Use of phenylpropanolamine phosphate in the preparation of a medicament for the treatment of myocardial fibrosis

Abstract

The present application relates to the technical field of drug research and development, and particularly relates to application of phenylpropanolamine phosphate in preparation of a drug for treating myocardial fibrosis. The present application explores the inhibitory effect of phenylpropanolamine phosphate BPP on activation of primary myocardial fibroblasts. In addition, the present application determines the effect of myocardial fibrosis in vivo by constructing a rat myocardial hypertrophy model, and determines that BPP can alleviate the fibrosis level in heart failure and relieve heart dysfunction by constructing a TAC-induced mouse heart failure model. The experimental results show that BPP can be used as a potential drug for relieving myocardial fibrosis. The present application has good application value for providing a new therapeutic drug for heart diseases accompanied by myocardial fibrosis.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to the application of phenylpropanol phosphate in the preparation of drugs for treating myocardial fibrosis. Background Technology

[0002] Myocardial fibrosis is a significant factor inducing arrhythmias and leading to heart failure. Currently, there is a lack of effective intervention strategies in clinical practice, making it a major challenge that urgently needs to be addressed. Cardiac fibroblasts play a crucial role as one of the main drivers. When activated by external stimuli such as soluble signals (Ang-II, TGFβ, etc.), they transform into myofibroblasts. These cells highly express α-SMA and secrete large amounts of extracellular matrix (ECM). Although the initial fibrotic response after myocardial injury is considered an adaptive and protective mechanism, long-term fibrosis leading to excessive ECM deposition exacerbates mechanical stiffness. This stiffness is a direct consequence of fibrosis, but it is also a powerful signal for activating cardiac fibroblasts. Therefore, inhibiting soluble signals and mechanical pathways is a key approach. Furthermore, diseases characterized by excessive extracellular matrix deposition in normal myocardial tissue structure result in a visibly enlarged heart, increased weight, dilation of all cardiac chambers, and increased ventricular wall thickness. Timely inhibition of excessive myocardial fibrosis may improve overall cardiac function. Therefore, research on the prevention and treatment of myocardial fibrosis has significant social and economic value.

[0003] Benzophenprochlorperidine phosphate (BPP) is a commonly used cough suppressant in clinical practice. Current technology has not found its application in cardiovascular diseases, let alone in myocardial fibrosis. Summary of the Invention

[0004] In view of this, the present invention proposes the application of BPP in the preparation of drugs for treating myocardial fibrosis. BPP can significantly reduce the degree of myocardial fibrosis and is a potential drug in the current field of myocardial fibrosis treatment.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of BPP in the preparation of drugs for treating myocardial fibrosis. Benzophenone phosphate inhibits cardiac fibrosis by inhibiting Ang-II-induced fibroblast activation.

[0006] Furthermore, the drug comprises phenylpropanol phosphate or a phenylpropanol phosphate composition.

[0007] In a second aspect, the present invention provides a medicament for treating myocardial fibrosis, comprising phenylpropionate phosphate.

[0008] Furthermore, the dosage form of the drug includes tablets, pills, capsules, oral liquids, or injections.

[0009] Furthermore, the drug is administered via injection, inhalation, skin, mucosa, or cavity.

[0010] The application of the BPP of the present invention in the preparation of drugs for treating myocardial fibrosis has the following advantages over the prior art: This invention systematically evaluated the therapeutic effect of BPP by constructing various mouse models of pulmonary fibrosis (such as Ang-II-induced and TAC-induced models). The results showed that BPP can significantly reduce the degree of myocardial fibrosis. Furthermore, in vitro experiments confirmed that BPP exerts its anti-fibrotic effect by inhibiting Ang-II-induced fibroblast activation. These findings provide important theoretical and experimental evidence for the repurposing of BPP for the treatment of myocardial fibrosis, and the results are reliable. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 This is a schematic diagram representing the expression levels of COL-1, COL-3, and α-SMA proteins in cells of each group detected by Western Blot. Figure 2 To perform quantitative and statistical analysis using ImageJ software; among which, Figure 2 In Figure A, Western blotting analysis was used to detect COL-1 levels in each group of cells. Figure 2 B represents the statistical analysis of COL-3 in cells from each group detected by Western blotting. Figure 2 C represents the statistical analysis of α-SMA in cells from each group detected by Western blotting; Figure 3 The mRNA expression levels of COL-1 and α-SMA were detected by RT-qPCR; among them, Figure 3 In section A, statistical analysis of COL-1 detected by RT-qPCR is presented. Figure 3 B represents the statistical analysis of α-SMA detected by RT-qPCR; Figure 4 To detect the expression level of α-SMA in cells of each group using immunofluorescence; Figure 5 To perform quantitative and statistical analysis using ImageJ software; Figure 6The collagen gel shrinkage assay was used to detect the cell contraction ability of CFs in each group. Figure 7 Representative images of the heart appearance, echocardiography, and Sirius red stained heart tissue sections of rats treated with Ang-II and BPP by gavage; Figure 8 Analysis of the calculated HW / BW and HW / TL results for heart weight, body weight, and tibia length parameters of rats in each group; among them, Figure 8 In section A, the HW / BW ratio results were calculated based on the heart weight, body weight, and tibia length parameters of rats in each group. Figure 8 In Figure B, the HW / TL results of the calculated parameters of heart weight, body weight, and tibia length of rats in each group are analyzed; Heart weight / Body weight (HW / BW) and Heart weight / Tibial length (HW / TL). Figure 9 The parameters LVEF, LVFS, LVIDs, and LVIDd were calculated and analyzed for M-mode echocardiography measurements in each group of rats; among them, Figure 9 In Figure A, the left ventricular ejection fraction (LVEF) was calculated based on M-mode echocardiography measurements of rats in each group, and the statistical analysis was performed. Figure 9 Figure B shows the statistical analysis of LVFS calculated from M-mode echocardiography measurements in each group of rats. Figure 9 In the middle C, the LVIDs were calculated and statistically analyzed based on the M-mode echocardiography measurements of rats in each group. Figure 9 The data in section D represents the M-mode echocardiography measurements of rats in each group, including LVIDd, statistical analysis, left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular internal dimension at end-systolic (LVIDs), and left ventricular internal dimension at end-diastolic (LVEDd). Figure 10 Western blot was used to detect the protein expression levels of cell fibrosis-related proteins COL-1, COL-3, α-SMA, and LOX in myocardial tissue. Figure 11 Representative images of the heart appearance, echocardiography, and Sirius red stained heart tissue sections from mice treated with TAC (aortic coarctation model) and BPP by gavage. Figure 12 Analysis was performed on the calculated HW / BW and HW / TL results of heart weight, body weight, and tibia length parameters for each group of mice; among them, Figure 12 In the middle, A represents the HW / BW statistical analysis. Figure 12 B represents the HW / TL statistical analysis; Figure 13 For each group of mice, the parameters LVEF, LVFS, LVIDs, and LVIDd were calculated and analyzed using M-mode echocardiography; among them, Figure 13 In Figure A, the LVEF parameters of each group of mice were calculated and analyzed using M-mode echocardiography. Figure 13 In Figure B, the LVFS parameters of each group of mice were calculated and analyzed using M-mode echocardiography. Figure 13 In the middle C, the parameters of LVIDs were calculated and analyzed by M-mode echocardiography of mice in each group. Figure 13 In the middle D, the LVIDd parameter was calculated and analyzed by M-mode echocardiography of mice in each group; Figure 14 Western blot was used to detect the protein expression levels of cell fibrosis-related proteins COL-1 (type I collagen), COL-3 (type III collagen), and α-SMA (α-smooth muscle actin) in myocardial tissue. Detailed Implementation

[0013] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] BPP is a commonly used antitussive in clinical practice, but its specific efficacy and mechanism in myocardial fibrosis have not been studied. In particular, whether it can inhibit fibroblast activation and reduce ECM deposition needs further verification.

[0015] In view of this, the present invention proposes the application of BPP in the preparation of drugs for treating myocardial fibrosis. BPP can significantly reduce the degree of myocardial fibrosis and alleviate TAC-induced cardiac dysfunction in mice, and is a potential drug in the current field of myocardial fibrosis treatment.

[0016] The technical solution of the present invention will be further explained below with reference to experiments.

[0017] Example 1

[0018] The BPP used in this invention was purchased from TargetMol China, and Ang-II was purchased from Shanghai MCE Company.

[0019] Experimental subjects: Cardiac fibroblasts from 2-3 day old Wistar rats were provided by Jinan Xingkang Biotechnology Co., Ltd.; C57BL / 6 mice and Wistar rats were purchased from Beijing Sibefore Biotechnology Co., Ltd. and Jinan Xingkang Biotechnology Co., Ltd., respectively.

[0020] The method for isolating and extracting primary rat neonatal myocardial fibroblasts is as follows: (1) Preparation stage: After cleaning the required experimental instruments, autoclave them for later use. Prepare an appropriate amount of collagenase II solution and CFs complete culture medium according to the requirements. Add an appropriate amount of Hank's Balanced Salt Solution (HBSS) to a 60 mm culture dish and pre-cool it on an ice plate.

[0021] (2) Heart removal: The anterior chest of 1-3 day old Wistar rats was disinfected by immersion in 75% alcohol. The thoracic cavity was cut open along the left side of the xiphoid process. The thoracic cavity was slightly squeezed to expose the entire heart. The heart was then picked up with forceps and placed in pre-cooled HBSS to wash away any residual blood.

[0022] (3) Pre-digestion: After the above operations, the heart is cut into pieces to 1 mm. 3 The tissue fragments were collected and transferred to an Erlenmeyer flask containing a rotor. Excess HBSS was aspirated, and 10 mL of collagenase II solution was added. The flask was sealed with aluminum foil and placed on a magnetic stirrer. The stirring speed was adjusted to 800 rpm, and the mixture was pre-digested in a cell culture incubator at 37°C for 5 min.

[0023] (4) Formal Digestion: Carefully aspirate the supernatant, retaining the tissue block, and add 10 mL of collagenase II solution again. Seal the container with aluminum foil and place it on a magnetic stirrer, adjusting the speed to 800 rpm, and digest in a 37℃ cell culture incubator for 1 h. After digestion, aspirate the upper cell suspension and add an equal volume of CFs complete culture medium to terminate the digestion. Centrifuge at 700 rpm for 5 min, discard the supernatant, and resuspend the cells in an appropriate amount of CFs complete culture medium. If there are any incompletely digested tissue blocks, add 10 mL of collagenase II solution to digest again until no tissue remains.

[0024] (5) Differential adhesion: After centrifugation and resuspending the collected cell suspension, filter it through a 100 μM filter to remove residual tissue debris, and then evenly spread it into a T75 culture flask. Transfer the culture flask to a 37℃ cell culture incubator and let it stand for 75 min. At this time, the fibroblasts have adhered. Discard the supernatant, rinse appropriately with PBS, add an appropriate amount of CFs complete culture medium and continue culturing for subsequent passage and use.

[0025] Western blot detection method.

[0026] (1) Gel preparation: Wash the gel preparation glass plate with distilled water, fix it vertically on the gel preparation frame, and add distilled water to check for leaks. Prepare the corresponding concentration of separating gel according to the instructions based on different molecular weight requirements, mix thoroughly, pour it between the two glass plates to 2 / 3 of the height, add 1 mL of isopropanol to remove air bubbles and flatten the liquid surface, and let it stand at room temperature for 30 min. Discard the isopropanol, add the prepared upper layer stacking gel, quickly insert a comb of appropriate size, and let it stand at room temperature for 30 min to solidify.

[0027] (2) Electrophoresis: Fix the prepared gel in the electrophoresis tank and add the prepared 1× electrophoresis buffer between the two plates. Thaw the protein samples and protein markers on ice, and slowly pull out the comb vertically. Add appropriate amounts of protein samples and protein markers according to the experimental groups. Fill the space between the glass plates and the entire electrophoresis tank with 1× electrophoresis buffer, and set the constant voltage to 80V to start electrophoresis. After about 60 minutes, when the protein markers show red bands, adjust the constant voltage to 120V until the protein samples reach the bottom of the separating gel.

[0028] (3) Transfer: Cut a PVDF membrane to a suitable size, immerse it in methanol for a few seconds to activate, and then immerse it in the prepared 1× transfer buffer. After electrophoresis, remove the gel between the two glass plates and install the transfer clamp in the following order: black plate, fiber pad, filter paper, gel, PVDF membrane, filter paper, fiber pad, white plate, taking care to avoid air bubbles. Place the transfer clamp in the transfer tank and fill it with 1× transfer buffer. Immerse the transfer tank in an ice-water mixture, connect the power supply, and adjust the constant voltage to 80V. The transfer time can be adjusted according to the molecular weight of the target protein.

[0029] (4) Sealing: After the PVDF membrane transfer is completed, immerse it in 5% skim milk powder prepared in advance and seal it on a shaker at room temperature for 1.5 hours.

[0030] (5) Primary antibody incubation: Discard the blocking solution, add PBST buffer and wash the PVDF membrane twice at room temperature on a shaker for 10 min each time. According to the protein marker indication and the molecular weight of the target protein, cut the required bands and place them in the corresponding pre-prepared primary antibodies. Incubate overnight on a shaker in a 4°C cold storage.

[0031] (6) Secondary antibody incubation: Recover the primary antibody into the original tube, add PBST buffer and wash twice on a shaker at room temperature for 10 min each time. Add the pre-prepared secondary antibody corresponding to the species of the primary antibody and incubate on a shaker at room temperature for 1 h. Recover the secondary antibody into the original tube, add PBST buffer and wash twice on a shaker at room temperature for 10 min each time.

[0032] (7) Development: Prepare ECL luminescent solution fresh according to the ECL luminescent solution instructions, with a ratio of A solution: B solution = 1:1. Place the PVDF membrane into the fully automated chemiluminescence image analysis system and add an appropriate amount of the well-mixed luminescent solution for development.

[0033] Primary fibroblasts were treated with 1 μM Ang-II and 10 μM BPP. Western blot results are shown below. Figure 1 and Figure 2 As shown, Ang-II induced significant activation of cardiac fibroblasts, manifested by significantly increased collagen deposition and expression levels of the fibroblast marker α-SMA protein. However, the addition of BPP significantly alleviated this phenomenon; that is, compared with the Ang-II-induced group, treatment with both Ang-II and BPP significantly reduced COL-1, COL-3, and α-SMA levels.

[0034] Example 2

[0035] RNA was extracted from cells.

[0036] (1) Cell lysis and phase separation: After discarding the culture medium, wash three times with PBS buffer to remove residual liquid. Then add 1 mL of RNAex reagent to each well of a six-well plate, mix thoroughly by pipetting, and transfer to RNase-free EP tubes. Add 200 μL of chloroform to each tube, vortex to mix, and let stand at room temperature for 5 min. After the system has separated into layers, centrifuge at 4 °C and 12,000 rpm for 15 min.

[0037] (2) RNA precipitation: After centrifugation, carefully aspirate the upper aqueous phase into a new RNase-free EP tube, avoiding disturbance of the middle layer. Add an equal volume of isopropanol, mix well, and incubate at -20℃ for 2 hours. Then centrifuge at 4℃ and 12000 rpm for 15 minutes, discard the supernatant, and retain the RNA precipitate.

[0038] (3) RNA washing and dissolution: Add 1 mL of pre-cooled 80% ethanol to the precipitate for washing, centrifuge at 4℃ and 12000 rpm for 5 min and discard the supernatant. Repeat twice. After drying the precipitate, add an appropriate amount of RNase-free water to dissolve it, mix well and then measure and record the RNA concentration.

[0039] RT-qPCR experiment.

[0040] Thermo Scientific's SYBR Green qRT-PCR Master Mix (2X) and ROX Solution provided mix were used. The relative expression levels of mRNA and miRNA were calculated using the 2-ΔΔCt method.

[0041] The qRT-PCR primers used are as follows: β-actin: Positive 5'-CGTTGACATCCGTAAAGACC-3' (SEQ ID NO:1) Reverse 5'-TAGAGCCACCAATCCACACA-3' (SEQ ID NO:2); COL-1: Positive 5'-CCAGCGGTGGTTATGACTTCA-3' (SEQ ID NO:3) Reverse 5'-TGCTGGCTCAGGCTCTTGA-3' (SEQ ID NO:4); a-SMA: Positive 5'-CATCCGACCTTGCTAACGGA-3' (SEQ ID NO:5) Reverse 5'-GTCCAGAGCGACATAGCACA-3' (SEQ ID NO:6); The results are as follows Figure 3 As shown, Ang-II significantly induced the activation of cardiomyocytes, as evidenced by a significant increase in the mRNA expression levels of COL-1 and α-SMA. However, compared to the Ang-II-induced group, co-treatment with Ang-II and BPP significantly alleviated the increase in the mRNA levels of both.

[0042] Example 3

[0043] Immunofluorescence staining procedure: (1) Slide preparation: Add the CFs cell suspension to a 24-well plate with a 13mm slide placed in it. After the slides adhere, group the cells according to the experimental requirements. After processing, discard the culture medium and wash three times with PBS.

[0044] (2) Fixation: Add 300 μL of 4% paraformaldehyde fixative to each well to ensure that the cell slide is completely covered. Fix for 15 min and then remove the fixative. Wash three times with PBS for 5 min each time.

[0045] (3) Permeability test: Remove residual PBS, add 0.3% Triton X-100 to each well, and incubate at room temperature for 5 min. Wash three times with PBS, 5 min each time.

[0046] (4) Blocking: Prepare a 10% donkey serum blocking solution using PBS, add 300 μL to each well, incubate at room temperature for 30 min, and then discard the blocking solution. Wash three times with PBS, 5 min each time.

[0047] (5) Primary antibody incubation: According to the antibody instructions, dilute the primary antibody with antibody diluent to an appropriate ratio, take out the slide with the cell side facing down, place it on the sealing film with 30 μL of primary antibody, and incubate overnight in a humidified box at 4°C.

[0048] (6) Secondary antibody incubation: Place the cell slides back into the 24-well plate and wash with PBS. Dilute the fluorescent secondary antibody corresponding to the species of the primary antibody with PBS at a ratio of 1:200, and incubate the fluorescent secondary antibody according to the procedure in (5) at room temperature in the dark for 40 min.

[0049] (7) Mounting: Place the cell slides back into the 24-well plate and wash three times with PBS for 5 minutes each time. Remove the slides and label them according to the experimental groups. Place the slides with the cell side down on a mounting medium containing 6 μL of DAPI-containing anti-fluorescence quenching solution, avoiding air bubbles as much as possible. Apply nail polish around the slides to fix them in place. Keep the entire process away from light. After the nail polish dries, the cell fluorescence intensity can be imaged using an upright fluorescence microscope, or the slides can be temporarily stored in a humidified chamber at 4°C in the dark until image capture.

[0050] The results are as follows Figure 4-5 As shown, Ang-II stimulation alone significantly induced an increase in the fluorescence intensity of α-SMA, indicating the presence of more activated fibroblasts. Compared to the Ang-II-inducing group alone, treatment with Ang-II and BPP together significantly reduced the fluorescence intensity of α-SMA.

[0051] Example 4

[0052] Collagen gel shrinkage experiment.

[0053] (1) Preparation of working solution: Add the required amount of collagen solution, 5×DMEM and neutralization solution to a pre-cooled sterile centrifuge tube in the order specified in the instructions, mix immediately to avoid generating bubbles, and keep the collagen gel on ice throughout the operation.

[0054] (2) Preparation of collagen lattice: Collect cells and resuspend them to 5×10⁻⁶. 6 Resuspend cells at 1 / mL. Mix 2 parts cell suspension with 8 parts cold collagen gel working solution, gently pipetting to ensure even distribution and avoid air bubbles. Add 0.5 mL of the cell-collagen mixture to each well of a 24-well plate and incubate at 37°C for 1 h.

[0055] (3) Group treatment: After collagen polymerization, 1 mL of culture medium was added to the top of each collagen lattice, and different groups were simultaneously treated with the drug. The groups were incubated in a 37°C incubator for 2 days, during which stress was generated in each group.

[0056] (4) Release the gel: Gently release the collagen gel from the side of the culture dish using a sterile spatula. At this time, the collagen gel begins to shrink. Take pictures at different times to record the size changes of each group of collagen gel.

[0057] according to Figure 6 The results showed that the enhanced cell contraction function induced by Ang-II was inhibited by BPP.

[0058] Example 5

[0059] The procedure for establishing a rat model of myocardial hypertrophy is as follows: Eight-week-old Wistar rats were used. Preoperatively, rat weight was measured, and the required dose of Ang-II was precisely calculated based on weight and administration rate. The Ang-II was diluted with physiological saline, and under sterile conditions, the diluted Ang-II (250 mg / kg / min) was infused into the pump. The control group received an equal volume of physiological saline. The Ang-II-filled osmotic pump was immersed in sterile physiological saline at 37°C for 12 hours to activate the drive system. After activation, the rats were anesthetized with isoflurane inhalation. Hair between the scapulae on the back of the rats was removed with depilatory cream and the area was wiped clean. The surgical area was then disinfected with 75% alcohol and iodine-soaked cotton balls. An incision was made between the scapulae, and a subcutaneous space the size of the osmotic pump was created. The Ang-II-containing osmotic pump was implanted on one side away from the incision. Sham-operated rats underwent the same procedure, but the implanted osmotic pump contained an equal volume of physiological saline. The incision was sutured with 4-0 sutures, disinfected with iodine-soaked cotton balls, and the rats were allowed to recover naturally. After surgery, rats were randomly divided into Sham group (sham operation group), Sham+BPP group, Ang-II group, and Ang-II+BPP group according to whether they received drug treatment. BPP was treated by gavage (25mg / kg) starting the day after surgery and continued for 6 weeks.

[0060] Echocardiography is used to assess cardiac function.

[0061] After the animal experiments, each group of animals was placed in an anesthesia induction chamber and anesthesia was induced using isoflurane gas. Ultrasound coupling agent was applied to the electrodes of a 37°C constant-temperature heating plate. The anesthetized animals were fixed to the heating plate in a supine position, ensuring their limbs were secured to the electrodes for heart rate monitoring. A mask was inserted into their mouth and nose to maintain anesthesia. Hair removal cream was applied to the chest of the animals to remove excess hair and fully expose the skin. Ultrasound coupling agent was applied to the projection of the heart onto the body surface. The probe position and orientation were adjusted to obtain a standard parasternal long-axis section, and the long-axis results were recorded using a small animal high-frequency ultrasound imaging system. The probe was then rotated 90° counterclockwise to acquire short-axis images. The obtained images were measured and the left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular internal dimension at end-systolic (LVIDs), and left ventricular internal dimension at end-diastolic (LVEDd) were calculated. The average value of all parameters was collected from at least three consecutive cardiac cycles.

[0062] Steps for Sirius red staining of myocardial tissue; (1) Place the slices in a 60℃ oven and bake for 2 hours. Then, perform dewaxing and hydration treatment in sequence: first immerse in environmentally friendly dewaxing and clarifying solution for 20 minutes, then place them in anhydrous ethanol, 95%, 90%, 80% and 75% ethanol for 5 minutes each. After that, wash with distilled water 3 times (5 minutes each time) to complete the dewaxing to water treatment.

[0063] (2) Prepare the iron hematoxylin staining solution according to the instructions, add the solution and stain for 5 minutes, then rinse with distilled water for 20 seconds to remove excess staining solution. Then, rinse with tap water for 5 minutes to reverse the staining, and rinse three times with distilled water (10 seconds each time). Next, add Sirius red staining solution and stain for 10 minutes, then rinse quickly with distilled water to remove excess staining solution. Finally, dehydrate gradually starting with 75% ethanol, and after clearing with an environmentally friendly dewaxing solution, mount the slide with neutral resin.

[0064] Figure 7-8The results showed that the Ang-II model group had significantly enlarged hearts, with significantly increased heart weight / body weight (HW / BW) and heart weight / tibial length (HW / TL) ratios, which were significantly alleviated by BPP gavage treatment. Furthermore, cardiac function indicators in each group were detected by small animal ultrasound, and the M-mode echocardiogram results were analyzed. Figure 7 , Figure 9 The results showed that the Ang-II model group had significantly increased ventricular wall thickness, with significantly reduced systolic and diastolic diameters. However, BPP treatment alleviated the progression of Ang-II-induced myocardial hypertrophy. Paraffin sections of cardiac tissue from each group were stained with Sirius red. Figure 7 The results were consistent with in vitro experiments, showing significant collagen deposition in the model group, which was inhibited by BPP. Next, myocardial tissue from each group of rats was extracted, and the expression levels of fibrotic proteins were detected by Western blot. Figure 10 The results showed that the Ang-II model group exhibited significant fibrosis, characterized by increased collagen deposition, increased LOX levels, and significantly elevated α-SMA levels. Compared to the model group, the protein levels of these indicators were significantly reduced after BPP gavage treatment, further reflecting that BPP can inhibit fibroblast activation and thus exert an anti-fibrotic effect. In summary, the above results indicate that BPP significantly improved the level of Ang-II-induced myocardial fibrosis in rats in vivo.

[0065] Example 6

[0066] The mouse aortic constriction model was constructed as follows: Eight-week-old C57BL / 6 mice were anesthetized preoperatively by intraperitoneal injection of 1.25% aphthylamine (0.2 mL / 10 g). The mice were then immobilized in a supine position with their limbs and head fixed. Hair was removed from the anterior chest using depilatory cream and the area was wiped clean to fully expose the surgical area. The surgical area was then disinfected with 75% alcohol and iodine-soaked cotton balls. An incision was made along the midline of the neck, approximately 1 cm along the midline of the sternum. The sternum was opened using a chest retractor, and surrounding muscle tissue, blood vessels, and trachea were bluntly dissected to expose the surgical field. Next, the thymus and adipose tissue obscuring the aortic arch were bluntly dissected to fully expose them. After determining the ligation position, a 7-0 microsurgical suture was carefully threaded through the aortic arch. A 27G needle was placed parallel to the blood vessel and the suture was tightened. The needle was quickly withdrawn, leaving a narrowing ring of the needle's thickness on the aortic arch to construct the TAC model. The sham-operated group mice underwent the same steps but without the tightening of the suture to narrow the aortic arch. Postoperatively, the sternum and skin were sutured with 4-0 sutures, and the area was disinfected with iodine-soaked cotton balls. The mice were allowed to recover naturally. After surgery, the mice were randomly divided into four groups (Sham group, Sham+BPP group, TAC group, TAC+BPP group, and TAC+valsartan group) according to whether they received drug treatment, with 6 mice in each group. One week after surgery, mice were given oral administration of BPP (50 mg / kg) and valsartan (16 mg / kg) for 5 weeks.

[0067] Figure 11-14 The results showed the effect of BPP on TAC-induced heart failure. Mice underwent TAC surgery and sham surgery. After a one-week recovery period, mice were treated with BPP (50 mg / kg) or valsartan (16 mg / kg) by gavage for 5 weeks. The control group was given an equal volume of physiological saline. Figure 11-12 The results showed that HW / BW and HW / TL were significantly elevated in the TAC model group, and significant relief was achieved by BPP gavage treatment, with effects similar to valsartan. Furthermore, to further determine the impact of BPP on cardiac function, small animal ultrasound examination was performed. Figure 11 , Figure 13 M-mode echocardiography results showed a significant decrease in LVEF in the TAC model group, indicating the occurrence of heart failure. Furthermore, LVIDs, LVIDd, and calculated LVFS were all significantly reduced in the model group, reflecting a significant decline in left ventricular systolic function caused by increased stress due to collagen deposition. Compared to the model group, these indicators significantly improved after BPP gavage, with effects similar to valsartan, further demonstrating that BPP can inhibit TAC-induced myocardial fibrosis and improve cardiac dysfunction. This result was also confirmed by... Figure 11 Supported by Sirius red staining of cardiac tissue. Figure 14The results showed consistent changes in the levels of COL-1, COL-3, and α-SMA proteins in myocardial tissue as detected by Western blot. In summary, these results indicate that BPP significantly alleviated myocardial fibrosis and cardiac dysfunction in TAC-induced heart failure in mice in vivo, with effects similar to valsartan.

[0068] In summary, BPP significantly inhibits myocardial fibrosis both in vivo and in vitro, and can improve TAC-induced cardiac dysfunction.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

[0070] Example 7

[0071] As a clinical application of BPP, it can be developed into a potential drug for the treatment of myocardial fibrosis.

[0072] The drug may be a single-component drug containing a therapeutically effective amount of BPP, or a pharmaceutical composition containing other components such as a pharmaceutically acceptable carrier.

[0073] BPP can be used as the sole active ingredient in this drug, or it can be used in combination with other drug ingredients used to prevent myocardial fibrosis.

[0074] As a specific embodiment of the use of two or more drug components in combination, the drug is a pharmaceutical composition containing a therapeutically effective amount of BPP and / or other drug components for preventing myocardial fibrosis.

[0075] The phrase "pharmaceutically acceptable" as used in this invention refers to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers are well known in the art and include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient, including, for example, aqueous solutions (such as water or physiologically buffered saline) or other solvents or mediators (such as glycols, glycerin, oils (such as olive oil), or injectable organic esters). Excipients may be selected, for example, to achieve delayed release of the drug or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions may be in the form of dosage units, such as tablets, capsules (including dispersible capsules and gelatin capsules), granules, powders, solutions, syrups, suppositories, injections, etc. As used herein, the term "effective amount" refers to a therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and involves an adequate amount of medicine to provide the desired effect. Therefore, the term "therapeutic effective amount" refers to a therapeutic amount sufficient to produce a specific effect when administered to a typical subject. In various contexts, effective amount as used herein also includes amounts sufficient to delay the development of a disease condition, alter the course of a disease condition (e.g., but not limited to, slowing the progression of a disease condition), or reverse a disease condition. It should be understood that many methods are known in the art for determining an effective amount for a given application. For example, pharmacological methods for dosage determination can be used in a therapeutic context. In the context of therapeutic or prophylactic application, the amount of composition administered to a subject will depend on the type and severity of the disease and individual characteristics such as general health status, age, sex, weight, and tolerance to the drug. It also depends on the extent, severity, and type of the disease. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. For example, a therapeutically effective amount of BPP can be determined by reference to its currently safe dosage administered to cancer patients for the treatment of myocardial fibrosis and through clinical investigation. Appropriate and effective dosage also needs to take into account treatment factors such as drug formulation, individual constitution, weight, age, disease progression, and administration site.

Claims

1. The application of phenylpropanol phosphate in the preparation of drugs for cardiac fibrosis, characterized in that, Benzophenazine phosphate inhibits cardiac fibrosis by suppressing Ang-II-induced fibroblast activation.

2. The use of phenylpropanolamine phosphate according to claim 1 in the preparation of drugs for cardiac fibrosis, characterized in that, The drug includes phenylpropionate phosphate or a combination of phenylpropionate phosphate.

3. The use of phenylpropanolamine phosphate according to claim 2 in the preparation of drugs for cardiac fibrosis, characterized in that, The dosage forms of the drug include tablets, pills, capsules, oral liquids, or injections.

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