Application of panax notoginseng outer vesicle in preparation of drugs for preventing and improving heart disease

Abstract

The application discloses application of Panax notoginseng exovesicle in preparation of a medicine for preventing and treating or improving heart diseases. The plant exovesicle is low in toxicity, high in biocompatibility, biodegradable, and cannot induce immunogenicity, and is high in yield and can be prepared on a large scale. A large number of experimental researches prove that the Panax notoginseng exovesicle has a significant heart disease prevention and treatment effect. Therefore, the application is favorable for promoting development and application of Panax notoginseng fresh medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of Panax notoginseng vesicles in the preparation of drugs for the prevention or improvement of heart disease. Background Technology

[0002] Heart disease is a leading cause of death and disability worldwide, constituting a huge healthcare burden. Heart disease includes cardiomyopathy, arrhythmia, valvular heart disease, and heart failure. Among these, cardiomyopathy is a heterogeneous group of myocardial diseases; in addition to primary factors, various secondary factors can also lead to structural and functional damage to the myocardium. For example, obesity, a chronic metabolic disease caused by multiple factors, is considered an independent risk factor for heart disease. With improved living standards, the obese population is increasing, and the incidence of obesity-related cardiomyopathy is also rising. It manifests as myocardial hypertrophy and fibrosis, systolic or diastolic heart failure, and ultimately leads to cardiac lesions.

[0003] Currently, clinical treatments for cardiomyopathy and even heart failure include ACEIs, beta-blockers, and digitalis. Conventional drug therapy can improve symptoms, but it has no significant effect on long-term survival. Furthermore, long-term use can easily lead to cardiotoxicity and arrhythmias, and accelerate myocardial cell necrosis. Therefore, there is an urgent need to explore a safer and more effective drug.

[0004] Plant exovesicles have been extensively studied as a novel drug delivery platform due to their low toxicity, high biocompatibility, biodegradability without inducing immunogenicity or carrying pathogens, and high yield for scalable production. However, there are few reports on the use of plant exovesicles for the treatment of heart disease. Therefore, developing a safe and efficient plant exovesicle for the treatment of heart disease is of great significance. Summary of the Invention

[0005] To address at least some of the technical problems existing in the prior art, this invention develops a plant-derived exovesicle drug that is low in toxicity, highly biocompatible, biodegradable, does not induce immunogenicity, has high yield, and can be prepared on a large scale. This drug can solve the problems of cardiotoxicity and accelerated myocardial cell necrosis present in existing drugs for treating heart disease. Specifically, this invention includes the following:

[0006] In a first aspect, the invention provides the use of Panax notoginseng vesicles in the preparation of medicaments for the prevention or improvement of heart disease and its symptoms.

[0007] In some embodiments, the use of the Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention, treatment or improvement of heart disease and its symptoms, wherein the heart disease includes cardiomyopathy, myocardial ischemia, myocardial infarction, ischemia-reperfusion injury, heart failure and / or arrhythmia.

[0008] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention, treatment or improvement of heart disease and its symptoms, wherein the cardiomyopathy includes cardiomyopathy caused by hypertension, obesity or overexcitation.

[0009] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention, treatment, or improvement of heart disease and its symptoms, wherein the Panax notoginseng vesicles are prepared by the following method:

[0010] (1) Take fresh roots or rhizomes of Panax notoginseng, homogenize them, and then centrifuge them at a differential speed to obtain crude extract;

[0011] (2) The crude extract was purified by density gradient centrifugation to obtain the Panax notoginseng exovesicles.

[0012] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of drugs for the prevention, treatment or improvement of heart disease and its symptoms, wherein the particle size of the Panax notoginseng vesicles is 200-250 nm and the Zeta potential value ranges from -40 mV to -10 mV.

[0013] In some embodiments, the use of the Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention or improvement of heart disease and its symptoms, wherein the prevention or improvement includes at least one of the following:

[0014] (1) Reduce the amount or activity of lactate dehydrogenase and / or creatine kinase isoenzymes;

[0015] (2) Reduce or inhibit the effects of oxidative stress;

[0016] (3) Increase or restore the mitochondrial membrane potential of cardiomyocytes;

[0017] (4) Reduce or restore the phosphorylation level and / or protein expression level of cGAS-STING signaling pathway proteins;

[0018] (5) Restore lipid metabolism function;

[0019] (6) Reduce or inhibit myocardial damage;

[0020] (7) Improve or restore heart function.

[0021] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of drugs for the prevention, treatment or improvement of heart disease and its symptoms, wherein the cGAS-STING signaling pathway protein includes at least one of cGAS, STING, NF-κB, TBK1 and IRF3.

[0022] In a second aspect, the invention provides a pharmaceutical composition for preventing or improving heart disease and its symptoms, comprising Panax notoginseng exovesicles.

[0023] In some embodiments, the pharmaceutical composition according to the present invention further comprises a pharmaceutically acceptable carrier.

[0024] A third aspect of the present invention provides a method for reducing the amount and / or activity of lactate dehydrogenase and / or creatine kinase isoenzymes in cardiomyocytes in vitro, comprising the step of contacting Panax notoginseng exovesicles with the cardiomyocytes.

[0025] This invention has conducted extensive experimental research at the cellular and tissue levels, and has found that the outer vesicles of Panax notoginseng have the effect of preventing and treating heart disease. Therefore, this invention provides a theoretical basis and technical support for promoting the development and application of fresh Panax notoginseng, and has a great promoting effect on the transformation of basic research results. Attached Figure Description

[0026] Figure 1 The image shows a transmission electron microscope image of the outer vesicles of Panax notoginseng.

[0027] Figure 2 The results of NTA detection for Panax notoginseng exovesicles are shown.

[0028] Figure 3 Figures A and B show the particle size and potential measurement results of Panax notoginseng vesicles, respectively.

[0029] Figure 4 The high-performance liquid chromatogram of the mixed reference standard of Panax notoginseng saponins is shown.

[0030] Figure 5 The results of the analysis of saponin components in the outer vesicles of Panax notoginseng are shown.

[0031] Figure 6 Figures A and B show the standard curves for ginsenosides Rb1 and Rd, respectively.

[0032] Figure 7 The results of H9C2 cells taking up Panax notoginseng exovesicles are shown.

[0033] Figure 8 Figures A and B show the effects of different concentrations of PA and PDNs on the viability of H9C2 cells, respectively. Figure C shows the effects of PA and different concentrations of Panax notoginseng vesicles on the viability of H9C2 cells.

[0034] Figure 9 Figures A and B show the effects of Panax notoginseng vesicles on the myocardial injury markers LDH and CK-MB, respectively.

[0035] Figure 10Figures A and B show the qualitative and quantitative results of the effect of Panax notoginseng vesicles on ROS in H9C2 cells.

[0036] Figure 11 Figures A and B show the qualitative and quantitative results of the effect of Panax notoginseng vesicles on the mitochondrial membrane potential of H9C2 cells, respectively.

[0037] Figure 12 This study demonstrates the regulatory effect of Panax notoginseng exovesicles on proteins in the cGAS-STING signaling pathway of H9C2 cells.

[0038] Figure 13 A, B, C, D, and E show the quantitative results of the regulation of the proteins cGAS, STING, NF-κB, TBK1, and IRF3 in the cGAS-STING signaling pathway of H9C2 cells by Panax notoginseng vesicles, respectively.

[0039] Figure 14 The results of the insulin tolerance test in HFD mice are shown, where A shows the blood glucose levels of each treatment group and B shows the blood glucose clearance capacity of each treatment group.

[0040] Figure 15 The results of the glucose tolerance test in HFD mice are shown. A shows the blood glucose levels of each treatment group, and B shows the blood glucose handling capacity of each treatment group.

[0041] Figure 16 The results of the blood lipid experiment in HFD mice are shown, where A and B show the concentrations of TG and TC in serum, respectively.

[0042] Figure 17 The study showed that Panax notoginseng vesicles have a protective effect against myocardial injury in HFD mice. In this study, A and B show the levels of LDH and CK-MB in each treatment group, respectively.

[0043] Figure 18 The echocardiogram results of different groups of mice are shown.

[0044] Figure 19 Figures A and B show the effects of Panax notoginseng vesicles on cardiac function indicators EF% and FS% in HFD mice, respectively.

[0045] Figure 20 A shows the effect of Panax notoginseng vesicles on H9C2 cell viability, B shows the effect of ANGII on H9C2 cell viability, and C shows the effect of co-treatment with Panax notoginseng vesicles and ANGII on H9C2 cell viability.

[0046] Figure 21 Figures A and B show the effects of Panax notoginseng vesicles on LDH and CK-MB in ANGII model cells, respectively.

[0047] Figure 22 The effect of Panax notoginseng exovesicles on the cell size of the ANGII model was shown.

[0048] Figure 23 The effect of Panax notoginseng vesicles on ROS in ANGII model cells was shown.

[0049] Figure 24 The effects of Panax notoginseng vesicles on myocardial injury in ISO model mice are shown. A shows the changes in body weight of mice in each treatment group, B shows the cardiac index of each treatment group, and C shows the Masson staining results of each treatment group. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] application

[0054] One aspect of the present invention provides the use of Panax notoginseng vesicles in the preparation of medicaments for the prevention, treatment, or improvement of heart disease and its symptoms. The heart disease includes, but is not limited to, cardiomyopathy, myocardial ischemia, myocardial infarction, ischemia-reperfusion injury, heart failure, and arrhythmia. The heart disease specifically refers to cardiomyopathy. In a preferred embodiment, the heart disease is cardiomyopathy caused by obesity. In another preferred embodiment, the heart disease is cardiomyopathy caused by hypertension. In yet another preferred embodiment, the heart disease is cardiomyopathy caused by over-excitation.

[0055] In this invention, "plant extracellular vesicles" and "plant-derived extracellular vesicles" can be used interchangeably. They are nanoscale membrane-structured vesicles actively secreted by plant cells, with a diameter ranging from 30 to 500 nm. They have a lipid bilayer structure and contain active ingredients such as proteins, nucleic acids, and plant-specific metabolites. They are secreted through exosomes, microvesicles, and apoptotic bodies and are widely present in plant roots, stems, leaves, and other tissues and secretions.

[0056] In this invention, "prevention or improvement" refers to improving the condition before or after the onset of a disease or functional disorder. This improvement or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). The treatment or improvement includes at least one of the following cardiomyopathy-related conditions: reducing the amount or activity of lactate dehydrogenase and / or creatine kinase isoenzymes; reducing or inhibiting the effects of oxidative stress; increasing or restoring the mitochondrial membrane potential of cardiomyocytes; decreasing or restoring the phosphorylation level and / or protein expression level of cGAS-STING signaling pathway proteins; restoring lipid metabolism function; alleviating or inhibiting myocardial damage; and improving or restoring cardiac function. The cGAS-STING signaling pathway proteins include, but are not limited to, cGAS, STING, NF-κB, TBK1, and IRF3.

[0057] In a preferred embodiment, the Panax notoginseng exovesicles of the present invention are prepared by the following method:

[0058] (1) Take fresh roots or rhizomes of Panax notoginseng, homogenize them, and then centrifuge them at a differential speed to obtain crude extract;

[0059] (2) The crude extract was purified by density gradient centrifugation to obtain the Panax notoginseng exovesicles.

[0060] In a further preferred embodiment, the Panax notoginseng exovesicles of the present invention are prepared by the following method:

[0061] (1) Take the roots or rhizomes of Panax notoginseng with a water content of not less than 70%, homogenize them, and then obtain the crude extract by gradient ultracentrifugation.

[0062] (2) The crude extract is enriched with 1 M-2 M sucrose or with 45%-60% sucrose to obtain the Panax notoginseng exovesicles.

[0063] In step (1) of this invention, the roots or rhizomes of Panax notoginseng are first sliced ​​or cut into pieces and homogenized at low temperature in a buffer solution. Unless otherwise specified, the low temperature in this invention refers to 0-10°C, for example, 0, 2, 4, 6, 8, or 10°C. Subsequently, the homogenized liquid is filtered, and a protein inhibitor is added to adjust the pH to neutral (preferably 6-8, for example, 6, 6.5, 7, 7.5, or 8). Then, a low-temperature gradient centrifugation is performed at 400-10000 g. Here, gradient centrifugation means centrifuging at speeds of 400, 800, and 10000 g sequentially for 5-40 min, preferably 10-30 min, for example, 10, 15, 20, 25, or 30 min. Collect the supernatant and then perform low-temperature ultracentrifugation at a speed of not less than 100,000 g (e.g., not less than 110,000, 120,000, 130,000, 140,000, 150,000, or even greater than 150,000 g), and collect the precipitate to obtain the crude extract. Further dissolve the crude extract with excipients, preferably in a buffer, to obtain the crude extract solution.

[0064] In step (1) of the present invention, the buffer is preferably Tris-HCl, more preferably 0.5-50 mM Tris-HCl, and even more preferably 1-40 mM (e.g. 5-40 mM, 10-30 mM, 15-25 mM, 18-22 mM) Tris-HCl, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mM.

[0065] In step (2) of the present invention, enriching the crude extract with 1 M-2 M sucrose means performing low-temperature high-speed centrifugation (preferably 4°C, not less than 100,000 g) together with 1 M sucrose and 2 M sucrose for 0.1-2 h, preferably 0.5-1.5 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 h, and taking the intermediate liquid after centrifugation. Alternatively, enrichment with 45%-60% sucrose can be achieved. This means purifying the crude extract of Panax notoginseng with a 45% and 60% sucrose concentration gradient, collecting liquids from different sucrose layers, washing the sucrose with the aforementioned concentration of Tris-HCl, and then centrifuging at low temperature and high speed (preferably 4°C, not less than 100,000 g) for 0.1-2 h, preferably 0.5-1.5 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 h.

[0066] In a preferred embodiment, the Panax notoginseng raw material is Panax notoginseng with a moisture content of not less than 70%, rather than dried Panax notoginseng. The specific criteria for determining whether Panax notoginseng has a moisture content of not less than 70% or dried Panax notoginseng are known in the art. For example, the moisture content can be determined by drying according to the moisture determination method (General Rule 0832, Method II) in the Chinese Pharmacopoeia (2020 edition). Specifically, Panax notoginseng with a moisture content of not less than 70% refers to Panax notoginseng that has been sampled, pulverized, and dried at 105°C to constant weight, with a measured moisture content of not less than 70%, preferably 70%-85%, and even more preferably 75%-80% (w / w).

[0067] In one specific embodiment, the Panax notoginseng exovesicles obtained by the present invention have a distinct double-layer membrane structure on the outside, and are generally in the form of saucer-like or hemispherical structures of varying sizes, possessing an intact structure (containing little or no fragments), with a yield of not less than 480 mg / kg, and a particle size of 200-250 nm, preferably 200-245 nm, even more preferably 200-240 nm, further preferably 200-235 nm, and more preferably 200-230 nm, for example 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 nm. nm, the Zeta potential ranges from -40 mV to -10 mV, preferably from -38 mV to -11 mV, even more preferably from -36 mV to -12 mV, further preferably from -34 mV to -13 mV, more preferably from -32 mV to -14 mV, for example -32, -31, -30, -29, -28, -27, -26, -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14 mV.

[0068] In a preferred embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

[0069] In this invention, the treatment and improvement of heart disease are achieved by administering a therapeutically effective amount of the Panax notoginseng vesicles to the subject. Subjects include, but are not limited to, mammals, including but not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0070] In this invention, there are no particular limitations on the method of administration of the drug. Representative methods of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) and local administration. Accordingly, the drug of this invention can be formulated into various clinically acceptable dosage forms, including oral dosage forms, injectable dosage forms, and local administration dosage forms.

[0071] The effective dose for prevention and treatment mentioned in this invention refers to the pharmaceutically recognized effective dosage, that is, the amount of active compound (i.e., Panax notoginseng exovesicles) is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the Panax notoginseng vesicles is typically 0.01-1000 mg / Kg, preferably 0.1-500 mg / Kg, even more preferably 1-400 mg / Kg, further preferably 10-300 mg / Kg, more preferably 20-200 mg / Kg, more preferably 30-150 mg / Kg, and even more preferably 40-120 mg / Kg, for example 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 mg / Kg. It can be administered as a single dose once daily, or divided into multiple doses daily, or used at intervals.

[0072] Pharmaceutical Composition

[0073] In one aspect, the present invention provides a pharmaceutical composition for preventing or improving heart disease and its symptoms, comprising Panax notoginseng exovesicles. In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0074] In this invention, pharmaceutically acceptable carriers are used to transport or deliver a drug from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., Panax notoginseng exovesicles) and does not harm the patient. The pharmaceutically acceptable carriers include at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, dicalcium phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; disintegrants include, but are not limited to, croscarmellose sodium, croscarmellose, surfactants, and low-substituted hydroxypropyl cellulose; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium dodecyl sulfate, micronized silica gel, and talc; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, and aspartame; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0075] method

[0076] One aspect of the present invention provides a method for reducing the amount and / or activity of lactate dehydrogenase and / or creatine kinase isoenzymes in in vitro cardiomyocytes, comprising the step of contacting Panax notoginseng vesicles with the cardiomyocytes in vitro. This method is a non-therapeutic method and can be used for drug screening, drug structure optimization, disease mechanism research, etc.

[0077] In a preferred embodiment, the method of the present invention includes: (1) obtaining cardiomyocytes; (2) performing in vitro cell expansion culture; (3) contacting the expanded cardiomyocytes with the Panax notoginseng vesicles described in the present invention; and (4) detecting the amount and / or activity of lactate dehydrogenase and / or creatine kinase isoenzymes in the cardiomyocytes.

[0078] Example 1

[0079] This embodiment illustrates the preparation of Panax notoginseng vesicles and their role in obesity-induced myocardial injury.

[0080] 1. Experimental Methods

[0081] 1.1 Extraction and characterization of Panax notoginseng exovesicles

[0082] 1.1.1 Extraction of exovesicles from fresh Panax notoginseng

[0083] Take an appropriate amount of Panax notoginseng with a moisture content of not less than 70%, wash it clean with distilled water, and air dry it. Cut the Panax notoginseng into slices 3-5 cm thick. Homogenize it in a homogenizer with 1*PBS (calcium and magnesium-free). The temperature is controlled at 4℃ throughout the homogenization process. Pass the homogenate through a silk cloth to obtain Panax notoginseng homogenate. Immediately add protease inhibitors (leucopeptide, PMSF, sodium azide). Then adjust the pH of the Panax notoginseng homogenate to 7.0 with 1 M Tris-HCl. Centrifuge at 4℃, 400 g, 800 g, and 10000 g for 20 min in sequence and collect the supernatant. Ultracentrifuge at 4℃ for 100000 g and collect the precipitate after 1 h. Dissolve the precipitate with 20 mM Tris-HCl to obtain crude Panax notoginseng extract.

[0084] The crude extract of Panax notoginseng was centrifuged together with 1 M sucrose and 2 M sucrose (4℃, 100000 g, 1 h), and the middle layer liquid was collected to obtain the Panax notoginseng vesicle solution.

[0085] 1.1.2 Characterization of Panax notoginseng exovesicles

[0086] The morphology of the exovesicles of Panax notoginseng was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown, they are uniform in shape, with a distinct double membrane structure on the outside, and the whole structure is a saucer-like or hemispherical structure of varying sizes, which is consistent with the microscopic identification characteristics of extracellular vesicles.

[0087] The particle count in the Panax notoginseng exovesicle solution was determined using NTA, and the results are as follows: Figure 2 As shown, the number of particles obtained from Panax notoginseng exovesicles purified with 1-2 M sucrose was 5*10-1. 12 particles / mL.

[0088] The particle size and potential of the exovesicles of Panax notoginseng were measured, and the results are as follows: Figure 3 As shown, the average diameter of the outer vesicles of Panax notoginseng is 213 nm, and the Zeta potential ranges from -22.0 to 8.04 mV.

[0089] 1.1.3 Compositional Analysis of Panax notoginseng Vesicles

[0090] Referring to the 2020 edition of the Chinese Pharmacopoeia, high performance liquid chromatography (HPLC) was used to determine the five main saponin components in the outer vesicles of Panax notoginseng: notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd.

[0091] Preparation of reference solutions: Accurately weigh appropriate amounts of each reference standard and dissolve them in chromatographic grade methanol to prepare solutions containing ginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd at concentrations of 2.7, 2.65, 2.5, 2.5, and 2.2 mg·mL, respectively. -1 The reference stock solutions were filtered through a 0.22 μm filter membrane and set aside. The high-performance liquid chromatography (HPLC) results for each reference standard are shown below. Figure 4 As shown.

[0092] Sample solution preparation: Weigh 94.5 mg of trehalose and dissolve it in 5 mL of Panax notoginseng vesicles to a final concentration of 50 mM. Quickly freeze the solution in liquid nitrogen and place it in a lyophilizer to make lyophilized powder. Dissolve 100 mg of lyophilized powder in 1 mL of chromatographic methanol, sonicate for 30 min, and filter using a 0.22 μm filter membrane.

[0093] Chromatographic conditions: Agilent ZORBAX SB-Aq high performance liquid chromatography column (5 μm, 4.6 × 250 mm).

[0094] Mobile phase: acetonitrile (A) - 0.05% phosphate buffer (B); column temperature: 30℃; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 203 nm; gradient elution: 0-20 min, 20% A; 20-45 min, 20%-46% A; 45-55 min, 46%-55% A; 55-60 min, 55% A.

[0095] HPLC analysis was performed on the components of a mixed reference standard of Panax notoginseng saponins and Panax notoginseng exovesicles. The results showed that the exovesicles mainly contained ginsenoside Rb1 and ginsenoside Rd monomers. Furthermore, Panax notoginseng saponin R1, ginsenoside Rg1, and ginsenoside Re monomers were not detected. (See results below.) Figure 5 As shown.

[0096] A standard curve was plotted using ginsenoside Rb1 and ginsenoside Rd. (See curve for details.) Figure 6 The equations of the curves are: y = 2015.6x + 11.455, R... 2 =1 and y=3853.8x+6.7054, R 2 =1, indicating a good linear relationship between ginsenoside Rb1 in the range of 0-2.5 mg / mL and ginsenoside Rd in the range of 0-2.2 mg / mL. After three parallel experiments, the saponin content of ginsenoside Rb1 in Panax notoginseng vesicles was measured to be 1 μg / mg, the saponin content of ginsenoside Rd was 0.5 μg / mg, and the total saponin content was 0.15%, indicating that Panax notoginseng vesicles contain only trace amounts of saponin components.

[0097] 1.2 Uptake of Panax notoginseng exovesicles by H9C2 cells

[0098] PKH26 is a specialized membrane labeling probe with a long aliphatic tail, enabling stable insertion into the lipid regions of cell membranes, making it suitable for in vitro cell labeling. PDNs were labeled with the red fluorescent dye PKH26, and H9C2 cells were cultured in confocal dishes at a density of 4 × 10⁻⁶ cells per well. 4 Cells were seeded into plates and cultured for 24 h. After co-culturing H9C2 cells with PKH26-labeled PDNs for 24 h, the cell nuclei were stained with DAPI, and finally the images were taken under a laser scanning confocal microscope.

[0099] 1.3 H9C2 Cytotoxicity Assay

[0100] The concentration of proteins in the exovesicles of Panax notoginseng was determined using a BCA kit. H9C2 cells were cultured in DMEM medium at 37°C in a 5% CO2 incubator. During the logarithmic growth phase, the cells were injected at a rate of 4 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of cells / well in 96-well plates. After 24 h, the culture medium was discarded, and PDNs (5, 10, 20, 40, 60, 80, 100, 200 μg / mL) and PA (40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, 800 μmol / L) were added. An in vitro myocardial injury model was established using 100 μM PA. The cell treatment groups were divided into 5 groups: blank control group (Control), model group (100 μMPA), and Panax notoginseng vesicle treatment group (5, 10, 20 μg / mL). After 24 h of cell culture, the supernatant was discarded, and CCK-8 reagent was added (10 μL of reaction solution per 100 μL of culture medium), and the cells were incubated at 37°C for 1 h. After incubation, the absorbance of each reaction well was measured at 450 nm using a microplate reader, and the relative growth rate of each group of cells was calculated.

[0101] Cell viability = (absorbance of drug-treated group - absorbance of blank group) / (absorbance of blank control group wells - absorbance of blank group) × 100% (Drug-treated group: cell culture with different concentrations of Panax notoginseng vesicles; blank control group: cell culture without Panax notoginseng vesicles; blank group: no cell culture, only culture medium).

[0102] 1.4 Detection of LDH and CK-MB, markers of myocardial injury in H9C2 cells

[0103] Lactate dehydrogenase (LDH) is present in various tissues and organs of the human body, while creatine kinase isoenzyme (CK-MB) is mainly distributed in the myocardium. When myocardial injury occurs, it can serve as a major biochemical marker for detecting myocardial damage. (H9C2 cells, 6×10⁶) 4 Cells were seeded into 6-well plates and cultured for 24 h, then divided into 5 groups: blank group, PA model group, and Panax notoginseng vesicle drug administration group (5, 10, and 20 μg / mL), and incubated together for 24 h. During the experiment, after collecting cell samples, 0.3 mL of PBS was added to each cell sample, and the cells were sonicated in an ice-water bath, centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. The protein concentration of the supernatant was measured, and the LDH and CK-MB detection kits were tested according to the instructions. After the reaction, the absorbance of each reaction well was measured at 450 nm using a microplate reader. Reagents were prepared according to the instructions of the Nanjing Jiancheng LDH kit. Serum samples were prepared according to the instructions, with blank control wells, standard wells, assay wells, and control wells set up. The LDH content in serum was calculated using the formula: LDH (U / L) = (A assay - A control) / (A standard - A blank) * C standard * N dilution factor * 1000. According to the instructions of the Shanghai CK-MB ELISA kit, the OD value of each well was measured at 450 nm using an ELISA reader. A standard curve was established by subtracting the OD value of the blank well from the OD value of the standard well, and the corresponding CK-MB content was obtained.

[0104] 1.5 Reactive Oxygen Species Experiment

[0105] H9C2 cells 6×10 4 Cells were seeded per well in 6-well plates and cultured for 24 h. Five groups were formed: a blank control group, a PA model group, and a group receiving Panax notoginseng vesicle treatment (5, 10, and 20 μg / mL), and all were incubated together for 24 h. The ROS level of H9C2 cells was detected using a reactive oxygen species (ROS) detection kit. The fluorescent probe DCFH-DA can penetrate the cell membrane and be oxidized by intracellular ROS into fluorescent DCF, which was detected using a fluorescence microscope.

[0106] In situ probe loading: Dilute DCFH-DA 1:1000 with blank culture medium, discard the original culture medium in each well, add 1 mL of diluted DCFH-DA to each well, place in a 37℃ constant temperature incubator for 30 min, discard DCFH-DA, wash three times with PBS, add 2 mL of serum-free culture medium to each well, and take pictures under a fluorescence microscope in the dark for detection.

[0107] 1.6 JC-1 Experiment

[0108] Cell treatment followed the same method as the in situ loading of probes with reactive oxygen species. According to the JC-1 kit instructions, the JC-1 stock solution was diluted to the required concentration with staining buffer or PBS. After discarding the original culture medium, 1 mL of culture medium and 1 mL of JC-1 staining working solution were added to each well. The wells were shaken and placed in a 37°C incubator for 20 min. After incubation, the culture medium was discarded and the cells were washed twice with JC-1 staining buffer (1X). Culture medium was added to each well and the cells were photographed and observed under a fluorescence microscope in the dark. Quantitative analysis was performed using ImageJ.

[0109] The mitochondrial membrane was labeled with the fluorescent dye JC-1. JC-1 forms a polymer and emits red fluorescence when the mitochondrial membrane potential is high, and exists as a monomer and emits green fluorescence when the membrane potential is low. Therefore, the ratio of red to green fluorescence intensity (Red / Green) is a direct indicator for evaluating the mitochondrial membrane potential.

[0110] 1.7 Immunoblotting detection of relevant protein levels in cells

[0111] H9C2 cells were fed at a rate of 8 × 10 4 Cells were seeded in small dishes at a density of 1 cell / well and cultured for 24 h. Different concentrations of Panax notoginseng exovesicles were added, and after 24 h, cells were collected and total protein was extracted using RIPA lysis buffer. Protein concentration was determined using a BCA kit and adjusted to be consistent. The prepared samples were subjected to SDS-PAGE gel electrophoresis, transferred to nitrocellulose (NC) membranes, and placed in PBS buffer containing 5% skim milk powder. The membranes were blocked at room temperature for 1 h, and the corresponding primary antibodies (all diluted 1:1000) were added and incubated overnight at 4°C. The membranes were then incubated with HRP-labeled secondary antibodies (all diluted 1:1000) at room temperature for 1 h. The membranes were then developed using ECL chemiluminescence solution in a gel imaging system.

[0112] 1.8 Animal Experiments

[0113] Wild-type male C57BL / 6 mice (6-8 weeks old, weighing approximately 20 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and first acclimatized for one week in the SPF-grade mouse house of the Animal Center of Changchun University of Traditional Chinese Medicine. During this period, the ambient temperature was controlled at a constant 22℃, and a 12-hour light-12-hour dark circadian rhythm was adopted, with free access to food and water. The experiment lasted for 16 weeks. For the first 8 weeks, the mice were randomly divided into two groups: a control group fed a low-fat diet with 10% fat content, and an HFD group fed a high-fat diet with 60% fat content. Both groups were provided with ordinary tap water. Mice with a body weight greater than 20% were selected as the HFD model group for the experiment. Starting from week 9, the mice were divided into five groups: control group (10% fat + 20 mM Tris-HCl), HFD group (60% fat + 20 mM Tris-HCl), positive control group (60% fat + lovastatin), low-dose group (60% fat + 5 mg / mL PDNs), and high-dose group (60% fat + 10 mg / mL PDNs). The mice were weighed weekly, and at the end of the experiment, the mice's hearts were weighed, and the cardiac index (heart weight (mg) / body weight (g)) was calculated.

[0114] 1.9 Insulin and Glucose Tolerance

[0115] Glucose tolerance test: Mice were fasted for 14-16 hours with ample water. A 20% glucose solution prepared with physiological saline was administered intraperitoneally at a dose of 2 g glucose / kg mouse body weight. The tail tip of the mouse was quickly punctured with a lancet, and the first drop of blood was discarded. The basal blood glucose level at 0 minutes was measured using a glucometer. Hemostasis was achieved by gently applying pressure after blood collection. Blood glucose levels were measured at 15, 30, 60, and 120 minutes after injection. A curve was plotted with time on the x-axis and blood glucose level on the y-axis. The area under the curve (AUC) was calculated.

[0116] Insulin tolerance: Mice were fasted for 4-6 hours, and regular insulin was diluted with sterile PBS and injected intraperitoneally at a dose of 0.75-1.0 U insulin / kg mouse body weight. The basal blood glucose level at 0 minutes after injection was measured. Blood glucose was measured at 15, 30, 60, 90, and 120 minutes after injection, and the blood glucose change curve was plotted as in the GTT.

[0117] 1.10 Detection of TC and TG levels in mice using the kit

[0118] After establishing the model as described in Section 1.8, blood was obtained by collecting blood from the eyeball. The blood was collected in a centrifuge tube and allowed to stand at room temperature (or 4°C) for 30-60 minutes to allow it to coagulate naturally. Then, it was centrifuged at 3000 rpm for 15 minutes at 4°C. The pale yellow liquid on top was the serum. It was carefully aspirated into a new EP tube and tested and calculated according to the kit.

[0119] 1.11 Effects on cardiac function in mice

[0120] After feeding mice for 16 weeks, the hair on the mice's chest was removed with depilatory cream. The mice were then anesthetized with isoflurane and fixed on the operating table. A small amount of coupling gel was applied to the mice's chests, and then high-frequency ultrasound was used to perform cardiac ultrasound examinations. Data such as left ventricular ejection fraction (EF%) and left ventricular fractional shortening (FS%) were collected. Among these, EF% and FS% are the most critical indicators for assessing overall systolic function.

[0121] 1.12 Statistical Methods

[0122] All data represent at least three independent experiments and are expressed as mean ± SEM. Statistical calculations were performed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA). Statistical comparisons were performed using one-way ANOVA and Dunnett's post-hoc test. P < 0.05 was considered statistically significant.

[0123] 2. Experimental Results

[0124] 2.1 Cellular uptake

[0125] The uptake of Panax notoginseng vesicles by H9C2 cells is as follows: Figure 7 As shown, the results indicate that Panax notoginseng vesicles can be taken up by H9C2 cells.

[0126] 2.2 Cytotoxicity

[0127] The results are as follows Figure 8 As shown, different concentrations of the drug did not exhibit cytotoxicity within a certain range. Ultimately, 5, 10, and 20 μg / mL were selected as the low, medium, and high concentrations for the drug treatment group, and 100 μmol / L was chosen as the concentration for the PA injury model.

[0128] 2.3 Myocardial Injury Markers

[0129] The results of myocardial injury marker detection are as follows Figure 9 As shown, PA can induce elevated levels of LDH and CK-MB. After administration of Panax notoginseng exovesicles, it was found that they significantly reduced the release of LDH and CK-MB, H9C2 cell damage markers, indicating that Panax notoginseng exovesicles have a significant protective effect against H9C2 cell damage. Therefore, Panax notoginseng exovesicles can achieve cardioprotection by alleviating cardiomyocyte damage and reducing the release of damage markers.

[0130] 2.4 Reactive Oxygen Species

[0131] The results of the reactive oxygen species experiment are as follows Figure 10As shown, PA significantly increased the level of ROS, a product of oxidative stress, in H9C2 cells, while PDNs treatment significantly reduced the increase in ROS, indicating that PDNs can inhibit PA-induced oxidative stress.

[0132] 2.5 Membrane potential

[0133] The results of H9C2 cell mitochondrial membrane potential detection are as follows: Figure 11 As shown, in the PA-treated group, the intensity of red fluorescence in the cells gradually decreased, while the intensity of green fluorescence significantly increased, and the red-green ratio decreased, indicating that PA led to a significant decrease in mitochondrial membrane potential. After administration of Panax notoginseng vesicles, bright red fluorescence was observed, while green fluorescence was weaker, and the red-green ratio was high, indicating that the cells had a high mitochondrial membrane potential and were in a healthy state. That is, Panax notoginseng vesicles can improve or restore the mitochondrial membrane potential of H9C2 cells.

[0134] 2.6 cGAS-STING signal path

[0135] Immunoblotting results as follows Figure 12 and 13 As shown, Panax notoginseng vesicles have a regulatory effect on cGAS-STING signaling pathway proteins (such as NF-κB, P-TBK1, TBK1, P-IRF3, etc.) in PA-treated H9C2 cells. Panax notoginseng vesicles improved the upregulation of cGAS and its downstream factor STING caused by PA damage. The results indicate that Panax notoginseng vesicles exert cardioprotective effects by inhibiting the cGAS-STING signaling pathway.

[0136] 2.7 Glucose tolerance

[0137] Results of insulin and glucose tolerance tests in HFD mice as follows: Figure 14 and 15 As shown, at the experimental endpoint, the fasting blood glucose and fasting insulin levels of mice in the high-fat diet group were significantly higher than those in the normal control group, indicating that the high-fat diet successfully induced obesity, hyperinsulinemia, and elevated fasting blood glucose in mice, indicating that the obesity model was successfully established. Among them, H-PDNs were 10 mg / ml Panax notoginseng vesicles, and L-PDNs were 5 mg / ml Panax notoginseng vesicles.

[0138] To directly assess insulin sensitivity in peripheral tissues, an insulin tolerance test was performed in this embodiment. After intraperitoneal injection of insulin, blood glucose levels in mice began to decrease. However, the magnitude and rate of decrease in blood glucose were greater in the normal control group than in the high-fat diet group, and significantly greater in the Panax notoginseng vesicle treatment group than in the high-fat diet group. Within 30-120 minutes post-injection, blood glucose levels in the high-fat diet group remained significantly higher than in both the control group and the Panax notoginseng vesicle treatment group.

[0139] To assess the body's ability to process glucose, an intraperitoneal glucose tolerance test was conducted in this embodiment. After intraperitoneal injection of glucose, the blood glucose levels of mice in each group reached their peak at 0-30 minutes. However, throughout the experiment, the blood glucose levels of mice in the high-fat diet group were significantly higher than those in the normal control group at all time points. After administration of Panax notoginseng vesicles, the blood glucose levels of mice in the high-fat diet group were lower than those in the model group.

[0140] Area under the curve: Quantitative analysis of the area under the curve for insulin and blood glucose revealed that the AUC of the high-fat diet group was significantly greater than that of the normal control group, and the group treated with Panax notoginseng vesicles recovered the metabolic dysfunction caused by high-fat diet.

[0141] 2.8 Blood lipids

[0142] The levels of total cholesterol and triglycerides in the serum of mice fed a high-fat diet for 16 weeks were measured, and the results are as follows: Figure 16 As shown, compared with the normal control group, the serum TG and TC concentrations of mice in the high-fat diet group were significantly higher than those in the normal control group. The administration of Panax notoginseng exoves reduced the levels of both, indicating that Panax notoginseng exoves can reduce the lipid metabolism disorder induced by the HFD diet in mice.

[0143] 2.9 Myocardial Injury Markers

[0144] Results of myocardial injury markers, such as Figure 17 As shown, the results indicate that HFD can cause an increase in the levels of LDH and CK-MB in mouse serum. After administration of Panax notoginseng exovesicles, it was found that they could significantly reduce the levels of LDH and CK-MB in the serum of HFD mice, indicating that Panax notoginseng exovesicles have a significant protective effect against myocardial injury in HFD mice.

[0145] 2.10 Cardiac Function

[0146] Echocardiogram results as follows Figure 18 and 19 As shown, the experimental results indicated that the high-fat diet group mice experienced pathological remodeling of the heart, with left ventricular dilation and myocardial hypertrophy, while the mice treated with Panax notoginseng vesicles recovered their cardiac function.

[0147] Example 2

[0148] This embodiment demonstrates the role of Panax notoginseng vesicles in myocardial damage caused by hypertension.

[0149] 1. Cytotoxicity

[0150] The effect of Panax notoginseng vesicles on the cytotoxicity of H9C2 cells was detected using the CCK-8 assay. Different concentrations of Panax notoginseng vesicles were co-cultured with H9C2 cells for 24 h to assess their cytotoxic effects on H9C2 cells. The results are as follows: Figure 20As shown in Figure A, different concentrations of Panax notoginseng vesicles had little toxic effect on H9C2 cells. Concentrations of 10 and 20 μg / mL were selected as the low and high concentrations for the drug treatment group. Next, to evaluate the optimal induction concentration, H9C2 cells were treated with different concentrations of ANGII for 24 hours, divided into five groups: control group, 0.5 μmol / L, 0.75 μmol / L, 1 μmol / L, 2 μmol / L, and 4 μmol / L. The optimal concentration for the in vitro ANGII injury model was determined, and the results are shown below. Figure 20 As shown in Figure B, different concentrations of ANGII all had a damaging effect on H9C2 cells, with significant differences starting from 0.75 μmol / L. The optimal concentration for the in vitro ANGII damage model was selected as 1 μmol / L. Finally, to verify the therapeutic effect of Panax notoginseng vesicles on ANGII-induced H9C2 cells, the experimental groups were: control group, 1 μmol / L ANGII group, 1 μmol / L ANGII + PDNS 5 μg / mL, 1 μmol / L ANGII + PDNS 10 μg / mL, and 1 μmol / L ANGII + 20 μg / mL. The treatment groups were co-cultured with 1 μmol / L ANGII and three concentrations of Panax notoginseng vesicles (low, medium, and high) for 24 h. It was found that Panax notoginseng vesicles could promote cell proliferation of H9C2 cells after ANGII-induced damage. The results are shown in Figure B. Figure 20 As shown in C.

[0151] 2. Markers of myocardial injury

[0152] The expression of myocardial injury markers LDH and CK-MB was detected using a kit, and the results are as follows: Figure 21 As shown, ANGII can induce elevated levels of LDH and CK-MB. After administration of Panax notoginseng exovesicles, it was found that they significantly reduced the release of LDH and CK-MB, markers of H9C2 cell damage. The data indicate that Panax notoginseng exovesicles have a significant protective effect against H9C2 cell damage. Panax notoginseng exovesicles can achieve cardioprotection by alleviating cardiomyocyte damage and reducing the release of damage markers.

[0153] 3. Cell volume

[0154] H9C2 cells (4×10) 4Cells (per well) were cultured in confocal dishes for 24 h, followed by ANGII modeling and drug administration, and then co-incubated for another 24 h. For the experiment, the stock solution was freshly prepared using 1xPBS buffer and diluted to a working concentration of 80-100 nM. The original culture medium was removed, and the cells were washed twice with preheated 1xPBS (pH 7.4) at 37°C. The cells were fixed with 4% formaldehyde solution at room temperature for 10 min. They were then washed 2-3 times with PBS at room temperature, 10 min each time. The cells were permeabilized with 0.5% Triton X-100 solution at room temperature for 5 min. They were then washed 2-3 times with PBS at room temperature, 10 min each time. 200 μl of TRITC-labeled phalloidin working solution was incubated at room temperature in the dark for 30 min. The cells were washed 3 times with PBS, 5 min each time. The nuclei were stained with 200 μl of 100 nM DAPI solution for approximately 5 min. The cells were washed 3 times with PBS and mounted with mounting medium. Finally, the cells were observed and photographed under a fluorescence microscope. To assess the effect of PDNs treatment on ANGII-induced cell size, phalloidin staining was used to evaluate cell surface area. The results are as follows: Figure 22 As shown, ANGII induction resulted in an increase in the surface area of ​​H9C2 cells compared to the control group. However, treatment with Panax notoginseng exovesicles at concentrations of 10 µg / mL and 20 µg / mL reduced the ANGII-induced increase in cell volume.

[0155] 4. Reactive oxygen species

[0156] The fluorescent probe DCFH-DA was used to detect reactive oxygen species levels in H9C2 cells, and the results are as follows: Figure 23 As shown, ANGII significantly increased ROS levels in H9C2 cells, while treatment with Panax notoginseng exovesicles significantly reduced the increase in ROS. The data indicate that Panax notoginseng exovesicles can inhibit ANGII-induced oxidative stress.

[0157] Example 3

[0158] This embodiment illustrates the role of Panax notoginseng vesicles in myocardial injury caused by overexcitation.

[0159] Wild-type male C57BL / 6 mice (6-8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The experiment lasted 14 days. The blank control group received intraperitoneal injection of physiological saline, while the other model injury groups received intraperitoneal injection of ISO (10 mg / kg / day). Treatment was administered via gavage. After the mice stabilized, they were randomly divided into 5 groups: blank control group (20 mmol / L Tris-HCl), myocardial injury model group (ISO + 20 mmol / L Tris-HCl), positive control group (ISO + lovastatin 1 mg / kg / day), low-dose group (ISO + 5 mg / mL PDNs), and high-dose group (ISO + 10 mg / mL PDNs). After the experiment, the mice were anesthetized with sodium pentobarbital, and their heart tissue was harvested for testing. Body weight was recorded every other day, and serum and heart samples were collected after 14 days. The mouse heart was weighed, and the cardiac index (heart weight (mg) / body weight (g)) was calculated.

[0160] Fresh mouse heart tissue was fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Sections were dewaxed to water and then stained with prepared Weigert's iron hematoxylin staining solution. After thorough staining under a microscope, the tissue was differentiated with acidic ethanol differentiation solution and washed with water. Masson's blue solution was used for restaining, followed by staining with Ponceau S and fuchsin. During the above procedures, a weak acid working solution was prepared at a ratio of distilled water to weak acid solution of 2:1, and the tissue was quickly rinsed with the working solution. The tissue was then washed with phosphomolybdic acid solution, quickly rinsed with the prepared weak acid working solution, and directly immersed in aniline blue staining solution for staining, followed by a quick rinse with the prepared weak acid working solution. Finally, the tissue was dehydrated, cleared, and mounted.

[0161] The results are as follows Figure 24 As shown, Panax notoginseng vesicles can improve myocardial hypertrophy and reduce weight gain in mice. While ISO leads to myocardial fibrosis, Panax notoginseng vesicles can reduce the occurrence of myocardial fibrosis.

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of Panax notoginseng exovesicles in the preparation of a drug for preventing and treating myocardial injury, characterized in that, The particle size of the Panax notoginseng outer vesicle is 200-250 nm, and the Zeta potential value ranges from -32 mV to -14 mV.

2. The use of the Panax notoginseng exovesicle according to claim 1 in the preparation of a drug for preventing and treating myocardial injury, characterized in that, The myocardial injury includes myocardial injury caused by hypertension, obesity or overexcitation. 3.The use of the Panax notoginseng exovesicle according to claim 1 in the preparation of a drug for preventing and treating myocardial injury, characterized in that, The Panax notoginseng outer vesicle is prepared by the following method: (1) Take fresh roots or rhizomes of Panax notoginseng, homogenize at 0 10 ℃, and obtain a crude extract by 400-10,000 g differential centrifugation; (2) The crude extract is centrifuged at 4°C at a speed of not less than 100000 g for 0.1 hours with 1 M sucrose and 2 M sucrose. 2h, take the middle layer liquid to obtain the Panax notoginseng exovesicle.

4. The use of the Panax notoginseng exovesicle according to claim 1 in the preparation of a drug for preventing and treating myocardial injury, characterized in that, The efficacy of the drug at least includes one of the following cases: (1) reducing the amount or activity of lactate dehydrogenase and / or creatine kinase isoenzyme; (2) reducing or inhibiting the effect of oxidative stress; (3) increasing or restoring the mitochondrial membrane potential of cardiomyocytes; (4) reducing or restoring the phosphorylation level and / or protein expression level of cGAS-STING signaling pathway protein; (5) restoring blood lipid metabolism function; (6) reducing or inhibiting myocardial injury; (7) improving or restoring heart function.

5. The use of the Panax notoginseng exovesicle according to claim 4 for preparing a drug for preventing and treating myocardial injury, characterized in that, The cGAS-STING signaling pathway protein includes at least one of cGAS, STING, NF-κB, TBK1 and IRF3.

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