Application of verbascoside compound in preparation of medicine for improving cardiac function

Abstract

The invention discloses an application of a verbascoside compound in preparation of a medicine for improving a cardiac function, and belongs to the technical field of biological medicines. The verbascoside disclosed by the invention has certain effects of regulating the cardiac diastolic and systolic functions of model mice, improving cardiac hemodynamics and protecting cardiac functions, has relatively good activity of reducing myocardial fibrosis, and can prevent cardiovascular complications induced by diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a verbascoside compound in preparing a medicine for improving cardiac function. Background Art

[0002] Verbascoside is a phenylethanoid glycoside component, which is widely distributed in a variety of medicinal plants such as Orobanchaceae, Scrophulariaceae, Magnoliaceae, and Lamiaceae. Currently, the main source of Verbascoside is Cistanche deserticola, a plant of the Orobanchaceae family. Verbascoside has positive effects in neuroprotection, anti-tumor, anti-depression, anti-inflammatory, and regulation of blood sugar and blood lipids, and is widely used in the prevention and treatment of diabetes and kidney disease, especially db / db diabetic nephropathy.

[0003] Hyperglycemia and hyperlipidemia are the most common high-risk factors for cardiovascular diseases. Continuous hyperglycemia and hyperlipidemia will increase the heart load, causing heart dysfunction and increasing the risk of cardiovascular diseases such as myocardial infarction and heart failure. They can also damage the elasticity of renal blood vessels, increase the blood pressure of glomerular capillaries, impair glomerular filtration function, and lead to kidney damage. Therefore, dual protection of the heart and kidneys has become a key link in the comprehensive prevention and treatment system of DKD. Summary of the invention

[0004] The present invention provides an application of a verbascoside compound in the preparation of a drug for improving cardiac function. The verbascoside compound can reduce the area of ​​myocardial fibrosis, inhibit myocardial fibrosis, thereby inhibiting ventricular remodeling and improving cardiac function; and can effectively treat and prevent db / db diabetes and improve cardiac and renal function.

[0005] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0006] Application of a verbascoside compound in preparing a medicine for improving cardiac function.

[0007] Furthermore, the verbascoside compound includes verbascoside or a pharmaceutically acceptable salt thereof.

[0008] Furthermore, the amount of verbascoside added to the drug is 25-200 mg, preferably 150 mg.

[0009] Furthermore, the drug contains a pharmaceutically acceptable excipient.

[0010] Furthermore, the dosage form of the drug is selected from tablets, capsules, intravenous injections, intraperitoneal injections, inhalants, nebulizers, lyophilized agents, patches, gels, sprays or suppositories.

[0011] Furthermore, the verbascoside includes Cistanche deserticola verbascoside.

[0012] The use of the verbascoside compound of the present invention in the preparation of a drug for improving cardiac function has the following beneficial effects:

[0013] (1) Verbascum glycoside has certain effects on regulating cardiac systolic and diastolic function of model mice, improving cardiac hemodynamics, and protecting cardiac function. It has good activity in reducing myocardial fibrosis and can prevent cardiovascular complications induced by diabetes.

[0014] (2) Verbascoside can effectively regulate the levels of urinary creatinine and urinary albumin, downregulate ACR, thereby reducing proteinuria, and significantly reduce renal functional indicators such as BUN and UA, showing a good renal protective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 This is a comparison chart of urine volume and urine protein in each group of mice of the present invention;

[0017] Figure 2 Schematic diagram of renal function indexes of mice in each group of the present invention;

[0018] Figure 3 is the ultrasound image (long-axis view) of the kidney of each group of mice of the present invention;

[0019] Figure 4 is the renal artery blood flow spectrum of each group of mice in the present invention;

[0020] Figure 5 is the renal pulsatility index (PI) and renal resistance index (RI) of each group of mice in the present invention, Note: Compared with the WT group, # P<0.05; compared with the db / db group, * P<0.05, ** P < 0.01;

[0021] Figure 6 It is the cardiac ultrasound image (M-mode) of each group of mice in the present invention, Note: A is the normal control group (WT), B is the model group (db / db), C is the dapagliflozin group (db / db+Da), and D is the verbascoside intervention group (db / db+ACT);

[0022] Figure 7It is the cardiac function index of each group of mice in the present invention, Note: A is heart rate, B is cardiac output, C is ejection fraction, D is shortening fraction, E is stroke volume, F is left ventricular weight, G is left ventricular end-diastolic diameter, H is left ventricular end-systolic diameter, I is left ventricular end-diastolic volume, J is left ventricular end-systolic volume, K is left ventricular end-systolic anterior wall thickness, L is left ventricular end-diastolic anterior wall thickness, M is left ventricular end-systolic posterior wall thickness, N is left ventricular end-diastolic posterior wall thickness. Compared with the WT group, # P<0.05, ## P<0.01; compared with the db / db group, * P<0.05, ** P<0.01.

[0023] Figure 8 is the HW / BW level of mice in each group of the present invention, Note: Compared with the WT group, ns means no difference; Compared with the db / db group, * P < 0.05;

[0024] Fig. 9 HE staining and Masson staining of the mouse heart of the present invention, Note: Scale bar = 100 μm or 50 μm;

[0025] Fig.10 This is the TUNEL staining result of mouse heart tissue of the present invention (200X). Wherein TUNEL: apoptotic cell green fluorescence, DAPI: cell nucleus blue light, Merge: composite image; Note: yellow arrows are apoptotic cells, scale = 75 μm;

[0026] Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 WT was the normal control group, db / db was the model group, db / db+Da was the dapagliflozin group, and db / db+ACT was the verbascoside intervention group. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0029] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable salt" refers to salts prepared from compounds of the present invention and relatively nontoxic, pharmaceutically acceptable acids or bases.

[0030] The term "treat" as used herein refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects or side effects associated with the condition or one or more symptoms, effects or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0031] Example

[0032] 1.1 Experimental animals

[0033] 6-week-old male BKS-Lepr em2Cd479 / Nju (db / db, n = 30) mice and littermate male C57BL / Ksj (wide-type, n = 10) mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (SCXK (Su) 2018-0008) and housed in the Animal Experiment Center of Xinjiang Medical University (SYXK (Xin) 2018-0003).

[0034] 1.2 Grouping and Dosing

[0035] All mice were housed in an SPF environment with adequate feed and water, and normal circadian rhythm. They were weighed after 1 week of adaptive feeding. Thirty db / db male mice were randomly divided into a model group, a dapagliflozin group, and a verbascoside intervention group, with 10 mice in each group. Another 10 homologous male wide-type mice were used as a normal control group (see Figure 1). During the experiment, blood was collected from the tail tip of the mice every week, and fasting blood glucose was measured using a blood glucose meter. The diabetes model was successfully established when the fasting blood glucose was ≥16.7mmol / L, and the increase in kidney weight / body weight and 24h urine microalbumin was considered to be the standard for the DKD animal model.

[0036] The normal control group and the model group were gavaged with an equal volume of saline once a day; the dapagliflozin group was gavaged with 1.3 mg / kg dapagliflozin once a day (the intervention dose of dapagliflozin tablets was calculated based on the 10 mg / d for adults in the drug instructions, the standard weight of adults was 70 kg, and the conversion factor between mice and humans was 9.1. Therefore, the equivalent clinical dose for mice was 10 mg / d÷70 kg×9.1=1.3 mg / kg / d); the verbascoside intervention group was gavaged with 150 mg / kg once a day (the intervention dose was determined by the results of the preliminary experiment). The drug administration lasted for 12 weeks, and the animals were killed after 12 weeks of intervention for index detection and histopathological examination. All operations related to animal experiments were in compliance with animal experiment ethics and relevant regulations.

[0037] 1.3 Data processing

[0038] Graphpad Prism7.0 and SPSS22.0 software were used for statistical analysis. All measured data were expressed as mean ± standard deviation (x ± s). The differences between the two groups were compared with the t-test, and the differences among multiple groups were compared with one-way ANOVA analysis. A two-sided test was used, and P < 0.05 was considered statistically significant.

[0039] 1.4 Test and Inspection

[0040] 1.4.1 Mouse proteinuria and ACR detection

[0041] The collected urine of mice in the model group, dapagliflozin group, verbascoside intervention group, and normal control group was taken out from the -80℃ refrigerator, and after returning to room temperature, the microalbuminuria (MAU) content of mice was determined by ELISA, and the urinary albumin concentration was calculated according to the instructions of the kit. The urinary creatinine (UrineCreatinine, Ucr) level of mice in each group was determined by the kit, and the ratio of urinary microalbumin to urinary creatinine (Albumin / UrineCreatinine Ratio, ACR) (mg / g) = MAU (mg / L) / Ucr (μmol / L) was calculated according to formula I;

[0042] The formula I is: ACR (mg / g) = MAU (mg / L) / Ucr (μmol / L).

[0043] like Figure 1 As shown in the results, with the continuous change of the degree of DKD pathology, the urine volume of mice in each group increased over time. Compared with the normal control group, the urine volume of mice in the model group increased significantly (P<0.01), the Ucr and MAU contents of mice increased significantly, and the ACR level increased significantly. Compared with the model group, the verbascoside intervention group and the dapagliflozin group could significantly reduce the urine volume of mice, reduce the Ucr level and MAU content, and thus downregulate the ACR level (P<0.05).

[0044] 1.4.2. Detection of mouse renal function indexes

[0045] The renal function indices such as serum creatinine (Scr), blood urea nitrogen (BUN), and blood uric acid (UA) of mice in the model group, dapagliflozin group, verbascoside intervention group, and normal control group were measured using an ELISA instrument according to the instructions of the kit, and the creatinine clearance (Ccr) (mL / kg / min) was calculated according to Formula II.

[0046] Formula II: Ccr(mL / kg / min)=Ucr(μmol / L)×urinary volume(mL / kg / min) / Scr(μmol / L).

[0047] like Figure 2 As shown in the figure, compared with the normal control group mice, the model group mice lost renal function due to renal damage, resulting in increased BUN and UA levels, significantly increased Scr and Ccr, and increased renal load. Compared with the model group, the levels of BUN, UA, Scr and other indicators of the mice in the dapagliflozin group were significantly reduced, and the effect of improving renal function indicators was outstanding. Verbascoside can effectively reduce the levels of BUN and UA in model mice, downregulate Scr and Ccr, and show good potential for antagonizing DKD.

[0048] 1.4.3 Renal Ultrasound Examination

[0049] (1) Five mice were randomly selected from each of the model group, dapagliflozin group, verbascoside intervention group, and normal control group for renal ultrasound examination;

[0050] (2) Place the mouse in an inhalation anesthesia induction box, anesthetize the mouse with isoflurane, and fix it on a constant temperature platform (isoflurane is maintained at a low dose for continuous inhalation);

[0051] (3) Use an electric razor and depilatory cream to remove the hair on the right lower abdomen of the mouse to obtain a clear image;

[0052] (4) Apply a sufficient amount of preheated ultrasound coupling agent and place the mouse in a supine position, that is, in the transverse imaging plane, the head should be located behind the image, the tail should be facing the observer, and the left side of the mouse should be on the right side of the screen;

[0053] (5) The probe is moved from the lower end of the chest cavity to the lower abdomen to locate the measurement area. The right kidney is located at the tail of the liver and adjacent to it.

[0054] (6) The image can be optimized by adjusting the field of view to fit the kidney, focus, and gain. Locate the widest middle portion of the kidney and center the organ on the screen. When imaging in the supine position, the animal holder can be tilted to bring the kidney closer to the probe and eliminate shadows caused by the intestines.

[0055] (7) Measure the kidney length in the long axis section under B-mode. Identify the intrarenal blood vessels under Doppler-mode, locate the interlobar arteries or arcuate arteries, set the pulsed Doppler mode, obtain 3-5 consecutive similar waveforms, measure the relevant parameters of intrarenal blood flow velocity: peak systolic velocity (PSV), end-diastolic trough velocity (EDV), mean velocity (MV), and calculate the renal resistance parameters: renal pulsatility index (PI) and renal resistivity index (RI). [83,84] The calculation formula is as follows:

[0056] PI = (PSV-EDV) / MV;

[0057] RI = (PSV-EDV) / PSV;

[0058] (8) Wipe off the medical ultrasonic coupling agent, remove the animal from the constant temperature operating table, put it back into the mouse box, and monitor the animal until it is fully awake.

[0059] from Figure 3 , Figure 4 , Figure 5 It can be seen that the overall morphology of the kidneys of mice in the normal control group was normal. Compared with the normal control group, the kidney volume of db / db mice in the model group increased, and the arrow indicated the presence of hydronephrosis. Compared with the model group, the kidney volume of the verbascoside intervention group decreased, and no hydronephrosis occurred. RI is related to renal cortical perfusion. When RI increases, renal cortical perfusion decreases, intrarenal pressure gradually increases, the kidney is prone to ischemia, and the risk of acute kidney injury (AKI) increases. Compared with the model group, the RI and PI of the verbascoside intervention group were significantly reduced (P<0.05).

[0060] 1.4.4 Cardiac Ultrasound Testing

[0061] (1) Five experimental mice were randomly selected from each of the model group, dapagliflozin group, verbascoside intervention group, and normal control group and underwent echocardiography one week before the end of the experiment;

[0062] (2) Gently place the mouse in an inhalation anesthesia induction box. After anesthetizing the mouse with isoflurane, fix it on a constant temperature platform (continue to maintain the inhalation dose and pay attention to monitoring the mouse's heart rate);

[0063] (3) Use animal-specific depilatory cream to remove the hair on the left chest wall of the animal to ensure a clear image. The specific area is from the level of the supraclavicular fossa to the level of the xiphoid process, and to the mid-axillary line on both sides;

[0064] (4) After placing the mouse in the supine position, dynamic adjustments are made based on the heart position and image quality of each mouse;

[0065] (5) Apply ultrasonic coupling agent to the chest wall and use a 40MHz high-frequency ultrasonic probe for measurement. Since the parasternal acoustic window is large, it is convenient for inspection and can detect most of the heart structures. Therefore, the probe is placed on the left chest of the mouse, ensuring that the probe is facing the mouse head. The probe is rotated to the right so that the long axis of the probe is parallel to the long axis of the mouse's left ventricle, and the left ventricular long-axis image is collected; the probe is rotated to the left, and the probe is kept vertically positioned along the long axis to obtain the left ventricular short-axis image. After obtaining the image, keep it still and adjust the trackball to position the detection point from the level of the left ventricular papillary muscle to the center of the left ventricular cavity, and measure the M-mode echocardiogram; since it is easy to obtain a clear cardiac ultrasound image of the mouse at this level, this section is used to measure the left ventricular anterior-posterior diameter, left ventricular anterior-posterior wall thickness, etc.

[0066] (6) Record images for analysis. Measure the heart rate, left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular stroke volume, ejection fraction and other cardiac function parameters of the mice.

[0067] The results are as follows Figure 6 and Figure 7 The results showed that there was no significant difference in heart rate among the groups of mice, indicating that the anesthesia effect of mice was consistent. The heart morphology and size of the mice in the normal control group were symmetrical, the cardiac output and ejection fraction were normal, the anterior and posterior walls of the ventricles were clear, and there was no obvious dilatation. Compared with the mice in the normal control group, the anterior wall of the ventricles of the mice in the model group was unclear, the ventricular diameter increased, and the LVDd and LVDs increased, and the EF and FS were significantly reduced, indicating that the heart contractile function was weakened. Compared with the mice in the model group, the EF and FS of the verbascoside intervention group were significantly increased, and the LVDd and LVDs were significantly reduced (P<0.05), which reduced the degree of heart dilatation, indicating that verbascoside can reduce ventricular remodeling after myocardial ischemia and improve cardiac function.

[0068] The left ventricular weight of the model group mice increased, and the DKD lesions induced myocardial dysfunction or myocardial hypertrophic remodeling. When the state of myocardial hypertrophy was transformed from the compensatory stage to the decompensated stage, the incidence of myocardial dysfunction increased rapidly. Myocardial hypertrophy causes insufficient myocardial contractility, which weakens the heart's contractile function. Echocardiography shows changes in cardiac function parameters, specifically a decrease in cardiac ejection fraction, a decrease in short-axis shortening rate, and an increase in cardiac residual blood volume, which causes an increase in the left ventricular systolic diameter. From another perspective, myocardial fibrosis can lead to increased ventricular wall stiffness. When the load exceeds the potential energy that the heart can withstand during diastole, the diastolic function is impaired. Echocardiography specifically shows a substantial increase in the left ventricular end-diastolic diameter. The db / db model group mice of the present invention showed an increase in left ventricular end-diastolic pressure, which in turn increased the left ventricular end-systolic pressure, resulting in a decrease in cardiac output. The significant reduction in the maximum rate of rise and fall of left ventricular pressure is a signal that the heart function is in the decompensated stage. When verbascoside was used to intervene in the model mice, the mice's cardiac systolic and diastolic function and cardiac output were greatly improved. Therefore, verbascoside has a more obvious effect on improving DKD-induced cardiac dysfunction.

[0069] 1.4.5 Effects of Verbascoside on Heart Mass / Body Mass of db / db Mice

[0070] Heart weight / body weight (HW / BW) is a key reference index for cardiac hypertrophy research. The body weights of mice in the model group, dapagliflozin group, verbascoside intervention group, and normal control group were measured daily using an animal weighing scale;

[0071] like Figure 8 As shown in the data, compared with the mice in the normal control group, the HW / BW of the mice in the model group increased, but there was no statistical difference; compared with the mice in the model group, the HW / BW of the mice in the dapagliflozin group was significantly reduced after dapagliflozin intervention (P<0.05), while the HW / BW of the mice in the verbascoside intervention group decreased, approaching the HW / BW level of the mice in the normal control group, and the cardiac load was reduced accordingly.

[0072] 1.4.6 Heart HE and Masson staining

[0073] (1) HE staining

[0074] The heart slices of mice in the model group, dapagliflozin group, verbascoside intervention group and normal control group were baked in a 60°C oven for 30-40 min, and then the following operations were performed in sequence: 5 min xylene I reaction, 10 min xylene II reaction, 5 s anhydrous ethanol, 5 s 95% ethanol, 5 s 90% ethanol, 5 s 80% ethanol, and finally rinsed with distilled water for several times to obtain pre-treated slice samples;

[0075] The pre-treated sections were stained with reagent I nuclear stain for 10 minutes, rinsed with distilled water for 2 minutes, then differentiated with 1% hydrochloric acid ethanol and then blued with PBS. Then, the sections were stained with reagent II pulp stain for 30 seconds, rinsed quickly with distilled water several times, and finally dehydrated and fixed in a standard way. Pathological images were collected after sealing with neutral gum.

[0076] In the treatment, xylene I is a mixture of xylene and anhydrous ethanol in a ratio of 1:1, and xylene II is xylene; reagent I is a hematoxylin staining solution; and reagent II is an eosin staining solution.

[0077] like Fig. 9 HE results showed that compared with the normal control group, the number of cardiomyocytes in the model group was reduced, some collagen fibers were fused with cardiomyocytes, and the degree of myocardial fibrosis was more obvious. Compared with the model group, dapagliflozin showed good activity in repairing cardiomyocytes in the dapagliflozin group, and the cardiomyocytes of the mice in the verbascoside intervention group were evenly distributed, and the nuclear condensation was reduced.

[0078] (2) Masson staining

[0079] Heart sections of mice in the model group, dapagliflozin group, verbascoside intervention group and normal control group were dewaxed to water: 20 minutes in dimethylbenzene I and dimethylbenzene II, 5 minutes in anhydrous ethanol, 3 minutes in 95% ethanol, 90% ethanol and 80% ethanol, and then rinsed with distilled water. Weigert iron hematoxylin staining for 8 minutes, gently rinsed with distilled water. 1% hydrochloric acid alcohol differentiation for 6 seconds, ultrapure water light wash for 2 minutes; then use ponceau acid fuchsin fixation for 6 minutes, and ultrapure water light rinse. Then use phosphomolybdic acid solution for 5 minutes, no need to wash with water, gently wipe off the excess phosphomolybdic acid solution. Use aniline blue staining for 4 minutes (do not wash). Rinse the sections with 1% glacial acetic acid aqueous solution in the staining box, observe and control under a microscope, and stop when there is no blue; finally, perform dehydration and fixation, gently shake off the solution on the slide, seal with neutral gum, and place in a fume hood to dry before pathological mapping.

[0080] Fig. 9 The results of Masson staining showed that compared with the normal control group, the cardiomyocytes in the model group showed fibrotic changes and the myocardial collagen content increased; compared with the model group, the verbascoside intervention group and dapagliflozin group could inhibit myocardial fibrosis by reducing the area of ​​myocardial fibrosis, thereby inhibiting ventricular remodeling and improving cardiac function.

[0081] 1.4.6 TUNEL staining of cardiomyocyte apoptosis

[0082] (1) Fixed

[0083] The heart sections of mice in the model group, dapagliflozin group, verbascoside intervention group and normal control group were dewaxed and hydrated. After heating in an oven for 30 minutes, the following steps were performed: 10 minutes of xylene I and xylene II reaction, 5 minutes of anhydrous ethanol and 95% ethanol reaction, and finally 3 minutes of 90% ethanol, 80% ethanol, and 70% ethanol light soaking. After fixation, the sections were placed in a light-proof staining box filled with PBS and rinsed several times (depending on the reaction), 5 minutes each time.

[0084] (2) Transparency

[0085] After rinsing, add 50 μL of proteinase K working solution on each slice and react in a wet box at room temperature for 20 minutes for permeabilization. Place the permeabilized tissue slices in a light-proof staining box containing PBS and wash gently 3 times at a frequency of 5 minutes.

[0086] (3) Marking

[0087] The slices cleaned in step (2) are processed into a positive control group and a negative control group;

[0088] Positive control group treatment: 100μL DNase I Buffer working solution was added to the slices of the positive control group and equilibrated in a wet box at room temperature for 5 minutes. Gently remove the liquid on the slices, then add 100μL DNase I working solution, incubate in a wet box at room temperature for 10 minutes; then take out the slices and put them in a light-proof staining box filled with PBS and wash them gently 3 times at a frequency of 5 minutes. Then add 100μL of equilibration solution and equilibrate at room temperature for 10 minutes. Use absorbent paper or filter paper to remove the equilibration solution on the slices, and then add 35μL equilibration solution + 10μL fluorescent labeling solution + 5μL TdT enzyme;

[0089] Negative control group treatment: 100 μL DNase I Buffer working solution was added to the slices and equilibrated in a wet box at room temperature for 5 minutes. Gently remove the liquid on the slices, then add 100 μL DNase I working solution, incubate in a wet box at room temperature for 10 minutes; then take out the slices and put them in a light-proof staining box filled with PBS and wash them gently 3 times at a frequency of 5 minutes. Then add 100 μL of equilibration solution and equilibrate at room temperature for 10 minutes. Use absorbent paper or filter paper to remove the equilibration solution on the slices, then add 35 μL equilibration solution + 10 μL fluorescent labeling solution, put all the slices in a wet box, seal the surrounding with sealing film, and move them into a 37°C incubator for reaction for 60 minutes.

[0090] (4) Re-staining

[0091] After labeling, the sections of the positive control group and the negative control group were placed in a light-proof staining box containing PBS and rinsed 3 times, 5 minutes each time. Excess liquid on the sections was removed with absorbent paper or filter paper, and 1 drop of DAPI was added to each section, incubated in the dark for 5 minutes, and the cell nuclei were counterstained. Then, the sections were placed in a light-proof staining box containing PBS and rinsed 4 times, 5 minutes each time. The liquid on the sections was removed, and the sections were sealed with a sealing medium containing an anti-fluorescence quencher.

[0092] (5) Microscopic examination and analysis

[0093] Observation under a fluorescence microscope showed that the maximum excitation wavelength of AF488 was 495 nm and the maximum emission wavelength was 519 nm (green fluorescence). After collecting the image, the image was processed and analyzed using Image J software. The results are shown in the figure. Fig.10 shown.

[0094] Fig.10 It shows that the activation of nuclease is accompanied by the occurrence of cell apoptosis. At the same time, DNA is cut and the breaks can be catalyzed by enzymes to bind to green fluorescent probes. The intensity and distribution of green fluorescence can be used to evaluate the apoptotic changes in cardiac tissue. TUNEL staining results show that Fig.10 As shown by the yellow arrows in the middle, a very small amount of apoptosis occurred in the hearts of mice in the normal control group and the model group, while no apoptosis occurred in the dapagliflozin group and the verbascoside intervention group. This is because DKD has a long course and involves many organs, causing mostly organic damage. The apoptosis that occurs during the course of the disease appears and disappears quickly and is transient, resulting in TUNEL staining being unable to capture apoptotic cells in time.

[0095] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0096] Finally, it should be noted that the embodiments disclosed in the present invention are only preferred embodiments of the present invention, which are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a verbascoside compound in the preparation of a drug for improving cardiac function.

2. The use according to claim 1, characterized in that: The verbascoside compound includes verbascoside or a pharmaceutically acceptable salt thereof.

3. The use according to claim 2, characterized in that: The amount of verbascoside added to the medicine is 25-200 mg.

4. The use according to claim 1, characterized in that: The medicament comprises a pharmaceutically acceptable excipient.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt is selected from lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, iron salts, and amine salts.

6. The use according to claim 1, characterized in that: The dosage form of the drug is selected from tablets, capsules, intravenous injections, intraperitoneal injections, inhalants, atomizers, lyophilized agents, patches, gels, sprays or suppositories.

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