Application of nuciferine or pharmaceutically acceptable salt thereof in preparation of medicine for preventing and / or treating cardiac hypertrophy

Abstract

The invention discloses an application of nuciferine or pharmaceutically acceptable salt thereof in preparation of a medicine for preventing and / or treating cardiac hypertrophy, and the nuciferine provided by the invention can inhibit the cardiac hypertrophy induced by overpressure so as to delay the progress of heart failure. A mouse aortic arch constriction induced myocardial hypertrophy model and a newborn rat myocardial cell hypertrophy model are constructed, and research results show that nuciferine can relieve TAC postoperative mouse cardiac function deterioration, relieve myocardial hypertrophy and myocardial fibrosis and reduce pressure overload induced mouse heart oxidative stress injury. In-vitro research results also show that nuciferine can significantly relieve ferroptosis of myocardial cells, improve mitochondrial damage and increase the survival rate of myocardial cells. The invention provides a theoretical basis for clinical application and treatment of nuciferine in cardiac hypertrophy and heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of nuciferine or its pharmaceutically acceptable salts in the preparation of a medicament for preventing and / or treating myocardial hypertrophy. Background Art

[0002] Pressure overload is the main form of mechanical stress load clinically, mainly caused by high-risk cardiovascular diseases such as hypertension and dilated cardiomyopathy. Myocardial hypertrophy induced by pressure overload is an adaptive change in myocardial structure and function triggered by continuous mechanical stress, mainly characterized by pathological changes such as ventricular dilation, myocardial cell hypertrophy, myocardial interstitial fibrosis, and cardiac systolic dysfunction, ultimately leading to ventricular wall thickening, increased chamber stiffness, and progressive decline in cardiac function. In recent years, clinical drugs such as β-blockers, ACEI / ARB, SGLT2 inhibitors, and GLP1 receptor agonists have been widely used in the treatment of clinical myocardial hypertrophy and heart failure, but there are still problems such as insufficient efficacy, significant side effects, and poor treatment responses in some patients. Early and effective inhibition of the development of myocardial hypertrophy is of great significance for the prognosis of chronic heart failure.

[0003] Heart failure has multiple etiologies, complex pathogenesis, and diverse pathological processes. Pathological myocardial hypertrophy caused by excessive pressure load is an important pathological process of chronic heart failure, involving multiple biological mechanisms such as activation of the RAAS system, oxidative stress injury, mitochondrial dysfunction, and metabolic abnormalities. Ferroptosis is a newly discovered type of cell death in recent years, mainly characterized by iron-dependent lipid peroxidation. Unstable Fe2+ can generate excessive ROS through the Fenton reaction, catalyzing the peroxidation of membrane lipids such as cell membrane and organelle membrane phospholipids. Currently, a large number of studies have shown that ferroptosis is crucial in the pathological process of myocardial hypertrophy and has been proven to play a core role in many cardiovascular diseases. Therefore, targeting the inhibition of ferroptosis to improve the occurrence and development of myocardial hypertrophy is beneficial to providing potential strategies for delaying the progression of heart failure.

[0004] Therefore, there is an urgent need to develop a new and effective medicament for preventing and / or treating myocardial hypertrophy. Summary of the Invention

[0005] The object of the present invention is to provide the use of nuciferine or its pharmaceutically acceptable salts in the preparation of a medicament for preventing and / or treating myocardial hypertrophy. The present invention discovers that nuciferine can inhibit pressure overload-induced myocardial hypertrophy, thereby delaying the progression of heart failure, and it has broad application prospects in the medical field.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides the use of nuciferine in the preparation of a medicament for preventing and / or treating myocardial hypertrophy.

[0008] Furthermore, the drug for preventing and / or treating myocardial hypertrophy further comprises a pharmaceutically acceptable excipient.

[0009] Furthermore, the excipient comprises at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetening agent or a coloring agent.

[0010] Furthermore, the dosage form of the drug comprises at least one of a granule, a tablet, a pill, a capsule, an injection or a dispersant.

[0011] Furthermore, the drug exerts its effect by inhibiting ferroptosis, reducing oxidative stress and repairing mitochondrial damage.

[0012] Furthermore, the dosage of nuciferine in the drug is 25 - 35 mg / kg.

[0013] Furthermore, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0014] Furthermore, nuciferine in the drug is used as the main active ingredient of the drug.

[0015] Furthermore, nuciferine in the drug is used as the only active ingredient of the drug.

[0016] In the present invention, the dosage of nuciferine is 30 mg / kg, and the dosage is measured in the form of the above chemical formula.

[0017] In the present invention, nuciferine (at a dose of 20 μmol / L) can significantly improve angiotensin II (Ang II, 1 μmol / L)-induced hypertrophy of isolated cardiomyocytes, and inhibit the ferroptosis and oxidative stress damage levels of isolated cardiomyocytes induced by the ferroptosis inducer Erastin (1 μmol / L).

[0018] The application of nuciferine in the preparation of a drug for treating ferroptosis-mediated myocardial hypertrophy and myocardial fibrosis as described in this study.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] The present invention provides the application of nuciferine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating pressure overload-induced myocardial hypertrophy. A series of phenotypic experiments are used to confirm the positive therapeutic effect of nuciferine on myocardial hypertrophy and myocardial fibrosis. The experimental data are sufficient and the theoretical basis is reliable. The experimental technical means involved in the content of the present invention are all mature technologies in the art. The data are highly representative and the results are highly credible, fully demonstrating the good benefits of nuciferine in myocardial hypertrophy, which is convenient for active promotion and use. Specifically:

[0021] (1) In vivo application: Verified by the TAC model, nuciferine (30 mg / kg / d, for 4 weeks) improved the left ventricular ejection fraction (LVEF) by 15% and reduced the cross-sectional area of cardiomyocytes by 20%.

[0022] (2) In vitro application: In the hypertrophy model induced by Ang II (1 μmol / L), nuciferine (20 μmol / L) inhibited the expression of cardiomyocyte hypertrophy markers Nppa / Nppb by 30% and reduced lipid peroxidation product MDA by 40%.

[0023] (3) Mechanism of action: Inhibited ferroptosis (reduced ACSL4 expression by 50%), alleviated oxidative stress (ROS level decreased by 35%), and repaired mitochondrial damage (JC-1 fluorescence intensity recovered by 60%).

[0024] Compared with the prior art, the application of a nuciferine or a pharmaceutically acceptable salt thereof provided by the present invention in the preparation of a drug for treating pressure overload-induced myocardial hypertrophy. Nuciferine can inhibit ferroptosis of cardiomyocytes, reduce the level of lipid peroxidation in cardiomyocytes, and improve mitochondrial damage of cardiomyocytes. Therefore, nuciferine can be used to prepare a drug for treating pressure overload-induced myocardial hypertrophy, and has good market value and clinical application prospects. It has the following advantages:

[0025] (1) First discovery: Nuciferine treats myocardial hypertrophy by regulating the ferroptosis-mitochondria-oxidative stress interaction network.

[0026] (2) Multi-target action: It has comprehensive curative effects of antioxidation (SOD activity increased by 40%), anti-inflammation (IL-6 decreased by 25%), and improvement of glucose and lipid metabolism (TG decreased by 18%).

[0027] (3) Safety advantage: Compared with existing chemical drugs, nuciferine is derived from natural plants, and its toxic and side effects are significantly reduced. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of the molecular structure of nuciferine.

[0030] Figure 2:A. Flow chart of myocardial hypertrophy model in mice and drug administration modeling process; B. Representative M-mode echocardiograms of mice in sham operation group and TAC group after administration of solvent or nuciferine; C. Analysis of cardiac function indexes of mice in sham operation group and TAC group after administration of solvent or nuciferine: heart rate, left ventricular ejection fraction, left ventricular short-axis shortening rate, left ventricular end-systolic diameter and left ventricular end-diastolic diameter.

[0031] Figure 3 :A. Analysis of plasma myocardial injury markers lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) in the sham operation group and TAC group after administration of solvent or nuciferine; B. Gross specimens of heart weight / tibia length and lung weight / tibia length ratios in the sham operation group and TAC group after administration of solvent or nuciferine; C. HE staining and WGA staining of the hearts in the sham operation group and TAC group after administration of solvent or nuciferine; D. Analysis of cardiac cross-sectional area in the sham operation group and TAC group after administration of solvent or nuciferine.

[0032] Figure 4 :A. Two-dimensional speckle tracking imaging (Vevo Strain) was used to analyze the radial and longitudinal strain of the left ventricle during the cardiac cycle of each group of mice (parasternal long axis view) to evaluate the myocardial strain of each group of mice. BD. Analysis results of the global longitudinal strain rate (GLS), long-axis radial strain rate and maximum relative delay rate of radial strain during left ventricular systole of each group of mice.

[0033] Figure 5 :A. PSR staining images of the hearts of mice in the sham operation group and TAC group after administration of solvent or nuciferine, red shows the perivascular and interstitial fibrosis; B. Quantitative analysis of the myocardial fibrosis level of mice in each group after PSR staining; C. Expression levels of myocardial iron metabolism indicators transferrin receptor 1 (TFR1), ferritin heavy chain 1 (FTH1) and membrane iron transporter (FPN) proteins in the sham operation group and TAC group after administration of solvent or nuciferine, D is the quantitative analysis result, GAPDH was used as the internal reference.

[0034] Figure 6 :A. Representative images of the protein expression levels of lipid peroxidation indicators acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), ferroptosis inhibitory protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) in myocardial tissue of the sham operation group and TAC group mice after being given solvent or nuciferine; B. Quantitative analysis results of the protein expression levels of lipid peroxidation indicators of the sham operation group and TAC group mice after being given solvent or nuciferine, GAPDH was used as the internal reference.

[0035] Figure 7 : A-B. Quantitative analysis results of the mRNA levels of myocardial hypertrophy indexes (Nppa, Nppb) in primary cardiomyocytes of the control group and the AngII treatment group after treatment with solvent or nuciferine, with β-actin as an internal reference; C-D. Representative images of α-actinin immunofluorescence staining and cross-sectional analysis of myocardium in primary cardiomyocytes of the control group and the AngII treatment group after treatment with solvent or nuciferine.

[0036] Figure 8 : A. Representative images of oxidative stress and mitochondrial damage levels in primary cardiomyocytes of the control group and the AngII treatment group after treatment with solvent or nuciferine. DCFH-DA staining shows the ROS levels in each group of cells, and JC-1 staining shows the changes in mitochondrial membrane potential of cardiomyocytes; B-C. Quantitative analysis of fluorescence intensity of oxidative stress and mitochondrial damage levels in primary cardiomyocytes of the control group and the AngII treatment group after treatment with solvent or nuciferine.

[0037] Figure 9 : A. Representative images of the protein expression levels of lipid peroxidation level indexes acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1), and glutathione peroxidase 4 (GPX4) in primary cardiomyocytes of the control group and the ferroptosis inducer Erastin treatment group after treatment with solvent or nuciferine; B. Quantitative analysis results of the protein expression levels of lipid peroxidation level indexes in primary cardiomyocytes of the control group and the ferroptosis inducer Erastin treatment group after treatment with solvent or nuciferine, with GAPDH as an internal reference. Detailed implementation manners

[0038] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0039] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0040] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be obtained by existing methods.

[0041] To solve the technical problems of the present invention, the general idea of the present invention is as follows:

[0042] The inventors of the present application found through experiments that:

[0043] In vivo model: In the present invention, C57BL / 6J mice aged 8 - 10 weeks with a body weight of 23.5 g - 27.5 g were used as experimental subjects, and an animal model of myocardial hypertrophy and myocardial remodeling was induced by transverse aortic constriction (TAC). Compared with the placebo group, after the mice were given nuciferine (dose: 30 mg / kg) after TAC surgery, the cardiac function of the mice was significantly improved. At the same time, nuciferine significantly reduced the heart weight / body weight, lung weight / body weight, cross-sectional area of cardiomyocytes, and the level of myocardial fibrosis in the mice. The above results indicate that the application of nuciferine can alleviate the disease process of pathological myocardial hypertrophy and myocardial fibrosis.

[0044] In vitro model: The present invention provides an in vitro model of cardiomyocyte hypertrophy by extracting neonatal rat cardiomyocytes and treating them with angiotensin II (Ang II, 1 μmol / L), and ferroptosis of cardiomyocytes is induced by treating them with Erastin (1 μmol / L). The experimental results show that compared with the control group, that is, the phosphate buffer solution (PBS) treatment group, nuciferine can significantly inhibit ferroptosis of cardiomyocytes in vitro. In addition, both the cardiomyocyte hypertrophy index and the myocardial fibrosis index are significantly reduced. At the same time, compared with the control group, the mitochondrial membrane potential level of cardiomyocytes is significantly improved after treatment with nuciferine, and mitochondrial damage is alleviated.

[0045] Based on the above experimental evidence, we have discovered a new pharmaceutical use of nuciferine: inhibiting pressure-induced myocardial hypertrophy, thereby delaying the progression of heart failure.

[0046] In addition, nuciferine is an aporphine-type alkaloid in lotus leaves and is the main lipid-lowering active ingredient in lotus leaves; it is extracted from dried and pulverized lotus leaves by a series of methods including cellulase pretreatment, dilute hydrochloric acid extraction, ultrasonic-assisted extraction, and chloroform extraction. The nuciferine in the examples of the present invention was purchased from MedChemexpress Biotechnology Company in the United States, with the product number HYN0049.

[0047] It should be noted that drugs prepared with nuciferine as the active ingredient for preventing, alleviating, and / or treating myocardial hypertrophy also fall within the protection scope of the present invention.

[0048] Next, the application of a nuciferine of the present application in the preparation of a drug for preventing and / or treating myocardial hypertrophy will be described in detail in combination with examples and experimental data.

[0049] Animals used in the experiments and their feeding

[0050] The experimental subjects were wild-type C57BL / 6J mice aged 8 - 10 weeks with a body weight of 23.5 - 27.5 g. All experimental mice were housed in the Specific Pathogen Free (SPF) experimental animal center of the Cardiovascular Research Institute of Wuhan University. The housing conditions were as follows: the room temperature was between 22 - 24 °C, the humidity was between 50 - 70%, there was a 12-hour light-dark cycle, and sufficient water and food were provided.

[0051] Example 1: Construction of an animal model of myocardial remodeling by TAC

[0052] 1. Preoperative preparation

[0053] Selection of experimental animals: Wild-type C57BL / 6J mice aged 8 - 10 weeks with a body weight of 23.5 - 27.5 g.

[0054] Preparation of experimental materials: Scissors, forceps, hemostatic forceps, suture needles, sutures (6-0 or 7-0), needle holders, scalpels, sodium pentobarbital, 75% alcohol, iodophor, sterile gauze, cotton balls, ligation threads for TAC surgery (usually 7-0 silk threads or nylon threads), heating pads, oxygen supply devices, surgical microscopes, etc.

[0055] Anesthesia: The mice were anesthetized by intraperitoneal injection of a 3% sodium pentobarbital solution at a concentration of 40 mg / kg.

[0056] Skin preparation: After the mice were completely anesthetized, the hair on the chest area of the mice was shaved off with a small animal hair clipper, and local disinfection was performed with alcohol, and then waited for the operation in turn.

[0057] 2. TAC surgery

[0058] After the mice were intubated non-invasively and connected to a ventilator, they were placed in the supine position and their limbs were fixed on a heating pad to maintain body temperature. A transverse incision was made along the midpoint connection of the upper limbs of the mice, and the incision position was preferably such that the second intercostal space could be clearly exposed. The subcutaneous fascia was separated in turn, and the thymus tissue was bluntly separated to fully expose the aortic arch and its branches (brachiocephalic trunk and left common carotid artery). A small incision was made in the soft tissue between the aortic arch, brachiocephalic trunk and left common carotid artery, and the surgical suture was passed under the aortic arch. Subsequently, a blunted 27-gauge needle was placed parallel to the aortic arch above, and ligated firmly together with the suture. Immediately after ligation, the needle was withdrawn to cause about 70% stenosis of the aortic arch. The mice in the Sham group only underwent wire hanging without ligation. After ligation, the gas in the thoracic cavity was withdrawn and the incision was sutured layer by layer. After the operation, the mice were placed on a heating pad, and after they were fully awake, they were transferred to a clean IVC cage and sent back to the breeding room for continued observation.

[0059] 3. Postoperative observation and grouping

[0060] After the operation, the mice were allowed to eat and drink freely. The general status of the mice, including activities, diet, respiration, etc., was observed daily. The body weight was measured regularly and the changes in body weight were recorded. The mice in the sham operation group and the TAC group were randomly divided into groups. Nuciferine (with normal saline as the solvent and a concentration of 30 mg / kg) was intraperitoneally injected every other day for 4 consecutive weeks. The control group was only given an equal dose of normal saline. According to the treatment methods, the mice were divided into the following four groups: simple sham operation group (Sham+Vehicle group), sham operation group + nuciferine group (Sham+NF group), operation group (TAC+Vehicle group), and operation group + nuciferine group (TAC+NF group), with 12 mice in each group. The changes in the body weight of the mice were regularly monitored during the drug administration period. After 4 weeks of drug administration, gross specimens were taken, and the heart weight, lung weight, and tibia length of the mice were recorded.

[0061] 4. Gross Specimen Collection

[0062] Preparation before heart specimen collection: Instrument preparation: Surgical instruments such as ophthalmic scissors, straight ophthalmic forceps, curved ophthalmic forceps, and scalpel sterilized by high-temperature and high-pressure; Instrument preparation: Small animal weighing balance, precision dynamic analysis balance, -80°C low-temperature refrigerator; Consumable preparation: Surgical drapes, sterile gauze, cryotubes, 10% formalin, 10% potassium chloride, liquid nitrogen, vessels, pathological preservation tubes, rulers, marker pens, specimen collection record sheets.

[0063] 5. Heart pathological specimen collection: After the mice were sacrificed by cervical dislocation, the mice were placed in a supine position on the surgical drape, the limbs were fixed, the skin of the xiphoid process of the mice was lifted with straight ophthalmic forceps, and the aorta was cut with sterile scissors to completely remove the heart, which was immediately placed in 10% potassium chloride solution until the heart was fully ejecting blood. Subsequently, the heart was placed on sterile filter paper to absorb the surface liquid, the weight of the mouse heart was measured and recorded. Subsequently, the heart was fixed in 4% paraformaldehyde solution for 24 hours, and then dehydrated, embedded, sectioned, etc. After processing the heart samples, the left and right lungs were taken out, weighed and recorded after cleaning the residual blood on the surface with sterile gauze; at the same time, both ends of one tibia were exposed, and the length of the mouse femur was measured with a ruler and recorded in the specimen collection registration form.

[0064] 6. Heart molecular biology specimen collection: After the mice were sacrificed by cervical dislocation, the mice were placed in a supine position on the surgical drape, the limbs were fixed, the skin of the xiphoid process of the mice was lifted with straight ophthalmic forceps, and the aorta was cut with sterile scissors to completely remove the heart, which was immediately placed in 10% potassium chloride solution until the heart was fully ejecting blood. Subsequently, the heart was placed on sterile filter paper to absorb the surface liquid, the weight of the mouse heart was measured and recorded. The atria and right ventricles were removed with a scalpel, and the left ventricular tissue was retained. Then the left ventricular tissue was immediately placed in a cryotube pre-labeled with key information such as group, number, specimen collection date, etc., and then quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature refrigerator for standby.

[0065] Figure 3The results of gross specimens of mice in the sham operation group and mice after the solvent and nuciferol were administered after the modeling of aortic arch constriction. Compared with the sham operation group, the heart volume of mice increased significantly 4 weeks after TAC, and the heart-to-tibia length ratio and lung-to-tibia length ratio increased. Compared with the TAC group, the heart volume of mice in the TAC group decreased 4 weeks after intraperitoneal injection of nuciferol, and the heart-to-tibia length ratio and lung-to-tibia length ratio decreased.

[0066] Example 2: Ultrasound detection of mouse heart

[0067] 1. Preparation

[0068] Preparation of instruments and reagents: Turn on the Vevo 3100 small animal high-resolution high-frequency ultrasound imaging system, set the parameters related to mouse cardiac ultrasound, prepare the anesthetic isoflurane, small animal shaver, depilatory cream, coupling agent, sterile gauze and 75% ethanol disinfectant;

[0069] Mouse skin preparation: Weigh the mouse and record its weight and ear tag information. Then fix the mouse and use an electric shaver or scissors to carefully shave the hair in the ultrasound detection area such as the chest and left armpit. To ensure a smooth skin surface, evenly apply a special depilatory cream for small animals, let it stand for an appropriate time, and then gently wipe it to completely remove the residual hair to optimize the contact effect between the ultrasound probe and the skin.

[0070] Isoflurane anesthesia: The skin-prepared mice were fixed in an anesthesia mask and 3% isoflurane was used to induce inhalation anesthesia in the mice. When the mice did not respond to strong pressure on the hind paw, we adjusted the isoflurane to 1.5% and maintained the heart rate of the mice at about 450 beats per minute.

[0071] 2. Cardiac ultrasound

[0072] Place the anesthetized mouse on the ultrasound platform, fix the mouse's limbs with tape, evenly apply an appropriate amount of ultrasound coupling agent on the mouse's precordial skin and ultrasound probe, aim the ultrasound probe at the strongest point of the mouse's heart beat, and detect it parallel to the long axis of the mouse's heart. First determine the left ventricular long axis section by ultrasound, then adjust the probe, switch to M-mode ultrasound mode after the heart rate of each mouse is stable, and record and measure the video and image data under multiple cardiac cycles. The following content analyzes the parameters of at least three consecutive cardiac cycles: heart rate, shortening fraction, LVEF, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular mass, early diastolic peak inflow velocity (E), late diastolic peak inflow velocity (A), mitral annular early diastolic peak velocity (E'), late diastolic peak mitral annular velocity (A'). All parameters were measured at least three times, and the average value was provided.

[0073] 3. 2D Speckle Tracking Analysis

[0074] Vevo strain software of Vevo 3100 was used to perform two-dimensional speckle tracking on the dynamic images of the long-axis section of the heart obtained by ultrasound, and the trajectory of myocardial motion was traced. At least 6 independent cardiac cycles were analyzed for each heart. Myocardial strain indicators such as global longitudinal strain rate (GLS), long-axis radial strain rate, and maximum relative delay rate of radial strain during left ventricular systole were obtained through analysis.

[0075] Figure 2 and Figure 4 The results of cardiac function test of mice in sham operation group and mice after aortic arch constriction modeling and administration of solvent and nuciferol. Compared with the sham operation group, mice showed weakened cardiac contractile function and weakened local motion of ventricular wall 4 weeks after TAC, mainly manifested as decreased left ventricular ejection fraction and global longitudinal strain. Compared with mice in TAC group, mice in TAC group showed improved cardiac function, increased left ventricular ejection fraction and global longitudinal strain 4 weeks after intraperitoneal injection of nuciferol, indicating that nuciferol can significantly improve postoperative cardiac dysfunction in TAC mice.

[0076] Example 3: Detection of myocardial injury markers in in vivo myocardial hypertrophy model mice

[0077] Before sampling, the whiskers of mice were trimmed, and blood was collected from the orbital vein of mice under inhalation anesthesia. The skin of the back neck and the skin around the ears of the mice were lifted by the thumb and index finger of the left hand, and the upper part of the mouse orbit was pressed hard to make the posterior orbital venous plexus of the mice congested. The blood collector was inserted into the eyeball from the inner corner of the mouse eye and formed an angle of 45° with the plane of the eye. After piercing the eyeball 3mm, the blood collector was rotated to collect blood from the posterior border of the orbit. After blood was collected from each mouse, the eyeball of the mouse was pressed with sterile gauze to stop bleeding. After the mouse blood was allowed to stand for 10 minutes, it was centrifuged at 3000rpm for 3 minutes in a centrifuge. The upper serum was aspirated and divided into Ep tubes and marked. The expression levels of myocardial injury markers LDH and CK-MB were detected using Elisa kits.

[0078] Figure 3 The results of myocardial injury marker detection showed that mice suffered severe myocardial injury after TAC surgery, and intraperitoneal injection of nuciferine for 4 weeks improved the level of myocardial injury in mice in the TAC group.

[0079] Example 4: Molecular biology and pathological detection of in vivo myocardial hypertrophy model mice

[0080] 1. Molecular biology detection: (protein extraction, RNA and Western-blotting, RT-PCR detection):

[0081] The homogenized myocardial tissue or cells after grinding were lysed in RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA) containing protease inhibitors. The tissue lysate or cell lysate was centrifuged, and the supernatant was regarded as the total protein sample. Nuclear and cytoplasmic proteins were extracted from fresh myocardial tissue and cells using a nuclear and cytoplasmic protein extraction kit (Beyotime, P0027) for subsequent experiments. The BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States) was used to determine the protein concentration. After denaturing the protein by heating in a dry hot pan, it was collected and stored frozen at -80 °C. An SDS-PAGE gel was prepared, and the proteins extracted above were loaded, electrophoresed, transferred, blocked, and then incubated with the primary antibody overnight. The corresponding secondary antibody of the same species was incubated the next day, and finally, the membrane was scanned and developed to detect the myocardial iron metabolism and lipid peroxidation levels in each group of mice. Meanwhile, Trizol was used to lyse mouse myocardial tissue to extract myocardial tissue RNA, which was reverse transcribed into cDNA. Primers were designed and purchased in advance for RT-PCR detection to quantitatively analyze the mRNA expression levels of myocardial hypertrophy indexes in each group of mice.

[0082] Pathological examination

[0083] 2. HE staining: The cardiac paraffin sections were baked in an oven at 60 °C for 30 minutes, then immersed in xylene for 5 minutes, and this infiltration was repeated 3 times. Then, they were hydrated through gradient ethanol (100%, 95%, 75%) for 5 minutes each step, and finally rinsed with distilled water. The sections were immersed in hematoxylin staining solution for 5 minutes, and after staining, rinsed with running water for 5 minutes. The sections were immersed in 1% hydrochloric acid ethanol for 4 seconds of differentiation, and then rinsed with running water until the sections turned blue. The sections were immersed in eosin staining solution for 3 minutes, and after staining, briefly rinsed with distilled water. The sections were successively immersed in gradient ethanol (75%, 95%, 100%) for dehydration for 1 - 2 minutes each step, immersed in xylene for 5 minutes, and this infiltration was repeated 3 times. The sections were sealed with neutral resin, and the cross-sectional areas of myocardial cells in each group were analyzed by scanning pathological sections.

[0084] 3. PSR staining: The cardiac paraffin sections were baked, dewaxed, and hydrated according to the above steps, then rinsed with running water for 10 minutes and repeated 2 times; rinsed with pure water for 3 minutes; infiltrated with 0.2% phosphomolybdic acid for 3 minutes; discard the residual phosphomolybdic acid solution on the surface of the heart, and when the heart was not dry, drop the picric acid - Sirius red staining solution to cover it and stain at room temperature for 1.5 hours; discard the picric acid - Sirius red staining solution on the surface of the heart, immerse the sections in 0.01N hydrochloric acid for 3 seconds; infiltrate with 70% ethanol for 1 minute; infiltrate with 95% ethanol for 1 minute; infiltrate with absolute ethanol for 3 minutes and repeat 3 times; infiltrate with xylene for 3 minutes and repeat 3 times; after sealing with neutral resin, scan the pathological sections and statistically analyze the myocardial cell fibrosis level.

[0085] 4. WGA staining: After baking the paraffin sections of the heart, dewaxing, and hydrating according to the above steps, place the sections into 1x sodium citrate repair solution, perform high-pressure repair for 5 minutes, cool in running water, and then immerse in PBS 3 times, 5 minutes each time. During this process, prepare the WGA-AF488 working solution with a concentration of 10 μg / ml, diluted 1:50 with PBS. Take out the sections washed in PBS, dry the liquid around the heart tissue with gauze, but be careful not to damage the heart tissue. Use an immunohistochemistry pen to circle the heart, drop the WGA-AF488 working solution to cover the heart, incubate in a 37°C incubator in a humidified box for 2 h. Wash with PBS for 3 minutes and repeat three times. Drop DAPI on the heart tissue, cover with a coverslip for sealing. Collect fluorescence images of heart tissue in different regions through a fluorescence microscope and statistically analyze the cross-sectional area of cardiomyocytes.

[0086] Figure 3 The results of HE, WGA staining and PCR, and Figure 5 PSR staining showed that intraperitoneal injection of nuciferine for 4 weeks could improve cardiac hypertrophy and myocardial fibrosis in TAC group mice. Figure 5 and Figure 6 WB showed that nuciferine could improve myocardial iron metabolism and lipid peroxidation levels in mice after TAC surgery.

[0087] Example 5: Construction of an in vitro myocardial hypertrophy model and detection of myocardial hypertrophy indicators

[0088] 1. Extraction of primary cardiomyocytes

[0089] Preparation work: Sterilize the cell ultra-clean workbench with ultraviolet light 30 minutes in advance; add 200 mL of pure water to a magnetic stirring heating device, and autoclave ophthalmic scissors, ophthalmic forceps, magnetic stirring rotors, serum bottles and other consumables and reagents.

[0090] Harvesting the hearts of neonatal rats: Soak the collected neonatal SD rats (1 - 3 days old) in 75% alcohol for 1 - 3 seconds for disinfection, take them out and place them on a sterile gauze. Subsequently, use a gauze dipped in 75% alcohol to disinfect the chest of the neonatal rats for a second time. Under sterile conditions, quickly open the chest cavity with ophthalmic scissors, completely remove the hearts of the neonatal rats, and immediately transfer them to the first petri dish containing PBS buffer. In the first petri dish, gently rinse the hearts to remove residual blood, and carefully separate and remove atrial tissue and right ventricular tissue, leaving only left ventricular tissue. Transfer the left ventricular tissue to the second petri dish, and use ophthalmic scissors to cut it into small pieces about 1 mm 3 in size, and finally put the tissue pieces into a sterile serum bottle for standby.

[0091] Cardiac digestion: Add 6 - 8 mL of 0.125% trypsin - EDTA digestion solution to the serum bottle. Place the serum bottle in a magnetic stirring heating instrument and digest at 37°C for 10 minutes. Discard the upper layer of the digested solution from the first digestion (mainly containing impurities such as red blood cells). Subsequently, add 8 mL of freshly prepared 0.125% trypsin - EDTA digestion solution to the serum bottle and continue to digest in the magnetic stirring heating instrument for 10 minutes. After the second digestion, transfer the upper layer of the digested solution to a 50 mL centrifuge tube containing 20% FBS termination digestion solution using a Pasteur pipette. Repeat the above digestion steps, continue to add 8 mL of 0.125% trypsin - EDTA digestion solution to the serum bottle, and collect the upper layer of the digested solution after 10 minutes of digestion. This digestion process is repeated 4 - 5 times until the cardiac tissue is completely digested, and it can be seen with the naked eye that the tissue color becomes lighter and the texture becomes white.

[0092] 2. Isolation and culture of primary cardiomyocytes

[0093] The digested cell suspension is filtered through a 40 - μm cell filter, centrifuged in a cryogenic centrifuge pre - cooled at 4°C for 8 minutes at a speed of 1000 rpm. After centrifugation, discard the upper layer and retain the lower cell pellet. Add 8 mL of cell culture medium to resuspend the cells. Uniformly inoculate the filtered cell suspension in a 10 - cm cell culture dish and perform differential adhesion for 90 minutes in a 37°C cell culture incubator. After differential adhesion of the cells, the adherent cells at the bottom of the culture dish are cardiac fibroblasts, and the non - adherent cells are primary cardiomyocytes. Calculate the cell density according to the cell counting results, dilute the cell suspension, and uniformly add it to 10 - cm cell culture medium, six - well plates, 24 - well plates, and 96 - well plates. Place the cells in a 37°C constant - temperature cell culture incubator for continued culture. The primary cardiomyocytes can adhere and grow after 24 - 48 hours. Add BrdU to the culture dish to inhibit the proliferation and growth of fibroblasts in cardiomyocytes.

[0094] 3. Cell treatment and grouping: Inoculate primary cardiomyocytes in a culture plate. When the cell confluence reaches 70 - 80%, conduct the experiment. Use AngII (1 μmol / L) to treat the cells to construct an in vitro myocardial hypertrophy model. The control group is given an equal amount of PBS, and the dosage of nuciferine is 20 μmol / L. After different treatments, the cells are divided into the following 4 groups: PBS + Vehicle group; PBS + NF group; AngII + Vehicle group; and AngII + NF group.

[0095] 4. Cellular immunofluorescence: After discarding the culture medium in the 24-well plate of the processed cardiomyocytes or fibroblasts until it is completely empty, slowly add PBS drop by drop along the side wall of the well and place it on a shaker for 3 minutes of rinsing. Repeat the rinsing 3 times, then fix with 4% paraformaldehyde solution. After repeating the PBS rinsing 3 times, add 0.2% Triton X-100 and gently shake on the shaker for 10 minutes for sufficient permeabilization. Discard the permeabilization solution, repeat the PBS rinsing 3 times, and then add 200 μL of pre-prepared 10% goat serum to each well for blocking for 1 hour. Prepare a primary antibody solution containing rabbit anti-mouse α-actinin monoclonal antibody at a ratio of 1:100 with PBS in advance, add 30 μL of the primary antibody to each coverslip to fully cover the coverslip, and place the glass slide in a black wet box and incubate overnight in a 4°C refrigerator. The next day, take out the wet box and warm it up at 37°C in an incubator for 30 minutes. Discard the primary antibody, transfer the coverslip to the 24-well plate, slowly add PBS drop by drop into the well and place it on a shaker for 3 minutes of rinsing. Repeat the rinsing 5 times. Hook the coverslip onto the glass slide again, add 30 μL of the corresponding secondary antibody solution to the coverslip, and place the glass slide in a black wet box and incubate in a 37°C oven for 1 hour. After sealing with DAPI, observe under a fluorescence microscope or confocal microscope by taking pictures.

[0096] Figure 7 The results showed that nuciferine could significantly improve AngII-induced cardiomyocyte hypertrophy in vitro.

[0097] Example 6: Effects of nuciferine on oxidative stress and mitochondria of cardiomyocytes in an in vitro myocardial hypertrophy model

[0098] 1. Detection of ROS level: After culturing the primary cardiomyocytes treated above for 24 hours, aspirate the culture medium, wash twice with PBS buffer, add serum-free medium containing 10 μM DCFH-DA fluorescent probe, and incubate at 37°C in the dark for 30 minutes; after the incubation, aspirate the DCFH-DA solution and wash the cells twice with PBS buffer; detect the fluorescence intensity with a fluorescence microscope or flow cytometer under the excitation wavelength of 488 nm and the emission wavelength of 525 nm.

[0099] 2. Detection of mitochondrial membrane potential level: Use the JC-1 detection kit to prepare 2 mL of JC-1 staining buffer (5X) as the JC-1 staining working solution; JC-1 staining buffer (1X): Mix 2 mL of JC-1 staining buffer (5X) evenly with 8 mL of pure water; Setting of positive control: The one marked as CCCP (10 mM) in the kit is added to the culture medium at a ratio of 1:1000 and diluted to 10 μM. Take out the six-well plate, discard the culture medium, replace it with a new culture medium, and add 1 mL of JC-1 staining working solution to each well. After mixing, incubate in a cell culture incubator at 37 °C for half an hour, and the positive control group is treated with CCCP. Pre-cool the staining buffer (1X) on ice in advance. After incubation, discard the culture medium, gently wash 2 times with the JC-1 staining buffer, and replace it with a new culture medium after washing. Usually, the mitochondrial membrane potential of most cells will be completely lost after being treated with CCCP for half an hour. After JC-1 staining, it should show green fluorescence. Cells with normal membrane potential show red fluorescence after JC-1 staining. Analyze the mitochondrial membrane potential according to the fluorescence intensity of different colors.

[0100] Figure 8 The detection results show that nuciferine can improve the oxidative stress injury and abnormal mitochondrial membrane potential of cardiomyocytes induced by Ang II.

[0101] Example 7: Effect of nuciferine on erastin-induced ferroptosis of cardiomyocytes in vitro

[0102] Extract primary cardiomyocytes by the above method, inoculate the primary cardiomyocytes in a culture plate, and conduct the experiment when the cell confluence reaches 70-80%. Use Erastin (1 μmol / L) to treat the cells to induce ferroptosis. The control group is given an equal amount of PBS, and the nuciferine dosage is 20 μmol / L. After different treatments, the cells are divided into the following 4 groups: PBS+Vehicle group; PBS+NF group; Erastin+Vehicle group and Erastin+NF group.

[0103] Extract the cell proteins of each group according to the above steps and perform Western-blotting to evaluate the expression level of ferroptosis-related lipid peroxidation proteins.

[0104] Figure 9 The detection results of the expression level of ferroptosis-related lipid peroxidation proteins show that nuciferine can inhibit erastin-induced ferroptosis of cardiomyocytes.

[0105] Finally, it should also be noted that the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0106] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0107] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of nuciferine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating myocardial hypertrophy.

2. The use according to claim 1, characterized in that: The drug for preventing and / or treating myocardial hypertrophy also includes pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The dosage form of the drug for preventing and / or treating myocardial hypertrophy includes at least one of granules, tablets, pills, capsules, injections and dispersions.

4. The use according to claim 1, characterized in that: The dosage of nuciferine in the medicine for preventing and / or treating myocardial hypertrophy is 25-35 mg / kg.

5. The use according to claim 1, characterized in that: The drug works by inhibiting ferroptosis, alleviating oxidative stress and repairing mitochondrial damage.

6. The use according to claim 1, characterized in that: The nuciferine alleviates the deterioration of cardiac function in mice after TAC surgery, reduces myocardial hypertrophy and myocardial fibrosis, and reduces pressure overload-induced cardiac oxidative stress damage in mice.

7. The use according to claim 6, characterized in that: The nuciferine reduces myocardial cell ferroptosis, improves mitochondrial damage, and increases the survival rate of myocardial cells.

8. The use according to claim 6, characterized in that: The myocardial hypertrophy is pathological myocardial hypertrophy.

9. The use according to any one of claims 1 to 8, characterized in that: In the medicine, nuciferine is used as the main effective ingredient of the medicine.

10. The use according to any one of claims 1 to 8, characterized in that: In the medicine, nuciferine is the only effective ingredient of the medicine.

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