Application of sophora flavescens external vesicles in preparation of medicine for relieving myocardial fibrosis after myocardial infarction

Abstract

The invention relates to the field of traditional Chinese medicine, in particular to application of sophora flavescens external vesicles in preparation of a medicine for relieving myocardial fibrosis after myocardial infarction. Experiments discover that the sophora flavescens outer vesicles can significantly improve cardiac super EF and FS values of myocardial infarction mice, improve myocardial infarction areas of myocardial infarction group mice, and inhibit mRNA and protein expression levels of alpha-SMA, collagen I and collagen III, and show that the sophora flavescens outer vesicles can significantly improve cardiac dysfunction and myocardial injury caused by myocardial infarction of mice, and can significantly improve myocardial infarction of mice. Transdifferentiation and matrix collagen deposition of myocardial fibroblasts induced by TGF-beta1 are remarkably relieved, and the compound can be used for preparing drugs for relieving myocardial fibrosis after myocardial infarction and improving cardiac dysfunction and myocardial injury caused by myocardial infarction.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically the application of Sophora flavescens vesicles in the preparation of drugs to alleviate myocardial fibrosis after myocardial infarction. Background Technology

[0002] In the millennia-long practice of Traditional Chinese Medicine, the concept of "treating disease before it occurs" has played a positive role in disease prevention and treatment. Sophora flavescens (Ku Shen) has extensive medicinal value. Research on Sophora flavescens covers multiple fields. Early studies discovered its anti-tumor, antiviral, anti-inflammatory, and antioxidant effects. Experimental studies have also found that Sophora flavescens has antioxidant stress-relieving properties, improves mitochondrial dysfunction, affects apoptosis, and inhibits cell proliferation. Therefore, its application in the treatment of liver fibrosis, tumors, and diabetes has recently become a focus of widespread attention.

[0003] Myocardial infarction is a serious cardiovascular disease that threatens human health, with a high incidence and poor prognosis. Following myocardial infarction, ventricular remodeling eventually progresses to heart failure, severely impacting patients' quality of life and safety. In this chain of events, long-term chronic inflammation, oxidative stress, and cardiomyocyte energy deficiency leading to cardiomyocyte apoptosis and fibrosis are key pathological mechanisms. Therefore, inhibiting cardiomyocyte fibrosis, maintaining ventricular compliance, and stabilizing cardiac structure are of significant clinical importance in treatment.

[0004] However, there are currently no reports on the application of Sophora flavescens external vesicles in myocardial fibrosis after myocardial infarction. Summary of the Invention

[0005] The purpose of this invention is to provide new uses for the external vesicles of Sophora flavescens and expand their application areas.

[0006] In a first aspect, the present invention provides the use of Sophora flavescens vesicles in the preparation of a medicament for reducing myocardial fibrosis after myocardial infarction.

[0007] Furthermore, in the aforementioned applications, the external vesicles of Sophora flavescens inhibit the mRNA and protein expression levels of α-SMA, collagen I, and collagen III, thereby inhibiting fibroblast activation, transdifferentiation into myofibroblasts, and matrix collagen deposition.

[0008] In a second aspect, the present invention provides the use of Sophora flavescens vesicles in the preparation of a medicament for improving cardiac dysfunction and myocardial damage caused by myocardial infarction.

[0009] Furthermore, in the aforementioned applications, the external vesicles of Sophora flavescens improve echocardiographic EF and FS values ​​and reduce the area of ​​myocardial infarction.

[0010] A third aspect of the present invention provides the use of Sophora flavescens vesicles in the preparation of a medicament for treating myocardial infarction.

[0011] Furthermore, the drug reduces myocardial fibrosis after myocardial infarction.

[0012] Furthermore, the drug improves cardiac dysfunction and myocardial damage caused by myocardial infarction.

[0013] In a fourth aspect, the present invention provides a medicament for reducing myocardial fibrosis after myocardial infarction, wherein the medicament uses Sophora flavescens exovesicles as the sole active ingredient.

[0014] Furthermore, the drug also includes pharmaceutically commonly used excipients.

[0015] Furthermore, the Sophora flavescens exovesicles can be selected from conventional commercially available products. In an embodiment of the present invention, the Sophora flavescens exovesicles were purchased from Junli Biotechnology (Shanghai) Co., Ltd.

[0016] The advantages and beneficial effects of this invention are as follows:

[0017] The experiments of this invention found that Sophora flavescens exovesicles can significantly improve the echocardiographic ejection fraction (EF) and free surface velocity (FS) values ​​in mice with myocardial infarction, and improve the myocardial infarction area in mice with myocardial infarction. Sophora flavescens exovesicles can inhibit the mRNA and protein expression levels of α-SMA, collagen I, and collagen III, indicating that Sophora flavescens exovesicles can significantly improve cardiac dysfunction and myocardial damage caused by myocardial infarction in mice, and significantly reduce TGF-β1-induced transdifferentiation of myocardial fibroblasts and matrix collagen deposition. It can be used to prepare drugs to reduce myocardial fibrosis after myocardial infarction and improve cardiac dysfunction and myocardial damage caused by myocardial infarction. Attached Figure Description

[0018] Figure 1 Characteristics of the external vesicles of Sophora flavescens.

[0019] Figure 2 Sophora flavescens vesicles improve cardiac damage in mice with myocardial infarction.

[0020] Figure 3 Sophora flavescens exovesicles reduce TGF-β1-induced transdifferentiation of cardiomyocytes and matrix collagen deposition. Detailed Implementation

[0021] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0022] Example:

[0023] 1. Laboratory animals and main reagents

[0024] Experimental animals: SPF-grade male C57BL / 6 mice were selected and purchased from Changzhou Cavens Laboratory Animal Co., Ltd. The mice were 8-10 weeks old, of moderate weight, and in good health.

[0025] Feeding conditions: Adapt to feeding for 1 week under constant temperature (22±2℃), humidity (50±10%) and normal light conditions.

[0026] Main reagents:

[0027]

[0028] 2. Experimental Grouping

[0029] 2.1 Animal Experiment Grouping

[0030] Sham group: Mice received normal open-chest surgery but did not undergo myocardial infarction surgery.

[0031] Myocardial infarction model group (MI group): Mice will undergo myocardial infarction induction surgery.

[0032] Sophora flavescens exovesicle group (SFR-Exos): Mice underwent normal thoracotomy and were given Sophora flavescens exovesicles (purchased from Junli Biotechnology (Shanghai) Co., Ltd.) (10 mg / kg) via intraperitoneal injection for 7 consecutive days.

[0033] Myocardial infarction model + Sophora flavescens vesicle treatment group (MI+SFR-Exos group): Mice underwent myocardial infarction induction surgery and were then given Sophora flavescens vesicles (10 mg / kg) via intraperitoneal injection for 7 consecutive days.

[0034] 2.2 Cell Experiment Grouping

[0035] Blank control group (Vehicle): Complete culture medium

[0036] Model group (TGF-β1): 10 ng / mL TGF-β1 + complete culture medium treatment for 48 h

[0037] Blank control group + Sophora flavescens exovesicle group (Vehicle + SFR-Exos): 100 μg / mL SFR-Exos + complete culture medium treatment

[0038] 48h.

[0039] Model group + Sophora flavescens exovesicle group (TGF-β1+ SFR-Exos): 100 μg / mL SFR-Exos + complete culture medium treatment for 48 h.

[0040] 3. Establishment of a mouse model of myocardial infarction

[0041] 3.1 Preoperative preparation

[0042] Fasting: Fasting is required for 12 hours before surgery, but water intake is permitted.

[0043] Anesthesia: Anesthesia was induced using isoflurane at an initial concentration of 5%, and then adjusted to 2% to maintain the anesthetized state after the mice were fully anesthetized.

[0044] 3.2 Surgical Procedure

[0045] Fixation and disinfection: The mouse was fixed in a supine position, and the surgical area on the left anterior chest was routinely prepared and disinfected.

[0046] Thoracotomy: Make a 1cm oblique incision on the left anterior chest where the apex of the heart beats most clearly (about 1cm away in mice). Bluntly dissect the chest wall muscles layer by layer, and quickly open the thoracic cavity from the 3rd or 4th intercostal space. Use hemostatic forceps to open the intercostal space.

[0047] Ligation of the coronary arteries: Gently compress the chest cavity to allow the heart to pop out of the opening, locate the left anterior descending coronary artery, and ligate it 1-2 mm from its root using a 7-0 suture. After ligation, observe a decrease in myocardial pulsation and a paleening of the myocardial tissue, indicating successful ligation.

[0048] Chest closure: Gently push the heart back into the chest cavity, quickly squeeze the chest cavity to expel air, close the chest cavity layer by layer, and suture the skin.

[0049] Postoperative care: After surgery, the mice were placed on a constant temperature mat to recover and were given intramuscular injections of penicillin for 3 consecutive days to kill bacteria and fight infection.

[0050] 4. Myocardial Injury Detection

[0051] 4.1 Cardiac Function Assessment: Eight weeks post-surgery, cardiac function in mice was assessed using a small animal ultrasound system. A high-resolution small animal ultrasound imaging system, a VisualSonics Vevo 2100 color Doppler ultrasound system equipped with an MS-40012 L probe, was used, typically operating at 30 MHz. Coupling gel was applied to the skin of the mouse's limbs and the cardiac region to prevent air bubbles from interfering with image quality. After obtaining clear two-dimensional images, the system was switched to M-mode ultrasound. The sampling line was adjusted to the correct position, ensuring it passed through the atrium, interventricular septum, and ventricle simultaneously to observe myocardial activity. The M-mode ultrasound mode button was pressed, and left ventricular systolic and diastolic motion curves were recorded for at least six cardiac cycles. Left ventricular ejection fraction (EF%) and left ventricular fractional shortening (FS%) were measured and calculated using M-mode echocardiography.

[0052] 4.2 HE staining: Heart tissue was taken for HE staining to observe the degree of myocardial tissue damage.

[0053] Experimental methods: ① Dewaxing paraffin sections: Sections were sequentially immersed in environmentally friendly dewaxing solution I for 20 min, environmentally friendly paraffin solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinsed with tap water. Frozen sections were then thawed and fixed: Frozen sections were removed from the -20°C freezer and brought to room temperature. They were fixed with tissue fixative for 15 min and then rinsed with running water. ② Hematoxylin staining: Sections were stained with hematoxylin for 3-5 min, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, and then stained with blue solution. ③ Eosin staining: Sections were sequentially immersed in 85% and 95% graded ethanol solutions for 5 min each, then stained with eosin for 5 min. ④ Dehydration and mounting: Sections were sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min for clearing. The sections were then mounted with neutral resin. ⑤ Microscopic examination and image acquisition and analysis.

[0054] 5. qRT-PCR detection of myocardial fibrosis-related genes

[0055] RNA was extracted from tissues, homogenized, centrifuged, and dissolved in chloroform, isopropanol, and anhydrous ethanol, respectively. Finally, 40 μL of DEPC water was added to dissolve the RNA, and the solution was stored at -80°C for later use. The reverse transcription reaction program was: 37°C for 60 min; 85°C for 5 min; 4°C for 5 min; and stored at -20°C. The prepared cDNA was then subjected to PCR amplification.

[0056] 6. Detection of myocardial fibrosis-related proteins

[0057] 6.1 Western Blot

[0058] Heart tissue was extracted using RIPA lysis buffer. Protein concentration was determined using a BCA protein assay kit. Total protein was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The cell membrane was blocked with 5% skim milk for 1 hour at room temperature, and the antibody was diluted according to the manufacturer's instructions and incubated overnight at 4°C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG / HRP secondary antibody for 1 hour at room temperature. The bands were observed using an enhanced chemiluminescence (ECL) Western blot assay kit, and protein quantification was performed using ImageJ.

[0059] 6.2 Immunofluorescence detection of α-smooth muscle actin expression

[0060] Heart tissue was taken for α-SMA staining to observe the degree of myocardial fibrosis.

[0061] Experimental method: Cell smears were placed in 6-well plates and then injected at a concentration of 1×10⁻⁶. 5Cells were seeded and cultured for 24 hours, then divided into groups. After 48 hours, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed with PBS, dried, permeabilized with 0.5% Tritium-10 for 15 minutes, blocked with 5% BSA at room temperature for 1 hour, and incubated with primary antibody overnight. The next day, the primary antibody was recovered, washed with PBS, and incubated with fluorescent secondary antibody at room temperature for 1 hour. The cells were then incubated with 200 μL of DAPI for 10 minutes, the cell slides were removed, the surrounding liquid was aspirated, the slides were removed, 10 μL of anti-fluorescence quencher was added, the cell slides were inverted on the slides, fixed, and photographed using a fluorescence microscope.

[0062] 7. Liver and kidney function tests in mice

[0063] For ALT, AST, BUN, and Crea, please refer to the kit's instructions.

[0064] 8. Data Statistics and Analysis

[0065] The Gtaphpad prism9 software package was used to process the data. When the econometric data conformed to a normal distribution and the homogeneity of variance was tested, the results were expressed as mean ± standard deviation. The numbers indicate that the t-test was used for comparisons between groups, and the one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0066] 9. Results

[0067] 9.1 Identification of Sophora flavescens exovesicles (SFR-Exos)

[0068] Figure 1 (A) Representative morphology of SFR-, with exosomes analyzed by transmission electron microscopy (TEM). (B) Size distribution of SFR-Exos detected by nanoparticle tracking analysis (NTA). (C) Representative immunofluorescence image of PKH67-labeled M2-EXO (green) internalized into cardiac fibroblasts.

[0069] 9.2 The external vesicles of Sophora flavescens significantly improved cardiac dysfunction and myocardial injury induced by myocardial infarction in mice.

[0070] Figure 2 (A) M-mode echocardiographic ejection fraction (EF%) and fractional shortening (FS%) values ​​of the heart in the myocardial infarction or sham surgery group. The results show that the external vesicles of *Sophora flavescens* significantly improved the echocardiographic EF and FS values ​​in mice with myocardial infarction (n=6). P <0.001. (B) HE staining was used to quantify the infarct area of ​​myocardial cells in heart sections of mice in the myocardial infarction and sham-operated groups. The external vesicles of *Sophora flavescens* significantly improved the myocardial infarction area in the myocardial infarction group (N=6, x40). P<0.001. Figure (C) After intervention with Sophora flavescens vesicles, there were no significant changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA).

[0071] 9.3 The external vesicles of Sophora flavescens significantly alleviated TGF-β1-induced transdifferentiation of cardiomyocytes and matrix collagen deposition.

[0072] Figure 3 (A) Cardiac fibroblasts were treated with 10 ng / mL TGF-β1 for 48 h. α-SMA staining revealed fibroblast activation and transdifferentiation into myofibroblasts. Treatment with Sophora flavescens vesicles significantly inhibited this process (scale bar 10 μm). (B) qrt-PCR analysis of α-SMA, collagen I, and collagen III mRNA expression levels showed that Sophora flavescens vesicles inhibited the mRNA expression levels of these genes (N=3). P <0.001. (C) Western blotting was used to detect the expression of α-SMA, collagen I, and collagen III proteins. The exovesicles of *Sophora flavescens* inhibited the expression levels of α-SMA, collagen I, and collagen III proteins. N=3. P <0.001.

[0073] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of Sophora flavescens exovesicles in the preparation of drugs to alleviate myocardial fibrosis after myocardial infarction.

2. The application of the Sophora flavescens vesicles according to claim 1 in the preparation of a drug to alleviate myocardial fibrosis after myocardial infarction, characterized in that, Sophora flavescens external vesicles inhibit fibroblast activation, transdifferentiation into myofibroblasts, and matrix collagen deposition.

3. Application of Sophora flavescens exovesicles in the preparation of drugs to improve cardiac dysfunction and myocardial damage caused by myocardial infarction.

4. Application of Sophora flavescens external vesicles in the preparation of drugs for treating myocardial infarction.

5. The application of the Sophora flavescens vesicles according to claim 4 in the preparation of a drug for treating myocardial infarction, characterized in that, The drug reduces myocardial fibrosis after myocardial infarction.

6. The application of the Sophora flavescens vesicles according to claim 4 in the preparation of a drug for treating myocardial infarction, characterized in that, The drug improves cardiac dysfunction and myocardial damage caused by myocardial infarction.

7. A drug for reducing myocardial fibrosis after myocardial infarction, characterized in that, The drug described uses Sophora flavescens vesicles as its sole active ingredient.

8. The drug for reducing myocardial fibrosis after myocardial infarction according to claim 7, characterized in that, The drugs mentioned also include pharmaceutically commonly used excipients.

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