Application of pseudo-ginseng external vesicles in preparation of medicine for preventing or improving fatty liver disease

Abstract

The invention discloses application of pseudo-ginseng external vesicles in preparation of drugs for preventing or improving fatty liver diseases. Researches find that the pseudo-ginseng external vesicles can significantly reduce the levels of total cholesterol, triglyceride, asparaginic acid aminotransferase and alanine aminotransferase, down-regulate expression of fat synthesis related genes and inflammatory factors, improve lipid accumulation caused by fatty liver diseases, reduce body weight and liver-to-weight ratio, repair pathological damage of liver tissues and the like.

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, treatment or improvement of fatty liver disease. Background Technology

[0002] Fatty liver disease, such as metabolic dysfunction-associated fatty liver disease (MASLD), is a common chronic liver disease. In recent years, with the continuous rise in the global incidence of obesity and type 2 diabetes, the prevalence of fatty liver disease has also been increasing. Fatty liver disease is mainly caused by the pathological changes resulting from the accumulation of ectopic fat in hepatocytes. MASLD may further develop into metabolic dysfunction-associated steatohepatitis (MASH), a process that exacerbates liver inflammation and fibrosis, leading to cirrhosis, hepatocellular carcinoma, and extrahepatic complications such as atherosclerotic cardiovascular disease, chronic kidney disease, and osteoporosis. During MASH, the accumulation of large amounts of lipids mediates endoplasmic reticulum stress, oxidative stress, organelle dysfunction, gut microbiota dysbiosis, and ferroptosis, triggering lipotoxicity.

[0003] Currently, lifestyle interventions, including diet and exercise, are the preferred treatment for fatty liver disease. In many countries, the annual medical costs associated with this disease have reached hundreds of billions of dollars, demonstrating the significant health and socioeconomic burden it places on the world. However, due to the diverse mechanisms involved in fatty liver disease and the difficulties in conducting clinical trials, there is currently no universally accepted drug treatment.

[0004] Therefore, there is an urgent need to develop a new drug to prevent or improve fatty liver disease. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides the use of Panax notoginseng vesicles in the preparation of drugs for the prevention and treatment or improvement of fatty liver disease. Specifically, the present invention includes the following.

[0006] In a first aspect, the present invention provides the use of Panax notoginseng vesicles in the preparation of a medicament for the prevention or improvement of fatty liver disease.

[0007] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention or improvement of fatty liver disease, wherein the fatty liver disease includes fatty liver disease associated with metabolic dysfunction.

[0008] In some embodiments, the application of the Panax notoginseng vesicles according to the present invention in the preparation of drugs for preventing or improving fatty liver disease, wherein the Panax notoginseng vesicles are prepared by the following method: (1) Take fresh roots or rhizomes of Panax notoginseng, homogenize them, and then centrifuge them at a differential speed to obtain crude extract; (2) The crude extract was purified by density gradient centrifugation to obtain the Panax notoginseng exovesicles.

[0009] In some embodiments, the application of the Panax notoginseng vesicles according to the present invention in the preparation of drugs for the prevention or improvement of fatty liver disease, 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.

[0010] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of a medicament for the prevention or improvement of fatty liver disease is described, wherein the prevention or improvement is achieved by administering a therapeutically effective amount of the medicament to a subject.

[0011] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention or improvement of fatty liver disease, wherein the subjects include mammals.

[0012] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of medicaments for the prevention or improvement of fatty liver disease, wherein the mammals include humans.

[0013] In some embodiments, the use of Panax notoginseng vesicles according to the present invention in the preparation of drugs for the prevention and treatment or improvement of fatty liver disease, wherein the therapeutically effective amount of the Panax notoginseng vesicles is 0.1-500 mg / Kg.

[0014] A second aspect of the present invention provides a method for regulating hepatocyte function in vitro, comprising the step of contacting hepatocytes with Panax notoginseng exovesicles in vitro.

[0015] In some embodiments, the method for in vitro regulation of hepatocyte function according to the present invention includes at least one of the following: (1) Improve or alleviate cell damage caused by fat; (2) Reduce lipid accumulation in cells; (3) Reduce the levels of total cholesterol, triglycerides, aspartate aminotransferase and / or alanine aminotransferase in cells; (4) Downregulate the expression of lipid synthesis-related genes and / or inflammatory factors.

[0016] This invention has found that Panax notoginseng exovesicles can significantly reduce the levels of total cholesterol, triglycerides, aspartate aminotransferase and alanine aminotransferase, downregulate the expression of lipid synthesis-related genes and inflammatory factors, improve lipid accumulation caused by fatty liver disease, reduce body weight and liver weight ratio, reduce fat content, and repair pathological damage to liver tissue. Attached Figure Description

[0017] Figure 1 The effects of different concentrations of Panax notoginseng exovesicles on cell viability were shown.

[0018] Figure 2 The effect of Panax notoginseng exovesicles on the viability of palmitic acid-damaged cells was shown. PDNs-L represents low dose of Panax notoginseng exovesicles, PDNs-M represents medium dose of Panax notoginseng exovesicles, and PDNs-H represents high dose of Panax notoginseng exovesicles.

[0019] Figure 3 The results of Oil Red O detection in AML12 cells are shown.

[0020] Figure 4 The results of TG detection in AML12 cells are shown.

[0021] Figure 5 The results of lipid synthesis-related transcription factor detection in AML12 cells are shown, where A represents the relative mRNA expression level of ACCα and B represents the relative mRNA expression level of FAS.

[0022] Figure 6 The results of inflammatory factor detection in AML12 cells are shown.

[0023] Figure 7 The results show that Panax notoginseng vesicles can reduce the body weight of mice with metabolic dysfunction-related fatty liver disease. In the figure, A represents the body weight test result, and B represents the liver / body weight test result.

[0024] Figure 8 The results show that Panax notoginseng exovesicles (PDNs) reduced the fat and free fatty acid content in mice with metabolic dysfunction-related fatty liver disease. In the figures, A represents the results of MRI, B represents the results of fat content detection, and C represents the results of free fatty acid detection.

[0025] Figure 9 The results of lipid metabolism and liver function indicators in the serum of model mice are shown, where AD represents the detection results of TC, TG, ALT and AST, respectively.

[0026] Figure 10 The results of lipid metabolism and liver function indicators in the liver tissue of model mice are shown. AD represents the detection results of TC, TG, ALT and AST, respectively.

[0027] Figure 11 The results of H&E staining in the liver of the model mouse are shown.

[0028] Figure 12 The results of Oil Red O staining of the liver of the model mouse are shown. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 conditions associated with fatty liver disease: reducing weight gain and liver-to-body weight ratio caused by fatty liver disease; reducing fat content caused by fatty liver disease; reducing elevated levels of total cholesterol, triglycerides, aspartate aminotransferase, and / or alanine aminotransferase caused by fatty liver disease; improving or alleviating fat-induced cell damage; downregulating the expression of lipid synthesis-related genes and / or inflammatory factors; and repairing pathological liver tissue damage caused by fatty liver disease.

[0033] application One aspect of the present invention provides the use of Panax notoginseng vesicles in the preparation of a medicament for the prevention, treatment, or improvement of fatty liver disease. In a preferred embodiment, the fatty liver disease is metabolic dysfunction-related fatty liver disease.

[0034] In this invention, Panax notoginseng exovesicles are nanoscale membrane-structured vesicles actively secreted by Panax notoginseng, 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.

[0035] In a preferred embodiment, the Panax notoginseng exovesicles of the present invention are prepared by the following method: (1) Take fresh roots or rhizomes of Panax notoginseng, homogenize them, and then centrifuge them at a differential speed to obtain crude extract; (2) The crude extract was purified by density gradient centrifugation to obtain the Panax notoginseng exovesicles.

[0036] In a further preferred embodiment, the Panax notoginseng exovesicles of the present invention are prepared by the following method: (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. (2) The crude extract was enriched with 1 M-2 M sucrose to obtain the Panax notoginseng exovesicles.

[0037] 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 (0-10°C) 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). 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.

[0038] 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.

[0039] In step (2) of the present invention, enriching the crude extract with 1 M-2 M sucrose means subjecting the crude extract to low-temperature (0-10°C) high-speed centrifugation (preferably 4°C, not less than 100,000 g, for example not less than 110,000, 120,000, 130,000, 140,000, 150,000, or even greater than 150,000 g) for a centrifugation time of 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.

[0040] 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).

[0041] 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.

[0042] In a preferred embodiment, the medicament further comprises a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is involved in transporting or delivering the medicament 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 carrier includes 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.

[0043] In this invention, fatty liver disease is prevented or improved 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.

[0044] 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.

[0045] The effective dose for prevention and treatment described in this invention refers to a pharmaceutically recognized effective dosage, meaning the amount of the 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 exovesicles is typically 0.1-500 mg / kg, preferably 1-450 mg / kg, even more preferably 5-400 mg / kg, further preferably 10-350 mg / kg, more preferably 15-300 mg / kg, more preferably 20-250 mg / kg, and even more preferably 20-200 mg / kg. mg / Kg, for example 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 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, 1 18, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200 mg / kg. It can be administered as a single daily dose, divided into multiple daily doses, or at intervals.

[0046] method One aspect of the present invention provides a method for in vitro regulation of hepatocyte function, comprising the step of contacting hepatocytes with Panax notoginseng exovesicles in vitro. The regulation includes, but is not limited to, the following: improving or alleviating adipose-induced cell damage; reducing lipid accumulation in cells; lowering the levels of total cholesterol, triglycerides, aspartate aminotransferase, and / or alanine aminotransferase in cells; and downregulating the expression of lipid synthesis-related genes and / or inflammatory factors. The method of the present invention is a non-therapeutic method and can be used for drug screening, drug structure optimization, and disease mechanism research.

[0047] In a preferred embodiment, the method for in vitro regulation of hepatocyte function according to the present invention includes: (1) culturing hepatocytes to construct a fatty liver disease cell model; (2) contacting the fatty liver disease cell model with Panax notoginseng exovesicles in vitro; and (3) detecting the function of the fatty liver disease cell model, such as, but not limited to, the levels of total cholesterol, triglycerides, aspartate aminotransferase and / or alanine aminotransferase in the cells, cell damage, lipid accumulation in the cells, and expression of lipid synthesis-related genes and / or inflammatory factors. In this invention, the detection of hepatocyte function can be performed using methods and apparatus known in the art, and is not particularly limited thereto.

[0048] Example This embodiment illustrates the preparation and application of Panax notoginseng exovesicles.

[0049] 1. Preparation and characterization of Panax notoginseng exovesicles 1.1 Preparation method Take an appropriate amount of fresh Panax notoginseng, wash it clean with distilled water, and air dry it. Cut the Panax notoginseng into slices 3-5 cm thick, weigh them, and homogenize them 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 (1 mM leucopeptide, 100 mM PMSF, and 1 M sodium azide). Then adjust the pH of the Panax notoginseng homogenate to 7.0 with 1M Tris-HCl. Centrifuge at 4℃, 400 g, 800 g, and 10000 g for 20 min each time, and collect the supernatant. Centrifuge at 4℃ (150000 g) for 1.5 h to collect the precipitate. Dissolve the precipitate with 20 mM Tris-HCl to obtain crude Panax notoginseng extract. The crude extract of Panax notoginseng was centrifuged together with 1 M sucrose and 2 M sucrose (4℃, 150,000 g, 90 min) to collect the intermediate liquid. This intermediate liquid was then centrifuged with 20 mM Tris-HCl (4℃, 100,000 g, 60 min). The sucrose was washed off to obtain purified protein dendritic cells (PDNs), which were stored at -80℃. The concentration of PDNs was determined using a BCA protein concentration assay kit.

[0050] 1.2 Characterization The particle count of Panax notoginseng exovesicle solution was determined by NTA. The particle count of Panax notoginseng exovesicles purified with 1-2 M sucrose was 5*10⁻⁶ particles. 12 particles / mL.

[0051] The particle size and potential of the Panax notoginseng exovesicles were measured. The average diameter of the exovesicles was 213 nm, and the zeta potential ranged from -22.0 ± 8.04 mV.

[0052] Analysis of the saponin composition in the outer vesicles of Panax notoginseng showed that the saponin content of ginsenoside Rb1 was 1 μg / mg, the saponin content of ginsenoside Rd was 0.5 μg / mg, and the total saponin content was 0.15%, indicating that the outer vesicles of Panax notoginseng contain only trace amounts of saponin components.

[0053] 2. Experimental Methods 2.1 AML12 Cytotoxicity Assay AML12 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were cultured in a dedicated culture medium at 37°C in a 5% CO2 incubator. During the logarithmic growth phase, the cells were injected at a rate of 1.2 × 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 (2.5, 5, 10, 20, 40, 80, 100, 200 μg / ml) were added. An in vitro liver injury model was established using 120 μM palmitic acid. The cell treatment groups were divided into 5 groups: a blank control group (Control), a model group (120 μM palmitic acid), and PDNs-treated groups (10, 20, 40 μg / ml, denoted as PDNs-L, PDNs-M, and PDNs-H, respectively). 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.

[0054] 2.2 Oil Red O Detection in AML12 Cells Oil Red O is a lipid-soluble azo dye with strong lipophilicity. It can physically bind to and dissolve in intracellular neutral lipids (such as triglycerides and cholesterol esters), thus giving the lipid droplets a bright red or orange-red color. AML12 cells, 24 × 10⁻⁶ 4Cells were seeded in 6-well plates and cultured for 24 h, then divided into 5 groups: a blank control group, a model group, and a PDNs-treated group, and incubated together for 24 h. The culture medium was discarded, and the cells were gently washed 2-3 times with PBS to remove serum and dead cells. Sufficient 10% paraformaldehyde was added, and the cells were fixed at room temperature for 10 minutes. The fixative was discarded, and the cells were washed twice with PBS. Staining was performed using the Beyotime Oil Red O staining kit, and the cells were observed directly under an optical microscope. The lipid droplets appeared bright red or orange-red.

[0055] 2.3 Detection of AML12-TC and TG AML12 cells 2.4 × 10 5 Cells were seeded per well in 6-well plates and cultured for 24 h. The cells were divided into 5 groups: a control group, a model group, and a PDNs-treated group, all incubated for 24 h. The levels of TC and TG in AML12 cells were detected using a kit, and the results were calculated according to the kit instructions.

[0056] 2.4 qRT-PCR Cell processing followed the same method as Oil Red O detection. After cell culture, the culture medium was discarded, cells were washed with PBS, and lysis buffer (TRIzol) was added directly for lysis. RNA was extracted, and high-quality RNA was reverse transcribed into cDNA. Different primers were added, and amplification was performed using a real-time quantitative PCR instrument. -ΔΔCt The expression levels of each group were calculated using quantitative methods.

[0057] 2.5 Animal Experiments Wild-type male C57BL / 6 mice (6-8 weeks old) 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. They had free access to food and water. The experiment lasted for 16 weeks. In the first 8 weeks, the mice were randomly divided into five groups: control group: fed with a low-fat diet with 10% fat content; model group, positive drug group, low-dose PDNs group, and high-dose PDNs group: fed with a high-fat diet with 60% fat content; all were given ordinary tap water. Starting in week 9, mice were administered 0.2 ml of oral gavage daily. The treatment groups included a control group (20 mM Tris-HCl), a model group (20 mM Tris-HCl), a positive control group (3 mg / ml atorvastatin calcium tablets), a low-dose group (5 mg / ml PDNs, denoted as PDNs-L), and a high-dose group (10 mg / mL PDNs, denoted as PDNs-H). This continued until week 16, with weekly body weight recording. At the end of week 16, liver weight was recorded, and serum and tissue samples were collected for relevant testing.

[0058] 2.6 Effects of PDNs on Fat Distribution in MASLD Mice During week 16 of a high-fat diet, mice were anesthetized with isoflurane gas and placed in a small animal MRI system to detect the distribution of fat throughout the body and perform body composition analysis.

[0059] 2.7 Detection of free fatty acids in MASLD mice by PDNs After modeling was performed according to the method in Section 2.5, livers were collected, and the content of free fatty acids in mouse livers was detected using a kit.

[0060] 2.8 Detection of TG, TC, AST and ALT levels in mice using the kit After establishing the model as described in Section 2.5, liver cells were collected, and blood was obtained by collecting blood from the eye. The blood was collected in centrifuge tubes and allowed to stand at room temperature (or 4°C) for 2 hours to allow it to coagulate naturally. Then, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C. The pale yellow liquid on top was the serum, which was carefully aspirated into a new EP tube and tested and calculated according to the kit.

[0061] 2.9 HE staining of mouse liver After the mice were sacrificed, their livers were removed, fixed in paraformaldehyde, and then paraffin sections were prepared, dewaxed, rehydrated, stained with hematoxylin and eosin (HE), dehydrated and cleared, mounted, and observed and photographed under a microscope.

[0062] 2.10 Oil Red O staining of mouse liver Oil Red O is a lipid-soluble azo dye with a high affinity for neutral lipids (such as triglycerides and cholesterol esters) in tissues, dissolving them and giving the lipid droplets a bright red color. After euthanizing mice, liver tissue was quickly removed and rinsed thoroughly with pre-cooled physiological saline. The tissue was cut into pieces approximately 0.5 cm thick. Immediately, the tissue was placed in 4% paraformaldehyde and fixed at 4°C for 6–12 hours. After fixation, the fixative was rinsed off with PBS. The tissue was then sequentially immersed in 15% sucrose solution and 30% sucrose solution until it settled, followed by embedding and sectioning. Oil Red O working solution was prepared, and the dried sections were rinsed in 60% isopropanol for approximately 1 minute. The sections were then immersed in freshly prepared and filtered Oil Red O working solution and stained in the dark for 10 minutes. The sections were then rapidly transferred to 60% isopropanol for differentiation. Microscopic examination was maintained until the background was essentially colorless. Gently rinse the slides with a fine stream of running water for about 1 minute to thoroughly wash away isopropanol and excess stain. Counterstain the cell nuclei with hematoxylin for about 1-2 minutes. Let the slides return to blue under running water, then mount them with glycerol gelatin. Observe and photograph them immediately under an optical microscope.

[0063] 2.11 Statistical Methods All data represent at least three independent experiments and are expressed as mean ± SEM. Statistical computations 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 indicates a statistically significant difference.

[0064] 3. Experimental Results The concentration of PDNs protein was determined using a BCA kit. AML12 cells in logarithmic growth phase were used, and the concentration was determined at 1.2 × 10⁻⁶. 4 Cells were seeded per well in 96-well plates and cultured for 24 h. Different concentrations of PDNs were then added and co-cultured with AML12 cells for another 24 h to detect their cytotoxicity against AML12 cells. The results are as follows: Figure 1 As shown, different concentrations of the drug did not exhibit cytotoxicity within a certain range. Therefore, 10, 20, and 40 μg / ml were selected as the low, medium, and high concentrations for the drug treatment group in the cell experiments.

[0065] Next, palmitic acid and different concentrations of PDNs (10, 20, 40 μg / ml) were co-incubated with AML12 for 24 h. CCK-8 solution was added at a ratio of 10:1, and the cells were incubated in a cell culture incubator for 1 h. The absorbance was measured at 450 nm, and the relative growth rate of each group of cells was calculated. The results are as follows: Figure 2 As shown, PDNs can repair palmitic acid-induced damage to AML12 cells.

[0066] 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 PDNs; blank control group: cell culture without Panax notoginseng-derived extracellular vesicles; blank group: no cell culture, only culture medium).

[0067] The results are as follows Figure 3 As shown, no red lipid droplets were observed in the blank control group, and the cell morphology was normal. Compared with the blank control group, a large number of bright red lipid droplets appeared in the model group cells, with a significant increase in the number and size of the droplets, which were diffusely distributed in the cytoplasm. The experimental results indicate that palmitic acid successfully induced lipid accumulation in hepatocytes, establishing a hepatocyte lipid accumulation model. Under the action of PDNs, the number and area of ​​red lipid droplets were significantly reduced compared with the palmitic acid model group.

[0068] The results of the TG test are as follows Figure 4As shown, palmitic acid can cause an increase in triglyceride (TG) levels. After administration of PDNs, it was found that they significantly reduced triglyceride levels in AML12 cells. The results indicate that PDNs can alleviate palmitic acid-induced triglyceride accumulation in AML12 cells.

[0069] Results of lipid synthesis-related transcription factor detection: Figure 5 As shown, palmitic acid treatment significantly increased the expression of intracellular lipid synthesis-related genes, indicating that palmitic acid leads to lipid accumulation in hepatocytes. After administration of PDNs, the levels of related transcription factors decreased, therefore, PDNs reduced the expression of palmitic acid-induced lipid synthesis genes in AML12 cells at the transcriptional level.

[0070] The expression of inflammatory factors in AML12 cells is as follows: Figure 6 As shown, palmitic acid significantly increased the level of the inflammatory factor IL-6, and PDNs reduced the expression level of palmitic acid-induced inflammation-related genes in AML12 cells at the transcriptional level.

[0071] This embodiment constructs an in vivo high-fat diet model, and the results are as follows: Figure 7 As shown, the body weight and liver weight ratio of mice in the model group increased, while PDNs treatment reduced the increase in body weight and the liver-to-body weight ratio.

[0072] The results of the body fat content in mice are as follows Figure 8 As shown, abnormal fat accumulation occurred in the model group mice. Administration of PDNs significantly reduced fat accumulation in the model group mice. The red areas in the figure represent the locations of fat deposits. PDNs reduced the fat content in mice with metabolic dysfunction-related fatty liver disease in the model group. Simultaneously, free fatty acids significantly increased after a high-fat diet, and PDNs treatment reduced the model-induced increase in FFA.

[0073] To investigate the effects of a high-fat diet on lipid metabolism in mice, this study measured the levels of TC, TG, AST, and ALT in serum and liver tissue of mice fed a high-fat diet for 16 weeks. The results are as follows: Figure 9 and Figure 10 As shown, compared with the normal control group, the serum and liver function indicators of mice in the high-fat diet group were higher than those in the normal control group. After administration of PDNs, the levels of TC, TG, AST and ALT were reduced, indicating that PDNs can improve abnormal liver function in mice.

[0074] H&E results are as follows Figure 11As shown, compared with the control group, the model group showed sparse cytoplasm, mild edema, and vacuolar degeneration of hepatocytes; fatty degeneration of hepatocytes was observed in the liver tissue, manifested as round vacuoles of varying sizes in the cytoplasm. Administration of PDNs reduced vacuolar degeneration and fatty degeneration of hepatocytes, repairing the pathological damage to liver tissue induced by a high-fat diet.

[0075] To further demonstrate the protective effect of PDNs against fatty liver disease, this embodiment used Oil Red O for lipid accumulation analysis, and the results are as follows: Figure 12 As shown, compared to the blank control group, the model group showed a large accumulation of red lipid droplets, while the PDNs-treated group showed a significant reduction in red lipid droplets. These results indicate that PDNs can reduce fat accumulation in liver tissue induced by a high-fat diet.

[0076] 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 application of Panax notoginseng vesicles in the preparation of drugs for the prevention, treatment, or improvement of fatty liver disease, characterized in that, The size of the Panax notoginseng vesicles is 200-250 nm, and the Zeta potential ranges from -40 mV to -10 mV.

2. The application of the Panax notoginseng vesicles according to claim 1 in the preparation of drugs for preventing or improving fatty liver disease, characterized in that, The fatty liver disease mentioned includes fatty liver disease associated with metabolic dysfunction.

3. The application of the Panax notoginseng vesicles according to claim 1 in the preparation of drugs for preventing or improving fatty liver disease, characterized in that, The Panax notoginseng exovesicles are prepared by the following method: (1) Take fresh roots or rhizomes of Panax notoginseng, homogenize them, and then centrifuge them at a differential speed to obtain crude extract; (2) The crude extract was purified by density gradient centrifugation to obtain the Panax notoginseng exovesicles.

4. The application of the Panax notoginseng vesicles according to claim 1 in the preparation of drugs for preventing or improving fatty liver disease, characterized in that, The drug further includes a pharmaceutically acceptable carrier.

5. The application of the Panax notoginseng vesicles according to claim 1 in the preparation of drugs for preventing or improving fatty liver disease, characterized in that, The prevention or improvement is achieved by administering a therapeutically effective dose of the drug to the subject, wherein the therapeutically effective dose of the Panax notoginseng vesicles is 0.1-500 mg / Kg.

6. The application of the Panax notoginseng vesicles according to claim 5 in the preparation of drugs for preventing or improving fatty liver disease, characterized in that, The subjects included mammals.

7. The application of the Panax notoginseng vesicles according to claim 6 in the preparation of drugs for preventing or improving fatty liver disease, characterized in that, The mammals mentioned include humans.

8. A method for regulating hepatocyte function in vitro, characterized in that, It includes the step of bringing hepatocytes into contact with Panax notoginseng exovesicles in vitro.

9. The method for in vitro regulation of hepatocyte function according to claim 8, characterized in that, The regulation includes at least one of the following situations: (1) Improve or alleviate cell damage caused by fat; (2) Reduce lipid accumulation in cells; (3) Reduce the levels of total cholesterol, triglycerides, aspartate aminotransferase and / or alanine aminotransferase in cells; (4) Downregulate the expression of lipid synthesis-related genes and / or inflammatory factors.

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