Application of camel milk exosome in preparation of medicine for treating hepatic diseases

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of camel milk exosome in preparation of a medicine for treating hepatic diseases. The camel milk exosome reduces the liver injury by reducing tissue cell apoptosis, tissue fibrosis degree and necrosis rate. For a carbon tetrachloride induced rat acute hepatic failure model, camel milk exosome intervention can significantly improve pathological changes of hepatic tissue and reduce the activity of serum ALT, and the treatment effect is significantly superior to that of a non-administration model group. The invention further provides a medicine containing the camel milk exosome, the camel milk exosome is simple in preparation process, good in stability and high in safety, the prepared camel milk exosome can be used for relieving or treating acute hepatic failure, and a new strategy is provided for liver disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of life sciences, and in particular, to the application of camel milk exosomes in the preparation of drugs for treating liver diseases. Background Art

[0002] Acute liver failure (ALF) is a severe liver disease, which refers to the massive necrosis of hepatocytes or severe impairment of liver function within a short period (usually within 26 weeks), resulting in severe disorders or decompensation of the liver's synthetic, detoxifying, excretory, and biotransformation functions, presenting a group of clinical syndromes mainly manifested as jaundice, coagulation dysfunction, hepatic encephalopathy, etc.

[0003] In recent years, exosomes have become a hot research topic in the field of biomedicine. Exosomes are nanoscale vesicles secreted by cells, with natural drug-loading properties, low immunogenicity, and targeted delivery capabilities. They can carry various bioactive molecules such as proteins and nucleic acids, precisely regulating the functions of recipient cells. In the research on the treatment of liver diseases, plant- and animal-derived exosomes have shown unique potential, playing an active role in anti-inflammation, antioxidant, and anti-fibrosis aspects. For example, tea exosomes (CN114699464A) can effectively alleviate liver injury by inhibiting the expression of fibrosis genes; garlic exosomes (CN116836908A) can regulate the inflammatory pathway and reduce hepatocyte apoptosis in an acute liver failure (ALF) model. However, plant exosomes have obvious limitations. Their functions are greatly affected by species specificity, and the preparation process is complex, with difficult-to-improve yields, unable to meet the needs of large-scale clinical applications.

[0004] In contrast, animal-derived exosomes, such as bovine milk and camel milk exosomes, have become a more promising research direction due to their wide sources and stable components. Among them, camel milk exosomes have attracted the attention of many researchers due to their unique biocompatibility and high safety. Existing studies have shown that camel milk exosomes have cytotoxic effects on various cancer cell lines, while having relatively low toxicity to normal cells.

[0005] Although camel milk exosomes have shown certain advantages in related research, current studies mostly focus on their in vitro effects on liver cancer, and the research on other liver diseases and their mechanisms of action is still very scarce. At the same time, the isolation process, stability, and large-scale preparation technology of exosomes have not been fully optimized, greatly limiting their wide clinical application.

[0006] Based on the above situation, the present invention first proposes to apply camel milk exosomes to the treatment of acute liver failure. By optimizing the exosome purification process, highly pure camel milk exosomes can be obtained quickly and efficiently, and with the help of a rat liver failure model induced by carbon tetrachloride (CCl4), its effects in reducing serum transaminases (ALT, AST), inhibiting hepatocyte apoptosis and fibrosis are verified, opening up a new way for the treatment of acute liver failure. Summary of the Invention

[0007] To overcome the problems of limited drug selection and efficacy in the current treatment of acute liver failure, the present invention provides an application of camel milk exosomes in the preparation of drugs for treating liver diseases. The present invention first proposes to use camel milk exosomes to alleviate acute liver failure, providing a brand-new and safe treatment plan for liver protection.

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

[0009] <First aspect>

[0010] The present invention provides an application of camel milk exosomes in the preparation of drugs for preventing and / or treating liver diseases.

[0011] The camel milk exosomes are derived from camel milk whey.

[0012] The liver diseases include hepatitis, liver fibrosis, liver cirrhosis, or liver failure.

[0013] The liver failure is acute liver failure.

[0014] The camel milk exosomes reduce acute liver injury by reducing tissue cell apoptosis, tissue fibrosis degree and necrosis rate.

[0015] The preparation method of the camel milk exosomes includes the following steps:

[0016] S1. Centrifuge fresh camel milk at 10,000g - 15,000g for 20 - 40 minutes, and extract the middle layer liquid after centrifugation as whey;

[0017] S2. After filtering the whey through a filter membrane with a pore size of 70 - 100μm, centrifuge the filtrate at 100,000 - 120,000g for 1 - 2 hours, collect the gel-like precipitate at the bottom of the tube and perform fragmentation treatment;

[0018] S3. Resuspend the fragmented precipitate with a buffer solution (such as PBS buffer solution), centrifuge the resuspended solution at 100,000 - 120,000g for 1 - 2 hours, collect the gel-like precipitate at the bottom of the tube and perform fragmentation treatment;

[0019] S4. Resuspend the precipitate broken in S3 with a buffer solution (such as PBS buffer solution), and pass the resuspended solution through a coarse filter membrane (70 μm) and a sterile filter membrane (0.22 μm), and collect the filtrate to obtain exosomes.

[0020] The dosage of the camel milk exosomes is 1-10*10 12 particles / kg.

[0021] <The second aspect>

[0022] The present invention provides a drug for treating liver diseases, and the drug comprises camel milk exosomes and a pharmaceutically acceptable carrier.

[0023] The dosage forms of the drug include at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] By administering camel milk exosomes and applying them to rats with acute liver failure, it can very effectively relieve acute liver injury, including significantly reducing tissue cell apoptosis, reducing the degree of tissue fibrosis, and significantly reducing the necrosis rate, with excellent comprehensive performance, achieving the curative effect of protecting the liver. It plays a role through the following mechanisms:

[0026] 1. Apoptosis regulation: Camel milk exosomes can precisely regulate the apoptosis-related signal pathways in cells and significantly reduce the apoptosis rate of tissue cells. By inhibiting the expression of pro-apoptotic proteins and enhancing the activity of anti-apoptotic proteins at the same time, the stability of the intracellular environment is maintained, enabling hepatocytes to maintain normal physiological functions and survival states.

[0027] 2. Fibrosis process inhibition: Camel milk exosomes effectively reduce the degree of liver tissue fibrosis. It can inhibit the activation and proliferation of hepatic stellate cells and reduce the excessive deposition of extracellular matrix, thereby blocking the progression of liver fibrosis. By regulating the cytokine network, it promotes the degradation and remodeling of fibrous tissue, gradually restoring the normal structure of the liver tissue.

[0028] 3. Necrosis degree alleviation: Camel milk exosomes significantly reduce the necrosis rate of liver tissue. It improves the blood circulation of the liver, increases the oxygen supply and nutrient supply to hepatocytes, and reduces the damage to hepatocytes caused by ischemia and hypoxia. At the same time, it enhances the antioxidant capacity of hepatocytes, scavenges free radicals, and reduces the damage to cells caused by oxidative stress, thereby effectively protecting the liver tissue from further damage.

[0029] Overall, the therapeutic effect of camel milk exosomes is not only reflected in the improvement of pathological indicators, but more importantly, in the protection and repair of the overall liver function. Through the synergistic effect of the above-mentioned multiple mechanisms, the liver can maintain normal metabolic, detoxification and synthesis functions under the state of acute liver injury, promote the regeneration and repair of hepatocytes, and ultimately achieve the curative effect of protecting the liver, providing a safe and effective new strategy for the treatment of acute liver failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Other features, objects and advantages of the present invention will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0031] Figure 1 Particle size distribution result diagram of camel milk whey exosomes prepared for Example 1.

[0032] Figure 2 Transmission electron microscope test diagram of camel milk whey exosomes prepared for Example 1.

[0033] Figure 3 H&E staining diagrams of the livers of rats in the camel milk exosome treatment group and the control group provided in Example 2; the scale bar of the figure is 100 microns;

[0034] Figure 4 Analysis of the effect of camel milk exosomes provided in Example 2 on the change of H&E staining of rat liver.

[0035] Figure 5 TUNEL staining diagrams of the livers of rats in the camel milk exosome treatment group and the control group provided in Example 2; the scale bar of the figure is 100 microns;

[0036] Figure 6 Analysis of TUNEL staining of rat liver by camel milk exosomes provided in Example 2.

[0037] Figure 7 MASSON staining diagrams of the livers of rats in the camel milk exosome treatment group and the control group provided in Example 2; the scale bar of the figure is 100 microns;

[0038] Figure 8 Analysis of MASSON staining of rat liver by camel milk exosomes provided in Example 2.

[0039] Figure 9 Analysis of the change of serological indexes of rats by camel milk exosomes provided in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The present invention will be described in detail below with reference to embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0041] Example 1 Preparation of Camel Milk Exosomes and Physical Sign Detection

[0042] 1.1 Preparation of Camel Milk Exosomes

[0043] Step 1: Separate whey. Pour 500 mL of fresh camel milk into a high-speed centrifuge tube, centrifuge at 13,000 g for 30 minutes, take the middle layer liquid to obtain whey.

[0044] Step 2: Filter to obtain exosomes. After filtering the whey through a 70 μm filter membrane, the filtrate is dispensed into ultracentrifuge tubes, centrifuged at 100,000 g for 1.5 h, discard the supernatant, take the gel-like precipitate at the bottom of the tube, and crush it into pieces.

[0045] Step 3: Remove impurities. Transfer the lumpy precipitate from the previous step to a new ultracentrifuge tube, make up the PBS buffer to 50 mL, then centrifuge at 100,000 g at high speed for 1.5 h, discard the supernatant, take the gel-like precipitate at the bottom of the tube, and crush it into pieces.

[0046] Step 4: Dissolve and sterilize. Resuspend the lumpy precipitate with 40 mL of PBS buffer, fully dissolve it into a uniform liquid by pipetting or homogenizing, and then sequentially filter through a 70 μm filter to remove insoluble impurities and a 0.22 μm filter membrane for sterilization, and collect the filtrate to obtain a purified exosome solution.

[0047] 1.2 NTA Detection Results

[0048] For the NTA detection of exosomes, nanoparticle tracking analysis (NTA) technology uses laser scattering microscopy imaging technology to record the Brownian motion trajectories of nanoparticles in solution, and calculates the particle size and particle concentration through the Stokes-Einstein equation. This technology can measure particle sizes in the range of 30 - 1000 nm, so it can provide more accurate particle size data and is currently the most mainstream method for measuring exosome size.

[0049] The prepared exosomes are diluted with PBS at a volume ratio of 1:1000. Use a 1 ml syringe to suck 1 ml of the exosome dilution, slowly inject it into the NTA device pipeline, set the detection parameters, start the detection, and the results are as Figure 1 shown, the average particle size is 166.6 nm, and the concentration is 1.33×10 12 Particles / mL.

[0050] 1.3 Electron microscopy observation

[0051] Take 20 μl of the prepared exosome suspension sample in step 1.1 with a pipette, drop it onto the copper grid and let it adsorb naturally for 5 - 10 minutes, then use a filter paper strip to absorb the excess liquid droplets and let it dry slightly; take 20 μl of 2% phosphotungstic acid solution with a pipette, drop it onto the copper grid, and let it stand for 3 - 5 minutes for staining; use a filter paper strip to absorb the excess liquid droplets and dry it under an incandescent lamp; finally, observe and take pictures under a transmission electron microscope. The obtained transmission electron microscope micrograph is as Figure 2 shown, and this result is consistent with the NTA detection result.

[0052] Example 2 Effect of camel milk exosomes on carbon tetrachloride-induced acute liver failure

[0053] Carbon tetrachloride (CCl4) is a selective hepatotoxic drug. After entering the body, it is activated into free radicals such as trichloromethyl radical (CCl3) in the liver. The latter can directly damage the plasma membrane, initiate lipid peroxidation, destroy the membranous structure of hepatocytes, etc., resulting in hepatocyte degeneration, necrosis and the formation of liver fibrosis. The liver injury model induced by carbon tetrachloride is widely used in the research of the etiology, histology and liver function changes of liver necrosis, cirrhosis and liver fibrosis, as well as the evaluation of hepatoprotective drugs and medicinal plant extracts due to its simple operation and high repeatability.

[0054] 1. Experimental animals and reagents: 30 male SD rats, 7 - 8 weeks old, SPF grade. The experimental animals were purchased from Vital River Laboratories and raised in Hanshu Biomedical Co., Ltd. The rats were raised in an IVC system in a barrier facility.

[0055] The information of the drugs used is shown in Table 1:

[0056] Table 1

[0057]

[0058] Preparation of glutathione: Weigh an appropriate amount of GSH, dissolve it in PBS to prepare a 20 mg / ml solution. The animals in the GSH group were intraperitoneally injected with 5 ml / kg. Assuming the animal weight is 40 g, the administration volume is (40 g / 1000) * 5 ml / kg = 0.2 ml.

[0059] 2. Experimental grouping and dosing regimen

[0060] Randomly divide 30 mice into 5 groups: A - control group, B - acute liver failure model group, C - glutathione treatment group, D - exosome low-dose treatment group, E - exosome high-dose treatment group, with 6 mice in each group.

[0061] The specific grouping and treatment methods are shown in Table 2: Groups A and B were gavaged with an equal volume of PBS buffer, 5 ml / kg, twice a day; Group C was intravenously injected with the control drug GSH (100 mpk), 5 ml / kg, once a day; Group D was intravenously injected with low-dose exosomes (1*10 11 particles / kg), twice a day; Group E was intravenously injected with high-dose exosomes (1*10 12 particles / kg), twice a day. On the 3rd day after administration, an acute liver failure model was established. Among them, the negative control group was gavaged with an equal volume of normal saline once, 2.5 ml / kg; the other four groups were gavaged with 50% carbon tetrachloride (the volume ratio of carbon tetrachloride to olive oil is 1:1) once, 2.5 ml / kg. After CCl4 gavage, blood was collected at 12 h, 24 h, and 36 h to prepare serum. The whole blood was left standing at room temperature for 1-2 h, centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was taken to obtain serum. Serum was used for blood biochemical detection of ALT (alanine aminotransferase) and AST (aspartate aminotransferase); (about 100 μl of serum was required). Endpoint sample collection: Heart, liver, lung, kidney, and whole brain samples were taken. Half of each tissue sample was snap-frozen in liquid nitrogen and stored at -80°C, and the other half was fixed with 4% PFA.

[0062] Table 2 Animal grouping information and dosing regimen

[0063]

[0064] *: Assuming the animal weight is 300 g, the dosing volume is (300 g / 1000) * 5 ml / kg = 1.5 ml.

[0065] **: PO: Gavage; QD: Once a day; BID: Twice a day.

[0066] 3. Experimental results

[0067] 3.1 H&E staining

[0068] Hepatocyte samples of each test group in Step 2 were collected for H&E staining. The results are as Figure 3 shown. A is the control group with no obvious lesions; B is the acute liver failure model group, where a large number of hepatocytes show ballooning degeneration, severe necrosis, and a large number of inflammatory cell infiltrations; C is the glutathione treatment group, where hepatocytes show ballooning degeneration and necrosis, and inflammatory cell infiltration and aggregation can be seen; D is the low-dose exosome treatment group, where the ballooning degeneration of hepatocytes is reduced, and the spread of inflammatory cell infiltration can be seen; E is the high-dose exosome treatment group, with a small amount of fatty change, and inflammatory cells infiltrating the hepatic sinus without spreading. Therefore, the present invention shows that the treatment with camel milk exosomes helps to restore the hepatic tissue morphology of rats with liver failure.

[0069] 3.2. TUNEL staining

[0070] The hepatocyte samples of each experimental group in Step 2 were collected for TUNEL staining. The TUNEL staining technique is a commonly used method for detecting DNA fragmentation in apoptosis. Cells with positive TUNEL staining indicate DNA fragmentation, which is a key feature of apoptosis. By counting the number of positive cells, the proportion of apoptotic cells can be evaluated. The results of the TUNEL analysis are as Figure 4 shown. A is the control group with no positive reaction; B is the acute liver failure model group with positive cells; C is the glutathione treatment group with no significant decrease in the number of positive cells; D is the low-dose exosome treatment group with a decrease in the number of positive cells; E is the high-dose exosome treatment group with a decrease in the number of positive cells. Compared with the liver failure model group, the number of apoptotic hepatocytes in the exosome treatment group of rats decreased significantly, indicating that camel milk exosome treatment alleviated hepatocyte apoptosis in the state of acute liver injury.

[0071] 3.3 Masson staining

[0072] The liver tissue samples of each experimental group in Step 2 were collected for Masson staining ( Figure 7 ). As a classic connective tissue staining method, Masson staining provides us with an intuitive and effective means of fibrosis detection. In fibrotic tissue, Masson staining will show obvious red or purple areas. These areas represent the proliferation and deposition of collagen fibers, which are typical manifestations of fibrotic lesions. By observing the distribution and scope of these areas, the degree and scope of fibrosis can be preliminarily judged. The results of the Masson analysis of the liver tissue samples are shown (as Figure 5 ): A is the control group with no obvious lesions; B is the acute liver failure model group with a large amount of collagen fiber deposition; C is the glutathione treatment group with collagen fibers accumulating around the hepatic sinusoids and spreading outwards; D is the low-dose exosome treatment group with collagen fibers accumulating in an aggregated state; E is the high-dose exosome treatment group with a small amount of collagen fiber accumulation and no spread. The fibrosis ratio of the rat liver in the exosome treatment group was lower than that in the acute liver failure model group, indicating that camel milk exosome treatment can alleviate the degree of liver fibrosis.

[0073] 3.4 Serological indicators:

[0074] The serum samples of each experimental group in Step 2 were collected and tested for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) on a biochemical analyzer. The testing method:

[0075] 3.4.1 Experimental equipment

[0076] The experimental equipment is shown in Table 3:

[0077] Table 3

[0078]

[0079]

[0080] 3.4.2 Main experimental consumables

[0081] The experimental consumables are shown in Table 4:

[0082] Table 4

[0083] Consumables Manufacturer Item Number 1.5 ml centrifuge tube Servicebio EP-150-M 1000 ul pipette Dragonmed KE0037273 200 ul pipette Dragonmed YE3K030591 50 ul pipette Dragonmed DS35110 10 ul pipette Dragonmed KE0012951

[0084] 3.4.3 Composition and storage conditions of the kit

[0085] The kit information is shown in Table 5:

[0086] Table 5

[0087] Kit Name Manufacturer Item Number Batch Number AST BIOBASE 70910 10427010H ALT BIOBASE 70911 10323011H

[0088] Main components: R1, R2 (for specific composition, refer to the corresponding specification of the kit)

[0089] Storage conditions and shelf life: The kit in the original package is stored at 2 - 8°C and is valid for 12 months.

[0090] 3.4.4 Experimental procedures:

[0091] ① Preparation of working reagents: For the single reagent method, mix R1 and R2 according to the ratio in the instruction manual; for the double reagent method, use R1 and R2 separately.

[0092] ② Set the corresponding parameters on the automatic biochemistry analyzer; load the sample. The automatic biochemistry analyzer will perform the measurement automatically.

[0093] ③ Experimental results: Export the results after detection by the automatic biochemistry analyzer and perform data analysis.

[0094] 3.4.4 Detection results

[0095] The detection results are as Figure 9 , compared with the positive liver failure model group, the serum ALT activity of rats in the low - dose treatment group of camel milk exosomes decreased significantly, and AST showed a decreasing trend but no significant difference. Serological data indicate that camel milk exosomes treatment improved the liver function of rats with liver failure.

[0096] In summary, compared with group B (positive liver failure group), in groups D and E of the camel milk exosome administration group, the degree of hepatocyte apoptosis in the liver tissue ( Figure 6 ) and the degree of liver tissue fibrosis decreased significantly ( Figure 8 ), and the necrosis rate of the liver injury tissue decreased significantly ( Figure 4) The serum ALT activity was significantly reduced. The present invention discloses the application of camel milk exosomes in the preparation of drugs for preventing and / or treating liver diseases. The preparation process of the camel milk exosomes in the present invention is simple, has good stability, and high safety. The obtained camel milk exosomes can be used to relieve or treat acute liver failure, providing a new strategy for the treatment of liver diseases.

[0097] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Application of camel milk exosomes in the preparation of drugs for the treatment of liver diseases.

2. The use according to claim 1, characterized in that: The camel milk exosomes are derived from camel milk whey.

3. The use according to claim 1, characterized in that: The liver diseases include hepatitis, liver fibrosis, cirrhosis, or liver failure.

4. The use according to claim 3, characterized in that: The liver failure is acute liver failure, including acute liver failure caused by carbon tetrachloride.

5. The use according to claim 1, characterized in that: The method for preparing camel milk exosomes comprises the following steps: S1. Centrifuge fresh camel milk at 10,000 g to 15,000 g for 20 to 40 minutes, and extract the middle layer liquid after centrifugation as whey; S2, after the whey is filtered through a 70-100 μm pore size filter membrane, the filtrate is centrifuged at 100,000-120,000 g for 1-2 hours, and the gelatinous precipitate at the bottom of the tube is collected and crushed; S3, resuspend the crushed precipitate with buffer, centrifuge the resuspended solution at 100,000-120,000g for 1-2 hours, collect the gelatinous precipitate at the bottom of the tube and crush it; S4. Resuspend the broken precipitate from S3 with buffer, pass the resuspended solution through a coarse filter membrane and a sterile filter membrane, and collect the filtrate to obtain exosomes.

6. The use according to claim 1, characterized in that: The particle size of the camel milk exosomes ranges from 50 to 200 nm.

7. The use according to claim 1, characterized in that: The camel milk exosomes reduce acute liver damage by reducing tissue cell apoptosis, tissue fibrosis degree and necrosis rate.

8. The use according to claim 1, characterized in that: The dosage of camel milk exosomes is 1 to 10*10 12 particles / kg.

9. A drug for treating liver diseases, characterized in that: The medicine comprises camel milk exosomes and a pharmaceutically acceptable carrier.

10. The drug according to claim 9, wherein The dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

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