protein by immunofluorescence, selects the heme analog Znpp with fluorescent properties, and simultaneously constructs FLVCR2 si-RNA (constructed by Seven Innovation Biotechnology Co., Ltd.) and overexpression plasmid FLVCR2. OE (Constructed by Gemma Gene Company), and set up a blank control group, a Znpp group (Znpp only), a si-FLVCR2 group (transfected with small interference), and a si-FLVCR2+Znpp group (Znpp added to the si-FLVCR2 experimental group) to conduct heme uptake experiments. The fluorescence module of the microplate reader (fluorescence excitation wavelength set to 420 nm) was selected to detect the heme uptake after knocking down FLVCR2 protein.

[0066] Transfecting with small interfering or overexpression plasmids, specific preparation steps:

[0067] a. Take one enzyme-free EP tube and add... Opti-MEM is available for backup; FLVCR2 si-RNA (FLVCR2 OE The amount of plasmid added was Adding In an EP tube containing Opti-MEM, gently mix and let stand for 5-10 minutes (for transfection with the expression plasmid FLVCR2). OE At this time, this EP tube needs to be added separately. Lipo3000 transfection reagent);

[0068] b. Add to one enzyme-free EP tube Opti-MEM, add to it respectively The Lipo3000 transfection reagent was gently mixed and allowed to stand at room temperature for 5-10 minutes.

[0069] c. Mix the liquid in the EP tube from step a with the liquid in the EP tube from step b, and let stand at room temperature for 20 minutes to form a si-RNA / transfection reagent and overexpression plasmid / transfection reagent complex;

[0070] d. Using a pipette, add the si-RNA / transfection reagent and overexpression plasmid / transfection reagent complex obtained in c to the high-fat-treated cardiomyocytes. Gently shake to ensure that the si-RNA / transfection reagent complex, overexpression plasmid / transfection reagent and culture medium are completely mixed. Incubate in a CO2 incubator at 37°C for 24-36 hours and perform PCR detection. After 48-72 hours, detect the protein expression level.

[0071] The primer sequences involved include:

[0072] M217721-si FLVCR2-F: 5'-GGAUGCCUGAGACCAAA(dT)(dT)-3' (SEQ ID NO. 2);

[0073] M217721-si FLVCR2-R: 5'-UUUGGUCUCAGGAGCAUCC(dT)(dT)-3' (SEQ ID NO.3);

[0074] FLVCR2 OE -F: 5'-ATGGTGAATGAAGGTCCCAACC-3' (SEQ ID NO.4);

[0075] FLVCR2 OE -R: 5'-GCTGTGTCAGAGGATCATCTCTGA-3' (SEQ ID NO. 5).

[0076] like Figure 6 As shown, the FLVCR2 protein is present on the surface of cardiomyocytes, suggesting that it plays an important role in heme uptake by cardiomyocytes. Therefore, this invention utilizes small interference technology to intervene in the FLVCR2 protein, and simultaneously selects ZnPP (a heme analogue) with fluorescent properties to simulate heme uptake experiments, such as... Figure 7 As shown, knocking down FLVCR2 protein resulted in a significant decrease in ZnPP uptake by cardiomyocytes, as expected.

[0077] In summary, this further confirms the hypothesis of the present invention that the FLVCR2 protein mediates the uptake of excess free heme by cardiomyocytes, and the excess heme is taken up by the FLVCR2 receptor protein on the surface of cardiomyocytes.

[0078] (2) The present invention constructs knockdown and overexpression FLVCR2 cell models (the preparation steps are the same as in (1)). That is, after reducing or increasing the heme content in cardiomyocytes, the cardiomyocyte death is detected, the expression of pyroptosis-related indicators is detected by Western Blot, and the LDH content of each group is detected by extracting cell supernatant.

[0079] like Figure 8 As shown in the middle section, high glucose (HG) or high fat (Pal) alone cannot induce massive death of cardiomyocytes. However, the exogenous addition of heme to a high glucose and high fat diet will induce massive cell death.

[0080] To further clarify the relevant mechanisms of high glucose and high lipid combined with heme-induced cardiomyocyte death, such as... Figure 9 As shown, cell death was investigated under the conditions of high glucose (HG), high lipid (Pal), high lipid combined with heme (Pal+Heme), and high glucose combined with heme (HG+Heme). The experiment found that both high glucose combined with heme and high lipid combined with heme induced a large number of cell deaths, with high lipid combined with heme inducing more significant cell death. Based on this, we hypothesize that heme plays an important role in inducing cardiomyocyte death, and that cardiomyocyte death is mainly due to the combined effect of heme and high lipid.

[0081] To further clarify the modes of cardiomyocyte death, this invention employs pyroptosis inhibitors and necroptosis-apoptosis inhibitors for intervention, with the following results: Figure 10 As shown, necroptosis inhibitors cannot significantly inhibit heme-induced cardiomyocyte death combined with high glucose and high lipid levels. However, the addition of pyroptosis inhibitors significantly inhibits cardiomyocyte death. Based on the above experiments, it is shown that heme-induced cardiomyocyte death is mainly dominated by pyroptosis.

[0082] To further confirm the role of heme in inducing pyroptosis in cardiomyocytes, Western blotting was used to detect the expression levels of key pyroptosis markers GSDMD and Caspase-1 in eight groups: blank control, high glucose (HG), high lipid (Pal), heme (Heme), heme combined with high glucose (HG+Heme), heme combined with high lipid (Pal+Heme), high glucose and high lipid (HG+Pal), and heme combined with high glucose and high lipid (HG+Pal+Heme). Figure 11 As shown, heme alone, heme combined with high glucose, heme combined with high lipid, and heme combined with high glucose and high lipid all induce the expression of key pyroptosis markers GSDMD and Caspase-1 to varying degrees, further confirming the key role of heme in inducing cardiomyocyte pyroptosis.

[0083] Based on the above experiments confirming the important role of heme in inducing cardiomyocyte pyroptosis, this invention aims to verify that heme mediates cardiomyocyte pyroptosis through uptake and translocation into cardiomyocytes via the FLVCR2 protein. The FLVCR2 protein was knocked down using small interference technique, and the expression levels of key pyroptosis markers GSDMD, Caspase-1, GSDME, and the inflammatory factor IL-18 were detected by Western blotting. Figure 12 As shown, the pyroptosis markers were significantly overexpressed after the addition of exogenous heme, further confirming that the addition of exogenous heme induces pyroptosis in cardiomyocytes. When the expression of FLVCR2 protein was inhibited and exogenous heme was administered, the expression of pyroptosis-related proteins was significantly downregulated.

[0084] It is evident that the FLVCR2 protein enters cardiomyocytes by taking up heme, inducing pyroptosis. Inhibiting FLVCR2 protein expression can alleviate cardiomyocyte pyroptosis and may improve myocardial damage. In conclusion, excessive heme combined with high sugar and high lipid levels exacerbates cardiomyocyte damage, while reducing heme uptake can reduce cardiomyocyte damage.

[0085] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Use of an FLVCR2 protein inhibitor in the preparation of a medicament for treating diabetic cardiomyopathy, characterized in that, The FLVCR2 protein inhibitor is siRNA, with the sense strand sequence being 5'-GGAUGCCUGAGACCAAAdTdT-3' and the antisense strand sequence being 5'-UUUGGUCUCAGGAGCAUCCdTdT-3'.

2. The application according to claim 1, characterized in that, The amino acid sequence of the FLVCR2 protein is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The application reduces the uptake of heme in the heart by inhibiting the expression of FLVCR2 protein.

4. The application according to claim 1, characterized in that, The drug contains an FLVCR2 protein inhibitor and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients are selected from one or more of dispersants, diluents, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, or flavoring agents.

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