Application of GANT61 in preparation of medicine for preventing and / or treating sleep apnea complicated with heart failure

Abstract

The invention discloses application of GANT61 in preparation of a medicine for preventing and / or treating sleep apnea complicated with heart failure. Experiments show that the GANT61 can improve cardiac function reduction induced by intermittent hypoxia combined with angiotensin II, reduce ventricular remodeling and myocardial fibrosis induced by intermittent hypoxia combined with angiotensin II, inhibit the multiplication capacity of cardiac fibroblasts, inhibit differentiation of cardiac fibroblasts induced by intermittent hypoxia to myofibroblasts, improve the cardiac function reduction induced by intermittent hypoxia combined with angiotensin II, and improve the cardiac function reduction induced by intermittent hypoxia combined with angiotensin II. Intermittent hypoxia-induced cardiac fibroblasts are inhibited from secreting extracellular matrixes, and the weight, blood pressure, heart rate and renal functions are not affected.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the use of GANT61 in preparing a medicament for preventing and / or treating sleep apnea combined with heart failure. Background Art

[0002] GANT61, molecular formula: C 27 H 35 N5, the chemical structure is shown below:

[0003]

[0004] GANT61 specifically binds to the downstream transcription factors GLI1 and GLI2 of the Hedgehog signaling pathway, inhibiting the transcriptional activity mediated by canonical and atypical GLI1 / GLI2 and suppressing tumor cell proliferation.

[0005] Obstructive sleep apnea (OSA), a major form of sleep-disordered breathing, not only impacts patients' quality of life and mood but also severely impairs cardiovascular and neuroendocrine function. Clinical and epidemiological studies have confirmed that OSA is an independent risk factor for cardiovascular disease. OSA is highly common in patients with cardiovascular disease or those at risk for cardiovascular disease, such as those with obesity, hypertension, coronary artery disease, heart failure, or atrial fibrillation. The Sleep and Heart Health Study found that patients with OSA (AHI ≥ 11) had a 2.38-fold increased incidence of heart failure. The prevalence of sleep-disordered breathing in patients with heart failure with reduced ejection fraction (HFrEF) ranged from 47% to 81%, with OSA accounting for 12% to 53%. Among 224 patients with heart failure with preserved ejection fraction (HFpEF), OSA was found in 40% and CSA in 29%. Coexisting sleep apnea is one of the causes of refractory heart failure. Therefore, identifying appropriate treatments for patients with sleep apnea and heart failure is a crucial task in heart failure treatment.

[0006] The pathophysiological mechanisms of OSA include reduced and fragmented sleep, intrathoracic pressure fluctuations, hypoxia and reoxygenation cycles, and cardiovascular cell damage induced by sympathetic nervous system activation. Our previous studies have demonstrated that intermittent hypoxia in resting cardiac fibroblasts increases the synthesis and release of TSP1, an upstream activator of TGF-β, further promoting fibroblast activation and ECM protein secretion, ultimately promoting myocardial fibrosis. Myocardial fibrosis is often a pathological manifestation of end-stage heart failure and is irreversible. Therefore, preventing and inhibiting myocardial fibrosis is an important intervention for sleep apnea combined with heart failure.

[0007] Current treatments for heart failure primarily focus on blocking activation of the neuroendocrine system to prevent and delay ventricular remodeling. Conventional drug therapies include angiotensin receptor / neprilysin inhibitors (ANRi), angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), beta-blockers, aldosterone receptor antagonists, and sodium-glucose transporter-2 inhibitors (SGLT2i). However, patients with sleep-disordered breathing and heart failure often present with refractory or intractable heart failure, leading to repeated hospitalizations, decreased quality of life, and increased mortality. Conventional medications and oxygen therapy are currently ineffective for the consequences of sleep apnea. Furthermore, current research remains inconclusive on whether noninvasive positive pressure ventilation can improve symptoms in patients with heart failure and sleep apnea. Summary of the Invention

[0008] The purpose of the present invention is to provide a new medical use of GANT61.

[0009] The new medical use of GANT61 provided by the present invention is the use of GANT61 in preparing the following products;

[0010] 1) Medications for the prevention and / or treatment of obstructive sleep apnea (OSA);

[0011] 2) Drugs that prevent and inhibit myocardial fibrosis;

[0012] 3) Drugs for preventing and / or treating sleep apnea combined with heart failure.

[0013] The present invention demonstrates through experiments that GANT61 can improve the reduction in cardiac function induced by intermittent hypoxia combined with angiotensin II, reduce ventricular remodeling and myocardial fibrosis induced by intermittent hypoxia combined with angiotensin II, inhibit the proliferation ability of cardiac fibroblasts, inhibit the differentiation of cardiac fibroblasts into myofibroblasts induced by intermittent hypoxia, and inhibit the secretion of extracellular matrix by cardiac fibroblasts induced by intermittent hypoxia, and has no effect on body weight, blood pressure, heart rate and renal function.

[0014] Sleep-disordered breathing causes myocardial tissue remodeling, aggravates myocardial fibrosis, and thus leads to reduced cardiac function, which may be the main pathophysiological basis of patients with sleep-disordered breathing combined with heart failure. Therefore, inhibiting myocardial fibrosis as a target may provide a new treatment strategy for refractory heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Angiotensin II sustained-release pump (1000 ng / kg / min) was subcutaneously implanted in the back of male C57BL / 6 experimental group mice in Example 1 of the present invention, followed by intermittent hypoxia (O2 concentration 5%-21% circulation) for 8 hours per day. GANT61 experimental group mice were given GANT61 (50 mg / kg) intraperitoneal injection every 3 days, and control group mice were given an equal amount of normal saline intraperitoneal injection. After 28 days, the mouse heart ultrasound examination was performed. (A) Isoflurane anesthesia was maintained to keep the mouse heart rate at about 500 beats / minute, and the left ventricular short-axis section was obtained and the image was recorded. (B) The left ventricular ejection fraction of each group of mice was counted. The experimental results are expressed as mean ± standard error, and the t test was used for inter-group comparison, n = 6, ****p < 0.0001.

[0016] Figure 2 Male C57BL / 6 mice described in Example 2 of the present invention were subcutaneously implanted with an angiotensin II sustained-release pump (1000 ng / kg / min) in their backs. They were then subjected to intermittent hypoxia (O2 concentration 5%-21%) for 8 hours per day. The GANT61 experimental group received intraperitoneal injections of GANT61 (50 mg / kg) every 3 days, while the control group received an equal amount of saline. After 28 days, the specimens were immersed in formalin, and paraffin sections of the mouse hearts were prepared. (A) Ratio of heart weight to tibia length in each group of mice. (B) HE staining, scale bar 500 μm. WGA staining, scale bar 40 μm. (C) Average myocardial cell area in each group of mice. (D) Sirius red staining and Masson staining, scale bar 500 μm. (E) Collagen deposition area in each group of mice. The experimental results are expressed as mean ± standard error, and intergroup comparisons were performed using a t-test, n = 6, ***p < 0.001, ****p < 0.0001.

[0017] Figure 3 An angiotensin II sustained-release pump (1000 ng / kg / min) was subcutaneously implanted in the back of male C57BL / 6 mice in Example 3 of the present invention, followed by intermittent hypoxia (O2 concentration 5%-21% circulation) for 8 hours per day. The mice in the GANT61 experimental group were given an intraperitoneal injection of GANT61 (50 mg / kg) every 3 days, and the mice in the control group were given an intraperitoneal injection of an equal amount of normal saline. After 28 days, the mice were weighed, and their blood pressure and heart rate were measured, and mouse plasma was collected. (A) Body weight of mice in each group. (B) Systolic blood pressure, diastolic blood pressure and heart rate of mice in each group. (C) Levels of creatinine, uric acid and urea in the plasma of mice in each group. The experimental results are expressed as mean ± standard error, and the t test was used for comparison between groups, n = 6, ns, no statistical difference.

[0018] Figure 4Cardiac fibroblasts from Example 4 of the present invention were pretreated with GANT61 (20 μM) or saline, then cultured in normoxic or hypoxic incubators for proliferation assays. (A) Fibroblast proliferation assay. Images of cells stained with EdU fluorescence, captured by confocal microscopy. Green fluorescence indicates EdU-positive nuclei, and blue fluorescence indicates DAPI (scale bar, 100 μm). (B) Quantitative analysis of the percentage of EdU-positive cells in each group. (Data are shown as mean ± standard error, ***p < 0.001; two-way ANOVA, n = 3).

[0019] Figure 5 The cardiac fibroblasts in Example 5 of the present invention were pretreated with GANT61 (20 μM) or physiological saline, and then cultured in a normoxic or hypoxic incubator for cell immunofluorescence staining and gel contraction experiments. (A) Images of cell immunofluorescence staining taken with a confocal microscope, in which the green fluorescent marker is GLI1, the red fluorescent marker is Phalloidin, and the blue fluorescent marker is DAPI (scale bar, 20 μm). (B) The fluorescence intensity of GLI1 and the fluorescence intensity of stress fibers were quantified using Image J. (C) In the gel contraction experiment, the red dotted line in the figure marks the gel area of ​​each group. (D) The gel area was quantified using Image J to quantify the cell contractility. (Data are shown as mean ± standard error, ****p<0.0001; two-way ANOVA, n=5).

[0020] Figure 6 Cardiac fibroblasts from Example 6 were pretreated with GANT61 (20 μM) or saline, then cultured in normoxic or hypoxic incubators and subjected to Western blotting. (A) Western blotting assays were used to measure GLI1, Collage I, and α-SMA protein levels. (B) Quantification and statistical analysis of these proteins were performed using Image J software. (Data are shown as mean ± standard error, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-way ANOVA, n = 5). DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0022] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0023] In the following examples, GANT61 was purchased from GLPBIO, USA, with the product number GC10359.

[0024] In the following examples, male C57BL / 6 mice, 8-10 weeks old and weighing 25-27 g, were implanted subcutaneously with an angiotensin II sustained-release pump (1000 ng / kg / min) in their backs. Subsequently, the mice were placed in an oxygen-depleting chamber during the day for 6-8 hours of intermittent hypoxia (O2 concentration 5%-21% circulating). The GANT61 experimental group of mice received an intraperitoneal injection of GANT61 (50 mg / kg) every three days, while the control group received an intraperitoneal injection of an equal amount of normal saline. After four weeks, the mice were measured for indicators of cardiac function, including blood pressure, heart rate, ejection fraction, heart weight to tibial length, and myocardial tissue morphology staining.

[0025] Example 1: GANT61 improves intermittent hypoxia combined with angiotensin II-induced cardiac function impairment

[0026] Male C57BL / 6 mice aged 8-10 weeks were implanted subcutaneously with an angiotensin II sustained-release pump (1000 ng / kg / min) on their backs and then subjected to intermittent hypoxia (O2 concentration 5%-21% circulation) for 8 hours per day.

[0027] The specific procedure is as follows: Pump implantation: Dissolve angiotensin II powder (Abcam, #ab120183) in saline, thoroughly dissolve and mix. Fill a microosmotic pump (ALZA, Alzet 2004), immerse the pump in saline, and incubate in a cell culture incubator for approximately 48 hours. Before implantation, sterilize all instruments with autoclave. Anesthetize mice with isoflurane and place them in the prone position on a warming table. Shave the hair on the back of the neck and disinfect with iodine. Use scissors to make a transverse incision approximately 1 cm long on the back of the neck. Use vascular forceps to separate the skin from the subcutaneous tissue. Implant the micropump subcutaneously on the back of the neck. Suture the skin and disinfect the incision.

[0028] Intermittent hypoxia: Place the mice in an intermittent hypoxia chamber, open the nitrogen valve and air compressor pump, and set the program. The first stage is the hypoxia stage with an oxygen concentration of 6%, which is maintained for 6 minutes. The second stage is the normoxia stage with an oxygen concentration of 21%, which is maintained for 12 minutes. This cycle is repeated. The oxygen concentration in hypoxia fluctuates between 6% and 21% for 8 hours a day for a total of 28 days.

[0029] The mice in the GANT61 experimental group were given intraperitoneal injection of GANT61 (50 mg / kg) every 3 days, and the mice in the normal saline group were given intraperitoneal injection of an equal amount of normal saline. After 28 days, cardiac function was measured by cardiac ultrasound.

[0030] The results are as follows Figure 1 As shown in A and B, the ejection fraction of mice in the GANT61 group was significantly higher than that in the saline group, indicating that GANT61 can improve the hypoxia-angiotensin II-induced cardiac dysfunction.

[0031] Example 2: GANT61 reduces intermittent hypoxia combined with angiotensin II-induced ventricular remodeling and myocardial fibrosis

[0032] Ventricular remodeling and myocardial fibrosis are important pathological manifestations of heart failure. In order to further clarify the relationship between GANT61 and ventricular remodeling and myocardial fibrosis, the model was completed (male C57BL / 6 mice were subcutaneously implanted with an angiotensin II sustained-release pump (1000 ng / kg / min) on the back, followed by intermittent hypoxia (O2 concentration 5%-21% circulation) for 8 hours a day. The mice in the GANT61 experimental group were given GANT61 (50 mg / kg) intraperitoneal injection every 3 days, and the mice in the control group were given an equal amount of normal saline intraperitoneally. After 28 days), the heart samples of the two groups of mice were weighed and the mice were measured. The tibia length was measured, and then the mouse heart paraffin sections were collected for HE staining, WGA staining, Sirius red staining and Masson staining (HE staining: paraffin sections were routinely dewaxed to water, stained with hematoxylin solution for 5 minutes, and washed with distilled water to remove floating color; differentiated with differentiation solution for 3 minutes, washed with tap water for 1 minute; eosin staining for 2 minutes, washed with distilled water for 30 seconds; rapid dehydration (75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol for 2 seconds each), transparentized with xylene twice for 1 minute each time, sealed with neutral gum, and observed under a microscope. WGA staining: paraffin sections were routinely dewaxed to water; sections were immersed in Tris-EDTA buffer (pH 9.0), antigen retrieval was performed in a 95°C water bath for 30 min, and then naturally cooled to room temperature; the Tris-EDTA buffer was discarded, and the sections were washed twice with PBS, each time for 5 min; fixed with 10% neutral formalin fixative, and allowed to stand at room temperature for 15 min; the formalin fixative was discarded, and the sections were washed twice with PBS, each time for 5 min; the sections were permeated with ice-cold methanol and allowed to stand at room temperature for 10 min; the ice-cold methanol was discarded, and the sections were washed twice with PBS, each time for 5 min; the sections were permeated with 0.5% Triton X-100 and allowed to stand at room temperature for 10 min; the 0.5% Triton X-100 and PBS were washed twice, each for 5 minutes. The sections were blocked with 1% bovine serum albumin (BSA) at room temperature for 1 hour. The BSA was discarded and the sections were incubated with a mixture of WGA and DAPI (at an appropriate dilution) at 4°C overnight. The secondary antibody was discarded and the sections were washed twice with PBS, each for 5 minutes. The sections were mounted with anti-fluorescence quenching mounting medium containing DAPI and observed under a laser confocal microscope. Sirius red staining: Paraffin sections were routinely deparaffinized and dehydrated. Iron hematoxylin working solution was prepared by mixing iron hematoxylin stock solution with iron hematoxylin diluent in a 1:1 ratio (prepared immediately before use). The sections were placed in a staining box and stained with Sirius red solution for 10-20 minutes. The sections were then rinsed with distilled water for 30 seconds to remove excess stain. The sections were then rinsed with tap water for 5 minutes, then stained with Sirius red solution for 10-20 minutes. The sections were then rinsed with running water to remove the surface stain. Finally, the sections were dehydrated with anhydrous ethanol, transparentized with xylene, mounted with neutral gum, and observed under a fiberoptic microscope.Masson staining: Dewax the paraffin sections to water, place the sections in a 2.5% potassium dichromate mordant solution, incubate in a 60°C oven for 1 hour, and wash with tap water for 30 seconds until the yellow color on the tissue fades (at the same time, preheat the Ponceau acid fuchsin and 2.5% aniline blue solution in the oven); mix equal volumes of iron hematoxylin stock solution and iron hematoxylin diluent (pre-mixed and used immediately), place the sections in a staining box, add the mixture dropwise for 1 minute, and rinse with running water; differentiate the sections in a differentiation solution for about 1 minute until the cell nuclei appear gray-black and the background is almost colorless or light gray; rinse with tap water, drain the excess water on the sections slightly, place the sections in a staining box, and add a dropwise amount of Ponceau acid fuchsin solution. The tissue was stained for 6 minutes, at which point the tissue appeared bright red. If the red color was too light, the staining time could be extended appropriately. The sections were slightly drained (do not allow the sections to dry out), placed in a slide staining box, and soaked in 1% phosphomolybdic acid solution for 1 minute (this step is for differentiation, and differentiation is carried out until the collagen fibers are light red. The E solution time can be adjusted according to the desired staining depth, generally 1-2 minutes). The sections were slightly drained of the 1% phosphomolybdic acid solution and, without washing, directly stained with 2.5% aniline blue solution for about 1 minute. The sections were rinsed and differentiated with 1% glacial acetic acid for about 1 minute. Finally, the sections were dehydrated with anhydrous ethanol, transparentized with xylene, mounted with neutral gum, and observed under an optical microscope. The heart weight / tibia length of mice in the GANT61 group was significantly lower than that in the saline group ( ). Figure 2 A). The results are as follows Figure 2 As shown in B and C, the cross-sectional area of ​​myocardial cells in the GANT61 group was significantly lower than that in the saline group. In addition, Sirius red staining and Masson staining showed that the collagen deposition area in the GANT61 group was also significantly lower than that in the saline group ( Figure 2 D, E). The above results indicate that GANT61 can reduce ventricular remodeling and myocardial fibrosis induced by intermittent hypoxia combined with angiotensin II.

[0033] Example 3: GANT61 does not affect the body weight, blood pressure, heart rate, and renal function of mice

[0034] In order to determine whether GANT61 affects the basic vital signs and renal function of mice, the model was established (male C57BL / 6 mice were subcutaneously implanted with an angiotensin II sustained-release pump (1000 ng / kg / min) on the back, followed by intermittent hypoxia (O2 concentration 5%-21% circulation) for 8 hours a day. The mice in the GANT61 experimental group were given GANT61 (50 mg / kg) intraperitoneal injection every 3 days, and the mice in the control group were given an equal amount of normal saline intraperitoneally. After 28 days, the weight, blood pressure and heart rate of each group of mice were measured, and plasma was collected to measure renal function. Figure 3As shown in Figures AB, we found no statistically significant differences in body weight, systolic blood pressure, diastolic blood pressure, or heart rate between the GANT61 and saline groups. This indicates that GANT61 does not affect the weight, blood pressure, or heart rate of mice. Furthermore, there were no statistically significant differences in blood creatinine, uric acid, and urea levels between the two groups. These results demonstrate that GANT61 has no effect on the basic vital signs or renal function of mice.

[0035] Example 4: GANT61 inhibits the proliferation of cardiac fibroblasts

[0036] To explore the effect of GANT61 on fibroblast function, we isolated and cultured cardiac fibroblasts from mouse hearts. The cells were divided into normal saline + normoxia group, normal saline + intermittent hypoxia group, GANT61 + normoxia group, and GANT61 + intermittent hypoxia group. The EdU method was used to investigate the proliferation capacity of fibroblasts. The procedure was as follows:

[0037] Isolation and culture of mouse cardiac fibroblasts

[0038] 3-5 adult mice were killed by cervical dislocation. The hearts were immediately removed and placed in a culture dish containing cold PBS. In a sterile environment, the culture dish was placed on ice. The heart was cut open and the blood in the heart was cleaned. A new culture dish and clean PBS were replaced and the heart was washed twice. The heart was transferred to a new culture dish and cut into approximately 1 × 1 × 1 mm pieces using tissue scissors. 3 Transfer the chopped tissue pieces to a 50 mL centrifuge tube, add 25 mL of digestion solution, and digest the tissue in a 37°C biochemical incubator with constant stirring for 10 minutes; let the mixture stand for 1 minute, collect the supernatant, and transfer the supernatant to a new 50 mL centrifuge tube containing 2 mL of culture medium; add 25 mL of new digestion solution to the settled tissue pieces, and digest the tissue at 37°C with constant stirring for 10 minutes; let it stand for 1 minute, and collect the supernatant again; repeat the digestion of tissue pieces 7 to 10 times until The tissue blocks were basically dissolved; the collected supernatant was centrifuged at 300g for 5 minutes at 4°C; the cells were resuspended, and the cell suspension was transferred to a 10-cm culture dish and placed in a 37°C incubator containing 5% CO2; after 2 hours, the viable fibroblasts adhered to the wall and appeared as small dots under the microscope. The cells were washed twice with preheated PBS and supplemented with new culture medium. Then, the culture dish was placed in a 37°C incubator containing 5% CO2; after 2 to 3 days, the fibroblasts reached a density of approximately 90% and could be passaged for subsequent experiments.

[0039] Fibroblast proliferation assay

[0040] Fibroblast proliferation assay: Sterilized cell slides were spread on the bottom of a 12-well plate and mixed with cell suspension. When the cells grew to 40-50% density, the cells were pretreated with GANT61 (20 μM) or saline for 6 hours, and then cultured under normoxia or intermittent hypoxia for 48 hours. Prepare 2×EdU incubation working solution: add 2 μL of EdU storage solution (10 mM) to every 1 mL of complete culture medium to obtain 20 μM 2×EdU incubation working solution, and preheat it. In a half-medium exchange mode, remove half of the original culture medium from the culture plate and add an equal volume of preheated 2×EdU incubation working solution, and incubate for 2 h. Remove the culture plate, wash twice with PBS, add 4% paraformaldehyde to fix the cells, and let them stand at room temperature for 15 min. Remove the fixative, wash twice with PBS for 3-5 min each, add 0.5% TritonX-100 to permeabilize the cells, and let them stand at room temperature for 15 min. Remove the permeabilization solution, wash twice with PBS for 3-5 min each. Prepare the click reaction solution during cell fixation and permeabilization. Remove PBS, add click reaction solution, ensure that the reaction solution completely covers the cells, and incubate at room temperature in the dark for 30 minutes; remove click reaction solution, wash twice with PBS, each time for 3-5 minutes; remove PBS, dilute Hoechst 33342 staining solution with PBS buffer at a ratio of 1:1000, add to cover cells, and incubate at room temperature for 5 minutes; remove Hoechst 33342 staining solution, wash twice with PBS, each time for 3-5 minutes; add anti-fluorescence quenching mounting medium containing DAPI on the slide, and mount the slide; examine and analyze under confocal microscopy.

[0041] The results are as follows Figure 4 As shown in A and B, the proliferation ability of cardiac fibroblasts was significantly reduced after treatment with GANT61.

[0042] Example 5: GANT61 inhibits intermittent hypoxia-induced differentiation of cardiac fibroblasts into myofibroblasts

[0043] Research has shown that a key mechanism of cardiac remodeling is the activation of quiescent fibroblasts by external stimuli, leading to their proliferation and differentiation into myofibroblasts, characterized by the formation of stress fibers and expression of α-smooth muscle actin (α-SMA). To investigate the effect of GANT61 on fibroblast differentiation, cells were divided into four groups, as previously described, and immunofluorescence staining of fibroblasts was performed as follows:

[0044] Sterilized cell slides were spread on the bottom of a 12-well plate, and the mixed cell suspension was added. When the cells grew to a density of 30-40%, they were pretreated with GANT61 (20 μM) or normal saline for 6 hours. Subsequently, the culture dishes of the intermittent hypoxia group were placed in a cell hypoxia chamber, and the control group continued to be cultured in a normal oxygen chamber. After 48 hours, the well plate was taken out, the culture medium was discarded, and the cells were washed 3 times with PBS for 5 minutes each time; the cells were fixed with 4% paraformaldehyde and allowed to stand at room temperature for 10 minutes; the paraformaldehyde was discarded, the cells were washed 3 times with PBS for 5 minutes each time, the membrane was broken with 0.5% Triton X-100, and allowed to stand at room temperature for 10 minutes; the 0.5% Triton X-100, washed 3 times with PBS, 5 minutes each time; blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour; discarded BSA, incubated with primary antibody at appropriate dilution ratio at 4°C overnight; recovered primary antibody, washed 3 times with PBS, 5 minutes each time; incubated with fluorescent secondary antibody at appropriate dilution ratio at room temperature for 2 hours; discarded secondary antibody, washed 3 times with PBS, 5 minutes each time, and mounted with anti-fluorescence quenching mounting medium containing DAPI; observed and analyzed under laser confocal microscope. Figure 5 As shown in AC, GANT61 significantly inhibited the increase in GLI1 expression induced by intermittent hypoxia; at the same time, it inhibited the differentiation phenotype of myofibroblasts induced by intermittent hypoxia.

[0045] We then used a gel contraction assay to verify the contractile ability of myofibroblasts. The procedure was as follows: primary mouse cardiac fibroblasts were pretreated with GANT61 (20 μM) or saline for 6 hours, and the collagen matrix stock solution was diluted to 3 mg / mL with 0.1% acetic acid for later use; the pretreated cells were trypsinized, neutralized with culture medium, centrifuged at 1000 rpm, 4°C, for 5 minutes, and the cells were resuspended. 3 mg / mL collagen solution was diluted with the cell suspension and mixed gently with a pipette to avoid bubbles. The final concentration was 1 mg / mL. An appropriate volume of Add sodium hydroxide solution and gently pipette until the solution turns pink; immediately add the discolored mixture to a 24-well plate, avoiding bubbles, 500 μL per well; place it in an incubator and let it stand for 20-30 minutes until the collagen matrix solidifies; slide the tip of the pipette along the edge of the well plate for a week to separate the matrix from the well, add 500 μL of culture medium, place the hypoxic group culture dish in the cell hypoxia box, and continue to culture the control group in a normal oxygen box. After 48 hours, remove the well plate, observe the area of ​​the gel in each group and take pictures. The results are as follows Figure 5 As shown in Figures DE, the contractile ability of cardiac myofibroblasts was significantly reduced after GANT61 treatment.

[0046] Example 6: GANT61 inhibits intermittent hypoxia-induced extracellular matrix secretion in cardiac fibroblasts

[0047] As described above, cells were divided into four groups and subjected to protein immunoblotting experiments (discontinuous SDS-polyacrylamide gels were prepared, samples were loaded, and electrophoresis was performed using a Bio-Rad machine at 80 V to the upper edge of the separation gel and at 120 V to the lower edge of the separation gel; the gel was cut and transferred to a PVDF membrane using a Bio-Rad machine at a constant current of 200 mA for 2 h; the PVDF membrane was blocked with milk blocking solution at room temperature for 1 h; the membrane was cut and incubated with the prepared primary antibody at 4°C overnight; the antibody was recovered and the membrane was washed three times with TBST solution for 10 min each; the secondary antibody was diluted at a ratio of 1:5000 and incubated with the PVDF membrane at room temperature for 1 h; the membrane was washed three times with TBST solution for 10 min each; the PVDF membrane was placed in an exposure machine, chemiluminescent solution was added, the appropriate program was selected for exposure, and the image was stored; the grayscale value of the image bands was digitized using Image J analysis software, and the grayscale value of the target protein was divided by the grayscale value of the internal reference for correction).

[0048] The results are as follows Figure 6 As shown in the figure, compared with the normal saline group, GANT61 significantly inhibited the expression of GLI1 protein, type I collagen (Collage I) and α-SMA protein.

[0049] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of GANT61 in the preparation of a medicament for preventing and / or treating sleep apnea combined with heart failure.

2. Use of GANT61 in the preparation of drugs for preventing and / or treating obstructive sleep apnea.

3. Application of GANT61 in the preparation of drugs for preventing and inhibiting myocardial fibrosis.

4. The use according to claim 1, characterized in that The application is: application of GANT61 in preparing medicine for improving decreased cardiac function.

5. The use according to claim 1, characterized in that The application is: use of GANT61 in preparing drugs for reducing ventricular remodeling and myocardial fibrosis.

6. The use according to claim 1, characterized in that The application is: application of GANT61 in preparing a drug for inhibiting the proliferation ability of cardiac fibroblasts.

7. The use according to claim 1, characterized in that The application is: use of GANT61 in preparing a drug for inhibiting the differentiation of cardiac fibroblasts into myofibroblasts.

8. The use according to claim 1, characterized in that The application is: application of GANT61 in preparing a drug for inhibiting cardiac fibroblasts from secreting extracellular matrix.

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