Application of ITSA1 in preparation of medicine for treating or preventing cardiac remodeling or hypertension

Abstract

The invention relates to application of ITSA1 in preparation of a medicine for treating or preventing cardiac remodeling or hypertension, and belongs to the technical field of prevention of cardiovascular diseases. The method comprises the following steps: constructing a chronic intermittent hypoxia (CIH) animal model by using a mouse, performing blood pressure measurement 2 weeks, 4 weeks and 8 weeks before and during construction of the animal model, and performing cardiac ultrasound, Masson staining, alpha-SMA immunohistochemistry, WGA staining and the like after the model is finished. Experimental results show that the ITSA1 relieves blood pressure rise, accelerated heart rate, cardiac function, myocardial fibrosis and myocardial cell hypertrophy caused by chronic intermittent hypoxia. Experiments prove that the ITSA1 can effectively relieve blood pressure rise and cardiac remodeling caused by chronic intermittent hypoxia, and a foundation is laid for widening the clinical application range of the ITSA1.

Description

Technical Field

[0001] The present invention relates to application of ITSA1 in preparing drugs for treating or preventing cardiac remodeling or hypertension, and belongs to the technical field of cardiovascular disease prevention. Background Art

[0002] Cardiac remodeling refers to a series of structural and functional changes that occur in the heart under various pathological conditions (such as hypertension, myocardial infarction, heart failure, etc.). It is an adaptive response of the heart to long-term exposure to hemodynamic or non-hemodynamic factors. In the early stage of cardiac remodeling, it is mainly characterized by hypertrophy of myocardial cells, which has a compensatory effect and can maintain normal cardiac function without changes in interstitial components. When under stress conditions such as pressure overload for a long time, cardiac remodeling will become a decompensated state, with decreased cardiac function, accompanied by activation of cardiac fibroblasts, infiltration and activation of immune cells, increase in interstitial components, changes in components, and apoptosis of myocardial cells. When cardiac remodeling enters decompensation, the incidence of arrhythmia, myocardial infarction, congestive heart failure, sudden death, and other cardiovascular events can increase by 6-8 times. Preventing or delaying cardiac remodeling can effectively curb the progression of various cardiovascular diseases and the occurrence of heart failure, and improve the survival rate and quality of life of patients. Therefore, finding new methods to delay cardiac remodeling is the key to effectively reduce its high mortality rate in clinical practice.

[0003] Although existing cardiac remodeling treatments and drugs can delay or alleviate this process to a certain extent, they still have many limitations:

[0004] (1) Drug side effects: The drugs currently used to treat cardiac remodeling are usually 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 system infections. These side effects not only affect the patient's quality of life, but may also limit the long-term use of the drug.

[0005] (2) Limited efficacy: Although existing drugs can slow the progression of cardiac remodeling to a certain extent, their efficacy is still limited, especially in the late stages of the disease, when drugs are often unable to 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 requires that treatment plans be highly individualized, increasing the complexity and challenges of clinical treatment.

[0007] (4) Compliance issues: The treatment of cardiac remodeling usually requires long-term or even lifelong medication. Complex treatment plans and frequent medication adjustments may reduce patient compliance, thereby affecting the treatment effect. In addition, the side effects of drugs and the financial burden faced by patients may also weaken patients' willingness to take medication, further affecting the treatment effect.

[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 result in reduced efficacy or increased adverse reactions.

[0009] In summary, although the existing means and drugs for treating cardiac remodeling can alleviate the condition to a certain extent, there are still many limitations, including drug side effects, limited efficacy, individual differences, compliance issues, drug interactions, economic burden, and the risk of invasive treatment, etc. Therefore, the development of new, biosafe, and highly effective drugs that can alleviate or treat cardiac remodeling is of great significance to curb the progression of various cardiovascular diseases and the occurrence of heart failure.

[0010] ITSA1 is a small molecule benzotriazole compound (molecular formula: C l3 H 7 C l2 N 3O, molecular weight 292.12g / mol), can penetrate into cells, and was first discovered as an inhibitor of TSA (Trichostatin A). ITSA1 antagonizes TSA-induced cell cycle arrest, acetylation of histones and tubulin, and transcriptional activation; in addition, ITSA1 reverses the inhibitory effect of TSA on protein deacetylase activity. Based on the results of Stuart L. Schreiber's team at the Chemistry and Biochemistry Laboratory at Harvard University, the effect of ITSA1 in inhibiting TSA has the following characteristics: Inhibition of TSA-induced cell cycle arrest: TSA causes the cell cycle to 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 originally arrested by TSA can re-enter the cell cycle, showing a cell cycle distribution similar to that of untreated cells. This effect of ITSA1 is not achieved by directly destroying the structure of TSA, but it works after TSA induces cell cycle arrest. ITSA1 can offset the effect of TSA on the cell cycle for a long time; and the inhibitory effect of ITSA1 does not act directly on histone deacetylase, but it works after TSA acts. Further studies have shown that TSA activates the transcription of certain genes, but ITSA1 can inhibit this transcriptional activation. ITSA1 inhibits the transcriptional activation caused by TSA, not the expression of the gene itself. This feature suggests that ITSA1 may act on the downstream effect pathway caused by TSA. At the same time, TSA not only causes histone acetylation, but also causes acetylation of α-tubulin in the cytoplasm, and ITSA1 can inhibit TSA-induced tubulin acetylation and restore the acetylation level of tubulin to normal.

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

[0012] The first objective of the present invention is to provide an application of ITSA1 in the preparation of a drug for treating or preventing cardiac remodeling, and to provide a new substance that can effectively treat or prevent cardiac remodeling.

[0013] The second objective of the present invention is to provide the use of ITSA1 in the preparation of a drug for treating or preventing hypertension, and to provide a new use of ITSA1 in treating or preventing hypertension.

[0014] In order to achieve the above-mentioned purpose, the technical solution of the application of ITSA1 in the preparation of a drug for treating or preventing cardiac remodeling in the present invention is:

[0015] Use of ITSA1 in preparing a drug for treating or preventing cardiac remodeling, wherein the structural formula of ITSA1 is

[0016] The beneficial effect of the above technical solution is that the use of ITSA1 of the present invention in the preparation of drugs for the treatment or prevention of cardiac remodeling is an invention of a new use of a known product. The present invention first uses mice to construct an animal model of chronic intermittent hypoxia (CIH), and after the model construction is completed, cardiac ultrasound, Masson staining, α-SMA immunohistochemistry, WGA staining, etc. are performed. The statistical analysis results show that the results of Masson staining and α-SMA immunohistochemistry indicate that ITSA1 alleviates myocardial fibrosis caused by chronic intermittent hypoxia; the WGA staining results show that ITSA1 alleviates the enlargement of myocardial cells caused by chronic intermittent hypoxia. These results fully prove that ITSA1 can effectively improve cardiac remodeling and lay the foundation for broadening the scope of clinical application of ITSA1.

[0017] Furthermore, the present invention demonstrates that ITSA1 has no obvious adverse effects on the tissue morphology and function of the liver and kidneys of mice after 8 weeks of intervention, showing good biocompatibility and safety, which provides an important experimental basis 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 a recurring state of hypoxia, usually caused by sleep apnea syndrome, high altitude environment or chronic obstructive pulmonary disease. CIH can lead to a series of pathophysiological changes, including overactivation of the sympathetic nervous system, increased oxidative stress, enhanced inflammatory response and endothelial dysfunction, which can cause multiple damages to the cardiovascular system, including vascular endothelial damage, myocardial cell hypertrophy and myocardial fibrosis, and ultimately lead to cardiac remodeling.

[0019] As a further improvement, the drug is a drug for inhibiting cardiac fibrosis and / or myocardial cell 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 refinement, the cardiac remodeling is caused by chronic intermittent hypoxia.

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

[0023] In order to achieve the above-mentioned object, the technical solution of the application of ITSA1 in the preparation of a drug for treating or preventing hypertension in the present invention is:

[0024] Use of ITSA1 in preparing a drug for treating or preventing hypertension, wherein the structural formula of ITSA1 is

[0025] The beneficial effect of the above technical solution is that: the present invention first uses mice to construct a chronic intermittent hypoxia (CIH) animal model, and measures blood pressure before, during and after 2, 4 and 8 weeks of the animal model construction. The results show that ITSA1 has a significant effect in alleviating abnormal blood pressure caused by chronic intermittent hypoxia, especially showing a significant antihypertensive effect under long-term exposure conditions. The regulatory effect of ITSA1 on blood pressure provides a strong experimental basis and technical support for its potential application in the treatment of chronic hypoxia-related cardiovascular diseases, which lays 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 further contains a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 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 the blood pressure changes caused by chronic intermittent hypoxia at different time points in Example 2 of the present invention (wherein **** represents P<0.0001, and ns represents no statistically significant difference);

[0030] Figure 3 The protective effect of ITSA1 on cardiac dysfunction caused by chronic intermittent hypoxia in Example 3 of the present invention (wherein, 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 left ventricular posterior wall systolic and diastolic thickness 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 chronic intermittent hypoxia-induced collagen fiber proliferation in Example 4 of the present invention (wherein, the left figure is a Masson trichrome staining image of the mouse heart in different treatment groups, the right figure represents the statistical result graph, **** represents P<0.0001, *** represents P<0.001);

[0032] Figure 5The effect of ITSA1 intervention on myocardial cell enlargement caused by chronic intermittent hypoxia in Example 5 of the present invention (wherein the left figure is a WGA staining diagram of the mouse heart in different treatment groups, the right figure represents the statistical result diagram, **** 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 diagram of the heart of mice in different treatment groups, the right figure represents the statistical result diagram, **** represents P<0.0001);

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

[0035] Figure 8 This is a test of the functional indicators of the liver and kidney of mice in different treatment groups in Example 7 of the present invention (wherein, A is the change in the alanine aminotransferase level of mice in different treatment groups, B is the change in the aspartate aminotransferase level of mice in different treatment groups, C is the change in the blood urea nitrogen level of mice in different treatment groups, D is the change in the blood creatinine level of mice in different treatment groups, and ns represents no statistically significant difference). DETAILED DESCRIPTION

[0036] In the prior art, chronic intermittent hypoxia leads to hypertension and cardiac dysfunction (cardiac remodeling). The present invention proves through experiments that ITSA1 plays an important role in intervening in cardiac dysfunction caused by chronic intermittent hypoxia, not only improving cardiac function, but also alleviating myocardial hypertrophy and myocardial fibrosis caused by CIH.

[0037] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; however, these embodiments are only examples and do not constitute any limitation on the scope of the present invention. The details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0038] Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments and experimental examples are briefly introduced as follows:

[0039] Drug configuration:

[0040] ITSA1 was purchased from Selleck, CAS No. 200626-61-5, 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, intraperitoneal injection, 3 times / week, dissolved in DMSO, and stored at -80°C.

[0041] The data processing process in the embodiment of the present invention is as follows: SPSS26.0 software is used for statistical analysis, the experimental data are expressed as mean ± standard deviation, and independent sample t test is used for statistical analysis. The test level is α = 0.05, and P < 0.05 is statistically significant.

[0042] Specific examples of the use of ITSA1 of the present invention in preparing drugs for treating or preventing cardiac remodeling or hypertension:

[0043] The present invention first constructs a chronic intermittent hypoxia model mouse. Compared with normal mice, the model mouse has impaired heart function and increased blood pressure. When ITSA1 is used for intervention, its blood pressure is reduced and heart function is significantly improved. The specific real-time operation is as follows:

[0044] Example 1 Method for constructing a chronic intermittent hypoxia animal model

[0045] This example establishes a chronic intermittent hypoxia mouse model to study the effects of a hypoxic environment on the physiological and biochemical reactions of mice and the effects of related treatments. The model simulates chronic intermittent hypoxia conditions by controlling oxygen concentration and exposure time using the Oxycycler A84 system from Biospherix, USA. The specific process and oxygen parameter control method are as follows: Figure 1 As shown, the specific implementation operations are as follows:

[0046] 1. Animal model construction process

[0047] The mice in the chronic intermittent hypoxia group were placed in a hypoxic environment chamber, and the Oxycycler A84 system was used to achieve precise oxygen concentration control. The specific steps in the model construction process are as follows:

[0048] Phase 1: Hypoxic Exposure

[0049] In the initial stage, mice were exposed to oxygen periodically in a hypoxic box. 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 the organism.

[0050] Phase 2: Recovery

[0051] Subsequently, the mice began to be connected to oxygen, and the system gradually increased the oxygen concentration from 6% to 21%. When the oxygen concentration reached 21%, it maintained the normoxic state for 20 seconds.

[0052] Cycle and exposure mode: Each cycle lasts 4 minutes, including two stages: hypoxia and normoxia. 15 cycles are performed every hour, which means that the mice will experience 15 fluctuations in oxygen concentration every hour. The hypoxia exposure mode lasts 8 hours a day, and the cumulative exposure time reaches 8 weeks.

[0053] 2. Oxygen parameter control and monitoring

[0054] The Oxycycler A84 system precisely controls oxygen concentration, ensuring that each switch between hypoxia and normoxia meets the set oxygen fluctuation parameters. The system monitors oxygen concentration changes in real time and automatically adjusts to ensure that the periodic changes in the hypoxic environment are accurate. The oxygen concentration gradually decreases from 21% to 6% to 6%, and then increases from 6% to 21% and maintains for 20 seconds, ensuring that the mice are exposed to repeated hypoxic environments for a long time.

[0055] 3. Experimental cycle and exposure duration

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

[0057] Through the establishment of this model, the present invention can comprehensively evaluate the effects of chronic intermittent hypoxia conditions on physiological functions such as cardiovascular, metabolic and fibrosis, and provide a strong experimental basis for potential therapeutic 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 example evaluates the intervention effect of ITSA1 on blood pressure changes caused by chronic intermittent hypoxia (CIH). The non-invasive blood pressure measurement method of the mouse tail is used to systematically observe the blood pressure changes of four groups of mice under CIH conditions, and focuses on analyzing the role of ITSA1 in regulating blood pressure. The specific implementation operations are as follows:

[0060] 1. Experimental design and operation steps

[0061] In this example, mice were divided into the following four groups: control group, model group, ITSA1 intervention group and other experimental groups. The experiment mainly used tail blood pressure measurement (a non-invasive, non-invasive blood pressure monitoring technology that can accurately assess the blood pressure changes in mice). After 2 weeks, 4 weeks and 8 weeks of CIH treatment, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of each group of mice were measured, and the significance of blood pressure changes was evaluated by 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, intraperitoneally injected, 3 times / week, and the experimental period was 8 weeks.

[0063] 2. Experimental results and analysis

[0064] After 2 weeks of CIH treatment, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of mice in the model group increased significantly (e.g. Figure 2 The difference was statistically significant compared with the control group (P<0.0001, n=6). This indicates that CIH can lead to an increase in blood pressure in the short term, simulating the pathological characteristics 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 completely inhibit the changes in blood pressure caused by CIH.

[0065] At 4 and 8 weeks of CIH treatment, the blood pressure levels of mice in the model group continued to increase as the CIH treatment time prolonged, especially after 4 and 8 weeks, when the systolic blood pressure, diastolic blood pressure, and mean arterial pressure were significantly higher than those in the control group, indicating that the pathological characteristics of hypertension caused by CIH gradually worsened. However, mice in the ITSA1 intervention group showed significant improvements in blood pressure at these time points. Specifically, the systolic blood pressure, diastolic blood pressure, and mean arterial pressure of 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 This result indicates that ITSA1 can effectively alleviate the increase in blood pressure caused by CIH, especially under the condition of long-term exposure to CIH (4 weeks or more), the intervention effect is more significant.

[0066] In summary, the experimental results of this example show that ITSA1 has a significant effect in alleviating abnormal blood pressure caused by chronic intermittent hypoxia, especially showing a significant antihypertensive effect under long-term exposure conditions. The regulatory effect of ITSA1 on blood pressure provides a strong experimental basis and technical support for its potential application in the treatment of chronic hypoxia-related cardiovascular diseases, which lays an important theoretical foundation for further drug development and clinical application.

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

[0068] This example evaluates the intervention effect of ITSA1 on cardiac dysfunction caused by chronic intermittent hypoxia (CIH). Using echocardiography technology, the changes in cardiac function of four groups of mice under CIH conditions were systematically observed, and the role of ITSA1 in regulating cardiac function was analyzed in detail. The specific implementation operations are as follows:

[0069] 1. Experimental design and operation steps

[0070] In this embodiment, mice were divided into four groups: a control group, a model group, an ITSA1 intervention group, and other experimental groups (the specific grouping is described in Example 2). After 8 weeks of CIH treatment, the cardiac function-related indicators of each group of mice were detected using small animal echocardiography technology. Through echocardiography, we can non-invasively evaluate the heart rate, left ventricular structure and functional parameters of mice. The experimental data include heart rate, left ventricular mass, left ventricular posterior wall systolic thickness, left ventricular posterior wall diastolic thickness and ejection fraction. Through statistical analysis, the significance of changes in cardiac function of each group of mice was evaluated.

[0071] 2. Experimental results and analysis

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

[0073] However, mice in the ITSA1 intervention group showed significant improvements in these indicators. Specifically, after ITSA1 intervention, each key cardiac function parameter was improved to varying degrees, among which heart rate and left ventricular mass decreased significantly, and the difference was statistically significant compared with the model group (P<0.0001, Figure 3 The thickness of the left ventricular posterior wall during systole was significantly reduced, and the difference was statistically significant (P<0.01, Figure 3 The thickness of the left ventricular posterior wall during diastole was reduced, and the difference was statistically significant (P<0.05, Figure 3In addition, the ejection fraction was significantly improved, indicating that the heart's pumping function was restored, and the difference was statistically significant (P<0.01, n=6, Figure 3 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 exhibits a significant protective effect.

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

[0075] Example 4 Evaluation of the intervention effect of ITSA1 on chronic intermittent hypoxia-induced collagen fiber proliferation

[0076] In this example, Masson trichrome staining technique was used to analyze the distribution of collagen fibers and muscle fibers in the heart tissue of mice after chronic intermittent hypoxia (CIH) treatment to evaluate the improvement effect of ITSA1 intervention on CIH-induced cardiac fibrosis. The specific implementation operation is as follows:

[0077] 1. Principle of Masson trichrome staining

[0078] Masson trichrome staining is a classic histological staining method that can significantly distinguish the distribution of collagen fibers and muscle fibers. The principle is that the small molecule dyes in the staining reagent can penetrate densely structured and low permeability tissues, while the large molecule dyes can only enter loosely structured and high permeability tissues. Due to the large molecular weight of light green dye or aniline blue, the results of Masson staining are as follows: muscle fibers are stained red; collagen fibers are stained green or blue; cell nuclei are 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 the analysis of the degree of fibrosis.

[0079] 2. Masson trichrome staining experimental steps (the kit is D026-1-3 from Nanjing Jiancheng Technology Co., Ltd.)

[0080] Before staining, rinse the sections in 39°C warm water twice, 60 seconds each time. Subsequently, use R1 (nuclear stain, purple-red liquid) for 60 seconds, R2 (paste stain, dark red liquid) for 45 seconds, R3 (color separation liquid, yellow transparent liquid) for 7 minutes (it is best to observe the collagen fibers partially faded to light pink under the microscope), and R4 (counter stain, blue liquid) for about 5 minutes. R5 (rinsing liquid, colorless transparent liquid) is used to rinse for 30 seconds between the R1, R2, and R3 staining steps, and the R4 blue counter stain needs to be rinsed with anhydrous ethanol. Finally, the sections are naturally dried, sealed with neutral gum, and photographed under a microscope to observe tissue collagen deposition.

[0081] 3. Experimental results and analysis

[0082] Masson trichrome staining showed that the collagen fibers in the heart tissue of mice treated with CIH for 8 weeks increased significantly, and the muscle fibers were arranged in disorder, showing obvious characteristics of myocardial fibrosis. The heart tissue of mice in the ITSA1 intervention group showed a significant decrease in collagen fiber content; the distribution of collagen fibers and muscle fibers tended to be normal; and the structural integrity of myocardial tissue was significantly improved. Statistical analysis showed that ITSA1 could effectively alleviate the collagen fiber proliferation caused by CIH and significantly improve myocardial fibrosis, further verifying its anti-fibrotic effect (P<0.001, n=6, such as Figure 4 shown).

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

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

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

[0086] 1. Principle of WGA staining technology

[0087] WGA is a plant lectin that can specifically recognize and bind to N-acetylglucosamine residues and is widely used to mark cell membranes and extracellular matrices. Through WGA staining, the morphology and size of cell membranes and the relationship between cells can be clearly observed, thus providing important information for studying biological processes such as cell proliferation and hypertrophy.

[0088] 2. WGA staining experimental steps

[0089] The tissue sections were 4 μm, and the slides were placed in a 65°C slide oven for 1 hour, then immersed in xylene I and II for 10 minutes each, and gradient ethanol (100%, 95%, 80%, 60%) for dewaxing, each step for 3 minutes, and then washed with PBS for 5 minutes × 3 times, and the sections were placed in citrate buffer (pH = 6.0), heated in a microwave for 10 minutes for antigen repair, cooled at room temperature, and washed with PBS for 5 minutes × 3 times. Subsequently, they were treated with 0.2% Triton X-100 for 10 minutes, washed with PBS for 5 minutes × 3 times, stained with WGA (1:300), kept in dark at 4°C overnight, washed with PBS for 5 minutes × 3 times, mounted with DAPI, photographed, and analyzed with ImageJ.

[0090] 3. Experimental results and analysis

[0091] Through WGA staining, we observed that in the heart tissue of mice treated with CIH for 8 weeks, the cardiomyocytes were significantly enlarged and the cell morphology was irregular, showing a typical myocardial hypertrophy phenotype. After ITSA1 intervention, the enlargement of mouse cardiomyocytes was significantly alleviated. Specifically, the volume of cardiomyocytes was significantly reduced and the morphology was restored to a near-normal state; the cells were arranged more neatly, reducing the degree of myocardial hypertrophy. Statistical analysis results showed that the difference in cardiomyocyte enlargement between the ITSA1 intervention group and the model group was statistically significant (P<0.01, n=6, as shown in Figure 2). Figure 5 shown).

[0092] In summary, the results of this example show that ITSA1 can effectively alleviate myocardial hypertrophy caused by CIH, and its mechanism may be related to inhibiting excessive proliferation of myocardial cells and improving cell morphology. The 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 example uses smooth muscle actin (α-SMA) immunohistochemical staining technology to evaluate the intervention effect of ITSA1 on fibroblast proliferation caused by chronic intermittent hypoxia (CIH). The specific implementation operation is as follows:

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

[0096] α-SMA is an actin isoform specifically expressed in smooth muscle cells and myofibroblasts and is widely used to assess the degree of tissue fibrosis. Studies have shown that in a variety of 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] The tissue sections were 4 μm, washed with PBS for 5 min × 3 times, placed in 0.01 mol / L citrate buffer at 95 ° C for 10 min for antigen repair, cooled naturally, washed with PBS for 5 min × 3 times, and immersed in 3% H 2 O 2 The sections were incubated in 4% PBS for 10 min to block endogenous peroxidase, washed with PBS for 5 min × 3 times, permeabilized with 0.2% Triton X-100 for 10 min, washed with PBS for 5 min × 3 times, blocked with 10% goat serum for 1 h, and incubated with primary antibody, α-SMA antibody (1:6000); placed in a 4°C refrigerator overnight, rewarmed for 30 min, washed with PBS for 5 min × 3 times, enzyme-labeled secondary antibody against mouse / rabbit IgG, incubated at room temperature for 30 min, washed with PBS for 5 min × 3 times, developed with DAB, terminated with tap water, stained with hematoxylin for 20 s, placed in tap water for 10 min to turn blue, dried naturally, and sealed with neutral gum.

[0099] 3. Experimental results and analysis

[0100] After 8 weeks of CIH treatment, there was a significant increase in fibroblasts in the heart tissue of mice in the model group, and the area of ​​α-SMA-positive regions increased significantly, indicating that CIH caused cardiac fibrosis and fibroblast activation. In the ITSA1 intervention group, the area of ​​α-SMA-positive regions decreased significantly, indicating that ITSA1 can effectively inhibit the proliferation and transformation of fibroblasts. Specifically, in the heart tissue of mice in the ITSA1 intervention group, the area of ​​α-SMA-positive regions decreased significantly, indicating that ITSA1 can significantly reduce cardiac fibrosis. The degree of fibrosis was significantly reduced compared with the model group, and the difference was statistically significant (P<0.01, n=6, as shown in Figure 2). Figure 6 shown).

[0101] In summary, the results of this example show that ITSA1 can effectively slow down the proliferation and transformation of fibroblasts caused 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 an important scientific basis 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] The present invention proves through experiments that ITSA1 has an alleviating effect on myocardial remodeling, myocardial fibrosis, myocardial hypertrophy, and cardiac dysfunction caused by chronic intermittent hypoxia. This example evaluates the potential effects of ITSA1 on the morphology and function of mouse liver and kidney tissues after 8 weeks of intervention to ensure its safety and feasibility. The experiment systematically evaluated the relevant indicators of four groups of mice through histological detection and serum biochemical analysis, and the specific implementation operations are as follows:

[0104] 1. Histological observation

[0105] HE staining results showed that the liver and kidney tissue structures of the four groups of mice were clear, with no obvious pathological changes. The liver lobule structure was intact, and the glomeruli and renal tubules were normal, indicating that ITSA1 intervention had no significant damage to the tissue morphology of the main organs (such as Figure 7 shown).

[0106] 2. Liver function test

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

[0108] 3. Renal function test

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

[0110] In summary, the results of this example prove that ITSA1 has no obvious adverse effects on the tissue morphology and function of the liver and kidneys of mice after 8 weeks of intervention, showing good biocompatibility and safety. The above results provide important experimental basis 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 is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. Use of ITSA1 in the preparation of a drug for treating or preventing cardiac remodeling, characterized in that: The structural formula of ITSA1 is 2. The use of ITSA1 according to claim 1 in preparing a drug for treating or preventing cardiac remodeling, characterized in that: The drug is a drug for inhibiting cardiac fibrosis and / or myocardial cell hypertrophy.

3. The use of ITSA1 according to claim 2 in preparing a drug for treating or preventing cardiac remodeling, characterized in that; The medicine is a medicine for improving heart rate, alleviating myocardial hypertrophy and enhancing myocardial contractile function.

4. Use of ITSA1 according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing cardiac remodeling, characterized in that: The cardiac remodeling is caused by chronic intermittent hypoxia.

5. Use of ITSA1 according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing cardiac remodeling, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

6. Use of ITSA1 in the preparation of a drug for treating or preventing hypertension, characterized in that: The structural formula of ITSA1 is 7. The use of ITSA1 according to claim 6 in preparing a drug for treating or preventing hypertension, characterized in that: The hypertension is caused by chronic intermittent hypoxia.

8. Use of ITSA1 according to claim 6 or 7 in the preparation of a drug for treating or preventing hypertension, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

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