Application of sedum spectabile total flavone in preparation of medicine for treating pathological cardiac hypertrophy

Abstract

The invention provides application of sedum spectabile total flavonoids in preparation of a medicine for treating pathological cardiac hypertrophy. The sedum spectabile total flavone can inhibit abnormal hypertrophy of cardiac muscle cells, improve arrangement disorder and fibrosis of the cardiac muscle cells, relieve thickening of ventricular walls, improve the diastolic function, delay or reverse pathological cardiac hypertrophy and relieve mitochondrial dysfunction in cardiac muscle tissue with cardiac hypertrophy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of total flavonoids of Sedum spectabile Boreau in the preparation of a drug for treating pathological cardiac hypertrophy. BACKGROUND

[0002] Pathological cardiac hypertrophy is an adaptive change of the heart under the stimulation of pathological factors such as long-term pressure or volume overload, and its characteristics are the increase of myocardial cell volume, the enhancement of protein synthesis and the increase of heart mass. Although it can compensate for maintaining cardiac output in the early stage, it will eventually lead to myocardial fibrosis, energy metabolism disorder and diastolic dysfunction. It is commonly seen in patients with chronic overload such as hypertension and heart valve disease, and the main manifestations are decreased exercise tolerance, dyspnea, arrhythmia, and easy to progress to heart failure. At present, the clinical treatment is mainly to control the primary disease (such as reducing blood pressure, valve intervention) and improve symptoms, and the commonly used drugs include angiotensin converting enzyme inhibitors, beta blockers, etc. However, the existing means is difficult to effectively reverse the hypertrophic structure and intracellular metabolic abnormalities, and also cannot completely prevent its transformation into heart failure.

[0003] Mitochondria, as the core organelle of myocardial cell energy metabolism, its normal oxidative phosphorylation function is the basis for maintaining the stable supply of ATP in the process of cardiac contraction and relaxation. In pathological cardiac hypertrophy, although the number of myocardial cells does not decrease significantly, the structure and function of mitochondria have been damaged, manifested as decreased mitochondrial cristae density, reduced biosynthesis and decreased oxidative phosphorylation efficiency, resulting in insufficient energy generation. This energy deficiency not only limits the diastolic performance of the myocardium, but also further activates pathological signaling pathways, accelerating the process of myocardial fibrosis and remodeling. Therefore, the treatment strategy targeting at improving mitochondrial function and restoring myocardial energy metabolism balance is becoming a research direction for delaying or even reversing pathological cardiac hypertrophy. The main intervention methods in this field at present include etiological control, application of neuroendocrine inhibitors, metabolic regulation drugs (such as trimetazidine) and lifestyle management. However, due to the complexity of its pathogenesis and large individual differences, there is still a lack of effective means to accurately improve myocardial metabolism and reverse structural abnormalities.

[0004] Total flavonoids of Sedum spectabile Boreau (TFP) are flavonoid active ingredients extracted from Sedum spectabile Boreau. Previous studies have confirmed that it has multiple pharmacological activities such as antioxidant, anti-inflammatory and metabolic regulation, and shows potential in improving glucose and lipid metabolism in metabolic diseases such as diabetes. At present, there is no research to systematically explore the role of total flavonoids of Sedum spectabile Boreau in pathological cardiac hypertrophy. Therefore, it is of great scientific significance and application prospect to further reveal the role of this component in improving myocardial energy supply and protecting mitochondrial structure and function, and to promote its clinical transformation in the prevention and treatment of pathological cardiac hypertrophy. SUMMARY

[0005] To address the problems existing in the background art, this invention provides the application of total flavonoids from Rhodiola rosea in the preparation of drugs for treating pathological myocardial hypertrophy. Total flavonoids from Rhodiola rosea can inhibit abnormal hypertrophy of cardiomyocytes, improve disordered arrangement and fibrosis of cardiomyocytes, reduce ventricular wall thickening, improve cardiac diastolic function, delay or reverse pathological myocardial remodeling, and alleviate mitochondrial dysfunction in hypertrophic myocardial tissue.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides the application of total flavonoids from Rhodiola rosea in the preparation of drugs for treating pathological myocardial hypertrophy.

[0007] According to the above scheme, the pathological myocardial hypertrophy is myocardial hypertrophy induced by chronic pressure overload.

[0008] According to the above scheme, the pathological myocardial hypertrophy is myocardial hypertrophy caused by aortic arch constriction.

[0009] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs that reduce abnormal hypertrophy of cardiomyocytes, improve disordered arrangement and fibrosis of cardiomyocytes, reduce ventricular wall thickening, improve cardiac diastolic function, and delay or reverse pathological myocardial hypertrophy.

[0010] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs for treating pathological myocardial hypertrophy mediated by mitochondrial structural and functional damage.

[0011] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs to alleviate mitochondrial damage and metabolic disorders in myocardial tissue with pathological myocardial hypertrophy.

[0012] According to the above scheme, the total flavonoids from Rhodiola rosea can alleviate mitochondrial damage by improving the mitochondrial oxidative phosphorylation function of myocardial tissue in pathological myocardial hypertrophy.

[0013] In a second aspect, the present invention provides a medicament for treating pathological myocardial hypertrophy, comprising total flavonoids from Rhodiola rosea and / or pharmaceutically acceptable salts thereof.

[0014] According to the above scheme, it also includes drug components that have a positive effect on the treatment of pathological myocardial hypertrophy when used simultaneously with total flavonoids of Rhodiola rosea, and / or pharmaceutically acceptable components that improve the stability of total flavonoids of Rhodiola rosea and / or pharmaceutically acceptable auxiliary components.

[0015] According to the above plan, the dosage forms of the drug are powder, granules, tablets, pills, capsules, oral liquid, gel, cream, spray or injection.

[0016] The beneficial effects of this invention are: 1. This invention is the first to apply total flavonoids from Rhodiola rosea to the preparation of products for the treatment of pathological myocardial hypertrophy. Total flavonoids from Rhodiola rosea can significantly improve the abnormal hypertrophy of cardiomyocytes caused by pathological myocardial hypertrophy, providing a theoretical basis for clinical treatment of pathological myocardial hypertrophy. 2. This invention provides the application of total flavonoids from Rhodiola rosea in the treatment of mitochondrial damage and metabolic disorders. Total flavonoids from Rhodiola rosea can improve mitochondrial damage in cardiomyocytes of pathological myocardial hypertrophy and increase the level of oxidative phosphorylation in cardiomyocytes, thus having clinical application value in the active treatment of myocardial mitochondrial dysfunction after myocardial hypertrophy. Attached Figure Description

[0017] Figure 1 In this paper, A and B represent the mRNA levels of hypertrophy markers in neonatal rat cardiomyocytes treated with solvent or total flavonoids from Sedum truncatum under isoproterenol (PE) stimulation. C and D represent the images of neonatal rat cardiomyocytes treated with solvent or total flavonoids from Sedum truncatum under PE stimulation and the quantitative analysis results of cell surface area, respectively. Figure 2 In the figure, A is a gross image of the heart of a mouse treated with solvent or total flavonoids of Sedum truncatum 4 weeks after sham surgery or TAC. B and C are WGA fluorescence staining image and cell cross-sectional area quantitative image, respectively. Figure 3 The images show the Masson staining results of heart sections from mice treated with solvent or total flavonoids from Sedum spectabile 4 weeks after sham surgery or TAC, where 3A is a Masson staining image (1X) and B is a quantitative image of collagen fibers stained by Masson staining. Figure 4 In Figure A, the mitochondrial morphology of the myocardial tissue of mice treated with solvent or total flavonoids of Sedum truncatum 4 weeks after sham surgery or TAC is shown by transmission electron microscopy. In Figure B, the percentage of intact mitochondrial cristae in the myocardial tissue of the present invention is quantitatively analyzed. Figure 5 The images show the echocardiographic results of mice in the sham-operated group and the TAC group after administration of the solvent or total flavonoids from Sedum truncatum in this invention. A represents the representative B-mode and M-mode echocardiograms of mice in the sham-operated group and the TAC group after administration of the solvent or total flavonoids from Sedum truncatum. B, C, D, and E represent the analysis results of cardiac ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVIDD), and left ventricular end-systolic diameter (LVIDS) for each group of mice, respectively. Figure 6In this invention, A and B represent the results of quantitative analysis of mitochondrial oxygen consumption curves and ATP production in neonatal rat cardiomyocytes treated with solvent or total flavonoids from Sedum truncatum, respectively, after 24 hours of culture in a control group (treated with PBS) and a PE-stimulated environment. The results were obtained by using the Seahorse Cell Energy Metabolism Analyzer. Detailed Implementation

[0018] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0019] The inventors discovered that total flavonoids from *Sedum spectabile* can alleviate abnormal hypertrophy of cardiomyocytes in pathological myocardial hypertrophy, thereby delaying the progression of heart failure. Through establishing a phenylephrine (PE)-stimulated neonatal rat model of cardiomyocyte hypertrophy and a mouse model of pathological myocardial hypertrophy induced by aortic arch constriction (TAC) surgery, they found that total flavonoids from *Sedum spectabile* significantly inhibited the hypertrophic response of cardiomyocytes and improved their mitochondrial oxidative phosphorylation function. Animal experiments showed that in the TAC-induced myocardial hypertrophy model, mice treated with total flavonoids from *Sedum spectabile* exhibited reduced heart weight index, less ventricular wall thickening, significantly reduced myocardial fibrosis area, and significant improvement in cardiac function. Simultaneously, total flavonoids from *Sedum spectabile* effectively reduced mitochondrial damage in hypertrophic myocardial tissue. Based on these findings, this invention provides the application of total flavonoids from *Sedum spectabile* in the preparation of drugs for treating pathological myocardial hypertrophy. Total flavonoids from *Sedum spectabile* have a good ameliorative effect on the structural and functional abnormalities of myocardium caused by pathological myocardial hypertrophy and can effectively prevent and treat accompanying mitochondrial damage.

[0020] In some specific embodiments of the present invention, the dosage of total flavonoids from Sedum spectabile in the animal model can be 5-20 mg / kg / day, preferably 10 mg / kg / day. Total flavonoids from Sedum spectabile (dose of 0.1 mg / ml) can significantly improve the dysfunction of mitochondrial oxidative phosphorylation in cardiomyocytes caused by stress overload.

[0021] In vivo model: This invention used 8-10 week old C57BL / 6J mice weighing 23.5g-27.5g as experimental subjects. A chronic pressure overload-induced myocardial hypertrophy model was induced in mice through transaortic arch stenosis (TAC). Small animal ultrasound, WGA staining, Masson staining, and transmission electron microscopy were used to evaluate changes in cardiac function, cardiomyocyte hypertrophy, fibrosis, and mitochondrial morphology in the hypertrophic mice. Results showed that after TAC surgery, mice treated with total flavonoids from *Sedum spectabile* (at a dose of 10mg / kg / d) showed a significant reduction in cardiomyocyte cross-sectional area, a significant decrease in fibrotic tissue, and a significant improvement in cardiac function. Simultaneously, total flavonoids from *Sedum spectabile* could alleviate mitochondrial damage in the myocardial tissue of mice with pathological myocardial hypertrophy.

[0022] In vitro model: This invention establishes a cardiomyocyte hypertrophy model by extracting cardiomyocytes from newborn rats and stimulating them with phenylephrine (PE). The mitochondrial oxidative phosphorylation function of cardiomyocytes after PE treatment was evaluated using biological techniques such as the Seahorse Cell Energy Metabolism Analyzer. Experimental results showed that, compared with the control group (PBS treatment group), total flavonoids from *Sedum spectabile* (dose 0.1 mg / ml) significantly improved the mitochondrial oxidative phosphorylation function of cardiomyocytes under PE stimulation.

[0023] Laboratory animals and their feeding The experimental subjects were wild-type C57BL / 6J mice aged 8-10 weeks and weighing 23.5-27.5g. All experimental mice were housed in the Specific Pathogen Free (SPF) Laboratory Animal Center of the Institute of Cardiovascular Diseases, Wuhan University. The housing conditions were as follows: room temperature between 22-24℃, humidity between 50-70%, 12-hour alternating light and dark lighting, and ample water and food were provided.

[0024] Example 1: Obtaining and Proportioning Total Flavonoids from Rhodiola Rosea 1. Acquisition: Total flavonoids (TFP) from Rhodiola rosea were purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number: 250903, product number: RH1126-5.5g, in the form of brown powder, with a purity ≥95%.

[0025] The extraction method for total flavonoids from *Sedum spectabile* is as follows: *Sedum spectabile* herb is pulverized and soaked twice in 75% ethanol (material-to-liquid ratio 1:20, soaking for at least 10 hours each time). After filtration, the ethanol extracts are combined and collected. The extracts are then adsorbed using DM30 macroporous adsorption resin, followed by gradient elution with 20%, 40%, 60%, 80%, and 90% ethanol, and the fractions are collected. Analyzing is performed using high-performance liquid chromatography (HPLC). The flavonoid-containing fractions are combined and concentrated to remove ethanol. The concentrate is loaded onto a C18 column and eluted with pure methanol. The methanol solution is concentrated to remove methanol, then concentrated to dryness and placed in a vacuum drying oven at 50℃ for 12 hours to obtain the total flavonoids from *Sedum spectabile*.

[0026] 2. Preparation: Take an appropriate amount of total flavonoids powder from *Sedum spectabile* and dissolve it in dimethyl sulfoxide (DMSO) to prepare a stock solution with a total flavonoid concentration of 25 mg / ml. Then, dilute the stock solution with physiological saline at a ratio of 1:50 to prepare a working solution with a total flavonoid concentration of 0.5 mg / ml. Administer the solution to mice in the corresponding groups at a dose of 0.5 ml / mouse (25 g) / day to achieve a dosage of 10 mg / kg / day. A similar procedure was followed when treating isolated myocardial cells in the corresponding groups to achieve a drug concentration of 0.1 mg / ml.

[0027] Example 2: Construction of a mouse model of myocardial hypertrophy (TAC) The mouse model of myocardial hypertrophy was established using aortic arch constriction (TAC). The model operation procedure is as follows: 1. Preoperative preparation (1) Anesthesia: First, weigh the mice and calculate the required amount of anesthetic (3% sodium pentobarbital) based on 90 mg / kg body weight. Administer the anesthetic via intraperitoneal injection and record the injection time. Successful anesthesia is defined as no obvious reaction to tail or toe clamping and the mouse being in good condition (generally, there is no obvious reaction about 10 minutes after injection; the optimal time for surgery is about 30 minutes after anesthesia, with the mouse showing a toe clamping reaction approximately 50 minutes after anesthesia).

[0028] (2) Surgical area preparation: Remove hair from the skin of the left chest, left lateral chest, and left forelimb axilla of the mouse. After shaving, wipe the surgical area with a damp gauze to remove the mouse hair, so as not to affect the surgical field.

[0029] (3) Endotracheal intubation: Secure the mouse's upper incisors to the inclined surface of the V-shaped plate with a rubber band, and quickly and accurately insert the endotracheal tube into the trachea through the glottis. Then, place the mouse in a right lateral decubitus position on a heating pad (the heating pad needs to be preheated). Connect the endotracheal tube to the ventilator and fix the mouse in place. If the rise and fall of the mouse's chest is consistent with the ventilator frequency, it indicates that the endotracheal intubation was successful.

[0030] 2. Transaortic arch stenosis (TAC) Mice were fixed in a supine position. After disinfection of the neck skin, an incision was made along the midline of the sternum. The thymus and surrounding tissues were bluntly dissected to fully expose the aortic arch. Using curved forceps, the aortic arch was gently separated at the junction with the brachiocephalic artery. A 7-0 suture needle with sutures was passed under the aortic arch and ligated to a 0.4 mm diameter acupuncture needle, which was then quickly withdrawn, creating a partial narrowing of the aortic arch. Successful modeling was indicated by significantly enhanced pulsation of the proximal vessel at the ligation site and a slightly paler color in the distal vessel. Postoperatively, the incision was sutured layer by layer. Penicillin was routinely administered intraperitoneally (100,000 units per mouse daily) for 3 consecutive days, and the incision skin was regularly disinfected to prevent infection.

[0031] 3. Postoperative care After TAC surgery, once the mice's spontaneous breathing stabilized and their response to pain stimuli recovered, they were transferred to pre-sterilized cages and provided with sterile bedding, feed, and drinking water. The rearing room was kept at a constant temperature and humidity, and the mice's mental state, activity level, and wound healing status were continuously observed.

[0032] 4. Group processing Mice in the sham-operated group (Sham group, with only thoracotomy to expose the heart and no other treatment) and the TAC group (a mouse model of pathological myocardial hypertrophy obtained by thoracotomy followed by aortic arch coarctation) were randomly assigned to two groups. For 10 consecutive days, two weeks post-surgery, mice received intraperitoneal injections of total flavonoids from *Sedum spectabile* (10 mg / kg / day in physiological saline containing 2% DMSO), while the control group received only the same dose of the solvent. Based on the treatment method, mice were divided into four groups: sham-operated group (Sham + solvent group), sham-operated group + total flavonoids from *Sedum spectabile* (Sham + TFP group), surgical group (TAC + solvent group), and surgical group + total flavonoids from *Sedum spectabile* (TAC + TFP group), with 15 mice in each group. Mice mortality was recorded daily. Echocardiography was performed at week 2 post-surgery to assess ventricular wall thickness and diastolic function; tissue samples were collected at week 4 post-surgery for Masson staining analysis of myocardial tissue to determine the degree of fibrosis.

[0033] Example 3: Pathological examination of a mouse model of pathological myocardial hypertrophy 1. Get materials (1) Preliminary work: Prepare a urine cup containing 20 mL of 10% formaldehyde and label it (mouse number, group, surgery type, and collection date). Place a petri dish filled with 10% KCl solution at the collection point. Turn on the analytical balance and zero it for later use. Weigh and euthanize the mice.

[0034] (2) Sample collection: The vascular pedicle below the atrial appendage was grasped with ophthalmic curved forceps, the heart was cut off, and quickly placed in a 10% KCl solution. After the heart stopped beating in diastole, it was placed on sterile gauze, the fluid in the heart chamber was gently squeezed out, the surface fluid was dried, the weight was weighed and recorded, the heart was placed in the corresponding urine cup, fixed for 48 hours, and then used for pathological examination.

[0035] (3) Related measurements and calculations: Remove the mouse heart and lungs, trim them, blot them dry with filter paper, weigh and record the weight. Cut open the skin at the tibia of the mouse's hind limb, measure and record the tibia length. Calculate the ratio of lung weight to body weight (LW / BW) and the ratio of heart weight to tibia length (HW / TL).

[0036] 2. Pathological examination 2.1 Preparation of paraffin-embedded specimen sections The main procedures include: trimming heart or lung tissue → embedding frame preparation → rinsing with running water → dehydration → clearing → wax infiltration → embedding → sectioning → spreading → air drying or baking for later use.

[0037] 2.2 WGA staining The specific steps are as follows: bake at 5℃ for 30 min → xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → rinse with running water for 10 min → double distilled water for 1 min → add EDTA or citrate antigen retrieval solution → autoclave retrieval → rinse with PBS 3 times → block → add an appropriate amount of fluorescently labeled WGA working solution → incubate at room temperature in the dark for 30-60 minutes → rinse with PBS in the dark → counterstain and mount the cell nuclei.

[0038] WGA staining as Figure 2 The results showed that treatment with total flavonoids from Rhodiola rosea after TAC surgery could significantly reduce cardiomyocyte hypertrophy.

[0039] 2.3 Masson's trichrome staining The specific steps are as follows: Baking at 55℃ for 30 minutes → Xylene for 2 minutes, 3 times → 100% alcohol for 1 minute → 95% alcohol for 1 minute → 70% alcohol for 1 minute → Rinse with running water for 10 minutes → Double distilled water for 1 minute → Weigert's iron hematoxylin stain for 5 minutes → Rinse with tap water for 5 minutes → Remove residual liquid → Differentiate with 1% hydrochloric acid alcohol for 4 seconds → Rinse with tap water for 5 minutes to return to blue → Stain with Ponceau S and acid fuchsin solution for 10 minutes → Rinse with distilled water for 5 minutes → Treat with phosphomolybdic acid aqueous solution for about 5 minutes → Counterstain with aniline blue solution for 5 minutes → Treat with 1% glacial acetic acid for 1 minute → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 seconds, 3 times → Xylene for 2 minutes, 3 times → Immediately cover with a glass slide while the xylene is still wet, and take pictures under a microscope.

[0040] Masson staining, such as Figure 3 As shown, the results showed that treatment with total flavonoids from Rhodiola rosea after TAC surgery could significantly reduce fibrosis in cardiac tissue.

[0041] Example 4: Transmission electron microscopy of myocardial tissue After euthanizing mice by cervical dislocation, the heart was removed within 3 minutes. Before sampling, a culture dish containing electron microscopy fixative was prepared in advance. After removing small pieces of myocardial tissue from the body, it was immediately placed in the culture dish and cut into small pieces in the fixative of the culture dish with a scalpel. The sampled tissue volume was controlled to be 1.0 mm × 1.0 mm × 1.0 mm cube. Care was taken to avoid mechanical damage such as squeezing with tweezers during sampling. The blade should be sharp to avoid bruising the tissue. After the myocardial tissue was removed, it was immediately placed in electron microscopy fixative and fixed at room temperature in the dark for 2 hours, and then transferred to 4°C for storage. Subsequently, an electron microscopy sample was prepared, and the morphology of mitochondria in the myocardial tissue was observed by transmission electron microscopy.

[0042] Transmission electron micrograph of mouse myocardial tissue as shown below Figure 4As shown, the results indicate that the mitochondria in the myocardial tissue of mice after TAC surgery were severely damaged, and a large number of mitochondrial cristae were destroyed. However, after intraperitoneal injection of total flavonoids from Rhodiola rosea for 2 weeks, the proportion of intact mitochondrial cristae in the myocardial tissue of the hypertrophic myocardial model mice increased from 7.6% to 12.7%, an increase of 4.1%, proving that total flavonoids from Rhodiola rosea can significantly improve mitochondrial damage in pathological hypertrophic myocardial model mice (TAC mice).

[0043] Example 5: Echocardiography to detect cardiac function in mice 1. Preliminary preparations (1) Preparation of the anesthesia machine: First connect the oxygen cylinder to the air inlet on the anesthesia machine, then unscrew the sealing cap of the drug delivery port on the anesthesia machine, quickly add isoflurane to the safe mark and then tighten the sealing cap. Unscrew the main valve on the oxygen cylinder, adjust the knob of the flow control valve, and maintain the outlet pressure at 0.2-0.3 MPa.

[0044] (2) Preparation of test mice: After the test mice are rapidly anesthetized with isoflurane, the hair in the chest area is shaved, and the head of the prepared mouse is inserted into the anesthetic catheter. The mouse is kept under stable anesthesia with 1.5-2.0% isoflurane.

[0045] 2. Echocardiography Anesthetized mice were placed on an ultrasound platform, their limbs secured with adhesive tape. A suitable amount of ultrasound coupling gel was evenly applied to the skin of the precordial region and the ultrasound probe. The ultrasound probe was aimed at the point of strongest cardiac contraction, parallel to the long axis of the mouse's heart. The long axis section of the left ventricle was first determined using ultrasound. The probe was then adjusted, and after each mouse's heart rate stabilized, the M-mode ultrasound was switched to record and measure video and imaging data over multiple cardiac cycles. Parameters from at least five consecutive cardiac cycles were analyzed: ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVIDD), and left ventricular end-systolic diameter (LVIDS). All parameters were measured at least five times, and the average value was provided.

[0046] Echocardiogram results as follows Figure 5 As shown, in a TAC-induced hypertrophic myocardial mouse model, TFP treatment improved ventricular systolic function (EF) and FS by approximately 60%; LVIDD, an indicator of ventricular dilatation, decreased from 5.5 mm to 4.8 mm, an improvement of approximately 12.7%, and LVIDS improved by approximately 28%. These results indicate that in a TAC-induced hypertrophic myocardial mouse model, TFP treatment significantly improved cardiac pumping function (EF and FS), while partially reversing cardiac enlargement.

[0047] Example 6: Detection of cardiomyocyte hypertrophy phenotype Primary cardiomyocytes were seeded in 24-well culture plates and cultured until the cell confluence reached 70-80%. After different treatments, the cells were divided into four groups: PBS + solvent group; PBS + TFP group; PE + solvent group; and PE + TFP group. Specific treatments were as follows: TFP was added to the PBS + TFP group and the PE + TFP group at a dose of 0.1 mg / ml, and then the cells were treated with PE for 24 hours to establish an in vitro cardiomyocyte hypertrophy model. The control group was cultured with PBS and solvent.

[0048] For the detection of cardiomyocyte hypertrophy markers, cardiomyocytes from each group were rapidly collected, immediately resuspended in pre-chilled PBS, and briefly centrifuged to remove residual culture medium. The supernatant was carefully discarded, and 500 μL of pre-chilled TRIzol strong lysis buffer was added to the cell pellet. The pellet was gently pipetted or vortexed to ensure complete lysis. The pellet was incubated at room temperature for 5 minutes to ensure complete dissociation of the nucleic acid-protein complex. Then, 100 μL of chloroform was added, the tube was tightly capped, and the mixture was vigorously shaken for 15-30 seconds. The pellet was incubated at room temperature for 2-3 minutes until the solution separated into layers. The pellet was centrifuged at 12000g for 15 minutes at 4°C. After centrifugation, three layers were observed: a colorless supernatant (aqueous phase containing RNA), a white intermediate layer (containing DNA and protein), and a pink organic phase. Approximately 200-250 μL of the supernatant was carefully transferred to a new RNase-free centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The mixture was gently inverted to mix. After incubating at room temperature for 10 minutes, the pellet was centrifuged at 12000g for 10 minutes at 4°C. A white gel-like RNA precipitate was observed at the bottom of the tube. Carefully discard the supernatant, add 1 mL of 75% ethanol and gently wash the precipitate. Centrifuge at 7500g for 5 minutes at 4℃, carefully aspirate the supernatant, and dry the precipitate in a cleanroom air conditioner for 3-5 minutes. Finally, dissolve the RNA precipitate in 20-50 μL LEPC water; if necessary, incubate at 55-60℃ for 5 minutes to promote dissolution. Detect RNA concentration and purity (ideal A260 / A280 value is 1.8-2.1) using a micro-spectrophotometer, and assess RNA integrity by agarose gel electrophoresis. Subsequent detection of hypertrophy markers can be performed using appropriate primers. Results of cardiomyocyte hypertrophy marker detection are shown below. Figure 1 As shown in A and 1B, PE stimulation significantly induced hypertrophic cardiomyocytes in newborn rats, while total flavonoids (TFP) from Rhodiola rosea could improve PE-induced myocardial hypertrophy.

[0049] α-actinin staining of cardiomyocytes was used to observe the degree of cardiomyocyte hypertrophy. First, cardiomyocytes grown in coverslips or glass-bottomed culture dishes were fixed with pre-chilled 4% paraformaldehyde at room temperature for approximately 15 minutes, followed by thorough washing with PBS. Next, the cells were permeabilized with PBS containing 0.1%-0.5% Triton X-100 to break the cell membrane, and washed again with PBS after 10-15 minutes at room temperature. Then, the cells were blocked with PBS containing 1%-5% BSA at room temperature for 1 hour to block non-specific binding. Subsequently, specific α-actinin primary antibody diluted with blocking buffer was added, and the cells were incubated overnight at 4°C. The next day, the primary antibody was recovered and thoroughly washed with PBS, followed by the addition of the corresponding fluorescently labeled secondary antibody, and incubation at room temperature in the dark for 1-2 hours, followed by washing with PBS in the dark. Finally, the cell nuclei were counterstained with DAPI for approximately 5 minutes, washed again, and mounted with anti-fluorescence quenching mounting medium. The cells were then observed under a fluorescence microscope or confocal microscope. Results are shown below. Figure 1 As shown in C and D, TFP can significantly improve cardiomyocyte hypertrophy stimulated by PE in vitro, with quantitative analysis showing a decrease in cardiomyocyte area of ​​approximately 50%.

[0050] Example 7: Effects of total flavonoids from Sedum spectabile on mitochondrial function in PE-stimulated primary cardiomyocytes 1. Primary neonatal SD rat cardiomyocyte culture (1) Ten Sprague-Dawley infant rats aged 1-3 days were disinfected below the neck with 75% alcohol. The heart was removed with ophthalmic scissors and microforceps and placed in a glass petri dish containing 10 ml of LDM / F12 solution. The above process was repeated for another rat.

[0051] (2) Wash the heart with DMEM / F12 medium and cut the heart into 1-2 mm pieces. 3 The fragments were transferred to the container holding the rotor. Remove DMEM / F12 from the serum bottle and add trypsin digestion solution. Digest at 120 rpm for 15 minutes, let stand for a few seconds, and discard the supernatant.

[0052] (3) Add trypsin digestion solution, rotate at 120 r / min, and digest for 15 min. Let stand for a few seconds, aspirate the supernatant, terminate digestion with DMEM / F12 medium containing 20% ​​fetal bovine serum, and store at 4°C. Repeat this step several times. When collecting the supernatant, try to collect as much as possible. When the tissue block turns white and becomes significantly smaller, stop digestion.

[0053] (4) Centrifuge the collected myocardial cell suspension at 1500 rpm for 8 min and discard the supernatant. Add an appropriate amount of culture medium to the centrifuge tube, gently pipette to resuspend the cells, and concentrate them into a 50 mL centrifuge tube. Filter the cell suspension through a 40 μm cell filter.

[0054] (5) Seed the cells in a 100 mm culture dish and allow them to adhere for 90 min. Then, filter the unadhered cell suspension. Add BrdU (final concentration 0.1 mM) to the total amount of cell suspension, mix well, and then add the mixture to a dish coated with 0.1% gelatin.

[0055] (6) Gently shake to disperse the cells, do not vortex. Incubate at 37°C and 5% CO2 for 48 hours, wash once with PBS, and change the culture medium.

[0056] (7) Cell grouping and treatment: Primary cardiomyocytes were seeded in 24-well culture plates and the experiment was conducted when the cell confluence reached 70-80%. After different treatments, the cells were divided into the following 4 groups: PBS + solvent group; PBS + TFP group; PE + solvent group; and PE + TFP group. The specific treatments were as follows: TFP was added to the PBS + TFP group and the PE + TFP group at a dose of 0.1 mg / ml. Then, the cells were treated with PE for 24 hours to construct an in vitro cardiomyocyte hypertrophy model. The control group was cultured with PBS and solvent. Mitochondrial function was then measured in each group of cells.

[0057] 2. Effects of total flavonoids from Rhodiola rosea on PE-stimulated mitochondrial function in primary cardiomyocytes Mitochondrial oxidative respiration rate (OCR) in neonatal rat cardiomyocytes was measured using a SeahorseXFe24 cell energy analyzer. Working concentrations of the required mitochondrial stress assay drugs (oligomycin A and FCCP) for primary cardiomyocytes were determined by dostotation. Primary cardiomyocytes were cultured in V-7 hippocampal plates at a density of ≥10⁵ cells per well. Before assay, primary cardiomyocytes were cultured for 1 hour at 37°C in a CO₂-free incubator (1 mM pyruvate + glucose-free Seahorse assay medium). A syringe was then loaded with 1.0 μM oligomycin, 1.0 μM FCCP, and 2.0 μM antimycin A. Mitochondrial oxidative respiration parameters, such as basal respiration, ATP production, maximum oxygen consumption, and respiratory potential, were recorded as mitochondrial oxygen consumption rate (OCR).

[0058] The Seahorse Cell Energy Metabolism Analyzer was used to detect the mitochondrial oxygen consumption curves (OCR) of cardiomyocytes in each group, as follows: Figure 6As shown. Basal respiration represents the rate at which cells consume oxygen through mitochondrial oxidative phosphorylation under basal conditions, reflecting the energy consumption level of cells to maintain basic physiological functions. ATP production: The portion of OCR that decreases after the addition of oligomycin (inhibiting ATP synthase) represents ATP production-related respiration, indicating the amount of oxygen consumed by mitochondria for ATP synthesis. The remaining OCR after the addition of rotenone and antimycin A (inhibiting the mitochondrial electron transport chain) represents oxygen consumption from non-mitochondrial sources. After PE stimulation, basal respiration, ATP synthesis, and maximal respiration of cardiomyocytes were significantly reduced, indicating mitochondrial dysfunction; while under PE conditions, basal respiration, ATP synthesis, and maximal respiration of cardiomyocytes treated with total flavonoids from Sedum truncatum were significantly improved, indicating that total flavonoids from Sedum truncatum significantly improved mitochondrial function of cardiomyocytes under PE stimulation.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of total flavonoids from Rhodiola rosea in the preparation of drugs for treating pathological myocardial hypertrophy.

2. The application according to claim 1, characterized in that, The pathological myocardial hypertrophy mentioned is myocardial hypertrophy induced by chronic pressure overload.

3. The application according to claim 2, characterized in that, The pathological myocardial hypertrophy mentioned above is myocardial hypertrophy caused by aortic arch constriction.

4. The application according to claim 1, characterized in that, The total flavonoids from Rhodiola rosea are used in the preparation of drugs that reduce abnormal hypertrophy of cardiomyocytes, improve disordered arrangement and fibrosis of cardiomyocytes, reduce ventricular wall thickening, improve cardiac diastolic function, and delay or reverse pathological myocardial hypertrophy.

5. The application according to claim 1, characterized in that, The application of the total flavonoids from Rhodiola rosea in the preparation of drugs for treating pathological myocardial hypertrophy mediated by mitochondrial structural and functional damage.

6. The application according to claim 5, characterized in that, The application of total flavonoids from Rhodiola rosea in the preparation of drugs to alleviate mitochondrial damage and metabolic disorders in pathological myocardial hypertrophy.

7. The application according to claim 6, characterized in that, The total flavonoids from Rhodiola rosea reduce mitochondrial damage by improving mitochondrial oxidative phosphorylation function in myocardial tissue with pathological myocardial hypertrophy.

8. A drug for treating pathological myocardial hypertrophy, characterized in that, Including total flavonoids from Sedum spectabile and / or its pharmaceutically acceptable salts.

9. The medicament according to claim 8, characterized in that, It also includes pharmaceutical components that, when used concurrently with total flavonoids from Rhodiola rosea, have a positive effect on the treatment of pathological hypertrophy, and / or pharmaceutically acceptable components that improve the stability of total flavonoids from Rhodiola rosea, and / or pharmaceutically acceptable adjuvant components.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug is powder, granules, tablets, pills, capsules, oral liquid, gel, cream, spray or injection.

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