Use of a ctsl inhibitor in the preparation of a medicament for treating fulminant myocarditis

Abstract

This invention provides the application of CTSL inhibitors in the preparation of drugs for treating fulminant myocarditis, specifically belonging to the field of biomedical technology. This invention discovered that CTSL levels are significantly elevated in the myocardial tissue of mice with fulminant myocarditis, and that CTSL inhibitors can be used to prepare drugs for treating fulminant myocarditis. Results show that CTSL inhibitors can improve cardiomyocyte damage, reduce cardiac inflammation, improve cardiac pathological remodeling and function, increase survival rate, and achieve treatment for fulminant myocarditis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CTSL inhibitors in the preparation of drugs for treating fulminant myocarditis. Background Technology

[0002] Myocarditis refers to inflammatory damage to the myocardium caused by various factors, ultimately leading to impaired cardiac function, including decreased systolic and diastolic function and arrhythmias. Fulminant myocarditis is the most severe and unique type of myocarditis, characterized by rapid onset and progression. Patients develop hemodynamic abnormalities (pump failure and circulatory failure) and severe arrhythmias within a short period, and may also experience respiratory failure and liver and kidney failure, resulting in a high early mortality rate. Fulminant myocarditis can be caused by infection, toxin / drug toxicity, and autoimmune diseases, with infection being the most important cause, and viruses being the most common pathogens.

[0003] Fulminant myocarditis causes severe damage to cardiomyocytes within a short period, leading to acute heart failure and malignant arrhythmias, resulting in an extremely high early mortality rate. Overactivation of the innate immune system and the formation of a cytokine storm are considered the core mechanisms of cardiomyocyte damage in fulminant myocarditis. However, because the molecular mechanisms by which the cytokine storm causes cardiomyocyte damage are not yet fully understood, currently, apart from immunomodulatory therapies (immunoglobulins) and immunosuppressive therapies such as glucocorticoids, there are no effective drugs to improve the survival rate and cardiac function of patients with fulminant myocarditis. Therefore, the development of drugs that block the cytokine storm from damaging cardiomyocytes could provide a new treatment option for patients with fulminant myocarditis. Summary of the Invention

[0004] The purpose of this invention is to provide the application of CTSL inhibitors in the preparation of medicaments for treating fulminant myocarditis. CTSL inhibitors can improve cardiomyocyte damage, reduce cardiac inflammation, improve cardiac pathological remodeling and function, thereby achieving the treatment of fulminant myocarditis.

[0005] This invention provides the application of CTSL inhibitors in the preparation of medicaments for treating fulminant myocarditis.

[0006] The present invention also provides the use of CTSL inhibitors in the preparation of medicaments for the treatment of viral myocarditis.

[0007] This invention also provides the use of CTSL inhibitors in the preparation of products that improve cardiomyocyte damage.

[0008] This invention also provides the application of CTSL inhibitors in the preparation of products that reduce programmed necrosis of cardiomyocytes.

[0009] This invention also provides the use of CTSL inhibitors in the preparation of products that improve cardiac function and / or hemodynamics.

[0010] This invention also provides the use of CTSL inhibitors in the preparation of products that improve cardiac inflammatory cell infiltration.

[0011] The present invention also provides the use of CTSL inhibitors in the preparation of products that reduce cardiac death.

[0012] This invention also provides the application of CTSL inhibitors in the preparation of products that improve cardiac pathological remodeling.

[0013] This invention also provides the use of CTSL inhibitors in the preparation of products that improve any one or more of the indicators described in ① to ⑦:

[0014] ① Long-axis and short-axis left ventricular ejection fraction;

[0015] ② Long-axis and short-axis left ventricular end-diastolic volume;

[0016] ③ Long-axis and short-axis left ventricular end-systolic volume;

[0017] ④ Left ventricular end-systolic pressure;

[0018] ⑤ Left ventricular end-diastolic pressure;

[0019] ⑥ Maximum rate of increase of left ventricular pressure;

[0020] ⑦ Maximum rate of decrease in left ventricular pressure.

[0021] Preferably, the CTSL inhibitor comprises Z-FY-CHO.

[0022] This invention provides the application of CTSL inhibitors in the preparation of drugs for treating fulminant myocarditis. Experimental results show that CTSL inhibitors have a therapeutic effect on viral-induced fulminant myocarditis, improving cardiomyocyte damage and fibrosis, inflammatory cell infiltration, and cardiac pathological remodeling and function. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The experimental flowchart for the CTSL inhibitor provided by this invention to improve cardiac function, survival rate, inflammatory cell infiltration and cardiac remodeling in fulminant myocarditis;

[0025] Figure 2 The following diagrams illustrate the results of lysosomal damage and programmed necrosis in the myocardial tissue of mice with fulminant myocarditis, provided by this invention. (A) is a flowchart of the experiment; (B) shows the mRNA expression level of CVB3 in myocardial tissue; (C) shows the protein levels of lysosomal cathepsins B, D, and L (CTSB, CTSD, CTSL) and the programmed necrosis marker protein (pMLKL) in myocardial tissue detected by Western blotting; (D) to (G) show the quantitative analysis results of CTSB, CTSD, CTSL, and pMLKL, respectively.

[0026] Figure 3 The results provided by this invention show that intraperitoneal injection of the CTSL inhibitor Z-FY-CHO reduced the mortality rate of mice with fulminant myocarditis, while intraperitoneal injection of the CTSB inhibitor CA-074-me and the CTSD inhibitor PepstainA did not reduce the mortality rate. Among them, (A) experimental flowchart; (B) survival rate of mice within 4 weeks.

[0027] Figure 4 The following diagrams illustrate the results of the present invention to verify the inhibitory effect of the CTSL inhibitor Z-FY-CHO on CTSL in myocardial tissue of mice with fulminant myocarditis; (A) experimental flowchart; (B) Western blotting results of the effect of Z-FY-CHO on CTSL expression; (C) quantitative analysis diagram of diagram (B); and (D) immunofluorescence staining results of CTSL in myocardial cells of four groups of mice.

[0028] Figure 5 The results of intraperitoneal injection of Z-FY-CHO significantly improving lysosomal damage and programmed necrosis of myocardial cells in mice with fulminant myocarditis are shown in the figure provided by the present invention. Among them, (A) is a representative electron micrograph of myocardial cell lysosomes in four groups of mice; (B) is a figure showing the lysosomal diameter; (C) is a figure showing the pMLKL protein level detected by Western blotting; (D) is a quantitative analysis figure of figure (C).

[0029] Figure 6 The results of intraperitoneal injection of Z-FY-CHO significantly improving the survival rate of mice with fulminant myocarditis provided by the present invention are shown in the figure; (A) experimental flowchart; (B) survival rate of mice within 4 weeks.

[0030] Figure 7The following images show the results of intraperitoneal injection of Z-FY-CHO significantly improving cardiac function in mice with fulminant myocarditis, provided by this invention. (A) is a representative long-axis echocardiogram image at 4 weeks; (B) to (D) are images of left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) at 1, 2, and 4 weeks, respectively; (E) is a representative short-axis echocardiogram image at 4 weeks; and (F) to (H) are images of LVEF, LVEDV, and LVESV at 1, 2, and 4 weeks, respectively.

[0031] Figure 8 The following figures illustrate the significant improvement in hemodynamics in mice with fulminant myocarditis after intraperitoneal injection of Z-FY-CHO, as provided by this invention. (A) Representative images of changes in left ventricular pressure (LVP) and the rate of increase / decrease of left ventricular pressure (dp / dt) at 4 weeks; (B) Heart rate statistics; (C) LVESP results; (D) LVEDP results; (E) Quantitative analysis results of +dp / dt; (F) Quantitative analysis results of -dp / dt.

[0032] Figure 9 The results of intraperitoneal injection of Z-FY-CHO significantly reducing inflammatory cell infiltration in mice with fulminant myocarditis provided by the present invention are shown in the figure; (A) is a representative image of cardiac inflammatory cell infiltration in mice with fulminant myocarditis after 1 week; (B) is a quantitative analysis figure of (A);

[0033] Figure 10 The following figures illustrate the results of intraperitoneal injection of Z-FY-CHO significantly improving myocardial fibrosis and cardiac remodeling in mice with fulminant myocarditis, provided by this invention. (A) Representative image of myocardial fibrosis in mice at 4 weeks of age; (B) Statistical results of (A); (C) MRNA expression levels of cardiac ANP in each group of mice at 4 weeks of age; (D) MRNA expression levels of cardiac BNP in each group of mice at 4 weeks of age; (E) MRNA expression levels of cardiac Col1a1 in each group of mice at 4 weeks of age; and (F) MRNA expression levels of cardiac GAL3 in each group of mice at 4 weeks of age. Detailed Implementation

[0034] This invention provides the application of CTSL inhibitors in the preparation of drugs for treating fulminant myocarditis. This invention discovers a new class of drugs for treating fulminant myocarditis—CTSL inhibitors. Lysosomes are organelles that primarily perform degradation functions in cells and participate in the regulation of many important cellular physiological functions. However, damage to the lysosomal membrane leads to the leakage of hydrogen ions, calcium ions, and hydrolases from the lysosomal lumen. Lysosomal membrane permeability (LMP) is one of the markers of lysosomal membrane instability, which can rapidly lead to lysosome-dependent cell death, including apoptosis, pyroptosis, and programmed necrosis, by releasing proteolytic enzymes (such as CTSB, CTSD, CTSL, etc.). This invention discovers that CTSL-specific inhibitors can significantly reduce cardiomyocyte damage in fulminant myocarditis, improve cardiac function and survival rate, exert a direct cardioprotective effect, and have a therapeutic effect on fulminant myocarditis. The CTSL inhibitors of this invention can be administered via intraperitoneal injection, making administration simple. In a specific embodiment, the CTSL inhibitor includes Z-FY-CHO.

[0035] This invention also provides the use of CTSL inhibitors in the preparation of drugs for treating viral myocarditis. In a specific embodiment, the CTSL inhibitor comprises Z-FY-CHO. This invention involves intramyocardial injection of a micro-dose (1×10⁻⁶) 5 A viral fulminant myocarditis model was constructed using PFU / mouse CVB3 virus, demonstrating that mice exhibited the phenotype of fulminant myocarditis (including myocardial inflammatory infiltration, severe reduction in cardiac function, and increased mortality), and demonstrating that CTSL inhibitors have a therapeutic effect on viral myocarditis (specifically fulminant myocarditis).

[0036] This invention also provides the application of CTSL inhibitors in the preparation of products that improve cardiomyocyte damage. Specifically, this invention provides the application of CTSL inhibitors in the preparation of products that reduce programmed necrosis of cardiomyocytes. In a specific embodiment, the CTSL inhibitor comprises Z-FY-CHO. Experimental results show that the CTSL inhibitor reduces the expression of pMLKL protein in myocardial tissue, indicating that it can block the intermediate step in lysosomal damage leading to programmed necrosis and can improve cardiomyocyte damage in fulminant myocarditis.

[0037] This invention also provides the use of CTSL inhibitors in the preparation of products that improve cardiac function and / or hemodynamics. In a specific embodiment, the CTSL inhibitor comprises Z-FY-CHO. Experimental results show that CTSL inhibitors can significantly improve long-axis and short-axis left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV); and can significantly improve left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase of left ventricular intraventricular pressure (+dp / dt) in mice with fulminant myocarditis at 4 weeks.max ), the maximum rate of decrease of left ventricular intraventricular pressure (-dp / dt) max ).

[0038] This invention also provides the use of CTSL inhibitors in the preparation of products that improve cardiac inflammatory cell infiltration. In a specific embodiment, the CTSL inhibitor comprises Z-FY-CHO. Experimental results show that CTSL inhibitors can improve the cardiac inflammatory cell infiltration area at 1 week.

[0039] This invention also provides the use of CTSL inhibitors in the preparation of products that reduce cardiac death. In a specific embodiment, the CTSL inhibitor comprises Z-FY-CHO. Experimental results show that CTSL inhibitors reduce mortality due to drastic decline in cardiac function, while CTSB and CTSD have no effect on reducing cardiac death.

[0040] This invention also provides the use of CTSL inhibitors in the preparation of products that improve cardiac pathological remodeling. In a specific embodiment, the CTSL inhibitor comprises Z-FY-CHO. In a specific embodiment, Z-FY-CHO can significantly improve myocardial fibrosis in mice with fulminant myocarditis. In a specific embodiment, the CTSL inhibitor can reduce the mRNA expression of the cardiac pathological remodeling genes ANP, BNP, Col1a1, and GAL3.

[0041] This invention also provides the use of CTSL inhibitors in the preparation of products that improve any one or more of the indicators described in ① to ⑦:

[0042] ① Long-axis and short-axis left ventricular ejection fraction;

[0043] ② Long-axis and short-axis left ventricular end-diastolic volume;

[0044] ③ Long-axis and short-axis left ventricular end-systolic volume;

[0045] ④ Left ventricular end-systolic pressure;

[0046] ⑤ Left ventricular end-diastolic pressure;

[0047] ⑥ Maximum rate of increase of left ventricular pressure;

[0048] ⑦ Maximum rate of decrease in left ventricular pressure.

[0049] In a specific embodiment, the CTSL inhibitor includes Z-FY-CHO.

[0050] To further illustrate the present invention, the application of the CTSL inhibitor provided by the present invention in the preparation of a medicament for treating fulminant myocarditis is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Experimental methods (experimental flowchart as follows) Figure 1 As shown):

[0053] 1. Construction and drug treatment of mice with fulminant myocarditis:

[0054] ① Establishment of a mouse model of fulminant myocarditis by intramyocardial injection of CVB3: First batch of mice ( Figure 2 Two groups were formed: a control group and a fulminant myocarditis (CVB3) group. Eight-week-old C57BL / 6j mice were used. Hair was removed from the skin of the anterior chest. After isoflurane inhalation anesthesia, the mice were fixed supine on a 37°C constant-temperature operating table. A transilluminator was placed near the anterior chest of the mouse. The skin above the xiphoid process was pulled back with forceps. The anterior chest was disinfected with 75% alcohol. A small incision, approximately 0.5–1 cm in length, was made in the fourth intercostal space on the left side of the sternum using ophthalmic scissors. The skin and subcutaneous tissue were cut, and the muscles and intercostal space were separated to expose the heart. 10 μL of CVB3 virus (1 × 10⁻⁶) was aspirated using a microsyringe. 7 (pfu / mL). Fix the heart position with your left hand to fully expose the anterior wall of the left ventricle. Then insert the needle into the myocardial tissue to a depth of 1–3 mm. After confirming no blood return upon aspiration, slowly inject the virus at a uniform and slow rate to avoid myocardial tissue damage or drug extravasation due to rapid injection. After injection, gently return the heart to its original position and check for bleeding. If bleeding occurs, gently apply pressure with a cotton ball or gauze to stop the bleeding. Then suture the intercostal muscles, subcutaneous tissue, and skin sequentially. Place the surgically operated mouse on a warming pad to allow it to gradually awaken, maintaining an ambient temperature of 30℃–35℃. Control group mice received an intramyocardial injection of 10 μL of phosphate-buffered saline (PBS).

[0055] ② Intraperitoneal injection of Z-FY-CHO, CA-074-me, and PepstainA: Weigh the mice precisely using a balance to determine the accurate dosage of the three drugs (5 mg / kg body weight). Draw the drugs into a 1 ml syringe, hold the mouse with your left hand, head down, body upside down. Disinfect the lower abdomen of the mouse 0.5 cm to the sides of the midline with 75% alcohol. Hold the syringe in your right hand and insert the needle into the skin, advancing 2-3 mm subcutaneously, with the needle inserted into the abdominal cavity at a 45-degree angle to the skin. Aspirate the syringe plunger; if no blood or fluid is returned, slowly inject the drugs.

[0056] The second batch of mice was divided into 5 groups: control group, fulminant myocarditis + DMSO group, fulminant myocarditis + Z-FY-CHO group, fulminant myocarditis + CA-074-me group, and fulminant myocarditis + PepstainA group. The injection dosage and method of DMSO were the same as those for CTSB, CTSD, and CTSL inhibitors. The day of intramyocardial injection of CVB3 was recorded as day 0. Starting from day 0, intraperitoneal injections were administered once daily for 2 weeks. Mice survival was recorded daily until day 28. The third batch of mice was divided into 4 groups: control + DMSO group, control + CTSL inhibitor group, fulminant myocarditis + DMSO group, and fulminant myocarditis + CTSL inhibitor group. One week after model establishment, Western blotting and immunofluorescence were used to detect CTSL protein levels in the cardiac tissue of each group of mice.

[0057] 2. Immunofluorescence staining of cardiac tissue: ① Dewaxing to water: Dewax paraffin sections to water according to the following steps: Environmentally friendly clearing agent I 10 min, environmentally friendly clearing agent II 5 min, environmentally friendly clearing agent III 5 min, anhydrous ethanol I 10 min, anhydrous ethanol II 5 min, anhydrous ethanol III 3 min, 95% ethanol I 1 min, 95% ethanol II 1 min, 90% ethanol 1 min, 80% ethanol 1 min, 70% ethanol 1 min. Wash twice with PBS, 5 min each time. ② Antigen retrieval: Place the dewaxed and hydrated paraffin sections in a heat-resistant tissue section staining rack containing pH=6.0 citrate antigen retrieval buffer (10mM citrate / sodium citrate, containing 0.05% Tween-20), and place it in a preheated 95±0.5℃ constant temperature water bath for heat-induced antigen epitope retrieval for 15 min (starting the timer from when the solution reaches the set temperature). After retrieval, remove the staining rack using heat-resistant gloves and allow it to cool naturally at room temperature for 20 min. Subsequently, wash twice with PBS, 5 min each time, to thoroughly remove any residual retrieval solution. ③ Permeabilization: Permeabilize the washed slides with 0.3% Triton-X100 permeabilization buffer for 15 min. Wash twice with PBS, 5 min each time. Blocking: Completely cover the sample with culture medium containing 5% serum and incubate on a shaker at room temperature for 1 h. Wash twice with PBS, 5 min each time, and blot away any surrounding moisture. ④ Incubation with CTSB (31718T, CST), CTSD (74089T, CST), CTSL (55914S, CST), and TroponinT (GB113806, Servicebio) primary antibodies: Accurately add the primary antibody working solution diluted with PBS buffer, ensuring complete coverage of the tissue area. Transfer the slides to a sealed humidified chamber and incubate overnight at 4°C. Warming: After removing the sample, warm it for 15 min. Wash twice with PBS, 5 min each time. ⑤ Secondary antibody incubation: Directly add AF488-labeled donkey anti-rabbit secondary antibody (34212ES60, Yeasen) or AF594-labeled donkey anti-rabbit secondary antibody (34206ES60, Yeasen) working solution prepared in PBS buffer to completely cover the sample. Incubate at room temperature in the dark for 1 hour. Wash twice with PBS, 5 min each time. ⑥ Nuclear staining: Add DAPI working solution to the sample and incubate at room temperature in the dark for 10 min. Wash twice with PBS, 5 min each time. ⑦ Mounting: Add DAPI-free anti-fluorescence quenching mounting medium and mount with a coverslip. ⑧ Photography: Take pictures and analyze using a laser confocal microscope.

[0058] 3. Detection of Lysosome Morphology and Diameter in Cardiac Cells: Mouse heart tissue was rapidly placed in pre-cooled fixative (2.5% glutaraldehyde, containing 0.1 mol / L phosphate buffer, pH 7.2–7.4) and fixed at 4°C for 2–4 hours. After rinsing with buffer, a series of dehydration steps were performed, typically using ethanol or acetone, with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol used sequentially, each for 15–30 minutes. After dehydration, the tissue was embedded in acetone or ethyl acetate, using an appropriate embedding agent (epoxy resin) for embedding and polymerization. After the embedded blocks solidified, they were cut into ultrathin sections with a thickness of approximately 50–70 nm using an ultramicrotome. The sections were placed on a copper grid, stained with uranium acetate and lead citrate, and observed using a transmission electron microscope. Ultrastructural images of lysosomes were obtained by adjusting parameters.

[0059] 4. Western Blotting of Myocardial Tissue: Mouse myocardial tissue was immediately placed in pre-chilled lysis buffer (RIPA lysis buffer, containing protease inhibitors and phosphatase inhibitors), homogenized with a tissue homogenizer, and lysed on ice for 30 minutes, vortexing every few minutes during lysis. Then, it was centrifuged at 4°C and 12,000 rpm for 15 minutes. The supernatant was collected as the protein extract, and the protein concentration was determined using the BCA method. SDS-PAGE electrophoresis was performed. An appropriate concentration of polyacrylamide gel was selected based on the molecular weight of the target protein. The protein sample and pre-stained protein marker were mixed and loaded into the gel wells, and the proteins were separated by electrophoresis. After electrophoresis, the proteins in the gel were transferred to a PVD membrane. After transfer, the membrane was blocked with 5% skim milk in TBST buffer for 1 hour. Primary antibodies CTSB (31718T, Cell Signaling Technology), CTSD (74089T, Cell Signaling Technology), CTSL (55914S, Cell Signaling Technology), pMLKL (#AF7420, Affinity), and MLKL (#DF7412, Affinity) (diluted appropriately) were added and incubated overnight at 4°C. After washing with TBST, the corresponding horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (GB23303, Servicebio) (diluted appropriately) was added and incubated at room temperature for 1–2 hours. After washing again, the membrane was developed using a chemiluminescence assay kit. The gray values ​​of the target protein bands were observed and analyzed using an imaging system to quantitatively analyze the protein expression level.

[0060] 5. Echocardiographic Measurements: Transthoracic echocardiography was performed at 1, 2, and 4 weeks after myocardial injection of CVB3. Echocardiography was performed by an experienced investigator unaware of the group assignments. Mice were anesthetized by inhalation of 3% isoflurane, with the isoflurane concentration then reduced to 1.0–1.5%, maintaining consistent heart rates across groups throughout. Two-dimensional and M-mode imaging was performed using a Vevo 2100 system. Preparation involved thoroughly cleaning the target imaging area with depilatory cream and cotton pads. Imaging began with the probe aligned along the long axis of the left ventricle (LV) to obtain a two-dimensional B-mode view. It was then rotated 90° clockwise to obtain a short-axis view of the LV, ensuring the papillary muscles were clearly visible. The system was switched from B-mode to M-mode to capture the image. After imaging, excess gel was removed, and the mice were allowed to recover in their cages. The left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) of echocardiography were analyzed using Vevo LAB 5.6.0 software.

[0061] 6. Hemodynamics: The hemodynamics of mice 4 weeks after myocardial injection of CVB3 were evaluated using a Millar tip pressure catheter. Mice were anesthetized with isoflurane inhalation and fixed supine on a 37°C constant-temperature operating table. The right common carotid artery was exposed. The Millar catheter was immersed in heparinized saline. Under magnification, a small vertical incision was made in the right common carotid artery using microscissors. The Millar catheter was inserted into the right common carotid artery and pushed into the left ventricular cavity for measurement, and the pressure was recorded in real time. Data were recorded and analyzed using a PowerLab system to obtain heart rate, left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), maximum rate of rise of left ventricular pressure (+dp / dtmax), and maximum rate of fall of left ventricular pressure (-dp / dtmax).

[0062] 7. HE staining of cardiac tissue sections: ① Dewaxing: Immerse the sections in xylene twice, 10-15 minutes each time, to remove paraffin. ② Hydration: Hydrate the sections sequentially in a gradient of alcohols, starting with 100% alcohol, then proceeding to 95%, 90%, 80%, and 70% alcohol, immersing for 2-3 minutes at each concentration, and finally rinsing with distilled water. ③ Staining: Immerse the sections in hematoxylin staining solution for 5-15 minutes. The specific time can be adjusted appropriately according to the staining solution concentration and tissue type. After staining, rinse with tap water to allow the section color to moderately return to blue. ④ Differentiation: Immerse the sections in 1% hydrochloric acid alcohol for several seconds to tens of seconds for differentiation. The differentiation time must be strictly controlled to remove excess hematoxylin from the cell nuclei and ensure clear staining of the cell nuclei. ⑤ Return to blue: Rinse the differentiated sections with tap water or immerse them in ammonia water for several seconds to allow the section color to return to blue. ⑥ Eosin staining: Immerse the blue-returned sections in eosin staining solution for 2-5 minutes to stain the cytoplasm and extracellular matrix red. ⑦ Dehydration and Clearing: After staining, the sections are sequentially dehydrated with a gradient of alcohols, starting with 70% alcohol, then 80%, 90%, 95%, and 100% alcohol, soaking for 2-3 minutes at each concentration. Finally, they are cleared twice in xylene, 5-10 minutes each time. ⑧ Mounting: After clearing, the sections are removed from the xylene, a suitable amount of neutral resin is added, and then a coverslip is placed on top, taking care to avoid air bubbles. The mounted sections are then placed in a ventilated area to air dry or dried in a low-temperature oven. Observation and analysis are then performed under a microscope.

[0063] 8. MASSON staining of cardiac tissue: After a series of dewaxing and rehydration steps, paraffin sections of the heart were stained with Masson's trichrome using a trichrome staining kit according to the manufacturer's protocol (Sigma-Aldrich, catalog number: HT 15). Images were scanned using a Pannoramic MIDI scanner (3DHISTECH) and viewed using CaseViewer software (3D HISTECH). The area of ​​fibrosis was quantitatively determined using ImageJ software.

[0064] 9. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction: Total RNA was extracted from heart tissue samples and purified using the RNAsimple Total RNA Kit (Qiagen) according to the manufacturer's instructions. Subsequently, PrimeScript with gDNA Eraser was used. TM Total RNA was reverse transcribed into cDNA using an RT kit (RR047A, TaKaRa). Gene expression levels were analyzed in triplicate using the SYBR Green PCR master mix in 10 μL of the reaction mixture, following the manufacturer's protocol (4472908, Thermo Fisher Scientific).

[0065] 10. Statistical Analysis: All quantitative data in this study are expressed as mean ± standard deviation (Mean ± SEM). Differences between two groups were compared using the Student's t-test (two independent samples), and differences among multiple groups were analyzed using one-way ANOVA combined with the Sidak method for multiple comparisons. All statistical tests were two-tailed hypothesis tests. GraphPadPrism 10.0 software (GraphPad Inc., USA) was used for data processing and visualization. A p-value < 0.05 was used as the threshold for statistical significance.

[0066] The experimental results of this invention demonstrate that Z-FY-CHO effectively alleviates lysosomal damage and programmed necrosis of cardiomyocytes in mice with fulminant myocarditis by inhibiting CTSL expression and activity, thereby improving cardiac function and increasing survival rate. Specific results are as follows:

[0067] 1. Verification that the CTSL inhibitor described in this invention can reduce the mortality rate of mice with fulminant myocarditis.

[0068] This invention uses intramyocardial injection of 10 μL (1×10⁻⁶) 5 A mouse model of fulminant myocarditis was established using the pfu(CVB3) method. Control mice were injected intramyocardially with 10 μL of phosphate-buffered saline (PBS). One week after modeling, the mRNA expression level of CVB3 in myocardial tissue was detected by PCR, and the protein levels of CTSB, CTSD, CTSL, and pMLKL in myocardial tissue were detected by Western blotting. The results of lysosomal damage and programmed necrosis in the myocardial tissue of fulminant myocarditis mice are as follows: Figure 2 As shown, (A) is the experimental flowchart; (B) is the result of CVB3 mRNA expression level in myocardial tissue, **P<0.01; (C) is the result of Western blotting detection of protein levels of CTSB, CTSD, CTSL and pMLKL in myocardial tissue; (D) to (G) are the results of quantitative analysis of CTSB, CTSD, CTSL and pMLKL, respectively, **P<0.01, ***P<0.001, ****P<0.0001; The results showed that the protein levels of CTSB, CTSD and CTSL in the myocardial tissue of mice in the fulminant myocarditis group were significantly increased, and the programmed necrosis marker protein pMLKL was significantly increased.

[0069] In this invention, 8-week-old C57BL / 6j mice were divided into 5 groups: control group, fulminant myocarditis + DMSO group (CVB3 + DMSO), fulminant myocarditis + CTSB inhibitor group (CVB3 + CA-074-me), fulminant myocarditis + CTSD inhibitor group (CVB3 + PepstainA), and fulminant myocarditis + CTSL inhibitor group (CVB3 + Z-FY-CHO). The day of intramyocardial injection of CVB3 was recorded as day 0. Starting from day 0, intraperitoneal injections were administered once daily for 2 weeks. The survival status of the mice was recorded daily until day 28. Results are as follows: Figure 3 As shown, (A) is the experimental flowchart; (B) is the survival rate of mice within 4 weeks. The results show that neither the CTSB inhibitor CA-074-me nor the CTSD inhibitor PepstainA can reduce the mortality rate, while the CTSL inhibitor Z-FY-CHO can reduce the mortality rate.

[0070] 2. To verify the inhibitory effect of Z-FY-CHO of the present invention on CTSL in myocardial tissue of mice with fulminant myocarditis.

[0071] In this invention, 8-week-old C57BL / 6j mice were divided into four groups: control + DMSO group, fulminant myocarditis + DMSO group (CVB3 + DMSO), control + CTSL inhibitor group (Control + Z-FY-CHO), and fulminant myocarditis + CTSL inhibitor group (CVB3 + Z-FY-CHO). One week after modeling, the CTSL protein level in the heart tissue of each group of mice was detected by Western blotting and immunofluorescence. Figure 4 To verify the inhibitory effect of the CTSL inhibitor Z-FY-CHO on CTSL in myocardial tissue of mice with fulminant myocarditis, the following diagrams are presented: (A) Experimental flowchart; (B) Results of Western blotting analysis of the effect of Z-FY-CHO on CTSL expression in myocardial tissue extracted one week after intraperitoneal injection of Z-FY-CHO (5 mg / kg) in mice; (C) Quantitative analysis of diagram (B); (D) Immunofluorescence staining analysis of CTSL in myocardial cells of four groups of mice, where Tropont T refers to troponin T. ****P<0.0001. The results show that the CTSL-specific inhibitor Z-FY-CHO significantly reduced CTSL expression in cardiac tissue. Figure 4 B in Figure 4 (D in the middle).

[0072] 3. Verify that the Z-FY-CHO of this invention reduces cardiomyocyte damage in mice with fulminant myocarditis.

[0073] To verify whether Z-FY-CHO can improve the damage of cardiomyocytes in mice with fulminant myocarditis, this invention observed the lysosomes of cardiomyocytes in the four groups of mice using transmission electron microscopy and detected the level of pMLKL protein, a marker of programmed necrosis, using Western blotting. Figure 5 Figure 1 shows the results of intraperitoneal injection of Z-FY-CHO significantly improving lysosomal damage and programmed necrosis in cardiomyocytes of mice with fulminant myocarditis. (A) Electron micrographs of cardiomyocyte lysosomes from four groups of mice, with red arrows indicating lysosomes; (B) Lysosome diameter, n = 15–17; (C) Western blotting analysis of pMLKL protein levels; (D) Quantitative analysis of figure (C). ****P < 0.0001. Electron microscopy results showed that the diameter of lysosomes in cardiomyocytes of mice with fulminant myocarditis was significantly increased, and their internal structure was significantly disrupted. Figure 5 A and Figure 5 (B) The level of pMLKL protein in myocardial tissue was significantly increased in fulminant myocarditis. Figure 5 C and Figure 5 (D in the text). Although Z-FY-CHO treatment did not significantly reduce cardiomyocyte lysosomal swelling, it significantly reduced the expression of pMLKL protein in myocardial tissue. Figure 5 A in Figure 5 The D in the figure indicates that Z-FY-CHO can block the intermediate step in lysosomal damage leading to programmed necrosis.

[0074] 4. Verify that the Z-FY-CHO described in this invention improves the survival rate, cardiac pathological remodeling, and function in mice with fulminant myocarditis.

[0075] Figure 6 Figure 1 shows the results of intraperitoneal injection of Z-FY-CHO significantly improving the survival rate of mice with fulminant myocarditis; (A) experimental flowchart; (B) mouse survival rate within 4 weeks. **P<0.01. By recording the survival status of mice daily, this invention found that the mortality rate of mice with fulminant myocarditis was as high as 40% within 4 weeks. Figure 6 A and Figure 6 In mice injected intraperitoneally with Z-FY-CHO (B), the mortality rate decreased to 12%. Figure 6 (B in the middle).

[0076] In this invention, long-axis and short-axis M-mode cardiac ultrasound were performed on four groups of mice at 1 week, 2 weeks and 4 weeks. Figure 7Figures showing the results of intraperitoneal injection of Z-FY-CHO significantly improving cardiac function in mice with fulminant myocarditis. (A) Representative long-axis echocardiogram image at 4 weeks; (B)–(D) images of left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) at 1, 2, and 4 weeks, respectively; (E) Representative short-axis echocardiogram image at 4 weeks; (F)–(H) images of LVEF, LVEDV, and LVESV at 1, 2, and 4 weeks, respectively. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no statistically significant difference. The results showed that, compared with the mice injected with CVB3+DMSO, the mice treated with intraperitoneal injection of Z-FY-CHO had significantly improved long-axis and short-axis left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) at 1, 2, and 4 weeks. Figure 7 A in Figure 7 (H in the text).

[0077] Figure 8 Figure 1 shows the hemodynamic results of mice with fulminant myocarditis significantly improved by intraperitoneal injection of Z-FY-CHO. (A) Representative images of changes in left ventricular pressure (LVP) and the rate of increase / decrease of left ventricular pressure (dp / dt) at 4 weeks; (B) Heart rate statistics; (C) LVESP; (D) LVEDP; (E) Quantitative analysis of +dp / dt; (F) Quantitative analysis of -dp / dt. ***P<0.001, ****P<0.0001, no statistically significant difference in ns. The hemodynamic results in mice showed that Z-FY-CHO did not affect heart rate, but significantly improved left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase of left ventricular pressure (+dp / dt) at 4 weeks in mice with fulminant myocarditis. max ), the maximum rate of decrease of left ventricular intraventricular pressure (-dp / dt) max ()( Figure 8 A in Figure 8 (F in the text)

[0078] Figure 9 Figure 1 shows the results of intraperitoneal injection of Z-FY-CHO significantly reducing inflammatory cell infiltration in mice with fulminant myocarditis; (A) is a representative image of cardiac inflammatory cell infiltration in mice with fulminant myocarditis at 1 week; (B) is a quantitative analysis of (A). ****P<0.0001, ns, no statistical difference. HE staining showed that Z-FY-CHO treatment significantly improved the cardiac inflammatory cell infiltration area at 1 week ( Figure 9 A and Figure 9 (B in the middle).

[0079] Figure 10 Figure 1 shows the results of intraperitoneal injection of Z-FY-CHO significantly improving myocardial fibrosis and cardiac remodeling in mice with fulminant myocarditis. (A) Representative image of myocardial fibrosis in mice at 4 weeks; (B) Statistical results of (A); (C) mRNA expression levels of cardiac ANP in each group of mice at 4 weeks; (D) mRNA expression levels of cardiac BNP in each group of mice at 4 weeks; (E) mRNA expression levels of cardiac Col1a1 in each group of mice at 4 weeks; (F) mRNA expression levels of cardiac GAL3 in each group of mice at 4 weeks. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no statistically significant difference. Z-FY-CHO significantly reduced the area of ​​myocardial fibrosis ( Figure 10 A and Figure 10 The mRNA expression of B) and cardiac pathological remodeling genes ANP, BNP, Col1a1 and GAL3 (in the B) Figure 10 C in Figure 10 These results indicate that the CTSL-specific inhibitor Z-FY-CHO significantly improved survival, inflammatory infiltration, cardiac function, and cardiac remodeling in mice with fulminant myocarditis, demonstrating that Z-FY-CHO can be used to treat fulminant myocarditis.

[0080] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Z-FY-CHO in the preparation of drugs for treating fulminant myocarditis; The Z-FY-CHO is used to improve cardiac function and / or hemodynamics; The cardiac function and / or hemodynamics include at least one of the following indicators: ① Long-axis and short-axis left ventricular ejection fraction; ② Long-axis and short-axis left ventricular end-diastolic volume; ③ Long-axis and short-axis left ventricular end-systolic volume; ④ Left ventricular end-systolic pressure; ⑤ Left ventricular end-diastolic pressure; ⑥ Maximum rate of increase of left ventricular pressure; ⑦ Maximum rate of decrease in left ventricular pressure.

2. The application according to claim 1, characterized in that, The Z-FY-CHO is also used to improve myocardial cell damage.

3. The application according to claim 1, characterized in that, The Z-FY-CHO is also used to reduce programmed necrosis of cardiomyocytes.

4. The application according to claim 1, characterized in that, The Z-FY-CHO is also used to improve cardiac inflammatory cell infiltration.

5. The application according to claim 1, characterized in that, The Z-FY-CHO is also used to reduce cardiac death.

6. The application according to claim 1, characterized in that, The Z-FY-CHO is also used to improve cardiac pathological remodeling.

Images