Use of itsa1 in the preparation of a drug for treating or preventing cardiac remodeling or hypertension

Abstract

The application relates to application of ITSA1 in preparation of drugs for treating or preventing cardiac remodeling or hypertension, and belongs to the technical field of cardiovascular disease prevention. A mouse is used to construct a chronic intermittent hypoxia (CIH) animal model, blood pressure is measured before and during the construction of the animal model, and at 2 weeks, 4 weeks and 8 weeks after the construction, and cardiac ultrasound, Masson staining, alpha-SMA immunohistochemistry, WGA staining and the like are carried out at the end of the model. The experimental results show that ITSA1 can alleviate the blood pressure rise, heart rate acceleration, cardiac function, myocardial fibrosis and myocardial cell hypertrophy caused by chronic intermittent hypoxia. The above experiments prove that ITSA1 can effectively alleviate the blood pressure rise and cardiac remodeling caused by chronic intermittent hypoxia, and lay a foundation for widening the clinical application range of ITSA1.

Description

Technical Field

[0001] This invention relates to the application of ITSA1 in the preparation of drugs for the treatment or prevention of cardiac remodeling or hypertension, and belongs to the field of cardiovascular disease prevention technology. Background Technology

[0002] Cardiac remodeling refers to a series of structural and functional changes in the heart under various pathological conditions (such as hypertension, myocardial infarction, and heart failure). It is an adaptive response of the heart to long-term hemodynamic or non-hemodynamic factors. Early cardiac remodeling is mainly characterized by cardiomyocyte hypertrophy, which has a compensatory function and can maintain normal cardiac function without changes in intercellular matrix components. Under prolonged stress conditions such as pressure overload, cardiac remodeling becomes decompensated, with decreased cardiac function, accompanied by activation of cardiac fibroblasts, infiltration and activation of immune cells, increased intercellular matrix, changes in its components, and cardiomyocyte apoptosis. Once cardiac remodeling enters decompensation, it can lead to a 6-8 fold increase in the incidence of arrhythmias, myocardial infarction, congestive heart failure, sudden death, and other cardiovascular events. Preventing or delaying cardiac remodeling can effectively curb the progression of various cardiovascular diseases and the occurrence of heart failure, improving patient survival rates and quality of life. Therefore, finding new methods to delay cardiac remodeling is key to effectively reducing its high mortality rate in clinical practice.

[0003] While existing treatments and medications for cardiac remodeling can slow down or alleviate this process to some extent, they still have many limitations:

[0004] (1) Drug side effects: Currently used drugs for treating cardiac remodeling are often accompanied by side effects of varying degrees. For example, angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists may cause cough, hyperkalemia, and decreased renal function; beta-blockers may cause bradycardia, hypotension, and fatigue; aldosterone antagonists may cause hyperkalemia and renal impairment; and sodium-glucose cotransporter inhibitors may increase the risk of genitourinary infections. These side effects not only affect patients' quality of life but may also limit the long-term use of the drugs.

[0005] (2) Limited efficacy: Although existing drugs can slow down the progression of cardiac remodeling to some extent, their efficacy is still limited, especially in the late stage of the disease, where drugs often cannot completely reverse or prevent the further development of cardiac remodeling.

[0006] (3) Individual differences: There are significant individual differences in patients’ responses to drugs. Some patients may not respond to certain drugs or may experience serious adverse reactions. This makes treatment plans highly individualized, increasing the complexity and challenge of clinical treatment.

[0007] (4) Adherence issues: Treatment for cardiac remodeling often requires long-term or even lifelong medication. Complex treatment protocols and frequent medication adjustments may reduce patient adherence, thereby affecting treatment outcomes. In addition, the side effects of medications and the economic burden faced by patients may also weaken their willingness to take medication, further affecting treatment outcomes.

[0008] (5) Drug interactions: Patients with cardiac remodeling often need to use multiple drugs in combination to control their condition, which increases the risk of drug interactions and may lead to reduced efficacy or exacerbated adverse reactions.

[0009] In summary, although existing treatments and medications for cardiac remodeling can alleviate symptoms to some extent, they still have many limitations, including drug side effects, limited efficacy, individual variability, adherence issues, drug interactions, economic burden, and the risks of invasive treatments. Therefore, developing new, biosafety-friendly drugs that can alleviate or treat cardiac remodeling is of great significance in curbing the progression of various cardiovascular diseases and the development of heart failure.

[0010] ITSA1 is a benzotriazole small molecule compound (molecular formula C). l3 H7C l2N3O (molecular weight 292.12 g / mol) can penetrate cells and was first discovered as an inhibitor of TSA (trichosmin A). ITSA1 antagonizes TSA-induced cell cycle arrest, histone and tubulin acetylation, and transcriptional activation; furthermore, ITSA1 reverses the inhibitory effect of TSA on protein deacetylase activity. Based on the results of Stuart L. Schreiber's team at the Laboratory of Chemistry and Biochemistry at Harvard University, the inhibitory effect of ITSA1 on TSA has the following characteristics: inhibition of TSA-induced cell cycle arrest: TSA causes cell cycle arrest in the G1 or G2 phase, while ITSA1 can reverse this arrest, allowing cells to enter the S phase normally for DNA replication. After the addition of ITSA1, cells that were previously TSA-arrested can re-enter the cell cycle, exhibiting a cell cycle distribution similar to untreated cells. The effect of ITSA1 is not achieved by directly disrupting the structure of TSA, but rather by acting after TSA-induced cell cycle arrest. ITSA1 can counteract the effects of TSA on the cell cycle for a long period. Furthermore, the inhibitory effect of ITSA1 does not directly act on histone deacetylases; it only takes effect after TSA action. Further research shows that TSA activates the transcription of certain genes, but ITSA1 can inhibit this transcriptional activation. ITSA1 inhibits the TSA-induced transcriptional activation, not the expression of the genes themselves. This characteristic suggests that ITSA1 may act on downstream effector pathways induced by TSA. Simultaneously, TSA not only causes histone acetylation but also α-tubulin acetylation in the cytoplasm, and ITSA1 can inhibit TSA-induced tubulin acetylation, restoring tubulin acetylation levels to normal.

[0011] In summary, most current research on ITSA1 focuses on its role as a TSA inhibitor, while there are currently no related studies or reports on its role and mechanism in alleviating cardiac remodeling. Summary of the Invention

[0012] The first objective of this invention is to provide the use of ITSA1 in the preparation of medicaments for the treatment or prevention of cardiac remodeling, and to provide a novel substance that can effectively treat or prevent cardiac remodeling.

[0013] A second objective of this invention is to provide the use of ITSA1 in the preparation of drugs for the treatment or prevention of hypertension, and to provide a new use of ITSA1 in the treatment or prevention of hypertension.

[0014] To achieve the above objectives, the technical solution for the application of ITSA1 in the preparation of drugs for treating or preventing cardiac remodeling in this invention is as follows:

[0015] The application of ITSA1 in the preparation of drugs for treating or preventing cardiac remodeling, wherein the structural formula of ITSA1 is [insert structural formula here].

[0016] The beneficial effects of the above technical solution are as follows: the application of ITSA1 in the preparation of drugs for treating or preventing cardiac remodeling is a novel use of a known product. This invention first establishes a chronic intermittent hypoxia (CIH) animal model using mice. After model establishment, echocardiography, Masson staining, α-SMA immunohistochemistry, and WGA staining are performed. Statistical analysis results show that Masson staining and α-SMA immunohistochemistry indicate that ITSA1 alleviates myocardial fibrosis caused by chronic intermittent hypoxia; WGA staining results show that ITSA1 alleviates cardiomyocyte enlargement caused by chronic intermittent hypoxia. These results fully demonstrate that ITSA1 can effectively improve cardiac remodeling, laying the foundation for broadening the clinical application of ITSA1.

[0017] Furthermore, this invention demonstrates that ITSA1 did not have significant adverse effects on the tissue morphology and function of the liver and kidneys in mice after 8 weeks of intervention, exhibiting good biocompatibility and safety. This provides important experimental evidence for the safety characteristics of ITSA1 and lays a solid foundation for its further development and application in the treatment of cardiovascular diseases.

[0018] Specifically, chronic intermittent hypoxia (CIH) refers to recurrent hypoxic states, typically caused by factors such as sleep apnea syndrome, high-altitude environments, or chronic obstructive pulmonary disease. CIH leads to a series of pathophysiological changes, including excessive activation of the sympathetic nervous system, increased oxidative stress, enhanced inflammatory response, and endothelial dysfunction. These changes cause multiple damages to the cardiovascular system, including vascular endothelial injury, cardiomyocyte hypertrophy, and myocardial fibrosis, ultimately leading to cardiac remodeling.

[0019] As a further improvement, the drug is a drug that inhibits cardiac fibrosis and / or cardiomyocyte hypertrophy.

[0020] As a further improvement, the drug is a drug that improves heart rate, reduces myocardial hypertrophy, and enhances myocardial contractile function.

[0021] As a further improvement, the cardiac remodeling is caused by chronic intermittent hypoxia.

[0022] As a further improvement, the drug also contains a pharmaceutically acceptable carrier.

[0023] To achieve the above objectives, the technical solution for the application of ITSA1 in the preparation of drugs for treating or preventing hypertension in this invention is as follows:

[0024] The application of ITSA1 in the preparation of drugs for the treatment or prevention of hypertension, wherein the structural formula of ITSA1 is as follows:

[0025] The beneficial effects of the above technical solution are as follows: This invention first uses mice to construct a chronic intermittent hypoxia (CIH) animal model, and measures blood pressure before, and at 2, 4, and 8 weeks during the model construction. The results show that ITSA1 has a significant effect in alleviating blood pressure abnormalities caused by chronic intermittent hypoxia, especially exhibiting a significant antihypertensive effect under long-term exposure conditions. The blood pressure-regulating effect of ITSA1 provides strong experimental evidence and technical support for its potential application in the treatment of cardiovascular diseases related to chronic hypoxia, laying an important theoretical foundation for further drug development and clinical application.

[0026] As a further improvement, the hypertension is caused by chronic intermittent hypoxia.

[0027] As a further improvement, the drug also contains a pharmaceutically acceptable carrier. Attached Figure Description

[0028] Figure 1 This describes the specific process of constructing the chronic intermittent hypoxia animal model in Example 1 of the present invention;

[0029] Figure 2 The intervention effect of ITSA1 on blood pressure changes caused by chronic intermittent hypoxia at different time points in Example 2 of the present invention (where **** represents P<0.0001, and ns represents no statistically significant difference);

[0030] Figure 3 This invention relates to the protective effect of ITSA1 against cardiac dysfunction caused by chronic intermittent hypoxia in Example 3 of this invention (where A represents the change in heart rate in different treatment groups, B represents the change in left ventricular mass in different treatment groups, C represents the change in systolic and diastolic thickness of the left ventricular posterior wall in different treatment groups, D represents the change in ejection fraction in different treatment groups, **** represents P<0.0001, *** represents P<0.001, ** represents P<0.01, * represents P<0.05, and ns represents no statistically significant difference).

[0031] Figure 4 This is the intervention effect of ITSA1 on collagen fiber hyperplasia induced by chronic intermittent hypoxia in Example 4 of the present invention (wherein, the left figure is the Masson trichrome staining of the heart of mice in different treatment groups, the right figure represents the statistical results, **** represents P<0.0001, *** represents P<0.001);

[0032] Figure 5The effect of ITSA1 intervention on cardiomyocyte enlargement caused by chronic intermittent hypoxia in Example 5 of this invention (wherein, the left figure is the WGA staining image of the heart of mice in different treatment groups, the right figure represents the statistical results, **** represents P<0.0001, *** represents P<0.001);

[0033] Figure 6 This is the intervention effect of ITSA1 on the increase of fibroblasts caused by chronic intermittent hypoxia in Example 6 of the present invention (wherein, the left figure is the α-SMA staining of the heart of mice in different treatment groups, the right figure represents the statistical results, **** represents P<0.0001);

[0034] Figure 7 HE staining images of the liver and kidney of mice in different treatment groups in Example 7 of this invention;

[0035] Figure 8 The functional indicators of the liver and kidneys of mice in different treatment groups in Example 7 of the present invention were detected (where A is the change in alanine aminotransferase level in mice in different treatment groups, B is the change in aspartate aminotransferase level in mice in different treatment groups, C is the change in blood urea nitrogen level in mice in different treatment groups, D is the change in blood creatinine level in mice in different treatment groups, and ns represents no statistically significant difference). Detailed Implementation

[0036] In the prior art, chronic intermittent hypoxia leads to hypertension and cardiac dysfunction (cardiac remodeling). However, this invention demonstrates through experiments that ITSA1 plays an important role in intervening in cardiac dysfunction caused by chronic intermittent hypoxia. It not only improves cardiac function but also alleviates myocardial hypertrophy and myocardial fibrosis caused by CIH.

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; however, these embodiments are merely examples and do not constitute any limitation on the scope of the present invention. Modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent manufacturers.

[0038] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following embodiments and experimental examples:

[0039] Drug preparation:

[0040] ITSA1 was purchased from Selleck, CAS No. 200626-61-5. It was dissolved in DMSO to prepare a stock solution (20 mg / mL) for later use. The working solution was prepared at 1 mg / mL. The dosage was 10 mg / kg, administered intraperitoneally, 3 times a week. It was dissolved in DMSO and stored at -80°C.

[0041] The data processing procedure in this embodiment of the invention is as follows: SPSS 26.0 software was used for statistical analysis. Experimental data are expressed as mean ± standard deviation, and independent samples t-test was used for statistical analysis. The significance level was set at α = 0.05, and P < 0.05 was considered statistically significant.

[0042] Specific embodiments of the application of ITSA1 of the present invention in the preparation of drugs for treating or preventing cardiac remodeling or hypertension:

[0043] This invention first constructs a mouse model of chronic intermittent hypoxia. Compared with normal mice, this model mice exhibit impaired cardiac function and elevated blood pressure. However, when ITSA1 is used for intervention, their blood pressure decreases and cardiac function significantly improves. The specific real-time operation is as follows:

[0044] Example 1: Method for constructing an animal model of chronic intermittent hypoxia

[0045] This embodiment establishes a chronic intermittent hypoxia mouse model to study the effects of hypoxic environment on the physiological and biochemical responses of mice and the efficacy of related treatments. The model simulates chronic intermittent hypoxia conditions by controlling oxygen concentration and exposure time using the Biospherix Oxycycler A84 system. Specific processes and methods for controlling oxygen parameters are as follows... Figure 1 As shown, the specific implementation steps are as follows:

[0046] 1. Animal model construction process

[0047] Mice in the chronic intermittent hypoxia group were placed in a hypoxic environment chamber, and the oxygen concentration was precisely controlled using the Oxycycler A84 system. The specific operational steps in the model construction process are as follows:

[0048] Phase 1: Hypoxia Exposure

[0049] In the initial phase, mice were periodically exposed to oxygen in a hypoxic chamber. During the first 150 seconds, nitrogen was introduced into the system to gradually reduce the oxygen concentration from 21% to 6%, and this low oxygen concentration (6%) was maintained for 30 seconds to simulate the effects of a hypoxic environment on organisms.

[0050] Phase Two: Recovery Period

[0051] Subsequently, the mice were introduced to oxygen, and the system gradually increased the oxygen concentration from 6% to 21%. When the oxygen concentration reached 21%, it was maintained in a normoxic state for 20 seconds.

[0052] The cycle and exposure pattern involved each cycle lasting 4 minutes, including both hypoxic and normoxic phases. Fifteen cycles were performed per hour, meaning the mice experienced 15 fluctuations in oxygen concentration per hour. This hypoxic exposure pattern lasted for 8 hours daily, accumulating to 8 weeks of exposure.

[0053] 2. Oxygen parameter control and monitoring

[0054] The Oxycycler A84 system precisely controls oxygen concentration, ensuring that each switch between hypoxia and normoxic conditions conforms to the set oxygen fluctuation parameters. The system monitors oxygen concentration changes in real time and automatically adjusts to ensure accurate periodic changes in the hypoxic environment. The oxygen concentration is gradually decreased from 21% to 6%, then increased from 6% to 21% and maintained for 20 seconds, ensuring long-term exposure of mice to repeated hypoxic conditions.

[0055] 3. Experimental period and exposure duration

[0056] The animal exposure period in this model was 8 weeks, with 8 hours of hypoxia exposure per day for a total of 8 weeks of hypoxia cycles. This embodiment, through periodic hypoxia exposure, can effectively simulate the long-term effects of chronic intermittent hypoxia on the physiological and biochemical responses of mice, providing a reliable experimental model for subsequent drug intervention.

[0057] By establishing this model, this invention can comprehensively evaluate the effects of chronic intermittent hypoxia on physiological functions such as cardiovascular, metabolic, and fibrosis, and provide strong experimental evidence for potential treatment strategies and drug development.

[0058] Example 2: Study on the intervention effect of ITSA1 on blood pressure changes under chronic intermittent hypoxia (CIH) conditions.

[0059] This embodiment evaluates the intervention effect of ITSA1 on blood pressure changes induced by chronic intermittent hypoxia (CIH). A non-invasive tail blood pressure measurement method was used to systematically observe blood pressure changes in four groups of mice under CIH conditions. The role of ITSA1 in regulating blood pressure was analyzed in detail. The specific implementation procedure is as follows:

[0060] 1. Experimental Design and Operation Procedures

[0061] In this embodiment, mice were divided into four groups: control group, model group, ITSA1 intervention group, and other experimental groups. The experiment primarily employed tail blood pressure measurement (a non-invasive, non-surgical blood pressure monitoring technique capable of accurately assessing blood pressure changes in mice). Systolic blood pressure, diastolic blood pressure, and mean arterial pressure were measured in each group after 2, 4, and 8 weeks of CIH treatment, and the significance of blood pressure changes was assessed through statistical analysis.

[0062] Among them, CON+DMSO was the normoxic solvent control group; CON+ITSA1 was the normoxic + ITSA1 treatment group; CIH+DMSO was the CIH exposure and solvent treatment control group; CIH+ITSA1 was the CIH exposure and ITSA1 treatment group. The ITSA1 dosage was 10 mg / kg, administered intraperitoneally, 3 times / week, and the experimental period was 8 weeks.

[0063] 2. Experimental Results and Analysis

[0064] Two weeks after CIH treatment, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of the model group mice were significantly increased (e.g., Figure 2 As shown in the figure, the difference was statistically significant compared to the control group (P<0.0001, n=6). This indicates that CIH can lead to a short-term increase in blood pressure, mimicking the pathological features of hypertension induced by chronic hypoxia. However, at the same time point, the blood pressure level of mice in the ITSA1 intervention group decreased slightly, but this improvement did not reach a statistically significant level (P>0.05), indicating that short-term intervention may not have completely suppressed the blood pressure changes induced by CIH.

[0065] At 4 and 8 weeks of CIH treatment, the blood pressure levels in the model group mice continued to rise with the extension of CIH treatment time, especially after 4 and 8 weeks, when systolic blood pressure, diastolic blood pressure, and mean arterial pressure were significantly higher than those in the control group, indicating that the pathological features of hypertension caused by CIH gradually worsened. However, the mice in the ITSA1 intervention group showed significant improvement in blood pressure at these time points. Specifically, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of the mice in the ITSA1 group were significantly lower than those in the model group, and the differences were statistically significant (P<0.0001, n=6). Figure 2 (As shown in the figure). This result indicates that ITSA1 can effectively alleviate the increase in blood pressure caused by CIH, especially under conditions of long-term CIH exposure (4 weeks or more), the intervention effect is more significant.

[0066] In summary, the experimental results of this embodiment demonstrate that ITSA1 has a significant effect in alleviating blood pressure abnormalities caused by chronic intermittent hypoxia, especially exhibiting a significant antihypertensive effect under long-term exposure conditions. The blood pressure-regulating effect of ITSA1 provides strong experimental evidence and technical support for its potential application in the treatment of chronic hypoxia-related cardiovascular diseases, laying an important theoretical foundation for further drug development and clinical application.

[0067] Example 3: Evaluation of the protective effect of ITSA1 against cardiac dysfunction induced by chronic intermittent hypoxia (CIH)

[0068] This embodiment evaluates the intervention effect of ITSA1 on cardiac dysfunction caused by chronic intermittent hypoxia (CIH). Echocardiography was used to systematically observe changes in cardiac function in four groups of mice under CIH conditions, with a focus on analyzing the role of ITSA1 in regulating cardiac function. The specific implementation procedures are as follows:

[0069] 1. Experimental Design and Operation Procedures

[0070] In this embodiment, mice were divided into four groups: a control group, a model group, an ITSA1 intervention group, and other experimental groups (specific grouping details are described in Example 2). After 8 weeks of CIH treatment, cardiac function-related indicators were measured in each group of mice using small animal echocardiography. Echocardiography allowed for non-invasive assessment of heart rate, left ventricular structure, and functional parameters. Experimental data included heart rate, left ventricular mass, systolic thickness of the left ventricular posterior wall, diastolic thickness of the left ventricular posterior wall, and ejection fraction. Statistical analysis was used to assess the significance of changes in cardiac function in each group of mice.

[0071] 2. Experimental Results and Analysis

[0072] Under CIH conditions, the model group mice exhibited significant abnormalities in cardiac function, specifically manifested as increased heart rate, a significant increase in left ventricular mass, and a significant increase in the thickness of the left ventricular posterior wall during both systole and diastole. These changes indicate that CIH leads to pathological alterations in the structure and function of the mouse myocardium.

[0073] However, the mice in the ITSA1 intervention group showed significant improvements in these indicators. Specifically, after ITSA1 intervention, all key cardiac function parameters improved to varying degrees, with heart rate and left ventricular mass significantly decreasing compared to the model group (P<0.0001). Figure 3 (As shown in A and B). The systolic thickness of the left ventricular posterior wall was significantly reduced, and the difference was statistically significant (P<0.01). Figure 3 As shown in Figure C, the diastolic thickness of the left ventricular posterior wall was reduced, and the difference was statistically significant (P<0.05). Figure 3(As shown in C). Furthermore, the ejection fraction significantly improved, indicating a recovery in cardiac pumping function; the difference was statistically significant (P<0.01, n=6). Figure 3 (As shown in D). These results indicate that ITSA1 can effectively alleviate cardiac dysfunction caused by CIH, especially in terms of myocardial remodeling, myocardial hypertrophy, and myocardial contractile function, where ITSA1 shows significant protective effects.

[0074] In summary, the experimental results of this embodiment demonstrate that ITSA1 has a significant effect in alleviating cardiac dysfunction caused by chronic intermittent hypoxia, particularly in improving left ventricular remodeling, reducing myocardial hypertrophy, and enhancing myocardial contractile function. The regulatory effect of ITSA1 on cardiac function provides strong experimental evidence and technical support for its potential application in the treatment of chronic hypoxia-related heart diseases. This lays an important theoretical foundation for further drug development and clinical application, and shows its significant application value in the field of cardiac function protection.

[0075] Example 4: Evaluation of the intervention effect of ITSA1 on collagen fiber hyperplasia induced by chronic intermittent hypoxia

[0076] This embodiment uses Masson's trichrome staining technique to analyze the distribution of collagen and muscle fibers in the heart tissue of mice after chronic intermittent hypoxia (CIH) treatment, in order to evaluate the effect of ITSA1 intervention on improving CIH-induced cardiac fibrosis. The specific implementation is as follows:

[0077] 1. Masson's trichrome staining principle

[0078] Masson's trichrome staining is a classic histological staining method that can clearly distinguish the distribution of collagen fibers and muscle fibers. Its principle lies in the fact that small-molecule dyes in the staining reagent can penetrate dense tissues with low permeability, while large-molecule dyes can only enter loose tissues with high permeability. Due to the large molecular weight of pale green dyes or aniline blue, the results of Masson staining are: muscle fibers stained red; collagen fibers stained green or blue; and cell nuclei stained blue-black. This method is mainly used to clarify the distribution of collagen fibers and muscle fibers in tissues, and is particularly suitable for analyzing the degree of fibrosis.

[0079] 2. Masson's trichrome staining procedure (reagent kit is Nanjing Jiancheng Technology Co., Ltd. D026-1-3)

[0080] Before staining, rinse the sections twice in 39°C warm water for 60 seconds each time. Then, stain sequentially with R1 (nuclear stain, purple-red liquid) for 60 seconds, R2 (paste stain, dark red liquid) for 45 seconds, R3 (separation solution, yellow transparent liquid) for 7 minutes (under the microscope, the collagen fibers should fade to a light pink), and R4 (counterstain, blue liquid) for approximately 5 minutes. Rinse with R5 (rinsing solution, colorless transparent liquid) for 30 seconds between each of the R1, R2, and R3 staining steps. R4 (blue counterstain) should be rinsed with anhydrous ethanol. Finally, allow the sections to air dry naturally, mount them with neutral resin, examine under a microscope, and photograph to observe collagen deposition in the tissue.

[0081] 3. Experimental Results and Analysis

[0082] Masson's trichrome staining revealed a significant increase in collagen fibers and disordered muscle fiber arrangement in the heart tissue of mice treated with CIH for 8 weeks, indicating obvious myocardial fibrosis. In the ITSA1 intervention group, the heart tissue showed a significant decrease in collagen fiber content; the distribution of collagen and muscle fibers tended to normalize; and the structural integrity of the myocardial tissue was significantly improved. Statistical analysis showed that ITSA1 could effectively alleviate CIH-induced collagen fiber hyperplasia and significantly improve myocardial fibrosis, further validating its anti-fibrotic effect (P<0.001, n=6, ...). Figure 4 (As shown).

[0083] In summary, the results of this embodiment demonstrate that ITSA1 improves the pathological changes of CIH-induced cardiac fibrosis by significantly reducing collagen fiber deposition. The Masson trichrome staining results provide direct evidence and experimental support for the application of ITSA1 in the treatment of chronic hypoxia-related cardiac fibrosis, offering a scientific basis for its clinical translation.

[0084] Example 5: Evaluation of the effect of ITSA1 intervention on cardiomyocyte enlargement caused by chronic intermittent hypoxia

[0085] This embodiment uses WGA (wheat germ lectin) staining technology to evaluate the intervention effect of ITSA1 on the enlargement of mouse cardiomyocytes induced by chronic intermittent hypoxia (CIH). The specific implementation procedure is as follows:

[0086] 1. Principles of WGA staining technique

[0087] WGA is a plant lectin that specifically recognizes and binds to N-acetylglucosamine residues and is widely used for labeling cell membranes and the extracellular matrix. WGA staining allows for clear observation of cell membrane morphology, size, and intercellular relationships, providing crucial information for studying biological processes such as cell proliferation and hypertrophy.

[0088] 2. WGA staining experimental procedure

[0089] Tissue sections were 4 μm in size. Slides were placed in a 65°C slide oven for 1 hour, then sequentially immersed in xylene I and II for 10 min each, followed by dewaxing in a series of ethanol solutions (100%, 95%, 80%, and 60%) for 3 min each. The sections were then washed with PBS for 5 min × 3 times. The sections were then placed in citrate buffer (pH 6.0) and microwaved for 10 min for antigen retrieval. After cooling to room temperature, the sections were washed with PBS for 5 min × 3 times. Subsequently, the sections were treated with 0.2% Triton X-100 for 10 min, washed with PBS for 5 min × 3 times, stained with WGA (1:300), incubated overnight at 4°C in the dark, washed with PBS for 5 min × 3 times, mounted with DAPI, photographed, and analyzed using ImageJ.

[0090] 3. Experimental Results and Analysis

[0091] WGA staining revealed significantly enlarged cardiomyocytes with irregular morphology in mouse heart tissue after 8 weeks of CIH treatment, exhibiting a typical phenotype of myocardial hypertrophy. ITSA1 intervention significantly alleviated this cardiomyocyte enlargement. Specifically, cardiomyocyte volume decreased significantly, restoring near-normal morphology; cell arrangement became more regular, reducing the degree of myocardial hypertrophy. Statistical analysis showed a statistically significant difference in cardiomyocyte enlargement between the ITSA1 intervention group and the model group (P<0.01, n=6, e.g., ...). Figure 5 (As shown).

[0092] In summary, the results of this embodiment demonstrate that ITSA1 can effectively alleviate cardiomyocyte hypertrophy caused by CIH, and its mechanism may be related to inhibiting excessive cardiomyocyte proliferation and improving cell morphology and structure. WGA staining results provide strong experimental support for the potential application of ITSA1 in the treatment of chronic hypoxia-related myocardial hypertrophy and cardiac dysfunction.

[0093] Example 6: Interventional effect of ITSA1 on fibroblast proliferation induced by chronic intermittent hypoxia

[0094] This embodiment uses smooth muscle actin (α-SMA) immunohistochemical staining to evaluate the intervention effect of ITSA1 on fibroblast proliferation induced by chronic intermittent hypoxia (CIH). The specific implementation procedure is as follows:

[0095] 1. Principle of α-SMA immunohistochemical staining

[0096] α-SMA is an actin subtype specifically expressed in smooth muscle cells and myofibroblasts, and is widely used to assess the degree of tissue fibrosis. Studies have shown that in various diseases such as cirrhosis, pulmonary fibrosis, and myocardial fibrosis, fibroblasts undergo myofibroblast transformation and begin to express α-SMA.

[0097] 2. α-SMA Immunohistochemical Staining Method

[0098] Tissue sections (4 μm) were washed 3 times with PBS for 5 min each, incubated at 95°C for 10 min in 0.01 mol / L citrate buffer for antigen retrieval, cooled naturally, washed 3 times with PBS for 5 min each, immersed in 3% H2O2 for 10 min to block endogenous peroxidase, washed 3 times with PBS for 5 min each, permeabilized with 0.2% Triton X-100 membrane for 10 min, washed 3 times with PBS for 5 min each, blocked with 10% goat serum for 1 h, incubated with primary antibody α-SMA antibody (1:6000), placed at 4°C overnight, thawed for 30 min, washed 3 times with PBS for 5 min each, encapsulated with enzyme-labeled secondary antibody anti-mouse / rabbit IgG, incubated at room temperature for 30 min, washed 3 times with PBS for 5 min each, developed with DAB, stopped with tap water, stained with hematoxylin for 20 s, placed in tap water for 10 min to return to blue, air-dried, and mounted with neutral resin.

[0099] 3. Experimental Results and Analysis

[0100] Eight weeks after CIH treatment, significant increases in fibroblasts and a marked increase in the area of ​​α-SMA-positive regions were observed in the heart tissue of mice in the model group, indicating that CIH induced cardiac fibrosis and fibroblast activation. In contrast, the area of ​​α-SMA-positive regions was significantly reduced in the ITSA1 intervention group, indicating that ITSA1 effectively inhibited fibroblast proliferation and transformation. Specifically, the area of ​​α-SMA-positive regions in the heart tissue of mice in the ITSA1 intervention group was significantly reduced, indicating that ITSA1 significantly alleviated cardiac fibrosis. The degree of fibrosis was significantly reduced compared to the model group, and the difference was statistically significant (P<0.01, n=6). Figure 6 (As shown).

[0101] In summary, the results of this embodiment demonstrate that ITSA1 can effectively slow down fibroblast proliferation and transformation induced by CIH and inhibit the progression of cardiac fibrosis. The α-SMA staining results provide strong experimental support for the potential application of ITSA1 in the treatment of chronic hypoxia-related cardiac fibrosis, and this finding provides important scientific evidence for the clinical application of ITSA1 as an anti-fibrotic therapeutic agent.

[0102] Example 7: Safety evaluation of ITSA1 on the structure and function of mouse liver and kidney tissues.

[0103] This invention experimentally demonstrates that ITSA1 has a mitigating effect on myocardial remodeling, myocardial fibrosis, myocardial hypertrophy, and cardiac dysfunction caused by chronic intermittent hypoxia. This embodiment evaluates the potential effects of ITSA1 intervention for 8 weeks on the morphology and function of mouse liver and kidney tissues to ensure its safety and feasibility. The experiment systematically evaluated relevant indicators in four groups of mice through histological examination and serum biochemical analysis. The specific implementation procedures are as follows:

[0104] 1. Histological observation

[0105] HE staining results showed that the liver and kidney tissues of all four groups of mice had clear structural textures and no obvious pathological changes. The liver lobules were intact, and the glomeruli and tubules were normal, indicating that ITSA1 intervention did not significantly damage the morphology of major organs (e.g., Figure 7 (As shown).

[0106] 2. Liver function test

[0107] Serum biochemical analysis showed no significant differences in liver function indicators, including alanine transaminase (ALT) and aspartate transaminase (AST) levels, among the four groups of mice. Statistical analysis indicated no statistically significant differences (P>0.05, n=6). Figure 8 (As shown in A and B).

[0108] 3. Kidney function test

[0109] Serum renal function indicators, including blood urea nitrogen (BUN) and creatinine (CRE), showed no significant differences among the four groups of mice. Statistical results indicated that ITSA1 intervention did not cause abnormal changes in renal function indicators, and the differences were not statistically significant (P>0.05, n=6). Figure 8 (As shown in C and D).

[0110] In summary, the results of this embodiment demonstrate that ITSA1 did not have significant adverse effects on the morphology and function of the liver and kidneys in mice after 8 weeks of intervention, exhibiting good biocompatibility and safety. These results provide important experimental evidence for the safety characteristics of ITSA1 and lay a solid foundation for its further development and application in the treatment of cardiovascular diseases.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of ITSA1 in the preparation of drugs for the treatment or prevention of cardiac fibrosis and / or cardiomyocyte hypertrophy caused by chronic intermittent hypoxia, characterized in that: The structural formula of ITSA1 is as follows: .

2. The application according to claim 1, characterized in that: The drug is used to improve heart rate, reduce myocardial hypertrophy, and enhance myocardial contractile function.

3. The application according to claim 1 or 2, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

4. The use of ITSA1 in the preparation of drugs for the treatment or prevention of hypertension caused by chronic intermittent hypoxia, characterized in that: The structural formula of ITSA1 is as follows: .

5. The application according to claim 4, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

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