Extracellular vesicles derived from poria cocos and use thereof in development and preparation of drugs for preventing or treating metabolic-related steatohepatitis

Abstract

This invention relates to the field of biomedical technology, disclosing extracellular vesicles derived from Poria cocos and their application in the development and preparation of drugs for the prevention or treatment of metabolic-associated steatohepatitis (MAS). The drugs improve liver tissue morphology and pathological lesions, regulate serum and liver lipid levels and inflammatory factor levels, and regulate mitochondrial structure and function, including alleviating mitochondrial ultrastructural damage, increasing the number of autophagosomes, improving oxidative stress and energy metabolism, promoting the expression of mitochondrial autophagy-related proteins, and reducing the production and efflux of Ox-mtDNA, thereby inhibiting the expression of NLRP3 inflammasome-related proteins. These drugs can improve MAS and provide a new direction for the development and research of drugs for the prevention and treatment of MAS.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to extracellular vesicles derived from Poria cocos and their application in the development and preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Non-alcoholic steatohepatitis (MASH) is the second leading cause of liver disease in the 21st century, and safe and effective drugs are still lacking. Therefore, the development of preventative and therapeutic drugs is of great significance. Current clinical treatment for MASH aims to prevent, delay, or reverse its histological changes and reduce the incidence of diseases such as cirrhosis. Treatment typically involves single-target drugs such as hepatoprotective agents, lipid-lowering drugs, insulin sensitizers, antioxidants, and anti-inflammatory drugs to treat MASH symptoms and improve related conditions.

[0004] Extracellular vesicles (EVs) are membrane-bound vesicles released by cells of prokaryotes, higher eukaryotes, and plants in an evolutionarily conserved manner. EVs possess a variety of significant pharmacological activities, offering insights for new drug development. Foreign literature, such as "Advanced research on extracellular vesicles based oral drug delivery systems," indicates that EVs can transport various cell-specific bioactive substances, including lipids, nucleic acids, and various proteins, from parent cells to other cells. As nanoscale vesicles, although some reports suggest the possible presence of homologous active small molecules within EVs, their trace amounts make it difficult to explain why the biological activity of EVs is clearly not solely related to homologous active small molecules. The main active ingredients in Rehmannia glutinosa are catalpol and verbascoside. Exosomes, catalpol, and verbascoside derived from Rehmannia glutinosa all have strong protective effects against LPS-induced acute lung inflammation. However, the protective effect of exosomes derived from Rehmannia glutinosa is stronger than that of catalpol and verbascoside. Furthermore, miR-7972 is the main component of exosomes derived from Rehmannia glutinosa.

[0005] The composition of EVs from different plant sources varies significantly, which may lead to different biological activities. Current research on EVs mainly focuses on their nucleic acids and proteins. Existing literature, "Methylation as an Acrucial Step in Plant MicroRNA Biogenesis," indicates that plant miRNAs can target genes in mammalian genomes, thus playing a role in cross-species communication. miR-7972, a novel plant exosomal miRNA extracted from fresh Rehmannia glutinosa, can improve lipopolysaccharide-induced acute lung injury and intestinal dysbiosis. There are significant differences in miRNAs among EVs from different sources. Existing literature, "Identification of Exosome-like Nanoparticle-Derived MicroRNAs from 11 Edible Fruits and Vegetables," shows that many edible plant-derived EVs possess anti-inflammatory properties. Transcriptome analysis of EVs from 11 different fruit and vegetable sources revealed that although EVs contain many miRNAs, the types and levels of miRNAs vary significantly between species. Proteins embedded on the exterior of EVs can bind to receptors on the surface of target cells, generating corresponding signals and exerting their effects; proteins encapsulated within EVs can be transported into target cells, thereby regulating cellular function and biological behavior. Proteins also differ among EVs from different sources. Lipids, as components of EVs, play a crucial role in maintaining their structure and function. The types and amounts of lipids in EVs from different sources also vary significantly. Grape-derived EVs are most abundant in phosphatidic acid (PA), which plays a role in cell proliferation and signal transduction; followed by phosphatidylethanolamine (PE), which regulates cell membrane curvature and plays a key role in cell membrane division and fusion. Ginger-derived EVs, in addition to PA, also contain digalactosyldiglyceride and monogalactosyldiglyceride, with small amounts of phosphatidylglycerol and PE remaining. Therefore, EVs can be considered as biologically active vesicles composed of different lipids, proteins, and nucleic acids, and their biological activity is mainly related to the lipids, nucleic acids, and various proteins they contain.

[0006] Poria cocos (Schw.) Wolf, a fungus belonging to the Polyporaceae family, is a dried sclerotium with diuretic, spleen-strengthening, and mind-calming properties. It is commonly used for spleen deficiency, edema, and restlessness. It is sweet and bland in nature and enters the heart, lung, spleen, and kidney meridians. Poria cocos' ability to promote diuresis and strengthen the spleen can effectively improve NASH symptoms. Clinically, it is frequently used to treat NASH caused by spleen deficiency and internal dampness, making it a frequently used traditional Chinese medicine for the prevention and treatment of NASH. Modern pharmacological studies have shown that it has pharmacological effects such as treating obesity, lowering blood lipids, protecting the liver, and anti-inflammation. However, whether Poria cocos-derived extracellular vesicles (PC-EVs) have an intervention effect on metabolic-related fatty liver disease remains unclear. Those skilled in the art cannot determine whether EVs derived from Poria cocos can treat the disease. The inventors of this application have, for the first time, demonstrated that EVs derived from Poria cocos can effectively alleviate MASH induced by a high-fat diet. Summary of the Invention

[0007] The purpose of this invention is to address the current lack of therapeutic drugs for non-alcoholic steatohepatitis (NAH). It provides extracellular vesicles derived from Poria cocos and their application in the preparation of drugs for the prevention or treatment of metabolic-related steatohepatitis, thus offering new therapeutic drugs and approaches for NHA and broadening the channels for NHA treatment.

[0008] The technical solution of the present invention is as follows:

[0009] This invention provides an extracellular vesicle derived from Poria cocos, which, under electron microscopy, is an irregular spherical vesicle with a double membrane structure, a particle size of 100-200 nm, a potential of -3.20±0.37 mV, and a protein distribution of 180-140 kDa, 80-45 kDa, and 35-25 kDa; it can improve metabolic-associated fatty liver disease.

[0010] According to a preferred embodiment, the steps include:

[0011] Peel and wash fresh Poria cocos, slice it, add 2-3 times the amount of pure water, juice it, collect the juice, centrifuge the collected juice at 1000×g for 10 min at 4℃; take the supernatant, centrifuge at 3000×g for 20 min at 4℃; take the supernatant, centrifuge at 10000×g for 30 min at 4℃; take the supernatant, centrifuge at 150000×g for 2 h at 4℃; discard the supernatant, add an appropriate amount of PBS, suspend it, and filter it through a 0.22μm filter to remove bacteria, thus obtaining Poria cocos-derived extracellular vesicles.

[0012] Another aspect of the present invention provides the use of Poria cocos-derived extracellular vesicles as described above in the development and preparation of medicaments for the prevention or treatment of metabolic-related steatohepatitis.

[0013] According to a preferred embodiment, the drug improves liver tissue morphology and pathological lesions, and regulates serum and liver lipid levels and inflammatory factor levels.

[0014] According to a preferred embodiment, the drug modulates mitochondrial structure and function, including alleviating damage to mitochondrial ultrastructure, increasing the number of autophagosomes, and improving oxidative stress and energy metabolism.

[0015] According to a preferred embodiment, the drug promotes the expression of mitophagy-related proteins and reduces the production and efflux of Ox-mtDNA.

[0016] According to a preferred embodiment, the drug inhibits the expression of NLRP3 inflammasome-related protein.

[0017] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic-associated steatohepatitis, characterized in that it comprises extracellular vesicles derived from Poria cocos as described above.

[0018] Compared with existing technologies, the advantages of this invention are:

[0019] 1. The application of extracellular vesicles derived from Poria cocos in the preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease. This is the first time that extracellular vesicles have been obtained from a fungal traditional Chinese medicine (Poria cocos).

[0020] 2. Application of Poria cocos-derived extracellular vesicles in the preparation of drugs for the prevention or treatment of metabolic-associated steatohepatitis: It was found that Poria cocos-derived extracellular vesicles can improve metabolic-associated steatohepatitis induced by a high-fat diet; it was revealed that PC-EVs regulate the PINK1 / Parkin pathway to mediate mitophagy, improve mitochondrial function, reduce Ox-mtDNA production and efflux, thereby inhibiting NLRP3 inflammasome activation and alleviating high-fat diet-induced MASH;

[0021] 3. The application of extracellular vesicles derived from Poria cocos in the preparation of drugs for the prevention or treatment of metabolic-related fatty liver disease is beneficial because the raw materials are widely available, the extraction method is simple, and large-scale preparation is possible. Attached Figure Description

[0022] Figure 1 The morphology of PC-EVs extracted in this application is shown under a transmission electron microscope.

[0023] Figure 2 Figure 1 shows the particle size and zeta potential analysis data of PC-EVs extracted in this application; Figure 2 shows the particle size distribution of the nanoparticles through tracking analysis; Figure 3 shows the particle size distribution of the nanoparticles through dynamic light scattering analysis; Figure 4 shows the zeta potential of the nanoparticles through dynamic light scattering analysis.

[0024] Figure 3The protein distribution of PC-EVs extracted in this application;

[0025] Figure 4 The lipid distribution of PC-EVs extracted in this application;

[0026] Figure 5 A shows the ingestion of PC-EVs in mice and their distribution in their organs; A is a graph showing the ingestion of PC-EVs in mice; B is a graph showing the distribution of PC-EVs in the organs of mice.

[0027] Figure 6 The effects of PC-EVs on body weight and food intake in MASH mice are shown in Figure A; Figure B shows the changes in mouse body weight; Figure C shows the average food intake of mice in the last week.

[0028] Figure 7 The effects of PC-EVs on the organs and adipose index of MASH mice are shown; where AE represents the index of each organ in mice; and FH represents the adipose index of each mouse.

[0029] Figure 8 The effects of PC-EVs on the morphology and pathology of liver tissue in MASH mice induced by a high-fat diet are shown in Figure A; Oil Red O staining image; Oil Red O staining quantitative statistical graph of lipid droplets; and H&E staining image.

[0030] Figure 9 The effects of PC-EVs on serum lipid metabolism-related indicators in MASH mice;

[0031] Figure 10 The effects of PC-EVs on the levels of TC, TG, ALT, AST, HDL-C, and LDL-C in the liver of MASH mice;

[0032] Figure 11 The effects of PC-EVs on serum and liver levels of TNF-α, IL-6, IL-1β and IL-18 in MASH mice;

[0033] Figure 12 The effects of PC-EVs on SOD, GSH, MDA and ROS in the liver of MASH mice;

[0034] Figure 13 The effects of PC-EVs on the activities of hepatic ATP synthase, Complex I, and Complex II in MASH mice;

[0035] Figure 14 The effects of PC-EVs on liver mitochondrial structure and autophagosomes (indicated by arrows) in MASH mice;

[0036] Figure 15The effect of PC-EVs on NLRP3 inflammasome-related proteins in MASH mice; where A represents mitochondrial-related proteins; BE represents quantitative data on mitochondrial autophagy-related proteins.

[0037] Figure 16 The effects of PC-EVs on Ox-mtDNA in the liver cytoplasm and mitochondria of MASH mice are shown in Figure A; Figure B shows the Ox-mtDNA content in the liver cytoplasm.

[0038] Figure 17 The effect of PC-EVs on NLRP3 inflammasome-related proteins in MASH mice is shown in Figure A; BE shows the quantitative figures of NLRP3 inflammasome-related proteins. Detailed Implementation

[0039] It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. All experimental materials used in the following embodiments are commercially available. In the quantitative experiments in the following embodiments, three replicates were performed, and the results were averaged.

[0040] Data from each group are expressed as mean ± standard deviation. GraphPad Prism 7 statistical software was used for data analysis. One-Way ANOVA was used for comparisons between groups, and Dunnet's t-test was used for pairwise comparisons among multiple groups. *P < 0.05 was considered statistically significant.

[0041] The Poria cocos used in this experiment was purchased from Pu'er, Yunnan Province, and identified by Professor Yu Jie as the dried sclerotium of the fungus *Poriacocos* (Schw.) Wolf, belonging to the genus *Poria* of the family Polyporaceae. The sample is preserved in the Key Laboratory of Traditional Chinese Medicine Prevention and Treatment of Metabolic Diseases at Yunnan University of Traditional Chinese Medicine.

[0042] Meaning of abbreviations:

[0043]

[0044]

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] In each data graph, NC represents the blank control group, MOD represents the model group, FC represents the fenofibrate capsule group, LPC-EVs represents the low-dose group (10 μg / mL), MPC-EVs represents the medium-dose group (50 μg / mL), and HPC-EVs represents the high-dose group (100 μg / mL).

[0047] Example 1: Separation and Characterization of PC-EVs

[0048] 1. Source of experimental reagents:

[0049]

[0050]

[0051] 2. Source of experimental equipment:

[0052] XPN-100 Ultraspeed Centrifuge Beckman Company, USA BS224 Electronic Balance Beijing Sellis Instrument Systems Co., Ltd. Allegra X-30 High-Speed ​​Centrifuge Beckman Company, USA 90Plus PALS Laser Particle Size Analyzer Brookhaven Company, USA ZetaVIEW Nanoparticle Tracking Analyzer Particle Metrix, a German company JEM-F200 Transmission Electron Microscope JEOL Corporation of Japan <![CDATA[PowerPac TM Basic type electrophoresis apparatus BioRad, Inc. (USA) LNG-T88 Benchtop Rapid Centrifugal Concentrator and Dryer Taicang Huamei Biochemical Instrument Factory

[0053] 3. PC-EVs isolation: Peel and wash fresh Poria cocos, slice it, add 2-3 times the amount of pure water, juice it, collect the juice, centrifuge the collected juice at 1000×g for 10 min at 4℃; take the supernatant, centrifuge at 3000×g for 20 min at 4℃; take the supernatant, centrifuge at 10000×g for 30 min at 4℃; take the supernatant, centrifuge at 150000×g for 2 h at 4℃; discard the supernatant, add an appropriate amount of PBS, suspend it, and filter it through a 0.22μm filter to remove bacteria, thus obtaining PC-EVs.

[0054] 2. Characterization: The morphology, size, particle size distribution, zeta potential, lipids and proteins of PC-EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), thin layer chromatography (TLC) and Coomassie brilliant blue method.

[0055] TEM showed that PC-EVs were typically "cup holders" shaped. Figure 1 As shown; the results of DLS and NTA measurements are as follows. Figure 2 As shown, the PC-EVs particle size ranged from 100 to 200 nm, with an average particle size of 161.4 ± 1.7 nm and a potential of -3.20 ± 0.37 mV. SDS-PAGE gel electrophoresis results showed that the PC-EVs contained protein components, as detailed below. Figure 3 As shown, the protein bands of PC-EVs are concentrated in the 80-45 kDa range, with some proteins also distributed in the 180-140 kDa, 80-45 kDa, and 35-25 kDa ranges, indicating the presence of many proteins of different molecular weights in PC-EVs; for example... Figure 4 TLC results showed that PC-EVs contained a range of lipid components of different sizes. They were identified as PC-EVs.

[0056] Example 2: Effects of PC-EVs on MASH mice induced by a high-fat diet.

[0057] I. Experimental Materials and Instruments

[0058] 1. Laboratory animals

[0059] 80 eight-week-old specific pathogen-free (SPF) healthy male Spraque-Dawley mice, weighing 20±2 g, were purchased from Beijing Spey Foster Biotechnology Co., Ltd., with the quality certificate number: SCXK(Beijing)2019-0010. All experimental animals were housed in the Animal Experiment Center of Yunnan University of Traditional Chinese Medicine. The experimental conditions and methods complied with the regulations of the Animal Ethics Committee of Yunnan University of Traditional Chinese Medicine and were approved by the ethics review of the Animal Experiment Center of Yunnan University of Traditional Chinese Medicine. The animal ethics review document number was: R-062023151.

[0060] 2. Medicinal material samples

[0061] Poria cocos-derived extracellular vesicles (prepared in Example 1); Fenofibrate capsules (Lipanthyl, FC) (produced by Laboratoire Fournier, France, import drug registration number: H20181239, batch number: 33020).

[0062] 3. Experimental reagents

[0063]

[0064]

[0065]

[0066] 4. Experimental instruments

[0067] BS224 Electronic Balance Beijing Sellis Instrument Systems Co., Ltd. ASP300S Automatic Dehydrator Leica GmbH, Germany CRYOSTAR NX5 Cryo Slicer Thermo Fisher Scientific EG1150H biological tissue embedding machine Leica GmbH, Germany G6012-2 Adhesive glass slide Wuhan Saiweier Biotechnology Co., Ltd. KZ-III-F High-Speed ​​Low-Temperature Tissue Morphing Instrument Wuhan Sewell Biotechnology Co., Ltd. SW-CJ-IFD type ultra-clean workbench Suzhou Antai Air Technology Co., Ltd. Ccentrifuge 5430R High-Speed ​​Refrigerated Centrifuge Eppendorf, Germany JEM-1400 Transmission Electron Microscope JEOL Corporation of Japan SpectraMax Plus Microplate Reader Molecular Devices, Inc. (USA) SuperMax 3000FA fluorescent microplate reader Shanghai Flash Spectrum Biotechnology Co., Ltd. Cobas c 311 Fully Automated Biochemical Chemistry Roche Diagnostics GmbH, Germany Tanon 5200 Fully Automated Chemiluminescence Image Analysis System Shanghai Tianneng Technology Co., Ltd. Milli-Q REFRENCE Pure Water System Millipore, USA

[0068] II. Experimental methods

[0069] 1. In vivo imaging of small animals to observe the uptake of PC-EVs by mice and their organ distribution

[0070] Preparation of Dir dye solution: ① Preparation of Dir stock solution: The Dir stock solution was prepared with DMSO at a concentration of 1 mM. After aliquoting, it was stored at -20°C to avoid repeated freezing and thawing; ② Preparation of Dir working solution: The stock solution was diluted with PBS to prepare a Dir working solution with a concentration of 100 μM.

[0071] Fluorescent labeling of PC-EVs: Take an appropriate amount of PC-EVs and add it to the pre-prepared Dir working solution (1 mL of PC-EVs suspension was added with 100 μL of Dir working solution for staining), mix well, react at room temperature in the dark for 30 min, and centrifuge at 150000×g for 2 h at 4°C; discard the supernatant, add an appropriate amount of PBS, and resuspend to obtain Dir-labeled PC-EVs.

[0072] Seven healthy male C57BL / 6J mice were acclimatized for 7 days and randomly divided into seven groups corresponding to 0, 1, 2, 4, 6, 12, and 24 hours, respectively. Hair removal cream was applied to the abdomen and above of the mice, and they were washed with warm water. They were then fed normally for one day to eliminate interference from hair and hair removal cream.

[0073] Small animal in vivo imaging: Before imaging, mice were fasted but allowed to drink water. At 0h, unlabeled PC-EVs were injected intraperitoneally as a blank control. The remaining 6 mice were given Dir-labeled PC-EVs. Mice were anesthetized with isoflurane mixed with oxygen (isofluorane concentration of 1-2%) at 0, 1, 2, 4, 6, 12 and 24h. The excitation and emission light (Ex = 748nm, Em = 780nm) were adjusted on the small animal in vivo imaging instrument to perform in vivo imaging.

[0074] Organ imaging: Euthanasia was performed using CO2 inhalation at 0, 1, 2, 4, 6, 12 and 24 hours, and the heart, liver, spleen, lungs and kidneys were removed for imaging.

[0075] Using Dir-labeled PC-EVs, we observed the uptake of PC-EVs in mice and their distribution in their organs. Figure 5 As shown in Figure A, when unlabeled PC-EVs were administered at 0 h, no fluorescent signal was observed in mice, indicating that PC-EVs themselves did not cause fluorescence interference. When Dir-labeled PC-EVs were administered at 0 h, different levels of fluorescent signals were detected in mice at 1, 2, 4, 6, 12, and 24 h, with the strongest fluorescence intensity at 4 and 6 h, followed by a decrease in fluorescence intensity over time. This indicates that PC-EVs can be absorbed and utilized by mice, with peak absorption occurring between 4 and 6 h. Figure 5 As shown in Figure B, organs were extracted and imaged at different time points. Except for 0h, when no fluorescence signal was observed, varying degrees of fluorescence signal were present at all other time points, mainly concentrated in the liver and spleen, indicating that PC-EVs may have a targeting effect on the liver and spleen. Furthermore, the liver fluorescence intensity was strongest at 4 and 6h, consistent with results obtained from in vivo imaging in mice.

[0076] 2. MASH mouse model replication, animal grouping, and drug administration

[0077] Sixty healthy male C57BL / 6J mice were acclimatized for 7 days and then randomly divided into 6 groups of 10 mice each (see Table 1 for animal grouping and drug administration). The mice were administered the drug via intraperitoneal injection once daily (1 mL) for 8 weeks. The normal control group was fed a normal diet, while the other groups were fed a high-fat diet. During the experiment, the daily food intake was recorded, and the body weight was recorded weekly.

[0078] Table 1 Animal grouping and administration

[0079]

[0080]

[0081] During the experiment, no mice died, and their diet was normal. Results after 8 weeks showed that the body weight of mice in all groups increased positively, with the model group having a significantly higher body weight than the normal group and the PC-EVs treatment group (P < 0.05, P < 0.01, P < 0.001). Figure 6 (AB) During the experiment, there was no significant difference in food intake among the rats in each group. Figure 6 C) indicates that PC-EVs do not affect the normal food intake of mice, but have a significant effect on the reduction of mouse weight.

[0082] 3. Sample Collection

[0083] 3.1 Blood Sample Collection

[0084] Mouse blood samples were collected from the abdominal aorta under pentobarbital anesthesia, allowed to stand at room temperature, and centrifuged at 3500 rpm for 15 min at 4°C. The supernatant was collected, aliquoted, and stored at -80°C.

[0085] 3.2 Calculation of Organ and Fat Index

[0086] After blood was collected from each group of mice, the white fat (WATg) of the heart, liver, spleen, lung, kidney, groin, white fat (WATi) of the epididymis, and brown fat (BAT) were removed. After washing with physiological saline, the organs were blotted dry with filter paper, weighed and recorded. Combined with the weight of each mouse, the organ and fat index were calculated: organ / fat index = organ / fat weight (g) / body weight (g) × 100%.

[0087] like Figure 7 As shown, compared with the normal group, the model group showed significantly increased liver, spleen, kidney, WATg, and WATi indices (P < 0.05, P < 0.01, P < 0.001), and significantly decreased heart and BAT indices (P < 0.05, P < 0.001). Compared with the model group, administration of fenofibrate significantly reduced liver, spleen, and WATg indices (P < 0.05, P < 0.001). Compared with the model group, administration of PC-EVs significantly increased heart and BAT indices (P < 0.05, P < 0.01, P < 0.001), and significantly reduced liver, spleen, WATg, and WATi indices (P < 0.05, P < 0.01, P < 0.001), indicating that PC-EVs have a significant ameliorative effect on liver and spleen enlargement and fat accumulation induced by a high-fat diet in MASH mice.

[0088] 4. Observation of liver tissue morphology and pathology, and observation of liver mitochondria and autophagosomes.

[0089] 4.1 Oil Red O staining

[0090] Liver tissue was collected and fixed in 4% paraformaldehyde. The tissue was then removed, embedded in OTC embedding medium, and frozen at -20°C for 30 min. The tissue was sliced ​​into 8 μm thick sections using a microtome, fixed onto glass slides, and stained with Oil Red O for 15 min. The sections were then removed, differentiated in 60% isopropanol for 5 s, and subsequently rinsed in pure water for 10 s. The sections were then counterstained with hematoxylin for 1 min, followed by rinsing in pure water for 10 s. Finally, the tissue sections were sealed with glycerol gelatin, and fat accumulation was observed under an optical microscope. Furthermore, the sections were scanned, and oil droplets in the liver tissue were quantitatively analyzed using ImageJ software.

[0091] 4.2 H&E staining

[0092] Liver tissue was collected and fixed in 4% paraformaldehyde; the tissue was removed and immersed in paraffin overnight; it was then embedded in an embedding machine and sectioned into 4 μm thick sections using a microtome; the sections were removed, stained with hematoxylin for 5 min, and then rinsed in pure water until the surface stain was removed; the sections were removed, differentiated in hydrochloric acid alcohol for 5 s, and then rinsed in pure water for 10 s; the sections were removed, stained with eosin for 5 s, and then rinsed in pure water until the surface stain was removed; the sections were removed, soaked in xylene for 2 min, and finally sealed with neutral resin. Pathological changes were observed under an optical microscope.

[0093] 4.3 Observation by transmission electron microscopy

[0094] Liver tissue was collected and fixed in electron microscopy fixative; the tissue was removed, rinsed with PBS buffer, and then dehydrated in a gradient of ethanol and acetone (20%, 30%, 50%, 70%, 90%, and 100%). The tissue was then embedded in EPON resin, sliced ​​into thin sections using a microtome, stained sequentially with uranyl acetate and lead citrate, and observed under a transmission electron microscope to observe the mitochondria and autophagosomes.

[0095] The results are as follows Figure 8 As shown in Figure A, compared with the normal group, the model group showed significant lipid droplet accumulation in the liver. Compared with the model group, administration of fenofibrate and PC-EVs significantly improved liver lipid droplet accumulation. Figure 8 As shown in Figure B, the quantitative analysis of lipid droplets revealed a significant increase in liver lipid droplets in the model group compared to the normal group (P < 0.001). Compared to the model group, administration of fenofibrate and PC-EVs significantly reduced liver lipids (P < 0.05, P < 0.01, P < 0.001).

[0096] like Figure 8As shown in Figure C, compared with the normal group, the model group showed increased hepatocyte swelling, degeneration, and necrosis, along with numerous round vacuolar lipid droplets of varying sizes and inflammatory infiltration. Compared with the model group, administration of fenofibrate and PC-EVs reduced hepatocyte swelling, degeneration, and necrosis, and also reduced the round vacuolar lipid droplets and inflammatory infiltration. In conclusion, PC-EVs can effectively improve the morphological and pathological lesions of liver tissue in MASH mice induced by a high-fat diet.

[0097] 5. Serum lipid level detection

[0098] Serum from each group of mice was collected, and the levels of TC, TG, HDL-C, LDL-C, ALT, and AST were measured using a fully automated biochemical analyzer.

[0099] like Figure 9 As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the serum levels of TC, ALT, AST, and LDL-C in the model group mice were significantly increased (P < 0.001), while the HDL-C level was significantly decreased (P < 0.001), and the TG level showed an increasing trend, indicating that a high-fat diet can induce abnormal lipid metabolism in MASH mice. Compared with the model group, after administration of fenofibrate, the serum levels of TG, ALT, and AST in mice were significantly decreased (P < 0.001), while the HDL-C level was significantly increased (P < 0.001), indicating that fenofibrate can improve lipid metabolism abnormalities caused by a high-fat diet. Compared with the model group, the levels of TC, TG, ALT and AST in the serum of mice were significantly reduced after administration of PC-EVs (P<0.05, P<0.01, P<0.001), while the level of HDL-C was significantly increased (P<0.001), and the level of LDL-C showed a decreasing trend. This indicates that PC-EVs can regulate lipid metabolism abnormalities induced by a high-fat diet in MASH mice.

[0100] 6. Detection of lipid markers in liver tissue

[0101] Weigh 0.1g of mouse liver, add 0.9mL of physiological saline, pre-cool the liver in a low-temperature tissue homogenizer, and homogenize it twice for 30s each time. After homogenization, centrifuge at 3000rpm for 10min at 4℃, and take the supernatant to detect the contents of BCA, TC, TG, ALT, AST, LDL-C and HDL-C.

[0102] like Figure 10As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the levels of TC, TG, AST, ALT, and LDL-C in the liver of mice in the model group were significantly increased (P < 0.001), and the level of HDL-C was significantly decreased (P < 0.001), indicating that high-fat diet induces lipid metabolism disorder in the liver of MASH mice. Compared with the model group, after administration of fenofibrate, the levels of TC, TG, AST, ALT, and LDL-C in the liver of mice were significantly decreased (P < 0.01, P < 0.001), while HDL-C showed an increasing trend, indicating that fenofibrate can improve the liver lipid metabolism disorder caused by high-fat diet. Compared with the model group, after administration of PC-EVs, the levels of TC, TG, AST, ALT, and LDL-C in the liver of mice were significantly decreased (P < 0.01, P < 0.001), while the level of HDL-C was significantly increased (P < 0.01), indicating that PC-EVs can regulate the liver lipid metabolism disorder caused by high-fat diet.

[0103] 7. Detection of serum and liver tissue inflammation-related markers

[0104] Serum samples from each group of mice were collected to directly measure the levels of inflammatory factors TNF-α, IL-6, IL-1β, and IL-18. Liver tissue samples were prepared using the same method as those for detecting lipid markers in liver tissue, and the levels of inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 in the liver were measured.

[0105] like Figure 11 As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the serum and liver levels of TNF-α, IL-6, IL-1β, and IL-18 in the model group mice were significantly increased (P < 0.05, P < 0.01, P < 0.001), indicating that high-fat diet induces inflammatory response in MASH mice. Compared with the model group, after administration of fenofibrate, the serum levels of TNF-α and IL-6 in mice were significantly decreased (P < 0.001), and the levels of IL-1β and IL-18 showed a decreasing trend; the liver levels of TNF-α, IL-6, and IL-1β were significantly decreased (P < 0.001), and the IL-18 level showed a decreasing trend, indicating that fenofibrate can improve the inflammatory response induced by high-fat diet. Compared with the model group, the levels of TNF-α, IL-6, IL-1β and IL-18 in the serum and liver of mice were significantly reduced after administration of PC-EVs (P<0.05, P<0.01, P<0.001), indicating that PC-EVs can improve the inflammatory response induced by a high-fat diet.

[0106] 8. Detection of oxidative stress-related indicators in liver tissue

[0107] The liver tissue sample preparation method is the same as that for liver tissue lipid index detection, measuring MDA, SOD, and GSH.

[0108] The content of ROS in liver tissue was measured by fluorescence spectrophotometry. Liver tissue homogenate was added to a fluorescent microplate (black plate), followed by 10 μM DCFH-DA probe. The mixture was incubated at 37°C in the dark for 30 min. Detection was performed using a fluorescent microplate reader with the excitation and emission light adjusted (Ex = 488 nm, Em = 530 nm).

[0109] like Figure 12 As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the activities of SOD and GSH in the liver of mice in the model group were significantly decreased (P < 0.01, P < 0.001), the ROS level was significantly increased (P < 0.001), and the MDA content showed an increasing trend, indicating that high-fat diet induced oxidative stress in the liver of MASH mice. Compared with the model group, after administration of fenofibrate, the activities of GSH in the liver of mice were significantly decreased (P < 0.001), the levels of MDA and ROS were significantly decreased (P < 0.001), and the SOD activity showed an increasing trend, indicating that fenofibrate can improve high-fat diet induced oxidative stress in the liver of MASH mice. Compared with the model group, the activities of SOD and GSH in the liver of mice were significantly increased after administration of PC-EVs (P<0.05, P<0.01), while the levels of MDA and ROS were significantly decreased (P<0.05, P<0.01, P<0.001), indicating that PC-EVs can alleviate oxidative stress in the liver of MASH mice induced by a high-fat diet.

[0110] 9. Detection of energy metabolism-related indicators in liver tissue

[0111] The liver tissue sample preparation method is the same as that for liver tissue lipid index detection, and ATP synthase, Complex I and II are measured.

[0112] like Figure 13 As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the Na+ level in the liver of mice in the model group was significantly lower. + -K + -ATPase, Ca 2+ -Mg 2+ - ATPase, Complex I, and II activities were significantly decreased (P < 0.05, P < 0.001), indicating abnormal liver energy metabolism in MASH mice induced by a high-fat diet. Compared with the model group, Na+ levels in the liver of mice administered fenofibrate were significantly lower. + -K + -ATPase and Ca 2+ -Mg 2+ -ATPase activity was significantly increased (P

[0113] <0.001), Complex I and II activities showed an increasing trend, indicating that fenofibrate can regulate high-fat diet-induced energy metabolism abnormalities in MASH mice. Compared with the model group, Na+ in the liver of mice administered PC-EVs increased significantly. + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, Complex I and II activities were significantly increased (P < 0.05, P < 0.05).

[0114] <0.001), indicating that PC-EVs can regulate energy metabolism abnormalities induced by a high-fat diet in MASH mice.

[0115] 10. Detection of oxidized mitochondrial DNA (Ox-mtDNA) in liver tissue cytoplasm and mitochondria

[0116] Weigh 0.1g of mouse liver, add 0.9mL of physiological saline, mince the tissue, homogenize 15 times in a glass homogenizer, collect the homogenate, and centrifuge at 11000×g for 10min at 4℃. The supernatant is hepatocyte cytoplasm, and the precipitate is hepatocyte mitochondria. Following the EasyPure Genomic DNA Kit procedure, extract DNA from the hepatocyte cytoplasm and hepatocyte mitochondria separately (mitochondrial lysis buffer should be added beforehand for mitochondrial DNA extraction). Add 500μL of [unspecified substance] to the cytoplasm or lysed mitochondria. Mix the BB2 solution thoroughly and incubate at room temperature for 10 min. After incubation, transfer the solution to a centrifuge column and centrifuge at 12000×g for 30 s at 4°C. Discard the effluent, add 500 μL of CB2 solution prepared in advance with anhydrous ethanol, and centrifuge at 12000×g for 30 s at 4°C. Discard the effluent, add 500 μL of WB2 solution prepared in advance with anhydrous ethanol, and centrifuge at 12000×g for 30 s at 4°C. Repeat this step once. Discard the effluent and centrifuge at 12000×g for 2 min at 4°C to completely remove residual WB2. Discard the centrifuge tube, place the centrifuge column in a clean centrifuge tube, add 100 μL of preheated EB (60-70°C) to the center of the column, let stand for 1 min, and centrifuge at 12000×g for 1 min at room temperature to elute the DNA. Repeat this step once. DNA samples extracted from hepatocyte cytoplasm and mitochondria were used to detect the content of 8-OHdG, which is the content of Ox-mtDNA.

[0117] like Figure 14As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the model group mice exhibited significant swelling, rupture, and blurring of the liver mitochondrial structure, with blank areas appearing in the mitochondrial matrix and a reduced number of autophagosomes. Compared with the model group, administration of fenofibrate and PC-EVs significantly improved the swelling, rupture, and blurring of the liver mitochondrial structure, and reduced the blank areas in the mitochondrial matrix, indicating that both fenofibrate and PC-EVs can alleviate the damage to the ultrastructure of mouse liver mitochondria caused by a high-fat diet. Furthermore, the number of autophagosomes increased, especially in the HPC-EVs group, suggesting that PC-EVs may also promote hepatic mitophagy to protect liver mitochondrial structure and function, thereby alleviating MASH caused by a high-fat diet.

[0118] 11. Western blot detection of mitophagy-related proteins and NLRP3 inflammasome-related proteins

[0119] 11.1 Protein Sample Preparation

[0120] Weigh 0.1g of mouse liver into a 1.5mL EP tube, add 1mL of pre-prepared tissue lysis buffer (RIPA:PMSF = 100:1), add 3 3mm zirconia grinding beads, pre-cool the grinder, and grind twice in the tissue grinder for 30s each time; after grinding, centrifuge at 12000rpm for 30min at 4℃; take the supernatant, determine its protein concentration, add tissue lysis buffer to the lowest protein concentration to level, then add 5× loading buffer, denature in a 100℃ metal bath for 10min; after cooling, aliquot and store at -80℃.

[0121] 11.2 SDS-PAGE gel electrophoresis

[0122] Prepare a 10% separating gel and quickly place it between two clean glass plates, to a height of approximately 70% of the glass plates. Then, fill the glass plates with isopropanol to prevent air bubbles and isolate them. Let it stand at room temperature. After the separating gel solidifies, recover the isopropanol and prepare a 5% stacking gel. Slowly add this to the separating gel to prevent air bubbles. Insert the comb and let it stand at room temperature. After the stacking gel solidifies, place it in the electrophoresis tank and add the pre-prepared 1× electrophoresis buffer. Remove the comb. Add 3 μL of 5× loading buffer to both sides, and then add 5 μL of marker and protein sample (50 μg loading volume) sequentially from left to right. After loading the samples, cover the electrophoresis tank, turn on the power, and perform electrophoresis at a constant voltage of 70V until the marker shows bands of each molecular weight. Then, perform electrophoresis at a constant voltage of 120V until the bottom edge of the separating gel is reached.

[0123] 11.3 Transfer (Wet Transfer)

[0124] After electrophoresis, remove the gel and place it in the instrument tray. Add the pre-prepared and pre-cooled 1× transfer buffer. Place the filter paper, sponge, and methanol-activated PVDF membrane into the tray to soak. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clamp starting from the black plate. Ensure that no air bubbles enter during the entire process. Clamp the transfer clamp and place it in the transfer tank. Transfer the membrane at a constant pressure of 280mA for 60 minutes at 4°C.

[0125] 11.4 Closed

[0126] After the transfer was completed, the PVDF membrane was removed and placed in 5% skim milk powder (prepared with 1×TBST solution) filtered through a 0.22μm filter membrane. The mixture was then placed on a shaker and shaken at a constant speed for 1 hour.

[0127] 11.5 Immunoblotting

[0128] Antibody dilution: Prepared using 5% skim milk powder filtered through a 0.22 μm filter membrane, containing PINK1 (1:2000), Parkin (1:2000), LC3 (1:2000), NLRP3 (1:5000), IL-1β (1:1000), Caspase-1 (1:4000), β-Actin (1:4000), Goat Anti-Mouse IgG (H+L) (1:5000), and Goat Anti-Rabbit IgG (H+L) (1:5000).

[0129] After blocking, wash the membrane three times with 1×TBST solution for 10 min each time; add an appropriate amount of primary antibody and incubate overnight at 4℃; after the primary antibody incubation is complete, recover the primary antibody and wash the membrane three times with 1×TBST solution for 10 min each time; add secondary antibody according to the different sources of the primary antibody and incubate at room temperature for 1 h; after the secondary antibody incubation is complete, recover the secondary antibody and wash the membrane three times with 1×TBST solution for 10 min each time.

[0130] 11.6 Image Acquisition

[0131] The chemiluminescent solution was uniformly added to the PVDF membrane, and images were acquired using a fully automated chemiluminescence image analysis system. The relative expression levels of proteins were analyzed using ImageJ software.

[0132] The results are as follows Figure 15As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the expression level of P62 protein in the liver of model group mice was significantly increased (P < 0.01, P < 0.001), while the expression level of PINK1 protein showed a decreasing trend, indicating that high-fat diet can induce abnormal mitophagy in the liver of MASH mice. Compared with the model group, after administration of fenofibrate, the expression level of PINK1 protein in the liver of mice was significantly increased (P < 0.001), the expression level of P62 protein was significantly decreased (P < 0.05), and the expression levels of Parkin and LC3Ⅱ / LC3Ⅰ proteins showed an increasing trend, indicating that fenofibrate can regulate the abnormal mitophagy in the liver of MASH mice induced by high-fat diet. Compared with the model group, the expression levels of PINK1, Parkin and LC3Ⅱ / LC3Ⅰ proteins in the liver of mice were significantly increased after administration of PC-EVs (P<0.05, P<0.01, P<0.001), while the expression level of P62 protein showed a decreasing trend, indicating that PC-EVs can regulate abnormal mitochondrial autophagy in the liver of MASH mice induced by a high-fat diet.

[0133] like Figure 16 As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the Ox-mtDNA content in the liver cytoplasm and mitochondria of mice in the model group was significantly increased (P < 0.001), indicating that a high-fat diet can induce mtDNA oxidation in the liver of MASH mice and release it into the cytoplasm. Compared with the model group, the Ox-mtDNA content in the liver cytoplasm and mitochondria of mice was significantly decreased after administration of fenofibrate (P < 0.01, P < 0.001), indicating that fenofibrate can regulate high-fat diet-induced mtDNA oxidation and Ox-mtDNA release in the liver of MASH mice. Compared with the model group, the Ox-mtDNA content in the liver cytoplasm and mitochondria of mice was significantly decreased after administration of PC-EVs (P < 0.001), indicating that PC-EVs can regulate high-fat diet-induced mtDNA oxidation and Ox-mtDNA release in the liver of MASH mice.

[0134] like Figure 17As shown, after 8 weeks of high-fat diet administration, compared with the normal group, the expression levels of NLRP3, Pro-Caspase-1, Cleared-Caspase-1, and Mature-IL-1β proteins in the liver of model group mice were significantly increased (P < 0.01, P < 0.001), indicating that high-fat diet can induce activation of the NLRP3 inflammasome in the liver of MASH mice. Compared with the model group, after administration of fenofibrate, the expression levels of NLRP3 and Pro-Caspase-1 proteins in the liver of mice were significantly decreased (P < 0.01, P < 0.001), and the expression levels of Cleared-Caspase-1 and Mature-IL-1β proteins showed a decreasing trend, indicating that fenofibrate can regulate the activation of the NLRP3 inflammasome in the liver of MASH mice induced by high-fat diet. Compared with the model group, the expression levels of NLRP3, Pro-Caspase-1, Cleared-Caspase-1 and Mature-IL-1β proteins in the liver of mice were significantly reduced after administration of PC-EVs (P<0.05, P<0.01), indicating that PC-EVs can regulate the activation of the NLRP3 inflammasome in the liver of MASH mice induced by a high-fat diet.

[0135] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. The application of extracellular vesicles derived from Poria cocos in the preparation of a drug for non-alcoholic steatohepatitis, characterized in that, The extracellular vesicles derived from Poria cocos were prepared using the following method: Peel and wash fresh Poria cocos, slice it, add 2-3 times the amount of pure water, juice it, collect the juice, centrifuge the collected juice at 1000×g for 10 min at 4℃; take the supernatant, centrifuge at 3000×g for 20 min at 4℃; take the supernatant, centrifuge at 10000×g for 30 min at 4℃; take the supernatant, centrifuge at 150000×g for 2 h at 4℃; discard the supernatant, add an appropriate amount of PBS, suspend it, and filter it through a 0.22 μm filter to remove bacteria, thus obtaining Poria cocos-derived extracellular vesicles.

2. The application as described in claim 1, characterized in that, The drug modulates mitochondrial structure and function, including alleviating damage to mitochondrial ultrastructure, increasing the number of autophagosomes, and improving oxidative stress and energy metabolism.

3. The application according to claim 1, characterized in that, The drug improves liver tissue morphology and pathological lesions, and regulates serum and liver lipid levels and inflammatory factor levels.

4. The application according to claim 1, characterized in that, The drug promotes the expression of mitophagy-related proteins and reduces the production and efflux of Ox-mtDNA.

5. The application according to claim 1, characterized in that, The drug inhibits the expression of NLRP3 inflammasome-related protein.

6. A pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, characterized in that, This includes extracellular vesicles derived from Poria cocos, which are prepared using the following method: Peel and wash fresh Poria cocos, slice it, add 2-3 times the amount of pure water, juice it, collect the juice, centrifuge the collected juice at 1000×g for 10 min at 4℃; take the supernatant, centrifuge at 3000×g for 20 min at 4℃; take the supernatant, centrifuge at 10000×g for 30 min at 4℃; take the supernatant, centrifuge at 150000×g for 2 h at 4℃; discard the supernatant, add an appropriate amount of PBS, suspend it, and filter it through a 0.22 μm filter to remove bacteria, thus obtaining Poria cocos-derived extracellular vesicles.

Images