Medicine for treating acute hepatic failure as well as preparation method and application thereof

Abstract

The invention provides a medicine for treating acute hepatic failure. The medicine comprises curcumin and extracellular vesicles derived from honey. The invention further provides a preparation method and application of the medicine. According to the invention, the extracellular vesicles are separated from the honey and are proved to have a remarkable anti-inflammatory effect, curcumin is loaded to the extracellular vesicles of the honey, the treatment effect on acute hepatic failure is excellent, and a new effective medicine is provided for clinic.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a drug for treating acute liver failure, its preparation method and uses. Background Art

[0002] Acute liver failure (ALF) is a severe liver disease, usually sudden, characterized by rapid progression, and has a mortality rate as high as 30%. Acetaminophen (APAP) overdose is considered the most common cause of ALF. In the case of overdose, APAP is metabolized into a large amount of N-acetyl-p-benzoquinone imine, which depletes glutathione, leading to the overproduction of reactive oxygen species and ultimately causing hepatocyte death.

[0003] However, the clinical treatment drugs for ALF are very limited. N-acetylcysteine is currently the only FDA-approved antidote for treating APAP-induced liver injury, but its treatment regimen is complex, including three different time phases and weight-based infusion, and often has adverse reactions due to the drug concentration. Therefore, there is an urgent need to develop new treatment strategies for APAP-induced ALF, and these strategies should have low toxicity and high efficiency.

[0004] Curcumin is a natural polyphenolic compound with various biological activities such as anti-inflammatory, antioxidant, and anticancer effects, and has a certain preventive and therapeutic effect on APAP-induced liver injury. However, due to the poor solubility and low bioavailability of curcumin, its clinical application is limited.

[0005] Extracellular vesicles (EVs) refer to particles secreted by cells with a lipid bilayer membrane structure and play an important role in cell-to-cell communication and the transport of bioactive molecules such as proteins, RNA, and other signaling molecules. EVs have been found in many common foods such as milk, grapes, broccoli, carrots, ginger, and apples. However, the research on honey-derived EVs is still limited. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a drug for treating acute liver failure, its preparation method and uses.

[0007] The present invention provides a drug for treating acute liver failure, and the drug comprises curcumin and honey extracellular vesicles.

[0008] Wherein, the drug is formed by mixing curcumin and honey extracellular vesicles; or curcumin is loaded into honey extracellular vesicles.

[0009] Wherein, the mass ratio of curcumin to honey extracellular vesicles is 1:1.

[0010] Among them, the method for preparing the honey extracellular vesicles includes: taking honey, centrifuging and diluting it for the first time, and then purifying it by low-speed centrifugation and ultra-high-speed centrifugation, and enriching it to obtain honey extracellular vesicles.

[0011] Among them, when preparing the honey extracellular vesicles, the honey is cauliflower honey; the method for the first centrifugation is: centrifuging at 200 g for 15 minutes at 4 °C; the dilution is carried out with PBS buffer solution.

[0012] Among them, when preparing the honey extracellular vesicles, the method for low-speed centrifugation is centrifuging at 10,000 g for 30 minutes at 4 °C;

[0013] The method for ultra-high-speed centrifugation purification is: first ultra-high-speed centrifuging at 100,000 g for 2 hours at 4 °C, then resuspending with PBS, and filtering through a filter membrane with a pore size of 200 nm;

[0014] The method for enrichment is: after diluting with PBS, ultra-high-speed centrifuging at 100,000 g for 2 hours at 4 °C.

[0015] The present invention also provides a method for preparing the above-mentioned drug. When loading curcumin into the honey extracellular vesicles, the loading method is as follows: uniformly mixing the honey extracellular vesicles and curcumin, and incubating at 37 °C for 1 hour.

[0016] The present invention also provides the use of the above-mentioned drug in the preparation of a drug for preventing and / or treating acute liver failure.

[0017] Among them, the acute liver failure is acetaminophen-induced acute liver failure.

[0018] Among them, the drug is a drug that inhibits pyroptosis and / or necroptosis

[0019] The inventors of the present invention isolated extracellular vesicles (honey extracellular vesicles, HEVs) from honey and proved that they have significant anti-inflammatory effects. Loading curcumin (Curcumin) into HEVs to form Cur@HEVs significantly improves stability and bioavailability. Compared with using curcumin or HEVs alone, Cur@HEVs show better efficacy in the treatment of APAP-induced ALF. In addition, compared with traditional drug delivery systems, HEVs, as a natural-source biological carrier, have higher safety and targeting properties and good application prospects.

[0020] In addition, programmed cell death (PCD) plays an important role in the occurrence and development of ALF, among which pyroptosis and necroptosis are two key forms of inflammatory cell death. The present inventors found that pyroptosis and necroptosis were significantly activated in the APAP-induced ALF mouse model. Cur@HEVs could significantly inhibit the expression of genes and proteins related to pyroptosis and necroptosis, and improve ALF by regulating programmed cell death (PCD).

[0021] The following specific embodiments in the form of the description of the drawings and examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. For those skilled in the art, any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. Brief Description of the Drawings

[0022] Figure 1 Characteristics of HEVs; (A) Isolation and purification process of HEVs. (B) Representative ultrastructure image of HEVs observed by transmission electron microscopy (TEM). (C) Size distribution of HEVs measured by nanoparticle tracking analysis (NTA). (D) Zeta potential of EVs measured by NTA. (E) Lysis effect of Triton X-100 (TX) on HEVs. (F) Representative example of a gel image of HEVs RNA. (RNA was loaded onto a 2.5% agarose gel after incubation with RNase (10 μg / ml) at 37 °C for 30 minutes (‘+’ indicates presence, ‘-’ indicates absence)). (G) Representative example of a gel image of HEVs protein. The protein was run on a 10% Bis-tris protein gel and visualized using Coomassie Brilliant Blue staining. For the data in this figure, 3 independent biological experiments were analyzed, and the results are expressed as mean ± standard error of the mean (SEM), *p < 0.05.

[0023] Figure 2 Biodistribution and effects in mice after oral administration of HEVs; (A) Mice were orally administered Cy7-labeled HEVs or control. (B and C) Representative IVIS images of normal mouse organs collected 3 hours and 6 hours after oral administration of Cy7-HEVs (10 10 particles / kg). Mice receiving PBS or Cy7 were used as controls. (D) A typical inflammatory mouse model was established by single intraperitoneal injection of LPS (10 mg / kg), followed by daily administration of 10 8 -10 10Treatment with HEVs at a dose of

[0024] Figure 3 Upregulation of necroptosis and pyroptosis in ALF patients and animal models; (A) Heatmap according to the z-score in GSE255834. (B) Immunostaining of GSDMD-NT and p-MLKL in ALF patients. (C) Pyroptosis and necroptosis in GSE255834. (D and E) After 48 hours of APAP treatment, the mRNA expression levels of pyroptosis genes (including Caspase-1, Gsdmd, Nlrp3) and necroptosis markers (including Ripk1, Ripk3, Mlkl) were detected, with β-actin as the internal reference. (F and G) Representative Western Blot results of pyroptosis markers (including p20, GSDMD-NT, and NLRP3) and necroptosis markers (including p-RIPK1, p-RIPK3, and p-MLKL) were detected, with β-actin as the protein loading control. For this figure, the data were analyzed in 3 independent biological experiments, and the results are presented as mean ± standard error of the mean (SEM), *p < 0.05, **p < 0.01, ***p < 0.001.

[0025] Figure 4 Cur@HEVs improved the stability and bioavailability of curcumin; (A) Representative TEM image of Cur@HEVs. (B) Zeta potential of EVs measured by NTA. (C) In vitro release curve of Cur in Cur@HEVs. (D) Schematic diagram of the animal experiment and Cy7-Cur@HEVs in the liver.

[0026] Figure 5Oral Cur@HEVs enhance the therapeutic effect of Cur administered in vivo; (A) Schematic diagram of the animal experiment. (B) Survival rates of mice treated with APAP and / or Cur@HEVs. (C) Detection of DNA breaks in the liver using the TUNEL assay (green), with DAPI (blue) as a counterstain. Representative H&E-stained liver tissue images. The dashed lines in the H&E images outline the areas of necrotic cell death. (D, E, and F) Serum total bilirubin, AST, and ALT levels in mice treated with APAP and / or Cur@HEVs. (G, H, and I) Serum UA, Bun, IL-1β, and TNF-α levels in mice treated with APAP and / or Cur@HEVs. For this figure, the data were analyzed in 3 independent biological experiments and the results are presented as mean ± standard error of the mean (SEM), *p < 0.05, **p < 0.0 , ***p < 0.001, #p < 0. , ##p < 0.01.

[0027] Figure 6 Oral Cur@HEVs limit necroptosis and pyroptosis in vivo; (A and B) The mRNA expressions of Nlrp3, Gsdmd, Caspase1, Ripk1, Ripk3, and Mlkl were detected. (C and D) The protein levels of NLRP3, GSDMD-NT, and p20, as well as p-RIPK1, p-RIPK3, and p-MLKL in mice treated with APAP and / or Cur@HEVs were measured, with β-actin as a protein loading control. (E and F) Immunostaining of pyroptosis and necroptosis markers in the livers of acute liver failure (ALF) mice. For this figure, the data were analyzed in 3 independent biological experiments and the results are presented as mean ± standard error of the mean (SEM), *p < 0.05, **p < 0.01, ***p < 0.001.

[0028] Figure 7 Oral Cur@HEVs are more beneficial to liver function than Cur or HEVs alone; (A - D) Serum total bilirubin, UA, AST, and ALT levels in mice of each group. (E) Serum IL-1β and TNF-α levels in mice of each group. For this figure, the data were analyzed in 3 independent biological experiments and the results are presented as mean ± standard error of the mean (SEM), *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01. Chow in the figure represents the control (the control is not treated with anything). * indicates comparison with Chow, and # indicates comparison with APAP.

[0029] Figure 8Oral Cur@HEVs was more effective in inhibiting cell death than Cur or HEVs alone; (A) Immunostaining of pyroptosis and necroptosis markers in the livers of mice in each group. (B and C) The mRNA expression of Nlrp3, Gsdmd, Caspase1, Ripk1, Ripk3, and Mlkl was detected. For this figure, the data were analyzed in 3 independent biological experiments, and the results are expressed as mean ± standard error of the mean (SEM), *p < 0.05, **p < 0.01, ***p < 0.001, ns represents no statistical difference. Chow in the figure represents the control, * refers to comparison with Chow, and # refers to comparison with APAP. Detailed implementation manners

[0030] The present invention will be further described in detail below in conjunction with embodiments, but the present invention is not limited thereto.

[0031] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0032] The experimental reagents and instruments used in the present invention are listed as follows: multi-functional cell culture incubator, low-temperature refrigerator, high-temperature sterilizer, electronic constant temperature water bath, constant temperature magnetic stirrer, timed horizontal shaker, CFX96 instrument, high-efficiency pure water system, low-speed centrifuge, ultra-high resolution upright fluorescence microscope, transmission electron microscope, nanoparticle tracking analyzer, electrophoresis instrument, chemical imager, micropipette, RIPA buffer, protease inhibitor, phosphatase inhibitor, BCA protein quantification kit, TritonX-100, RNase, SDS-PAGE gel, methanol, PVDF membrane, blocking solution, ECL chemiluminescence solution, related antibodies, Runningbuffer, Loadingbuffer centrifugal ultrafiltration tube, 1% phosphotungstic acid, electron microscopy copper mesh, phosphate buffer, 10% chloral hydrate, Protease, Collagenase D, DNase I, DAPI, fluorescence quantitative 8-well tube, SYBR qPCR Master Mix, SuperMix for qPCR kit, Trizol, DMEM medium, 1640 medium, various types of cell culture flasks, culture dishes, well plates, various types of pipette tips, various types of centrifuge tubes, various ELLSA kits.

[0033] Example 1 Preparation and effect of honey extracellular vesicles

[0034] I. Honey contains extracellular vesicles (HEVs)

[0035] To accelerate the clinical application of honey, we isolated HEVs from honey and detected them. The specific method is as follows:

[0036] 1. Sample preparation: Take 20 g of cauliflower honey (commercial or self-made cauliflower honey can be used). After pre-cooling overnight in a 4°C refrigerator, centrifuge at 200×g for 15 minutes. Take 2 ml of the supernatant and dilute it to 40 ml with 4°C PBS buffer.

[0037] 2. Centrifuge the diluent at 10,000×g for 30 minutes.

[0038] 3. Ultracentrifugation purification: Collect the supernatant and perform ultracentrifugation at 100,000×g for 2 hours. Wash the obtained nanoparticle precipitate with 4°C PBS, resuspend it in PBS, and filter it through a 200 nm pore size filter membrane.

[0039] 4. Nanoparticle enrichment: Dilute the fractionated particles with PBS and finally enrich the nanoparticles by ultracentrifugation at 100,000×g for 2 hours. (Note: All centrifugation operations are carried out at a temperature controlled at 4°C.)

[0040] This method reduces the sugar content in honey through pretreatment, and does not additionally increase the components of the solution such as polyethylene glycol, which is beneficial for oral administration and reduces gastrointestinal reactions. Compared with other ultracentrifugation protocols, this protocol simplifies the centrifugation process in the differential centrifugation part, greatly reducing the operation time, but does not affect the centrifugation effect. In addition, this protocol also adds a filtration step through a 200 nm pore size filter membrane, which can further purify HEV. Through the above means, this method can isolate a large amount of highly purified HEV.

[0041] 5. Detect the nanoscale characteristics of HEVs: After enriching the purified HEVs, disperse them, pick them up on a copper grid, add 1% phosphotungstic acid (PTA) and stain for 1 min, suck off the excess stain, and air dry. Image.

[0042] 6. NTA detection of the particle size and zeta potential of HEVs: Dilute the sample to an appropriate concentration with an appropriate buffer (such as PBS, distilled water, etc.). Add the diluted sample to the sample cell of the NTA device. Ensure that the sample cell is clean and free of bubbles. Start measuring the sample. The instrument will irradiate the particles in the sample with a laser, capture the scattered light of the particles in the solution, and generate a video image of the particles. Analyze the particle size, concentration distribution, and zeta potential through software.

[0043] The results are shown in Figure 1 .

[0044] First, HEVs were isolated from cauliflower honey ( Figure 1 A). Transmission electron microscopy (TEM) showed that HEVs had a membrane-enclosed vesicle-like structure ( Figure 1B). By nanoparticle tracking analysis (NTA), the diameter of HEVs was approximately 160 nm, and its zeta potential was -32.35 ± 0.7 mV( Figure 1 C and D). After treatment with the non-ionic detergent Triton X-100 (TX), the membrane structure was disrupted by breaking lipid-protein and lipid-lipid interactions, resulting in a significant decrease in the number of HEVs( Figure 1 E), indicating that HEVs are mainly membrane-enclosed particles. In addition, the naked RNA extracted from HEVs was easily degraded under the action of RNase( Figure 1 F). After separating the proteins of HEVs by SDS-PAGE, a large number of protein bands were observed( Figure 1 G). Therefore, it can be known that extracellular vesicles (EVs) do exist in honey. II. Oral administration of HEVs can alleviate inflammation in mice

[0045] 1. Evaluate the in vivo distribution of HEVs:

[0046] After normal mice were fasted for 12 hours, they were orally administered Cy7-labeled HEVs or control ( Figure 2 A). After anesthetizing the mice at 3 hours and 6 hours after oral administration, the signals in the organs were detected by the IVIS imaging system. Then the mice were sacrificed by excessive anesthesia to verify the fluorescence signals in the organs. The results showed that HEVs mainly accumulated in the liver, and no significant signals were observed in the organs of the PBS group( Figure 2 B and C).

[0047] 2. Evaluate the effect of HEVs on inflammation:

[0048] After normal mice were fasted for 12 hours, a typical inflammatory mouse model was established by single intraperitoneal injection of 10 mg / kg of LPS. Two days after modeling, HEVs (dose: 10^8 - 10^10 particles / kg) were administered daily for 3 consecutive days. After the mice were sacrificed by excessive anesthesia, mouse serum and liver were collected. The levels of IL-1β and TNF-α in the mouse serum were detected using an ELLSA kit. The RNA in the liver was extracted, reverse transcribed into cDNA, and then the expression of inflammation-related genes was detected.

[0049] Collect the serum of the model animals. Add an appropriate amount of antigen or antibody (usually 1 - 10 μg / mL) to the wells of the ELISA plate. Incubate the plate overnight at 4°C to ensure that the antigen binds fully to the well wall. Wash the plate wells 3 - 5 times and add the blocking solution and incubate for 30 minutes. Add the diluted serum to each well, incubate at room temperature for 1 hour, and wash the plate 3 - 5 times with the washing buffer. Use a secondary antibody that binds to the target antibody or antigen, incubate for 30 minutes, and wash the plate again with the washing buffer. Add an appropriate amount of enzyme substrate solution until the reaction is sufficient and the color change is obvious. Terminate the substrate reaction to prevent over-color development.

[0050] Levels of molecules related to qPCR analysis: Total RNA was extracted using the TRIzol method to avoid RNase contamination. After reverse transcribing the RNA, SYBR Green Master Mix, primers, and cDNA template were mixed, appropriate temperature conditions (denaturation, annealing, extension) were set, and fluorescence was monitored in real time. The data was analyzed.

[0051] Detection of the expression of related molecules by Western Blots: Liver samples were lysed using pre-cooled RIPA buffer, left standing on ice for 30 min, centrifuged at 12,000×g at 4°C for 15 min, the supernatant was taken, and the protein concentration was measured. 5×SDS loading buffer was added, and it was heated at 95°C for 5 min. Protein Marker and the samples were added to the loading wells, and electrophoresis was carried out at a constant voltage of 80–120 V until the bromophenol blue migrated to the bottom of the gel. The PVDF membrane was soaked in methanol for 1 min, immersed in the transfer buffer together with the gel, and the transfer membrane device was assembled for membrane transfer. It was blocked by shaking at room temperature in 5% skim milk for 1 h. After incubating with the related antibodies, ECL color development and imaging were performed.

[0052] To evaluate whether HEVs have an impact on inflammation in mice, we established a typical inflammatory mouse model by a single intraperitoneal injection of 10 mg / kg of LPS and administered HEVs (at a dose of 10^8 - 10^10 particles / kg) daily for 3 days ( Figure 2 D). The results showed that the levels of IL-1β and TNF-α in the serum of mice were significantly increased after LPS treatment, while HEVs significantly reduced the levels of these inflammatory factors ( Figure 2 E and F). At the same time, LPS induced an increase in the levels of AST, ALT, CREA, and UREA in the serum, while HEVs were able to improve these indicators ( Figure 2 G-J). In addition, LPS increased the expression of inflammation-related genes (Il-1β, Il-18, Il-6, and Tnf-α) in the liver, while HEVs significantly alleviated the expression levels of these genes ( Figure 2 K-N).

[0053] It can be seen that HEVs have significant anti-inflammatory activity and have potential in the treatment of various clinical inflammations.

[0054] Example 2 Improvement of ALF by HEVs loaded with curcumin (Cur@HEVs)

[0055] I. Pyroptosis and necroptosis are activated in ALF

[0056] To explore the role of pyroptosis and necroptosis in ALF, we analyzed the single-cell RNA sequencing data of ALF patients and APAP-induced ALF mouse models in the GEO database.

[0057] The results showed that, compared with the healthy control group, the expressions of pyroptosis- and necroptosis-related molecules (such as GSDMD, NLRP3, CASP1, RIPK1, RIPK3, and MLKL) were significantly upregulated in ALF patients ( Figure 3 A and B). In addition, in the APAP-induced ALF mouse model, the expression levels of pyroptosis- and necroptosis-related molecules (such as GSDMD-NT, NLRP3, caspase-1, RIPK1, RIPK3, and p-MLKL) in liver tissues were also significantly increased ( Figure 3 C-G).

[0058] It can be seen that pyroptosis and necroptosis are significantly activated in ALF.

[0059] II. Improvement of ALF by HEVs loaded with curcumin (Cur@HEVs)

[0060] 1. Construction of Cur@HEVs nanoparticles

[0061] To enhance the therapeutic effect of HEVs, we loaded curcumin into HEVs. After uniformly mixing according to the ratio of the protein mass of HEVs to the mass of curcumin at 1:1 and incubating at 37 °C for 1 hour, Cur@HEVs were formed ( Figure 4 A).

[0062] 2. Detection of the nanoscale characteristics of Cur@HEVs:

[0063] After enriching the purified Cur@HEVs, they were dispersed, fished onto a copper mesh, and stained with 1% phosphotungstic acid (PTA) for 1 min. The excess stain was aspirated and air-dried. Imaging was performed.

[0064] 3. Analysis of the drug release ability of Cur@HEVs (dynamic dialysis method):

[0065] Using a centrifugal ultrafiltration tube, the release medium passed through the nanoparticle suspension at a constant flow rate (such as 1 mL / min), and the effluent was collected. The effluent was collected at regular intervals, the drug concentration was measured, and a real-time release curve was plotted.

[0066] 4. In vitro verification of the function of Cur@HEVs:

[0067] In primary mouse hepatocytes and HepG2 cells, an APAP-induced cell injury model was constructed, and then HEVs, Cur, or Cur@HEVs were administered respectively. A cell viability detection kit was used to detect the changes in cell viability. After the treatment was completed, the culture supernatant and cells were collected, and ELISA was used to detect the release of inflammatory factors and Western Blots were used to detect the changes in the marker molecules of cell pyroptosis and apoptotic necrosis.

[0068] Cur@HEVs see Figure 4 A. Transmission electron microscopy (TEM) showed that Cur@HEVs still maintained an intact vesicle structure ( Figure 4 A and B). By high performance liquid chromatography (HPLC) analysis, the drug loading of curcumin in Cur@HEVs was 19.5 ± 0.8% ( Figure 4 C). In in vivo experiments, Cur@HEVs could significantly accumulate in the liver ( Figure 4 D).

[0069] 5. In vivo verification of the effect of Cur@HEVs:

[0070] After normal mice were fasted for 12 hours, an ALF model was induced by intraperitoneal injection of 300 mg / kg APAP, and then 10 10 particles / kg of Cur@HEVs were orally administered every three days for a total of two times. After the treatment was completed, the model was sacrificed by overdose anesthesia, and serum and liver were collected. ELISA was used to detect the release of inflammatory factors and blood biochemical indexes.

[0071] Collect the serum of the model animals. Add an appropriate amount of antigen or antibody (usually 1 - 10 μg / mL) to the wells of the ELISA plate. Incubate the plate overnight at 4°C to ensure that the antigen binds fully to the well wall. Wash the plate wells 3 - 5 times and add the blocking solution for incubation for 30 minutes. Add the diluted serum to each well and incubate at room temperature for 1 hour. Wash the plate 3 - 5 times with the washing buffer. Use a secondary antibody that binds to the target antibody or antigen, incubate for 30 minutes, and wash the plate again with the washing buffer. Add an appropriate amount of enzyme substrate solution until the reaction is sufficient and the color change is obvious. Terminate the substrate reaction to prevent over-color development.

[0072] In the APAP-induced ALF mouse model, oral administration of Cur@HEVs significantly improved the survival rate of the mice ( Figure 5 A and B). At the same time, Cur@HEVs significantly reduced the levels of AST, ALT, CREA, and UREA in the serum ( Figure 5 D - H), and improved the pathological damage of the liver tissue ( Figure 5 C). In addition, Cur@HEVs significantly inhibited the expression of inflammatory factors (such as IL-1β and TNF-α) in the liver ( Figure 5 I). These results indicate that Cur@HEVs can effectively improve ALF.

[0073] 6. Cur@HEVs improves ALF by inhibiting pyroptosis and necroptosis

[0074] To further explore the mechanism by which Cur@HEVs improves ALF, we detected the expression of proteins related to pyroptosis and necroptosis. The method is as follows:

[0075] (1) Analyze the levels of related molecules by qPCR: Extract total RNA using the TRIzol method to avoid RNase contamination. After reverse-transcribing the RNA, mix SYBR Green Master Mix, primers, and cDNA template, set appropriate temperature conditions (denaturation, annealing, extension), and monitor fluorescence in real-time. Analyze the data.

[0076] (2) Detect the expression of related molecules by Western Blots: Lyse liver samples using pre-chilled RIPA buffer, let stand on ice for 30 min, centrifuge at 12,OOOxg for 15 min at 4 °C, take the supernatant and measure the protein concentration. Add 5×SDS loading buffer and heat at 95 °C for 5 min. Load protein Marker and samples into the wells, perform electrophoresis at a constant voltage of 80–120 V until the bromophenol blue migrates to the bottom of the gel and then terminate. Soak the PVDF membrane in methanol for 1 min, immerse it in transfer buffer together with the gel, assemble the transfer device and perform transfer. Block in 5% skim milk by shaking at room temperature for 1 h. After incubating with related antibodies, develop and image using ECL.

[0077] (3) Observe the changes of related molecules in the liver by IHC: Fix liver tissues with 4% paraformaldehyde (PFA), dehydrate, and embed in paraffin for sectioning. Deparaffinize in xylene and rehydrate in different concentrations of alcohol. Heat-treat with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), let the sections cool naturally, and then wash with PBS or TBS buffer. Block the sections with 5% normal goat serum or bovine serum albumin (BSA) solution. Dilute the antibody specific to the antigen to an appropriate concentration, add it dropwise to the sections, incubate overnight, and wash. Use DAB (3,3'-diaminobenzidine) chromogenic reagent, usually incubate for several minutes until a visible color reaction is observed. Counterstain the sections with hematoxylin, usually incubate for 1–2 minutes, and then rinse with water. Dehydrate the sections successively through different concentrations of alcohol (70%, 95%, 100%), clear with xylene, mount with mounting medium, and cover with coverslips.

[0078] The results showed that Cur@HEVs significantly reduced the expression of pyroptosis- and necroptosis-related genes (such as GSDMD, NLRP3, CASP1, RIPK1, RIPK3, and MLKL) in the liver ( Figure 6 A-B). In addition, Cur@HEVs significantly inhibited the expression of GSDMD-NT, NLRP3, caspase-1, RIPK1, RIPK3, and p-MLKL in the liver ( Figure 6 C-F). These results indicate that Cur@HEVs improve ALF by inhibiting pyroptosis and necroptosis.

[0079] Evaluation of the Synergistic Effect of Cur@HEVs in the Treatment of ALF in Example 3

[0080] To compare the therapeutic effects of Cur@HEVs, HEV alone, or Cur on ALF, the following experimental method was adopted:

[0081] After normal mice were fasted for 12 hours, an acute liver failure (ALF) model was induced by intraperitoneal injection of 300 mg / kg acetaminophen (APAP). Subsequently, they were orally administered 10 10 particles / kg of HEVs (biological nanovesicles are usually weighed by protein mass, and the protein content of 10 10 particles of HEVs is 500 μg), 10 10 particles / kg of Cur@HEVs (where the mass of HEV and Cur is equivalent), or 5 g / kg curcumin (Cur) for treatment, once every three days, for a total of two times. After the treatment was completed, the models were sacrificed by overdose anesthesia, and serum and livers were collected. ELISA was used to detect the release of inflammatory factors and blood biochemical indexes.

[0082] Collect the serum of the model animals. Add an appropriate amount of antigen or antibody (usually 1 - 10 μg / mL) to the wells of the ELISA plate. Incubate the plate overnight at 4℃ to ensure that the antigen binds fully to the well wall. Wash the wells of the plate 3 - 5 times and add the blocking solution for incubation for 30 minutes. Add the diluted serum to each well and incubate at room temperature for 1 hour. Wash the plate 3 - 5 times with the washing buffer. Use the secondary antibody that binds to the target antibody or antigen, incubate for 30 minutes, and wash the plate again with the washing buffer. Add an appropriate amount of enzyme substrate solution until the reaction is sufficient and the color change is obvious. Terminate the substrate reaction to prevent overcoloring.

[0083] The results showed that APAP-induced ALF was manifested by elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and uric acid (UA). It is worth noting that only treatment with Cur@HEVs could significantly relieve the abnormalities of these indexes ( Figure 7 A - D). In addition, APAP increased the release of IL-1β and TNF-α, while single-agent treatment with Cur@HEVs could significantly improve this phenomenon ( Figure 7 E).

[0084] These data indicate that oral administration of Cur@HEVs can reduce APAP-induced hepatocyte damage, and the effect is significantly better than that of HEVs or Cur alone.

[0085] In vivo experiments further revealed that Cur@HEVs ameliorated ALF by inhibiting necroptosis and pyroptosis. In the livers of the APAP group, the expressions of pyroptosis- and necroptosis-related molecules (such as GSDMD, NLRP3, p-MLKL, and p-RIPK3) were upregulated. When HEV or Cur was used alone, there was no significant difference in the expression of related factors. Compared with the groups treated with HEV or Cur alone, the Cur@HEVs treatment group could significantly inhibit the expression of these molecules, and the results at the mRNA level also supported this conclusion( Figure 8 ).

[0086] It can be seen that Cur@HEVs alleviate acute liver failure by effectively inhibiting hepatic necroptosis and pyroptosis.

[0087] Programmed cell death (PCD) plays an important role in the occurrence and development of ALF, among which pyroptosis and necroptosis are two key forms of inflammatory cell death. Pyroptosis is mediated by Gasdermin D (GSDMD) and triggered by activating the NLRP3 inflammasome and caspase-1. Necroptosis is mediated by RIPK1, RIPK3, and MLKL. The present inventors found that pyroptosis and necroptosis were significantly activated in the APAP-induced ALF mouse model. Meanwhile, Cur@HEVs could significantly inhibit the expression of pyroptosis- and necroptosis-related genes and proteins, indicating that it improved ALF by regulating PCD. The results showed that Cur@HEVs could improve ALF by inhibiting pyroptosis and necroptosis.

[0088] In summary, the present invention demonstrated that honey-derived HEVs have significant anti-inflammatory effects and can ameliorate ALF by inhibiting pyroptosis and necroptosis. Loading curcumin into HEVs further enhanced its therapeutic effect. These findings provide an effective strategy for the treatment of ALF.

Claims

1. A drug for treating acute liver failure, characterized in that, The drug includes curcumin and honey extracellular vesicles.

2. The drug according to claim 1, characterized in that, The drug is formed by mixing curcumin and honey extracellular vesicles; or curcumin is loaded into honey extracellular vesicles.

3. The drug according to claim 1 or 2, characterized in that, The mass ratio of curcumin to honey extracellular vesicles is 1:

1.

4. The drug according to claim 1 or 2, characterized in that, The preparation method of the honey extracellular vesicles includes: taking honey, centrifuging for the first time, diluting, and then purifying by low-speed centrifugation and ultra-high-speed centrifugation, and enriching to obtain honey extracellular vesicles.

5. The drug according to claim 4, characterized in that, When preparing the honey extracellular vesicles, the honey is cauliflower honey; the method of the first centrifugation is: centrifuging at 200 g for 15 minutes at 4 °C; the dilution is carried out with PBS buffer.

6. The drug according to claim 4, characterized in that, When preparing the honey extracellular vesicles, the method of low-speed centrifugation is centrifuging at 10,000 g for 30 minutes at 4 °C; The method of ultra-high-speed centrifugation purification is: first ultra-high-speed centrifuging at 100,000 g for 2 hours at 4 °C, then resuspending with PBS, and filtering through a filter membrane with a pore size of 200 nm; The method of enrichment is: after diluting with PBS, ultra-high-speed centrifuging at 100,000 g for 2 hours at 4 °C.

7. According to the preparation method of the drug according to any one of claims 1-6, characterized in that When loading curcumin into honey extracellular vesicles, the loading method is as follows: uniformly mixing honey extracellular vesicles and curcumin, and incubating at 37 °C for 1 hour.

8. Use of the drug according to any one of claims 1-6 in the preparation of a drug for preventing and / or treating acute liver failure.

9. The use according to claim 8, wherein: The acute liver failure is acetaminophen-induced acute liver failure.

10. The use according to claim 8, characterized in that: The drug is a drug for inhibiting pyroptosis and / or necroptosis.

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