Application of liver tissue-derived extracellular vesicles in the treatment of alcoholic liver disease

Abstract

The present invention provides an application of liver-derived extracellular vesicles (EVnew and EVadult) in treating patients with alcoholic liver disease. The liver-derived extracellular vesicles of the present invention are EVs from rat livers and can effectively alleviate alcohol-induced liver damage in mice. Both EVnew and EVadult can reduce mitochondrial stress markers in the livers of ALD mice, inhibit the activation of M1 macrophages, promote M2 polarization, reduce the levels of proinflammatory cytokines, promote hepatocyte proliferation, reduce cell apoptosis, and improve histological results. Compared with EVadult, EVnew provides more obvious benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of liver tissue-derived extracellular vesicles in the treatment of alcoholic liver disease. Background Art

[0002] Alcoholic liver disease (ALD) is a chronic disease characterized by compulsive heavy drinking that follows a well-recognized disease progression pattern, including alcoholic fatty liver, alcoholic steatohepatitis (with or without progressive fibrosis), cirrhosis and its related complications, and hepatocellular carcinoma. Patients with ALD may develop an acute clinical syndrome called alcoholic hepatitis and have a poor prognosis. ALD is associated with significant morbidity and mortality, causing approximately 3.3 million deaths each year (5.9% of all deaths), which is a major public health challenge that requires urgent attention. Therefore, effective treatment strategies for ALD are particularly important. However, there is a lack of clinically effective therapeutic drugs, and alcohol abstinence and nutritional intervention remain the main means of ALD management. At present, researchers believe that multiple host factors affect the risk of ALD, including genetics, metabolic changes, epigenetic changes, oxidative stress, and inflammation. Based on the above mechanisms, the progress of ALD drug treatment in recent years has focused on anti-inflammatory and antioxidant agents, gut-liver axis regulation, and enhancing hepatocyte regeneration. However, challenges such as individual response, drug side effects, and safety still exist. For example, glucocorticoids and interleukin 1 (IL-1) receptor antagonists show limited benefits and risks, such as in acute kidney injury, highlighting the need for new effective treatments.

[0003] Extracellular vesicles (EVs) are small membrane vesicles secreted by cells, usually with a diameter between 30 and 150 nanometers, with unique biological properties such as low immunogenicity, strong stability, the ability to penetrate the cell membrane, and carrying rich biological information (such as proteins, lipids, and RNA). These characteristics make EVs play an important role in intercellular communication and signal transduction, and provide broad prospects for their application in disease treatment. In recent years, EVs from in vitro cultured cells (such as mesenchymal stem cells) have been shown to have certain tissue repair and disease treatment capabilities in the fields of skin repair, kidney injury, liver injury, respiratory diseases, nervous system diseases, musculoskeletal system, and cardiovascular diseases. However, there are significant limitations in the clinical translation of EVs extracted from in vitro cell culture. For example, traditional cell culture strategies have relatively low yields, high costs, and are time-consuming. In addition, repeated processing of large amounts of culture medium and the large amount of reagents / materials required for long-term cell culture may affect the phenotype of donor cells (especially primary cells), thereby impairing the function of EVs. In addition, due to the low differentiation potential and single lineage of cell lines, they are insufficient for contemporary exploratory and research medical research.

[0004] The present invention provides a therapeutic effect of EVs derived from the liver of a homologous heterologous animal on alcoholic hepatitis injury. EVs from different species may differ in structure and function, which provides new ideas for their application in disease treatment. In particular, homologous EVs may have unique advantages in cross-species drug delivery and immune regulation. These advantages may further enhance the clinical application of EV technology. Therefore, this study aims to explore new effective therapeutic strategies based on EVs materials. Summary of the invention

[0005] Before describing the present disclosure, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, because these methods and conditions can be varied. Unless otherwise specified, the terms used in the present disclosure should not be considered to be uncertain or unclear without special definitions, but should be understood according to the common meanings in the art. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and the purpose is not to limit. When a trade name appears in the present disclosure, it is intended to refer to the corresponding commodity or its active ingredient.

[0006] The present invention provides rat liver-derived extracellular vesicles (EVs), particularly the use of newborn rat EVs (EVnew) and adult rat EVs (EVadult) in alleviating liver damage associated with ALD in mice. Using a mouse model of ALD, how EVnew and EVadult affect liver function by tail vein injection was studied. Proteomics methods were used to analyze the protein differences between EVnew and EVadult, as well as the protein differences in mouse liver tissue after treatment. Histological and molecular biological methods were used to verify the therapeutic effect and study the mechanism. The results showed that EVs from rat livers were able to effectively alleviate alcohol-induced liver damage in mice. Both EVnew and EVadult can reduce mitochondrial stress markers in the livers of ALD mice, inhibit the activation of M1 macrophages, promote M2 polarization, reduce the levels of proinflammatory cytokines, promote hepatocyte proliferation, reduce cell apoptosis, and improve histological results. Compared with EVadult, EVnew provides more obvious benefits.

[0007] The present invention provides an application of liver-derived extracellular vesicles in preparing a medicine for treating patients with alcoholic liver disease.

[0008] In one embodiment, the use provided by the present invention is characterized in that the liver-derived extracellular vesicles are used to reduce mitochondrial stress in the liver of patients with alcoholic liver disease.

[0009] In one embodiment, the application provided by the present invention is that the liver-derived extracellular vesicles are used to reduce mitochondrial markers in the liver of patients with alcoholic liver disease, namely 8-hydroxy-2-deoxyguanosine and / or mitochondrial superoxide (MitoSOX).

[0010] In one embodiment, the application provided by the present invention is that the liver-derived extracellular vesicles are used to increase the expression of mitochondrial active substances peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), mitochondrial superoxide dismutase (SOD2) and / or glutathione peroxidase 1 (GPX1) in patients with alcoholic liver disease.

[0011] In one embodiment, the use provided by the present invention is characterized in that the liver-derived extracellular vesicles are used to regulate macrophage homeostasis in patients with alcoholic liver disease.

[0012] In one embodiment, the application provided by the present invention is characterized in that the liver-derived extracellular vesicles regulate macrophage homeostasis by downregulating pro-inflammatory macrophages in patients with alcoholic liver disease and / or increasing anti-inflammatory macrophages in patients with alcoholic liver disease.

[0013] In one embodiment, the use provided by the present invention is characterized in that the liver-derived extracellular vesicles reduce the expression of inducible nitric oxide synthase (iNOS) in patients with alcoholic liver disease and / or increase the expression of mannose receptor CD206 in patients with alcoholic liver disease.

[0014] In one embodiment, the application provided by the present invention is characterized in that the hepatic extracellular vesicles are used to reduce the protein expression of tumor necrosis factor-α (Tnfα) and / or interleukin 6 (IL6) involved in inflammatory response.

[0015] In one embodiment, the application provided by the present invention is characterized in that the extracellular vesicles derived from the liver of homologous xenogeneic animals are used to improve liver reproduction and regeneration in patients with alcoholic liver disease.

[0016] In one embodiment, the application provided by the present invention is characterized in that the liver-derived extracellular vesicles are selected from adult organisms or juvenile organisms of the same species; preferably, they are juvenile organisms; preferably, the organisms are humans or mice.

[0017] Terminology explanation:

[0018] As used herein, the terms "subject," "patient," or "subject" are used interchangeably. The terms "subject," "patient," or "subject" include any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the terms "subject," "patient," or "subject" are mammals. In some embodiments, the subject, patient, or subject is a mouse. In some embodiments, the subject, patient, or subject is a human.

[0019] The beneficial effects of the present invention are:

[0020] The extracellular vesicles provided by the present invention are derived from liver tissue. Different from extracellular vesicles from cell lines, the components contained in the extracellular vesicles from tissue sources are more complex.

[0021] The present invention successfully used rat liver-derived extracellular vesicles to treat diseases in mice, and the present invention proved that homologous and heterologous tissue extracellular vesicles can be used to treat diseases with good safety. In the future, it can be expanded to homologous and heterologous tissue exosomes for the treatment of human diseases.

[0022] The present invention uses exosomes derived from liver tissues of adult rats and newborn rats. Through comparison, it is found that the exosomes derived from newborn liver tissues have a better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Size and morphology diagram of EVnew and EVadult;

[0024] Figure 2 :Protein distribution in EVnew and EVadult;

[0025] Figure 3 :Comparison of activation activities of mitochondria-related proteins, cell proliferation-related proteins, and inflammatory response proteins in EVadult and EVnew;

[0026] Figure 4 : Biodistribution of EVs after administration;

[0027] Figure 5 :Changes of AST and ALT in ALD mice before and after EVnew and EVadult treatment;

[0028] Figure 6 : BODIPY and Oil Red O staining before and after EVnew and EVadult treatment showed the changes in the number and size of lipid droplets in the ALD group;

[0029] Figure 7 :Pathological changes of ALD mice after EVnew and EVadult injection;

[0030] Figure 8 : Cluster heat map analysis of protein profiles of ALD+EVnew group and ALD+EVadult group of ALD mice after EVnew and EVadult treatment;

[0031] Fig. 9 : Cluster 1 protein function;

[0032] Fig.10 : Cluster 2 protein function;

[0033] Fig.11 : Cluster 3 protein function;

[0034] Fig.12 : Cluster 3 protein differential analysis;

[0035] Fig.13 :Enrichment analysis results of GO terms and KEGG pathways of cluster 3 differentially expressed proteins;

[0036] Fig.14 :Changes in 8-hydroxy-2-deoxyguanosine and mitochondrial superoxide anion in ALD mice before and after EVnew and EVadult treatment;

[0037] Fig.15 :Changes of SOD2 and GPX1 in ALD mice before and after EVnew and EVadult treatment;

[0038] Fig.16 : Western blot analysis of mitochondrial activity in ALD mice before and after EVnew and EVadult treatment;

[0039] Fig.17 :Changes in M1 macrophages and M2 macrophages in ALD mice before and after EVnew and EVadult treatment;

[0040] Fig.18 : Western blot analysis of inflammatory molecules Tnfα and Il6 before and after EVnew and EVadult treatment of ALD mice;

[0041] Fig.19 :Changes in proliferation and apoptosis levels in ALD mice before and after EVnew and EVadult treatment;

[0042] Fig. 20 : Protein immunoblot analysis of BCL2 and BAX before and after EVnew and EVadult treatment of ALD mice. DETAILED DESCRIPTION

[0043] For the sake of clarity, the present disclosure is further illustrated with examples, but the examples do not limit the scope of the present disclosure. All reagents used in the present disclosure are commercially available and can be used without further purification.

[0044] 1. Materials and Methods

[0045] 1.1 Human specimens

[0046] Three healthy liver tissues and three ALD liver tissues were included. All six samples were from patients with large hepatic hemangiomas. The liver tissues were collected during surgical resection and immediately stored in liquid nitrogen. The inclusion criteria for healthy controls were patients with giant hepatic hemangiomas aged between 18 and 60 years, excluding all acute / chronic liver diseases (including viral hepatitis, metabolic fatty liver disease, autoimmune liver disease, genetic metabolic liver disease, etc.) and a history of drinking. The inclusion criteria for the disease group were patients with giant hemangiomas aged between 18 and 60 years who met the diagnostic criteria for ALD, excluding viral hepatitis, non-alcoholic fatty liver disease, autoimmune liver disease, genetic metabolic liver disease, and drug-induced liver damage. The clinical diagnostic criteria for alcoholic liver disease are as follows: a history of long-term drinking for more than 5 years, a daily equivalent ethanol intake of ≥40g for men and ≥20g for women; or a history of heavy drinking in the past two weeks (intake >80g / day). The conversion formula for ethanol volume is: g = volume of alcohol consumed (ml) * alcohol concentration (%) * 0.8.

[0047] 1.2 Animal experiments

[0048] C57BL / 6J male mice, SD adult male rats, and SD pregnant rats aged 6-8 weeks were obtained from the Animal Experimental Center of Sichuan University. They were housed under standard conditions at 22±2°C and a 12-h light-dark cycle. The mice were randomly divided into four groups (6 in each group): control group (Ctrl), ALD model group (ALD), ALD model plus adult rat liver EVs treatment group (ALD+EVadult), and ALD model plus neonatal rat liver EVs treatment group (ALD+EVnew). The ALD mouse model was established using the NIAAA model. Specifically, male C57BL / 6 mice were continuously fed with the Liber De-Carli diet (Dyets Biotechnology [Wu Xi] Ltd. China) for 2 weeks. In the first 5 days, the mice were given gradually increasing concentrations of alcohol (1%, 2%, 3%, 4%, and 5%), and then continued to be fed with 5% alcohol concentration for 9 days. On the 14th day, the mice were gavaged with 100% alcohol (5 g / kg). The control group was fed maltodextrin as an alcohol substitute for 14 consecutive days. When the mice began to be fed 5% alcohol, EVs treatment began, and 5g / kg of EVs were injected through the tail vein every other day. The ALD model group received an intravenous injection of 100μL PBS (phosphate buffered saline). Both groups of mice were euthanized on the 15th day. All animal experiments were reported in accordance with the ARRIVE guidelines 2.0.

[0049] 1.3 Extraction of EVs from liver tissue

[0050] Liver-derived exosomes (EVs) from adult male rats and neonatal rats were obtained separately. Specifically, adult rats and neonatal rats were euthanized individually, and fresh liver tissues were collected and cut into small pieces (1-2 mm) in a sterile low-temperature environment. The tissue fragments were placed in DMEM medium containing type II collagenase (1 mg / mL) and incubated at 37°C for 2 hours. After tissue dissociation, the solution was filtered through a 70 μm filter to remove large tissue fragments. The resulting filtrate was centrifuged at 300g for 10 minutes and then at 2000g for 20 minutes to remove cells and cell debris. The supernatant was then centrifuged at 118,000g for 2.5 hours to obtain exosome-rich vesicles. All centrifugation steps were performed at 4°C.

[0051] 1.4 Analysis of extracellular vesicle proteins and liver tissue protein profiles

[0052] Once the extracellular vesicles were obtained, they were lysed using RIPA lysis buffer (CST, USA) to extract proteins, and the protein concentration was quantified by the biuret method (Sigma, USA). 100 μg of fresh liver tissue was cut from each mouse and immediately placed in RIPA lysis buffer precooled to 4°C for tissue homogenization and lysis. After 30 minutes, the supernatant containing total liver tissue protein was extracted by centrifugation at 3000×g. The extracted protein was then enzymatically hydrolyzed using a modified filter-assisted sample preparation technique. Specifically, the protein was loaded onto a 30 kDa filter (Merck Millipore, Ireland) and centrifuged at 13,000×g for 15 minutes at 25°C. Subsequently, 8 M urea was replaced with 50 mM NH4CO3 buffer (Sigma, USA) and centrifuged at 13,000×g for 15 minutes. 2 μg of trypsin and 2 μg of Lys-C (Enzyme & Spectrum, China) were added and enzymatic hydrolysis was performed overnight at 37°C. Peptides were obtained by centrifugation at 13,000 × g for 15 min. Peptide concentrations were determined using a quantitative colorimetric peptide assay at 480 nm and subsequently lyophilized using SpeedVac. Peptides (approximately 500 ng) were analyzed using an OrbitrapFusion Lumos mass spectrometer (Thermo Fisher). Raw data files were aligned to the human UniProt database using Spectronaut software (v15.4, Biognosys).

[0053] 1.5 Histological analysis

[0054] Fresh liver tissue was cut into small cubes and fixed with 4% paraformaldehyde for 24 h at room temperature, then dehydrated, paraffin-embedded and sectioned. Paraffin sections were dewaxed, rehydrated and stained with H&E (G1120, Beijing Sohrab Technology Co., Ltd., China) and MASSON (G1340, Beijing Sohrab Technology Co., Ltd., China). In addition, sections were stained for TNF-α, IL-1β, SOD2 and 8-OHdG using immunohistochemistry kits (BD Bioscience, USA). The list of antibodies is shown in Table 1. Images were captured using a MAGSCANNER KF-PRO-002 scanner (Turkey). The remaining fresh liver tissue was coated with OTC (Sakura Finetek, USA), cut into 6 μm sections, air-dried and washed with PBS. Oil Red O staining (G1261, Beijing Sohrab Technology Co., Ltd., China) was performed in the dark for 10 min, followed by washing and nuclear counterstaining with Mayer's hematoxylin. Glycerol gelatin was used for mounting. Lipid droplet green fluorescence detection kit using BODIPY 493 / 503 (Beyotime, China) was performed in the dark for 20 min, followed by washing and blocking with anti-fluorescence quencher (Thermo Fisher, USA). Mitochondria staining in living cells was performed using MitoSOX TM Green superoxide indicator (Invitrogen, USA). Prepare the stock solution and working solution of MSR reagent according to the instructions, cover 200 μL of MSR reagent working solution on the fresh slice, and incubate at 37 ° C and 5% CO2 for 30 minutes. Gently wash 3 times with warm buffer and capture the image. In addition, another fresh slice was used for immunofluorescence. The tissue was blocked with a mixture of 3% bovine serum albumin, 1% TBS, 0.3% TritonTMX-100 and 5% goat serum for 1 hour, followed by incubation with primary antibody at 4 ° C overnight, and then incubated with secondary antibody at 37 ° C for 1 hour. The nucleus was stained with DAPI dihydrochloride (MedChemExpress, China). Imaging was performed using an OLYMPUSDP74 microscope, and the data were analyzed by ImageJ and QuPath.

[0055] Table 1. Antibody list

[0056]

[0057]

[0058] 1.6 In vivo EV bioluminescence imaging

[0059] An appropriate amount of EVs (about 10 μg) was dissolved in 500 μL of Dulbecco's PBS (DPBS). An appropriate amount of HPK67 (Sigma-Aldrich, USA) dye was dissolved in an equal volume of DPBS and gently pipetted to mix. The dye solution was added to the EVs mixture, quickly aspirated to mix, and incubated in the dark at room temperature for 5-10 minutes. The staining reaction was terminated by adding an equal volume of 1% BSA. The dye that was not bound to EVs was removed by washing. The stained EVs were injected into mice through the tail vein, and the heart, lungs, liver, spleen, pancreas and kidneys of the mice were isolated after 6, 12, 24 and 48 hours, respectively. The biodistribution of EVs was observed by a fluorescent imager (IVISSpectrum, Caliper Life Sciences, USA).

[0060] 1.7 Quantitative and statistical analysis

[0061] Data are presented as mean ± standard error of the mean (SEM), with individual data points included where necessary. Statistical tests were selected based on data distribution and variance, using two-tailed t-tests to compare two groups and one-way analysis of variance (ANOVA) with Bonferroni correction to compare multiple groups. Specific tests are detailed in the figure legends, and p < 0.05 indicates significance. Sample size followed guidelines, and allocation awareness was not blinded during the study.

[0062] Mitochondrial damage is more severe in the liver of ALD patients

[0063] To advance the treatment of ALD, we collected liver tissues from patients with and without ALD and analyzed the relevant characteristics. Compared with normal liver, ALD livers showed typical histopathological features, including obvious fatty degeneration, severe lobular inflammation, ballooning of Mallory-Denk bodies in hepatocytes, and fibrosis. In addition, immunostaining observed a significant increase in the protein levels of TNF-α, IL-1β, SOD2, and 8-OHdG in the livers of ALD patients. These data suggest that liver inflammation and mitochondrial damage play a key role in human ALD and may serve as potential therapeutic targets.

[0064] Example 1 Preparation and characterization of EVnew and EVadult

[0065] EVnew and EVadult were obtained by multi-step differential centrifugation. Electron microscopy imaging showed that the sizes of EVnew and EVadult were approximately 128nm and 139nm, respectively, and they exhibited a uniform spherical morphology ( Figure 1). A series of EVs markers, including CD63, Alix, and TSG101, were detected in these two types of EVs. In summary, EVnew and EVadult were successfully isolated and purified. To explore the differences between EVnew and EVadult, we further analyzed the obtained proteins. Proteomic analysis showed that EVnew and EVadult contained a variety of specific proteins ( Figure 2 ). In addition, protein network analysis of EVs showed that the activation of mitochondria-related proteins, cell proliferation-related proteins, and inflammatory response proteins was more significant in EVnew compared with EVadult ( Figure 3 ). These data revealed that the activation of mitochondrial stress-related molecules was more pronounced in EVnew.

[0066] Biodistribution of EVs after administration

[0067] To evaluate the biodistribution of EVs, EVnew and EVadult were labeled with HPK67 dye. These EVs were injected into ALD mice via the tail vein, and mice that received the same amount of free HPK67 dye served as controls. After administration, the mice were euthanized and their organs were removed for IVIS imaging. EVnew and EVadult mainly accumulated in the liver, pancreas, and kidneys, while there were no obvious signals in the organs of the free dye group. After injection, the fluorescence intensity of EVnew in these organs was no different from that of the EVadult group. It is worth noting that EVnew showed the strongest fluorescence in the liver at different time points ( Figure 4 ).

[0068] Example 3: Experiment on improving ALD mice with EVnew and EVadult

[0069] A typical ALD model was established and followed by daily tail vein injection of 5 g / kg EVs for 10 days as described previously. AST and ALT were significantly increased in the ALD group compared with the control group, but were effectively eliminated by EVnew or EVadult treatment ( Figure 5 Subsequently, BODIPY and Oil Red O staining showed that the number and size of lipid droplets increased significantly in the ALD group, while they decreased after EVnew or EVadult treatment ( Figure 6 ). In addition, the typical histopathological features of ALD were shown in the liver of mice, including obvious fatty degeneration, lobular inflammation and fibrosis. In contrast, the characteristics of ALD were significantly improved after EVnew or EVadult injection ( Figure 7 ). These data clearly demonstrate that treatment with EVnew or EVadult is effective in alleviating the progression of ALD.

[0070] Example 4 Comparative experiment on the effect of EVnew and EVadult on improving mitochondrial stress in ALD liver

[0071] To evaluate the mechanism of EVnew and EVadult treatment of ALD mice, mouse liver sample data were collected and a fully consistent mass spectrometry method was used. 4879 qualitative proteins were detected by proteomic analysis. To explore the differences in ALD mice after EVnew or EVadult treatment, we further analyzed the data by size heat map analysis of liver samples and classified them into three different clusters ( Figure 8 Cluster 1 focuses on proteins involved in metabolic processes ( Fig. 9 ), while cluster 2 contained multiple proteins related to transcription and proliferation ( Fig.10 Cluster 3 showed significant enrichment of mitochondrial-related proteins ( Fig.11 ). It is noteworthy that cluster 3 was significantly different between the ALD+EVnew group and the ALD+EVadult group. Therefore, we focused on cluster 3 and detected 410 qualitative proteins. 65 proteins showed significant differences between the ALD+EVnew group and the ALD+EVadult group ( Fig.12 ). Subsequently, GO term and KEGG pathway enrichment analysis performed in the two groups revealed significant differences in mitochondrial stress, inflammatory response, and apoptosis signaling pathways in the liver ( Fig.13 ). To further validate the data, mitochondrial function was examined. The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and mitochondrial superoxide anion (MitoSOX) were significantly increased in the ALD group compared with the control group. However, these levels decreased after treatment with EVnew or EVadult, with the effects of EVnew being superior to those of EVadult ( Fig.14 ). Compared with the control group, the resistance of ALD mouse hepatocytes to mitochondrial stress was significantly reduced, as manifested by a significant decrease in SOD2 and GPX1 expression, while a significant recovery was observed after EVs injection. Similarly, EVnew showed superior efficacy ( Fig.15 In addition, representative Western blots showed that multiple mitochondrial activities, such as PPAR-γ coactivator 1-α (PGC-1α), superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 (GPX1)) were significantly decreased in ALD livers, but significantly increased after EVnew or EVadult treatment ( Fig.16 ). Taken together, the results showed that EVnew and EVadult significantly limited liver mitochondrial stress, with EVnew performing better and further alleviating ALD.

[0072] Example 5: Experiment on EVnew and EVadult regulating macrophage homeostasis and reducing liver inflammation in ALD mice

[0073] Liver inflammation is a hallmark of ALD, and our previous proteomic data showed that both EVnew and EVadult could alleviate the disease in ALD mice. Therefore, additional examinations were performed to determine these changes. Macrophages are the most common non-parenchymal and inflammatory cells in the liver and are mainly divided into two categories: classical M1 (pro-inflammatory) and M2 (anti-inflammatory). Compared with the control group, the expression of inducible nitric oxide synthase (iNOS), an M1 marker, was significantly increased in the ALD group, while the level of CD206 (M2 marker) was significantly decreased. However, the activation of M1 macrophages was significantly downregulated in the ALD+EVnew group and ALD+EVadult group compared with the ALD group. No statistically significant differences were observed between the two intervention groups. In contrast, macrophages of the M2 phenotype showed a higher degree of activation after EVnew or EVadult treatment compared with the ALD group. In both groups, EVnew showed superior efficacy ( Fig.17 ). In addition, the protein levels of inflammatory molecules such as TNF-α and IL-6 were increased in the ALD group but decreased after EVnew or EVadult intervention. Interestingly, the efficacy of EVnew was superior to that of EVadult ( Fig.18 ). Overall, EVnew and EVadult have therapeutic effects on ALD by inhibiting liver inflammation, but the former is more effective.

[0074] Example 6 Experiment on promoting liver regeneration in ALD mice using EVnew and EVadult

[0075] Cell proliferation and apoptosis are important criteria for liver regeneration. Our previous proteomic data showed that both EVnew and EVadult significantly activated molecules related to cell proliferation. The level of Ki67, a commonly used biomarker for cell proliferation, was significantly lower compared with the control group. In contrast, a significant upregulation was observed after treatment with EVnew or EVadult, with EVnew showing a better effect. At the same time, TUNEL results of mouse liver samples showed that chronic alcohol feeding led to increased cell death in the ALD group, while apoptosis levels were effectively inhibited in both experimental groups ( Fig.19 ). Consistently, the protein level of BCL2 was decreased in the ALD group, while BAX was upregulated most significantly, and these effects were effectively ameliorated in the treatment group ( Fig. 20 ). These data indicate that administration of EVnew and EVadult promoted hepatocyte proliferation, inhibited apoptosis, and improved liver injury.

[0076] The EVnew and EVadult provided by the present invention inhibit mitochondrial stress, inflammation and apoptosis, and promote hepatocyte proliferation, thereby reversing hepatocyte damage caused by chronic alcohol exposure. The EVnew provided by the present invention may improve mitochondrial stress better than EVadult. The present invention provides a safe and effective treatment method for patients with alcoholic liver disease related to mitochondria.

Claims

1. Application of liver-derived extracellular vesicles in organisms in the preparation of drugs for treating patients with alcoholic liver disease.

2. The use according to claim 1, characterized in that: The liver-derived extracellular vesicles are used to reduce mitochondrial stress in the liver of patients with alcoholic liver disease.

3. The use according to claim 2, characterized in that: The liver-derived extracellular vesicles are used to reduce mitochondrial stress markers in the liver of patients with alcoholic liver disease, namely 8-hydroxy-2-deoxyguanosine and / or MitoSOX.

4. The use according to claim 1, characterized in that: The liver-derived extracellular vesicles are used to increase the expression of mitochondrial active substances PGC-1α, SOD2 and / or GPX1 in patients with alcoholic liver disease.

5. The use according to claim 1, characterized in that: The liver-derived extracellular vesicles are used to regulate macrophage homeostasis in patients with alcoholic liver disease.

6. The use according to claim 5, characterized in that: The liver-derived extracellular vesicles regulate macrophage homeostasis by downregulating pro-inflammatory macrophages in patients with alcoholic liver disease and / or increasing anti-inflammatory macrophages in patients with alcoholic liver disease.

7. The use according to claim 6, characterized in that: The liver-derived extracellular vesicles reduce the expression of iNOS in patients with alcoholic liver disease and / or increase the expression of CD206 in patients with alcoholic liver disease.

8. The use according to claim 1, characterized in that: The liver-derived extracellular vesicles are used to reduce the protein expression of TNF-α and / or IL-6 involved in inflammatory response.

9. The use according to claim 1, characterized in that: Hepatic extracellular vesicles for improving liver reproduction and regeneration in patients with alcoholic liver disease.

10. The use according to claim 1, characterized in that: The liver-derived extracellular vesicles are selected from adult organisms or juvenile organisms; preferably, the organisms are juvenile organisms; preferably, the organisms are humans or mice.

Images