Application of flufenidone in preparation of medicine for preventing or treating left heart failure

Abstract

The invention belongs to the technical field of biological medicine, and particularly provides application of flufenidone in preparation of a medicine for preventing or treating left heart failure. The research finds that the flufenidone has the effects of relieving myocardial cell hypertrophy under a heart failure cell model and relieving myocardial hypertrophy, fibrosis and heart function deterioration of an aortic constriction animal model. According to the present invention, further research results show that fluorofenidone is directly combined with SERCA2a through Q758, D812 and E917 residues of SERCA2a so as to inhibit recognition, combination and polyubiquitination effects of WWP1 on SERCA2a, such that the protein level and the activity of SERCA2a are stabilized so as to achieve the chronic left heart failure treatment purpose. Therefore, the flufenidone serving as an active ingredient has good application prospect and application value in research and development of novel medicines for treating chronic left heart failure.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the use of flufenidone in the preparation of drugs for the prevention or treatment of left ventricular failure. Background Technology

[0002] Left ventricular failure is the end-stage of cardiovascular disease, and its pathogenesis remains incompletely understood. Currently, neurohumoral dysregulation characterized by excessive sympathetic nerve excitation and overactivation of the renin-angiotensin-aldosterone system (RAAS) is considered the most critical driving mechanism in left ventricular failure. Cardiac cardiomyocyte sarcoplasmic reticulum Ca2+ 2+ -ATPase 2a (Sarcoplasmic Reticulum Ca 2+ Impaired sarcoplasmic reticulum calcium reuptake due to decreased ATPase 2a (SERCA2a) activity and levels is considered a hallmark pathophysiological feature of left ventricular failure, with myocardial hypertrophy and fibrosis being its core pathological characteristics. Although numerous studies have demonstrated the reduction of myocardial hypertrophy and fibrosis in animals through drugs and gene therapy, no drugs specifically approved for reducing these conditions are currently available for clinical use. Existing treatments primarily focus on correcting the underlying cause, improving symptoms, and inhibiting overactivation of systems such as the RAAS to delay ventricular remodeling, aiming to reduce hospitalization and mortality rates and improve quality of life and life expectancy.

[0003] According to the latest guidelines for the diagnosis and treatment of acute and chronic heart failure published by the European Society of Cardiology, angiotensin receptor-neprilysin inhibitors (ARNIs) or angiotensin-converting enzyme inhibitors (ACEIs), beta-blockers (BBs), mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors (specifically dapagliflozin and empagliflozin), and loop diuretics (when fluid retention occurs) are first-line drugs for the treatment of chronic left ventricular failure. ARNIs, ACEIs, BBs, and MRAs all alleviate symptoms in heart failure patients by regulating the RAAS system and the sympathetic nervous system. Loop diuretics primarily work by reducing fluid retention to decrease cardiac afterload. The benefits of SGLT2 inhibitors in heart failure are mainly achieved by inhibiting sodium-hydrogen exchange, thereby enhancing sodium-calcium exchange and increasing intracellular calcium ion concentration. Furthermore, digitalis drugs exert a positive inotropic effect by inhibiting sodium-potassium exchange in cardiomyocytes, thereby promoting sodium-calcium exchange and increasing intracellular calcium ion concentration. These drugs either directly reduce cardiac afterload, indirectly reduce cardiac afterload to varying degrees through neurohumoral regulation, or are based on changes in myocardial ion homeostasis and improved energy metabolism. However, in patients whose hearts have been under chronic overload and other pathogenic factors, the activity and content of myocardial contractile function-related proteins, represented by SERCA2a, have decreased in cardiomyocytes. These drugs have failed to directly protect or restore the function of these damaged proteins, and have not effectively corrected the imbalance of calcium ion homeostasis in cardiomyocytes.

[0004] As the dominant P-type ATPase in the sarcoplasmic reticulum of cardiomyocytes, a decrease in SERCA2a content and / or reduced activity significantly impairs the sarcoplasmic reticulum's ability to reuptake calcium ions. This leads to delayed cytoplasmic calcium reuptake and delayed cardiomyocyte relaxation, while also resulting in a substantial reduction in sarcoplasmic reticulum calcium reserves, causing a decrease in the amplitude and peak value of calcium transients, leading to excitation-contraction coupling disorders and reduced cardiomyocyte contractility. Therefore, finding drugs that target SERCA2a to stabilize its levels and activity is of great significance for the treatment of left ventricular failure. Summary of the Invention

[0005] The present invention aims to provide a drug targeting SERCA2a for the treatment of left ventricular failure.

[0006] The first objective of this invention is to provide the use of flufenidone in the preparation of medicaments for the prevention or treatment of left ventricular failure.

[0007] A second objective of this invention is to provide the use of flufenidone in the preparation of drugs for the prevention or treatment of myocardial hypertrophy.

[0008] A third objective of this invention is to provide the use of flufenidone in the preparation of medicaments for the prevention or treatment of aortic coarctation.

[0009] A fourth objective of this invention is to provide the use of flufenidone in the preparation of medicaments for the prevention or treatment of myocardial fibrosis.

[0010] The fifth objective of this invention is to provide a drug for the prevention or treatment of left ventricular failure.

[0011] The sixth objective of this invention is to provide a drug for the prevention or treatment of myocardial hypertrophy or myocardial fibrosis.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention demonstrates for the first time that fluorofenidone, used as a treatment for left ventricular failure, has the following effects: reducing cardiomyocyte hypertrophy in a heart failure cell model; directly acting on SERCA2a and increasing its protein level and activity; inhibiting the polyubiquitination degradation of SERCA2a by the E3 ubiquitin ligase WWP1 to stabilize its protein level; improving cardiac function in an animal model of aortic coarctation; reducing myocardial hypertrophy and fibrosis in an animal model of aortic coarctation; and inhibiting the polyubiquitination degradation of SERCA2a in the left ventricular myocardium of an animal model of aortic coarctation.

[0014] Therefore, the present invention provides the following application solutions:

[0015] Application of flufenidone in the preparation of drugs for the prevention or treatment of left heart failure.

[0016] Application of flufenidone in the preparation of drugs for the prevention or treatment of myocardial hypertrophy.

[0017] The use of flufenidone in the preparation of drugs for the prevention or treatment of aortic coarctation.

[0018] Application of flufenidone in the preparation of drugs for the prevention or treatment of myocardial fibrosis.

[0019] Application of flufenidone in the preparation of reagents that inhibit SERCA2a protein degradation or stabilize SERCA2a protein levels.

[0020] Application of flufenidone in the preparation of reagents to enhance the activity of SERCA2a protein.

[0021] As an alternative implementation, the drug or reagent includes a pharmaceutically acceptable salt or carrier.

[0022] This invention provides a medicament for the prevention or treatment of left ventricular failure, which contains flufenidone and a pharmaceutically acceptable salt or carrier.

[0023] This invention provides a medicament for the prevention or treatment of myocardial hypertrophy or myocardial fibrosis, which contains flufenidone and a pharmaceutically acceptable salt or carrier.

[0024] As an alternative implementation, the carrier is a liposome, micelle, dendritic macromolecule, microsphere, or microcapsule.

[0025] The present invention has the following beneficial effects:

[0026] This invention has revealed that flufenidone can reduce cardiomyocyte hypertrophy in a heart failure cell model and alleviate myocardial hypertrophy, fibrosis, and cardiac function deterioration in an aortic coarctation animal model. Further research shows that flufenidone directly binds to SERCA2a via the Q758, D812, and E917 residues of SERCA2a, inhibiting WWP1's recognition, binding, and polyubiquitination of SERCA2a, thereby stabilizing SERCA2a protein levels and activity, and ultimately achieving the goal of treating chronic left ventricular failure. Therefore, flufenidone, as an active ingredient, has promising application prospects and value in the development of novel drugs for the treatment of chronic left ventricular failure. Attached Figure Description

[0027] Figure 1 Figures show the results of using flufenidone to reduce cardiomyocyte hypertrophy in a heart failure cell model. Figures A and B show the mRNA levels of ANP and BNP in NRCMs after co-treatment with 50 μM PE and different concentrations of flufenidone for 72 h. Figures C and E show the Western blots of MYH7 and ANP in NRCMs after co-treatment with 50 μM PE and different concentrations of flufenidone for 72 h. Figures D and F show the grayscale values ​​of the MYH7 and ANP bands, respectively. Figure G shows the rhodamine-phalloidin staining of NRCMs after co-treatment with 50 μM PE and different concentrations of flufenidone for 72 h. Scale bar = 149.6 μM; ns indicates p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0028] Figure 2Figure 1 shows the results of flufenidone directly acting on SERCA2a and increasing its protein level and activity. Figure A shows the Western blotting (WB) of SERCA2a after co-treatment with 50 μM PE and different concentrations of flufenidone for 72 h; Figure B shows the statistical chart of WB band grayscale values; Figure C shows the results of co-treatment with 50 μM PE and different concentrations of flufenidone on NRCMs. After 72 hours, the SERCA2a enzyme activity was statistically analyzed. Figure D shows the results of the DARTS experiment (H9C2 cell lysates treated with 4mM flufenidone), Ctrl represents cells without streptomycin treatment, and 1:2400, 1:1200, 1:600, and 1:300 represent the ratio of added streptomycin to total protein. Figure E shows the statistical analysis of the band gray values. Figure F shows the results of the CETSA experiment (H9C2 cells treated with 4mM flufenidone). Figure G shows the Boltzmann S-shaped fitting curve of the band gray values. Compared with the Vehicle group at the same temperature, *p<0.05, **p<0.01; ns indicates p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 3 The results of using flufenidone to inhibit the polyubiquitination and degradation of SERCA2a to stabilize its protein level are shown in the figure. Figure A shows the mRNA level of SERCA2a after co-treatment of NRCMs with 50 μM PE and different concentrations of flufenidone for 72 h. Figure B shows the Western blot (WB) bands of SERCA2a after treatment of H9C2 alone or with 4 mM flufenidone at 10 μg / mL CHX for 24 h, 48 h, and 72 h. Figure C is a line graph showing the grayscale values ​​of the bands. Figure D shows the results of co-treatment of H9C2 with 50 μM PE and / or 4 mM flufenidone in the presence of 20 μM MG132. Six hours later, the results of the Co-IP experiment (cell lysate was immunoprecipitated with SERCA2a antibody, and then detected with Ubiquitin antibody and SERCA2a antibody respectively; IgG of the same species was used as a negative control) are shown in Figure E, which is a statistical graph of the gray values ​​of the bands; Figure F shows the bands of H9C2 cells treated with 4mM flufenidone or DMSO of the same concentration for 24 hours, followed by cell lysis and immunoprecipitation with SERCA2a antibody, and then stained with Coomassie brilliant blue; Figure G shows the secondary spectrum of WWP1 identified by immunoprecipitation tandem mass spectrometry; Figures H and J show the bands of H9C2 cells treated with 4mM flufenidone or DMSO of the same concentration. After 24 hours, the results of the Co-IP experiment (cell lysate was immunoprecipitated with SERCA2a antibody or WWP1 antibody, and then detected with WWP1 and SERCA2a antibodies respectively; IgG of the same species was used as a negative control) were obtained. The I-plot and K-plot are statistical graphs of the gray values ​​of the bands; ns indicates p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 4The diagram shows the results of WWP1 directly binding to SERCA2a and catalyzing its polyubiquitination and degradation; Figure A shows the results of Coomassie Brilliant Blue staining of bacteria transformed with prokaryotic expression plasmids before IPTG induction (BF). IPTG ), after (AF) IPTG Figure 1 shows the expression results of GST and GST-WWP1 proteins in whole bacterial lysate; Figures B and C show the results of GST and GST-WWP1 prokaryotic expression and purification, respectively, after which Coomassie brilliant blue staining was used to detect protein purification; Figure D shows the results of Flag-SERCA2a eukaryotic expression and purification, respectively, after which Coomassie brilliant blue staining was used to detect protein purification, where CL refers to whole bacterial / cell lysate, FT refers to flow-through buffer, W1 and W2 refer to the first and second washing buffers, and E1 and E2 refer to the first and second purified protein collection buffers; Figure E shows GST... Figure F shows the results of the pull-down experiment; Figure F shows the WB bands and their gray values ​​after 24 hours of medium change and 48 hours of sample collection from H9C2 cardiomyocytes transfected with two siRNAs-WWP1; Figure G shows the results of the Co-IP experiment and their gray values ​​after 48 hours of sample collection from HEK293T cells co-transfected with wild-type plasmid Myc-WWP1-WT or catalytically inactive mutant plasmid Myc-WWP1-C890A and wild-type plasmid Flag-SERCA2a-WT; ns indicates p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0031] Figure 5 Figure 1 shows the results of the interaction mode analysis between flufenidone and SERCA2a. Figure A shows the 3D chemical structure of flufenidone; Figure B shows the 3D structural model of SERCA2a and its four binding pockets suitable for flufenidone binding; Figure C shows the interaction mode of flufenidone and SERCA2a predicted by molecular docking, with the interaction region within the orange box, a magnified 3D binding schematic diagram on the upper right, where the purple-red represents the compound flufenidone, the blue-green represents the interacting amino acids, and the lower right shows a 2D binding schematic diagram.

[0032] Figure 6The diagram shows the results of the interaction between flufenidone and SERCA2a, where Q758, D812, and E917 residues on SERCA2a are involved. Figure A shows the Co-IP results of Flag-SERCA2a-WT and Flag-SERCA2a-Q758A / D812A / E917A, co-transfected with Myc-WWP1-WT into HEK293T cells for 48 hours, followed by treatment with flufenidone or the same concentration of DMSO and MG132 for 6 hours. The right side shows the statistical graph of the band gray values. Figure B shows the results of the Co-IP experiment of Flag-SERCA2a-WT,... Flag-SERCA2a-Q758A, Flag-SERCA2a-D812A, Flag-SERCA2a-E917A, and Flag-SERCA2a-Q758A / D812A / E917A were co-transfected with Myc-WWP1-WT into HEK293T cells for 48 hours, followed by treatment with flufenidone or the same concentration of DMSO and MG132 for 6 hours. The results of the Co-IP experiment and the statistical graph of the gray values ​​of the bands are shown. ns indicates p>0.05, *p<0.05, **p<0.01, ***p<0.001.

[0033] Figure 7 Figures showing the results of flufenidone improving cardiac function in mice with aortic coarctation; Figure A shows the M-mode echocardiograms of mice in the Sham, TAC, and Fluorofenidone groups on day 56 post-surgery; Figures B and C show the statistical results of LVEF and LVFS in each group on day 56 post-surgery; ***p<0.001.

[0034] Figure 8 Figure 1 shows the results of flufenidone in reducing myocardial hypertrophy in mice with aortic coarctation. Figure A shows the heart-to-tibia ratio on day 56 in the Sham, TAC, and Fluorofenidone groups; Figure B shows the heart-to-body ratio on day 56 in the Sham, TAC, and Fluorofenidone groups; Figure C shows microscopic photographs of left ventricular myocardial tissue sections stained with WGA on day 56 (scale bar = 50 μm); Figure D shows the cross-sectional area of ​​myocardial cells in each group measured by WGA staining. **p < 0.01, ***p < 0.001.

[0035] Figure 9The images show the results of fluorofenidone reducing myocardial hypertrophy and fibrosis in mice with aortic coarctation. Figures A and B show the mRNA levels of ANP and BNP in the left ventricular myocardial tissue of mice in the Sham, TAC, and Fluorofenidone groups, respectively. Figure C shows the Western blot (WB) bands of MYH6, MYH7, ANP, and BNP in the left ventricular myocardial tissue of each group of mice. Figures D, E, F, and G show the gray bands of MYH6, MYH7, ANP, and BNP, respectively. Statistical graphs of degree values; H graph is a microscopic photograph of MT-stained left ventricular myocardial tissue sections from each group of mice and a statistical graph of the area of ​​myocardial fibrosis region. The scale bar of the 100× graph is 400μm, and the scale bar of the 400× graph is 100μm; I graph, J graph, K graph and L graph are statistical graphs of the mRNA levels of COL1A1, COL3, FN1 and POSTN in the left ventricular myocardial tissue of each group of mice, respectively; *p<0.05, **p<0.01, ***p<0.001.

[0036] Figure 10 Figure 1 shows the results of flufenidone inhibiting the polyubiquitination and degradation of SERCA2a in the left ventricular myocardium of mice with aortic constriction. Figure 2 shows the statistical levels of SERCA2a mRNA in the left ventricular myocardium of mice in the Sham, TAC, and Fluorofenidone groups. Figure 3 shows the WB bands of SERCA2a in the left ventricular myocardium of each group of mice and their gray value statistics. Figure 4 shows the band diagrams and gray value statistics of the Co-IP experiment (immunoprecipitation with SERCA2a antibody and WWP1 antibody, respectively) of the left ventricular myocardium of each group of mice. ns indicates p > 0.05, **p < 0.01, ***p < 0.001. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] Neonatal rat cardiomyocytes (NRCMs) were extracted from 1-3 day old suckling mice. The extraction method was referenced in the literature: Jensen L, Neri E, Bassaneze V, De Almeida Oliveira NC, Dariolli R. LT, LevyD, Veronez D, Ferraz MSA, Alencar AM, Bydlowski SP, Cestari IA, KriegerJE. Integrated molecular, biochemical, and physiological assessment unravels keyextraction method mediated influences on rat neonatal cardiomyocytes. J CellPhysiol. 2018Jul; 233(7):5420-5430.》.

[0040] The reference for the Rhodamine-phalloidin staining assay is: "Li Qizhi, Yang Yeqiu, Xia Shu, et al. Exploration of a comprehensive experiment on microfilament labeling and observation of animal cytoskeleton [J]. Bulletin of Biology, 2021, 56(02):53-57."

[0041] Endoplasmic reticulum extraction kit (low-speed centrifugation method), manufactured by Beijing Solarbio Science & Technology Co., Ltd., catalog number EX2690.

[0042] Ultra-micro Ca-ATPase (tissue and ordinary cell) kit (Ca 2+ -ATPase assay kit), manufactured by Nanjing Jiancheng Biotechnology Research Institute, catalog number A070-4-2.

[0043] Flufenidone, CAS number 848353-85-5, has the following structural formula:

[0044]

[0045] Example 1: Flufenidone can reduce cardiomyocyte hypertrophy in a heart failure cell model.

[0046] Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are biomarkers for the diagnosis of heart failure, while MYH7 protein is an indicator of cardiac contractility. When heart failure or myocardial hypertrophy occurs, the expression of ANP, BNP, and MYH7 genes will be upregulated.

[0047] I. Experimental Methods

[0048] 1. Newborn rat cardiomyocytes (NRCMs) were extracted and seeded in six-well plates. After adhering culture for 36 h, they were starved for 24 h. Then, 50 μM phenylephrine (PE) and different concentrations of flufenidone (final concentrations of 1 mM, 2 mM, and 4 mM) were added and the cells were treated for 72 h. The protein levels of MYH7 and ANP in NRCMs were detected by Western blotting (WB experiment), and the gene expression levels of ANP and BNP in NRCMs were detected by real-time quantitative PCR (qPCR experiment).

[0049] 2. NRCMs were seeded in 24-well plates and cultured adherently for 36 hours. Then, they were starved for 24 hours and treated with 50 μM phenylephrine and different concentrations (including 1 mM, 2 mM and 4 mM) of flufenidone for 72 hours. Rhodamine-phalloidin staining was then performed on the NRCMs.

[0050] II. Experimental Results

[0051] We treated cardiomyocytes with 50 μM PE for 72 h to establish a heart failure cell model. The results of flufenidone reducing cardiomyocyte hypertrophy in the heart failure cell model are as follows: Figure 1 As shown, the results indicate that:

[0052] (1) Using qPCR to detect cardiomyocyte hypertrophy indices in a heart failure model, it was found that the mRNA levels of ANP and BNP in cardiomyocytes upregulated by PE treatment could be effectively reduced by flufenidone. Figure 1 (Figures A and B).

[0053] Verified by WB experiment ( Figure 1 (Figures C, D, E, and F) show that flufenidone treatment significantly reduces the levels of MYH7 and ANP proteins in cardiomyocytes.

[0054] (2) In addition, we used a rhodamine-labeled phalloidin probe for cardiomyocyte staining to assess hypertrophic changes. The results showed that flufenidone effectively reduced PE-induced cardiomyocyte hypertrophy. Figure 1 (G diagram).

[0055] Example 2: Flufenidone directly acts on SERCA2a and increases its protein level and activity.

[0056] I. Experimental Methods

[0057] 1. NRCMs were extracted and seeded in six-well plates. After adhering culture for 36 h, the plates were starved for 24 h. Then, 50 μMPE and different concentrations (including 1 mM, 2 mM and 4 mM) of flufenidone were added and treated for 72 h. The SERCA2a protein level of NRCMs was detected by Western blot.

[0058] 2. NRCMs were extracted and plated in 15cm culture dishes. After adhering to the plate for 36 hours, they were starved for 24 hours. Then, they were treated with 50μM PE and different concentrations (1mM, 2mM, and 4mM) of flufenidone for 72 hours. Following this, endoplasmic reticulum extraction and SERCA2a activity assays were performed on the NRCMs. Endoplasmic reticulum extraction was performed using an endoplasmic reticulum extraction kit (Solarbio) according to the manufacturer's instructions, using ultra-micro Ca... 2+ Use the Ca2+-ATPase assay kit to detect SERCA2a activity. Refer to the kit's instructions for specific detection methods.

[0059] 3. Rat cardiomyocyte H9C2 cells were seeded in 15cm culture dishes for Drug Affinity Responsive Target Stability (DARTS) assays. The specific steps were as follows: After routine medium changes and cell confluence, cells were lysed to obtain total protein solution. Protein concentration was determined using the BCA method. The total protein was evenly divided into two pre-labeled and pre-chilled 1.5mL EP tubes (groups A and C), and the total volume was recorded. DMSO (1 / 99 of the total volume) was added to group C, and an equal volume of 400mM flufenidone was added to group A. After thorough mixing, the tubes were incubated at room temperature with gentle shaking for 1 hour. The total protein from groups A and C was then evenly divided into six tubes, labeled A0, A1, A2, A3, A4 and C0, C1, C2, C3, C4, respectively. These tubes were then placed in a 25℃ constant-temperature metal bath for later use, and the total protein content in each tube after equal division was calculated. Streptomycin was dissolved in ultrapure water to prepare concentrated working solutions with ratios of 1:300, 1:600, 1:1200, and 1:2400 to the total protein. 2 μL of ultrapure water was added to tube A0, and the solution was mixed by pipetting. A timer was started. After 1 minute, 2 μL of ultrapure water was added to tube C0, and the solution was mixed by pipetting. Then, every minute, 2 μL of the corresponding concentrated working solution (A4 / C4—1:2400, A3 / C3—1:1200, A2 / C2—1:600, A1 / C1—1:300) was added to tubes A4, C4, A3, C3, A2, C2, A1, and C1, and the solution was mixed by pipetting. After 30 minutes, an appropriate amount of protease inhibitor was added to tube A0, the solution was mixed by pipetting, and the tube was placed on ice. Then, every 1 minute, appropriate amounts of protease inhibitor were added sequentially to C0, A4, C4, A3, C3, A2, C2, A1, and C1, mixed thoroughly by pipetting, and placed on ice. Finally, all treated samples were subjected to Western blotting to detect SERCA2a protein levels.

[0060] 4. Rat cardiomyocyte H9C2 cells were seeded in 18 15cm culture dishes for a Cellular Thermal Shift Assay (CETSA). The specific steps were as follows: the medium was changed routinely until the cells reached confluence. Nine dishes were treated with 4mM flufenidone for 1 hour (group A), and the other nine were treated with DMSO of the same concentration (group C). The cells in each dish were lysed to obtain total protein solution. Group A was divided into one tube, and group C into another tube, and the solutions were transferred to new 2mL EP tubes. After mixing by pipetting, the solutions were divided into 12×2 PCR tubes (pre-labeled with the treatment temperature and group, i.e., 37A / C, 40A / C, 43A / C, 46A / C, 49A / C, 52A / C, 55A / C, 58A / C, 61A / C, 64A / C, 67A / C, 70A / C), with 75μL in each tube. Cells in each tube were rapidly heated to a predetermined temperature for 3 min using an ABI gradient PCR instrument. After heating, the parameters were adjusted to 25℃ and the cells in each tube were cooled for 3 min. 20 μL of 1% NP-40 lysis buffer was added to each tube, and after thorough mixing, each tube was rapidly frozen in liquid nitrogen for 3 min. The tubes were then removed from the liquid nitrogen, thawed at 25℃ for 3 min, and then rapidly frozen in liquid nitrogen for another 3 min. This freeze-thaw cycle was repeated three times. Finally, the liquid from each tube was transferred to a labeled 1.5 ml EP tube and centrifuged at 4℃ and 20000 g for 20 min.

[0061] 60 μL of supernatant was aspirated from each tube for Western blotting to detect SERCA2a protein levels, with β-actin as an internal control and normalization performed using 37A and 37C as baselines. The Boltzmann-Sigmoid equation in GraphPad Prism was used to fit the data to obtain the apparent melting temperature Tm (i.e., the temperature at which the protein content drops to 50% according to the curve prediction) and the apparent aggregation temperature Tagg (i.e., the temperature at which the protein content just drops to the lowest level according to the curve prediction).

[0062] II. Experimental Results

[0063] (1) WB results show ( Figure 2 (Figures A and B) show that under co-treatment with different concentrations of flufenidone, the SERCA2a protein level induced by PE in NRCMs can be significantly restored, and the degree of restoration increases with increasing flufenidone concentration.

[0064] (2)Ca 2+ -ATPase activity assay showed ( Figure 2 (Figure C) shows that, compared with the heart failure model group with significantly reduced SERCA2a activity, co-treatment with different concentrations of flufenidone can increase SERCA2a activity to varying degrees.

[0065] (3) The DARTS experiment shows that ( Figure 2(Figures D and E) show that flufenidone can effectively antagonize the hydrolytic effect of streptococcal protease on SERCA2a in H9C2 cell lysate, which proves in vitro that SERCA2a is a target protein of flufenidone.

[0066] (4) The CETSA results, fitted using the Boltzmann S-curve, show that ( Figure 2 As shown in Figures F and G, the apparent melting temperature (Tm) of SERCA2a increased from 49.56℃ in the control group to 61.80℃ after flufenidone treatment, and its apparent aggregation temperature (Tagg) increased from 52℃ to 67℃. This indicates that, compared to the control group, SERCA2a in flufenidone-treated H9C2 cells exhibits a slower rate of thermal denaturation and precipitation, demonstrating under physiological conditions that SERCA2a is a target protein of flufenidone.

[0067] Example 3: Flufenidone directly binds to SERCA2a via Q758, D812, and E917, inhibiting the polyubiquitination and degradation of SERCA2a by the E3 ubiquitin ligase WWP1 to stabilize SERCA2a protein levels.

[0068] I. Experimental Methods

[0069] 1. NRCMs were extracted and seeded in a six-well plate. After adhering to the plate for 36 h, the plate was starved for 24 h. Then, 50 μMPE and different concentrations (including 1 mM, 2 mM and 4 mM) of flufenidone were added and treated for 72 h. The expression level of SERCA2a gene in NRCMs was detected by qPCR.

[0070] 2. H9C2 cells were seeded in six-well plates and treated with 10 μg / mL cyclohexane (CHX) alone or in combination with 4 mM flufenidone for 24 h, 48 h and 72 h. The SERCA2a protein level was then detected by Western blotting.

[0071] 3. H9C2 cells were seeded in 15cm culture dishes. After treating H9C2 cells with 50μM PE and / or 4mM flufenidone for 6h in the presence of 20μM MG132 proteasome inhibitor, Co-IP experiments were performed.

[0072] 4. H9C2 cells were seeded in 15cm culture dishes and treated with 4mM flufenidone or DMSO of the same concentration for 24h. The cells were then lysed and immunoprecipitated with SERCA2a antibody, followed by Coomassie brilliant blue staining and immunoprecipitation tandem mass spectrometry analysis.

[0073] 5. H9C2 cells were seeded in 15cm culture dishes and treated with 4mM flufenidone or DMSO of the same concentration for 24h. Then, Co-IP experiment was performed (cell lysate was immunoprecipitated with SERCA2a or WWP1 antibody, and then detected with WWP1 antibody or SERCA2a antibody, respectively).

[0074] 6. Construct prokaryotic expression plasmids for GST and GST-WWP1 proteins, as well as eukaryotic expression plasmids for GST and GST-WWP1 proteins. The prokaryotically expressed and purified GST and GST-WWP1 proteins, along with the eukaryotically expressed and purified Flag-SERCA2a protein, were subjected to an in vitro GST pull-down experiment.

[0075] 7. H9C2 cells were seeded in six-well plates and starved for 24 hours. Then, they were transfected with WWP1 siRNA. The medium was changed after 24 hours, and the samples were collected after another 48 hours for Western blot analysis.

[0076] 8. Construct wild-type plasmid Myc-WWP1-WT or catalytically inactive mutant plasmid Myc-WWP1-C890A, and co-transfect them with wild-type plasmid Flag-SERCA2a-WT into HEK293T cells. After 48 hours, collect samples for Co-IP detection.

[0077] 9. Molecular docking was used to simulate the optimal interaction mode between flufenidone and SERCA2a, and the key amino acid residues on SERCA2a that form hydrogen bonds with flufenidone in this mode were predicted.

[0078] 10. Based on the prediction results, mutant plasmids were constructed in which all key amino acid residues were simultaneously mutated to alanine, and mutant plasmids were constructed in which each key amino acid residue was individually mutated to alanine. After HEK293T cells were seeded in 15cm culture dishes, wild-type plasmid Flag-SERCA2a-WT or each point mutant plasmid was co-transfected with Myc-WWP1-WT into HEK293T cells. After 48h of transfection, the cells were treated with 4mM flufenidone or DMSO and MG132 for 6h, and then Co-IP was performed.

[0079] II. Experimental Results

[0080] (1) qPCR results showed that ( Figure 3 (Figure A) The co-treatment with flufenidone failed to alter the decrease in SERCA2a mRNA levels caused by PE, proving that this stabilizing effect is not due to the promotion of protein synthesis.

[0081] (2) WB results show ( Figure 3As shown in Figures B and C, after blocking intracellular translation with CHX, the presence of flufenidone significantly slowed down the degradation rate of SERCA2a.

[0082] (3) Co-IP results show ( Figure 3 (Figure D) PE treatment significantly upregulated the polyubiquitination level of SERCA2a in cardiomyocytes, while co-treatment with flufenidone significantly antagonized this upregulation, demonstrating that flufenidone inhibits the ubiquitination and degradation of SERCA2a.

[0083] (4) Visible after Coomassie brilliant blue staining ( Figure 3 (Figure F) Flufenidone treatment can reduce the binding degree between SERCA2a and interacting proteins.

[0084] (5) After screening by immunoprecipitation tandem mass spectrometry, Co-IP test proved that the intracellular E3 ubiquitin ligase WWP1 can bind to SERCA2a, and this binding can be inhibited by flufenidone. Figure 3 (G diagram, H diagram, I diagram, J diagram, K diagram).

[0085] (6) GST pull-down experiments performed after expression and purification in prokaryotes and eukaryotes proved that ( Figure 4 (Figures A, B, C, and E), WWP1 is directly linked to SERCA2a.

[0086] (7) The results of siRNA transfection showed that ( Figure 4 (See F-plot). When intracellular WWP1 levels are knocked down, SERCA2a levels can be significantly upregulated.

[0087] (8) Simultaneously, the co-transfection results of wild-type plasmid Myc-WWP1-WT or the catalytically inactive mutant plasmid Myc-WWP1-C890A with wild-type plasmid Flag-SERCA2a-WT showed that ( Figure 4 (G diagram) WWP1 overexpression significantly reduced the protein level of SERCA2a and significantly increased the polyubiquitination level of SERCA2a, while overexpression of the catalytically inactive mutant WWP1 failed to produce this effect, confirming that WWP1 can directly bind to SERCA2a and catalyze its polyubiquitination degradation.

[0088] (9) Molecular docking prediction Figure 5 The optimal interaction mode for flufenidone is in pocket 3 of SERCA2a, where it is predicted to bind directly to amino acid residues at positions 758, 812, and 917 on SERCA2a via hydrogen bonds.

[0089] (10) Co-IP results show ( Figure 6 Flufenidone inhibits the binding of WWP1 to SERCA2a and the polyubiquitination of SERCA2a by WWP1. Individual point mutations of amino acid residues 758, 812, and 917 of SERCA2a to alanine (denoted as Q758A, D812A, and E917A, respectively) weaken this inhibition to varying degrees. However, only when all three amino acid residues of SERCA2a are simultaneously mutated to alanine (denoted as Q758A / D812A / E917A) does this inhibition disappear. Therefore, residues Q758, D812, and E917 of SERCA2a are involved in the direct binding of flufenidone to SERCA2a.

[0090] Example 4: Flufenidone improves cardiac function in mice with aortic coarctation.

[0091] I. Experimental Methods

[0092] ① Wild-type C57BL / 6 mice (male, 22-25g, 6-8 weeks old) were housed in an SPF-grade barrier environment with constant temperature (24±1℃), constant humidity (55±5%) and a diurnal cycle (12h / 12h).

[0093] ② After weighing, anesthetizing, and preparing the skin, the mice were fixed and intubated and ventilated. After opening the chest and exposing the aortic arch, a pin was used as a mold to quickly tie a knot around the aortic arch and the pin to form a narrowing with a diameter of about 0.4 mm in the aortic arch between the brachiocephalic trunk and the left common carotid artery. The chest was closed layer by layer, the skin was sutured, and the area was disinfected. Ventilation was continued until resuscitation, thus establishing a mouse transverse aortic constriction (TAC) model.

[0094] ③ Sham surgery was performed on mice as a control. TAC mice were randomly divided into two groups. One group was given 250 mg / kg flufenidone by gavage daily from day 1 to day 56 after surgery (referred to as the Fluorofenidone group). The other group of TAC mice and mice that underwent sham surgery were given an equal volume of solvent by gavage daily from day 1 to day 56 after surgery (the solvent was a mixture of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% physiological saline, referred to as the TAC group and the Sham group, respectively).

[0095] ④ On the 56th day after surgery, transthoracic echocardiography was performed on mice in each group.

[0096] II. Experimental Results

[0097] Echocardiography results showed ( Figure 7Compared to the Sham group, the left ventricular ejection fraction (LVEF) and the percentage of left ventricular fractional shortening (LVFS) of mice in the TAC group were significantly reduced. Treatment with flufenidone significantly alleviated these changes, demonstrating that flufenidone treatment can effectively reduce the deterioration of cardiac function in mice with aortic coarctation.

[0098] Example 5: Flufenidone can reduce myocardial hypertrophy and fibrosis in mice with aortic coarctation.

[0099] MYH6 and MYH7 proteins are indicators of myocardial contractility. When heart failure or myocardial hypertrophy occurs, the expression level of the MYH6 gene decreases, while the expression level of the MYH7 gene increases.

[0100] I. Experimental Methods

[0101] ① Example 4: After echocardiography, each mouse was euthanized and its tissues were collected. The ventricular mass was weighed, the length of the left tibia was measured, and the ratio of ventricular mass to body weight (i.e., heart-body ratio) and the ratio of ventricular mass to left tibia length (i.e., heart-tibia ratio) were calculated.

[0102] ② At the midpoint of the line connecting the base and apex of the heart, a ring of ventricular tissue approximately 2 mm thick was excised from the harvested ventricle. After fixation, dehydration, and embedding, the tissue was cut into 5 μm thick sections. After sectioning, the sections were stained with wheat germ agglutinin (WGA) and Masson trichrome (MT). MT staining was used to calculate the fibrosis area: fibrosis area = (area of ​​blue-stained region / total area of ​​myocardial tissue) × 100%. WGA staining was used to measure the cross-sectional area of ​​cardiomyocytes. All parameters were measured using ImageJ software.

[0103] ③ After harvesting ventricular tissue for morphological analysis, the right ventricle portion of the remaining tissue was removed. The left ventricle and interventricular septum tissue were minced to a diameter of approximately 1 mm and divided into two parts. One part was used for tissue RNA extraction, and the other part was used for tissue total protein extraction. qPCR and WB experiments were performed. qPCR was used to detect the mRNA levels of ANP, BNP, COL1A1, COL3, FN1, and POSTN, while WB was used to detect the protein levels of MYH6, MYH7, ANP, and BNP.

[0104] II. Experimental Results

[0105] The results of the heart-to-tibia ratio and heart-to-body ratio measurements showed ( Figure 8(Figures A and B) In the TAC group mice, the heart-to-tibia ratio and heart-to-body ratio increased to 1.50 times and 1.61 times that of the Sham group, respectively, while flufenidone treatment significantly reduced the increase in heart-to-tibia ratio and heart-to-body ratio.

[0106] WGA staining results showed ( Figure 8 (Figures C and D) After treatment with flufenidone, the significantly increased cross-sectional area of ​​myocardial cells after TAC surgery can be significantly reduced.

[0107] qPCR results prove ( Figure 9 (Figures A and B) Compared to the Sham group, the mRNA levels of ANP and BNP in the left ventricular myocardial tissue of mice in the TAC group were significantly increased, while both indicators were significantly reduced after flufenidone treatment.

[0108] WB results show ( Figure 9 Figures C, D, E, F, and G show that in the TAC group, MYH6 protein levels were significantly downregulated, while MYH7, ANP, and BNP protein levels were significantly downregulated. After flufenidone treatment, MYH6 protein levels were significantly upregulated, while MYH7, ANP, and BNP protein levels were significantly downregulated. This indicates that flufenidone treatment can significantly slow the progression of myocardial hypertrophy in TAC mice.

[0109] MT staining results showed ( Figure 9 (H-plot) Eight weeks after TAC surgery, the area of ​​left ventricular myocardial fibrosis in mice was 4.61 times that of the Sham group, which decreased to 1.71 times that of the Sham group after flufenidone treatment. Simultaneously, the mRNA levels of COL1A1, COL3, FN1, and POSTN in the left ventricular myocardial tissue of mice in the TAC group were 3.29 times, 1.67 times, 5.81 times, and 1.50 times higher than those in the Sham group, respectively, while flufenidone treatment significantly reduced the upregulation levels of these fibrosis-related markers. Figure 9 (I diagram, J diagram, K diagram, L diagram).

[0110] Example 6: Flufenidone inhibits the polyubiquitination and degradation of SERCA2a in the left ventricular myocardium of mice with aortic constriction.

[0111] I. Experimental Methods

[0112] 1. The mRNA level of SERCA2a in the left ventricular myocardial tissue of mice obtained in Example 5 was detected by qPCR experiment.

[0113] 2. A small portion of the total protein solution extracted from the tissue in Example 5 was taken out and used for Western blotting to detect the SERCA2a protein level.

[0114] 3. The remaining total protein solution was divided into two tubes, and Co-IP experiments were performed on the two tubes using SERCA2a antibody and WWP1 antibody, respectively.

[0115] II. Experimental Results

[0116] qPCR and WB results showed that ( Figure 10 As shown in Figures A and B, compared to the Sham group, the mRNA and protein levels of SERCA2a in the left ventricular myocardial tissue of mice in the TAC group, which had already developed left heart failure, were significantly reduced. After treatment with flufenidone, the protein level of SERCA2a significantly recovered to the level of the Sham group, while its mRNA level showed no significant change compared to the TAC group. These results confirm that flufenidone stabilizes the protein level of SERCA2a by inhibiting its degradation rather than promoting its synthesis.

[0117] Co-IP experimental results show that ( Figure 10 (Figure C) Compared with the Sham group, the binding degree of WWP1 to SERCA2a and the polyubiquitination level of SERCA2a in the myocardial tissue of TAC group mice were significantly increased, while flufenidone treatment could significantly reduce these increases, proving that flufenidone can inhibit the binding of WWP1 to SERCA2a in the left ventricular myocardial tissue of TAC mice, thereby reducing the polyubiquitination degradation of SERCA2a.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of flufenidone in the preparation of drugs for the prevention or treatment of left heart failure.

2. Application of flufenidone in the preparation of drugs for the prevention or treatment of myocardial hypertrophy.

3. Application of flufenidone in the preparation of drugs for the prevention or treatment of aortic coarctation.

4. Application of flufenidone in the preparation of drugs for the prevention or treatment of myocardial fibrosis.

5. Application of flufenidone in the preparation of reagents that inhibit SERCA2a protein degradation or stabilize SERCA2a protein levels.

6. Application of flufenidone in the preparation of reagents to enhance the activity of SERCA2a protein.

7. The application according to any one of claims 1-6, characterized in that, The drug or reagent includes pharmaceutically acceptable salts or carriers.

8. A drug for the prevention or treatment of left ventricular failure, characterized in that, It contains flufenidone and a pharmaceutically acceptable salt or carrier.

9. A drug for preventing or treating myocardial hypertrophy or myocardial fibrosis, characterized in that, It contains flufenidone and a pharmaceutically acceptable salt or carrier.

10. The drug according to claim 8 or 9, characterized in that, The carrier is a liposome, micelle, dendritic macromolecule, microsphere, or microcapsule.

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