Application of hepatocyte-derived serum-free exosome to acute liver and kidney injury caused by sepsis

Abstract

The invention discloses application of a hepatocyte-derived serum-free exosome to acute liver and kidney injury caused by sepsis, and belongs to the technical field of biologication.The first research finds that the hepatocyte-derived serum-free exosome can effectively treat the acute liver and kidney injury caused by sepsis, and the hepatocyte-derived serum-free exosome can be used for treating the acute liver and kidney injury caused by sepsis. According to the application disclosed by the invention, the hepatocyte-derived serum-free exosome is injected into a sepsis acute liver and kidney injury model mouse, so that the contents of alanine transaminase (ALT), aspartate transaminase (AST) and urea nitrogen (BUN) in mouse serum are effectively reduced, and the effect of the hepatocyte-derived serum-free exosome on relieving acute liver and kidney injury caused by sepsis is disclosed; a new thought is provided for the treatment of sepsis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an application of hepatocyte-derived serum-free exosomes to acute liver and kidney damage caused by sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response caused by infection that can lead to dysfunction of multiple organs, including the liver and kidneys. The number of patients is increasing annually. The morbidity and mortality rates of sepsis are high worldwide. Sepsis-related acute liver and kidney injury is a complex pathophysiological process involving multiple mechanisms, and currently, ideal preventive and therapeutic measures for sepsis-related acute liver and kidney injury are lacking.

[0003] Exosomes are membrane vesicles secreted by cells, with a diameter usually between 30-150nm. Exosomes contain a variety of proteins, lipids, microRNAs and long non-coding RNA components. Almost all types of cells can produce and release exosomes to transmit information and exchange substances between cells, and play a variety of biological functions such as regulating cell proliferation, differentiation, migration, apoptosis and immune regulation.

[0004] Exosomes from different sources possess varying biological functions. For example, exosomes derived from mesenchymal stem cells have therapeutic effects such as tissue repair and immune regulation, while exosomes derived from tumor cells play a significant role in promoting tumor growth and metastasis. Hepatocyte-derived exosomes are nanoscale extracellular vesicles secreted by hepatocytes. Different hepatocyte-derived exosomes exhibit varying properties due to differences in physiological metabolism, immunogenicity, and the presence of stress-related proteins or miRNAs. Currently, there are no reports on the efficacy of serum-free liver-derived exosomes extracted from subclones of human hepatoblastoma cells in treating acute liver and kidney injury caused by sepsis. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide an application of serum-free exosomes derived from hepatocytes for acute liver and kidney damage caused by sepsis.

[0006] To achieve the above purpose, the present invention adopts the following technical methods:

[0007] In a first aspect, the present invention provides a use of serum-free exosomes derived from hepatocytes in the following (1) or (2):

[0008] (1) Preparation of drugs for treating acute liver injury caused by sepsis;

[0009] (2) Prepare drugs for treating acute kidney injury caused by sepsis.

[0010] The hepatocyte-derived serum-free exosomes are isolated and extracted from a subclone of human hepatoblastoma cells.

[0011] The term "hepatoblastoma cell subclone" refers to a single cell progeny population isolated from primary hepatoblastoma cells by single cell isolation technology.

[0012] Preferably, the single cell isolation technique is a limiting dilution method or a flow cytometry method.

[0013] The particle size of the hepatocyte-derived serum-free exosomes is 125-127 nm.

[0014] In a second aspect, the present invention provides a drug for preventing and treating acute liver and kidney damage caused by sepsis, wherein the drug contains serum-free exosomes derived from hepatocytes as an active ingredient.

[0015] The particle concentration of serum-free exosomes in the drug is (1-8)×10 11 particles / mL.

[0016] Preferably, the particle concentration of serum-free exosomes in the drug is 4.6×10 11 particles / mL.

[0017] The medicament may further include a pharmaceutically acceptable adjuvant component.

[0018] The adjuvant component is one or more of a filler, a sweetener, a binder, a lubricant, a disintegrant, a preservative and an antioxidant.

[0019] The dosage form of the drug is solution, lyophilized powder injection, capsule, tablet, powder or suppository.

[0020] Beneficial effects of the present invention:

[0021] The present invention discovered for the first time that serum-free exosomes isolated and extracted from a subclone of human hepatoblastoma cells can effectively treat acute liver and kidney injury caused by sepsis. The hepatocyte-derived serum-free exosomes used in the present invention have stable performance and are suitable for clinical transformation and drug development. By injecting hepatocyte-derived serum-free exosomes into mice with acute liver and kidney injury in sepsis model, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea nitrogen (BUN) in the mouse serum were effectively reduced, revealing the role of hepatocyte-derived serum-free exosomes in alleviating acute liver and kidney injury caused by sepsis, providing new ideas for the treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Exosomes observed under transmission electron microscopy.

[0023] Figure 2The figure shows the distribution of exosome particle size and concentration. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0025] As mentioned above, sepsis is a systemic inflammatory response. Currently, there is a lack of ideal prevention and treatment measures for sepsis-related acute liver and kidney damage. The development of engineered exosome technology is expected to become a new breakthrough in the multi-organ protective treatment of sepsis.

[0026] Based on this, the present invention proposes the use of serum-free exosomes derived from hepatocytes for the treatment of acute liver and kidney damage caused by sepsis. The serum-free exosomes, isolated and extracted from a subclone of human hepatoblastoma cells, effectively reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in the serum of mice with acute liver and kidney damage caused by sepsis, providing a new approach for the treatment of sepsis.

[0027] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0028] The human hepatoblastoma cells (HuH-6) used in the examples were purchased from Shanghai Guandao Bioengineering Co., Ltd.; the human umbilical cord mesenchymal stem cells used were purchased from Shanghai Kanglang Biotechnology Co., Ltd. The H710KJ serum-free culture medium used was purchased from Bomeida Life Sciences under the trade name CD 293 serum-free culture medium, model number is H710KJ.

[0029] All operations in the examples of the present invention comply with the Beijing Laboratory Animal Ethics Review Guidelines. Unless otherwise specified, the reagents and equipment used in the examples of the present invention are conventional reagents and conventional equipment in this technical field.

[0030] Example 1: Preparation of serum-free exosomes from hepatocytes

[0031] (1) Establishment of human hepatoblastoma cell subclones

[0032] Using a commonly used effective dilution method, vigorously growing human hepatoblastoma cells (HuH-6) were diluted and seeded into 96-well plates at an average of 1 cell per well. After 24 hours, the plates were observed under an inverted microscope. Wells containing only single cells were identified and labeled. After 10 days of culture at 37°C and 5% CO2, subclones of human hepatoblastoma cells with uniform morphology and good growth were selected.

[0033] (2) Preparation of serum-free exosomes from hepatocytes

[0034] Human hepatoblastoma cell subclones were expanded and cultured in H710KJ serum-free medium at 37°C and 5% CO2, with frequent medium changes and passages. When the cell count exceeded 10E8, they were inoculated into hollow fiber bioreactors for further expansion. Culture was terminated when protein secretion reached a standard of greater than 20 μg / ml, and the cell supernatant was collected and concentrated 20-fold.

[0035] The concentrated solution was sent to Huixin Biotechnology Co., Ltd. as a sample for exosome isolation and purification using the EXODUS system, where the exosomes were tested. The sample processing and isolation parameter settings are shown in Table 1.

[0036] The specific experimental steps for exosome isolation and purification are as follows:

[0037] 1. Centrifuge the sample at 4°C, 12,000 g for 30 min to remove microvesicles or protein aggregates, and collect the supernatant.

[0038] 2. Filter the sample with a 0.45 μm filter and take the supernatant.

[0039] 3. Filter the sample with a 0.22 μm filter to remove apoptotic bodies and microvesicles, and collect the supernatant.

[0040] 4. Place the EXODUS chip in the chip position and the filtered sample in the sample position.

[0041] 5. Set up the program on the EXODUS interface: enter the sample number, sample volume, select the chip type, and separation program.

[0042] 6. Start the automated exosome isolation and purification process. After the automated process is complete, press "Exit" to remove the chip and resuspend the purified exosomes in medical saline in a clean bench.

[0043] 7. Repeat the above steps to obtain exosome samples and test the exosomes. The test results are shown in Table 2.

[0044] Table 1: Sample preparation and separation parameters

[0045]

[0046] Table 2: Results of exosome isolation and purification experiments

[0047]

[0048] Comparative Example 1: Preparation of exosomes derived from mesenchymal stem cells

[0049] Human umbilical cord mesenchymal stem cells were cultured in serum-free medium (Shanghai Ezes AC-1001043). When the cells reached 80% confluence, fresh serum-free medium was replaced and cultured for another 48 hours. The supernatant (containing exosomes) was collected and centrifuged at 300×g for 10 minutes at 4°C to remove dead cell debris. The supernatant was then filtered through a 0.22μm filter and centrifuged at 10,000×g for 30 minutes at 4°C to remove large particles such as apoptotic bodies. The supernatant was then ultracentrifuged at 100,000×g for 70 minutes at 4°C and discarded. The pellet was resuspended in PBS and ultracentrifuged again, discarding the supernatant to obtain mesenchymal stem cell-derived exosomes.

[0050] Experimental Example 1: Effects of exosomes from different sources on acute liver and kidney injury caused by sepsis

[0051] 1. Experimental Methods

[0052] Experimental Animals: Forty SPF male ICR mice (18–22 g) were provided by Beijing Huafukang Biotechnology Co., Ltd. [Qualification Certificate No. 110322241102206363]. Animals were housed at the GLP Laboratory Animal Center of the Institute of Materia Medica, Chinese Academy of Medical Sciences, with free access to food and water. The animals were maintained at a temperature of 20–25°C, a relative humidity of 50 ± 10%, and light on every 12 hours with good ventilation. All experimental procedures adhered to the Beijing Municipal Laboratory Animal Ethics Review Guidelines.

[0053] After acclimating for 3 days, SPF male ICR mice were randomly divided into 7 groups (10 mice per group) based on body weight: blank control group, CCL4 (10 mg / kg) model group, positive drug bicyclol group (200 mg / kg), hepatocyte-derived serum-free exosomes group, and mesenchymal stem cell-derived exosomes group. The treatments for each group were as follows:

[0054] Blank control group: no treatment.

[0055] CCL4 (10 mg / kg) model group: Each mouse was intraperitoneally injected with 10 mg / kg CCL4 solution to induce septic acute liver and kidney damage.

[0056] Positive drug bicyclol group (200 mg / kg): Each mouse was first intraperitoneally injected with 10 mg / kg of CCL4 solution to induce acute liver and kidney damage caused by sepsis. One hour after the induction of acute liver and kidney damage caused by sepsis, bicyclol solution was then intraperitoneally injected with 200 mg / kg of bicyclol solution.

[0057] Hepatocyte-derived serum-free exosomes group: Each mouse was first intraperitoneally injected with 10 mg / kg CCL4 solution to induce acute liver and kidney damage caused by sepsis. One hour after the induction of acute liver and kidney damage caused by sepsis, 1.5×1010 The injection amount of exosome particles was calculated by intraperitoneal injection of the serum-free exosomes derived from hepatocytes prepared in Example 1.

[0058] Mesenchymal stem cell-derived exosomes group: Each mouse was first intraperitoneally injected with 10 mg / kg CCL4 solution to induce acute liver and kidney damage caused by sepsis. One hour after the induction of acute liver and kidney damage caused by sepsis, 1.5×10 10 The injection amount of exosome particles was calculated, and the mesenchymal stem cell-derived exosomes prepared in Comparative Example 1 were intraperitoneally injected.

[0059] After 24 hours of treatment, the levels of ALT, AST, and BUN (urea nitrogen) in the serum of mice in each group were detected. The test results are shown in Table 3.

[0060] 2. Experimental Results

[0061] Table 3: Detection of the effects of exosomes from different sources on acute liver and kidney injury caused by sepsis

[0062] Group Injection volume ALT(U / L) AST(U / L) BUN Blank control group - 40.51±4.25 137.59±25.26 9.3±1.59 <![CDATA[CCL4 model group]]> - <![CDATA[71.79±21.57 ** ]]> <![CDATA[237.11±50.92 ** ]]> <![CDATA[19.32±3.14 ** ]]> Bicyclol group 200mg / kg <![CDATA[53.45±16.23 # ]]> <![CDATA[182.51±26.25 # ]]> 16.02±3.01 Exosomes (serum-free) 1.5 billion / kg <![CDATA[51.11±18.1 ## ]]> <![CDATA[164.05±23.76 ## ]]> <![CDATA[12.08±2.71 ## ]]> Exosomes (mesenchymal) 1.5 billion / kg 68.14±16.19 <![CDATA[208.45±78.04 # ]]> 18.86±3.61

[0063] Note: **P<0.01, compared with the blank control group; #P<0.05, ##P<0.01, compared with the model group.

[0064] Experimental results showed that mesenchymal-derived exosomes had little therapeutic effect on acute liver and kidney injury, while injection of serum-free exosomes derived from hepatocytes into mice with acute liver and kidney injury caused by sepsis effectively reduced serum ALT, AST, and BUN levels. The results indicate that exosomes from different sources have significant differences in their therapeutic effects on acute liver and kidney injury caused by sepsis, and that serum-free exosomes derived from hepatocytes have the potential to alleviate acute liver and kidney injury caused by sepsis.

[0065] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Use of serum-free exosomes derived from hepatocytes in the following (1) or (2): (1) Preparation of drugs for treating acute liver injury caused by sepsis; (2) Prepare drugs for treating acute kidney injury caused by sepsis.

2. The use according to claim 1, characterized in that The hepatocyte-derived serum-free exosomes are isolated and extracted from a subclone of human hepatoblastoma cells.

3. The use according to claim 1, characterized in that The particle size of the hepatocyte-derived serum-free exosomes is 125-127 nm.

4. A drug for preventing and treating acute liver and kidney damage caused by sepsis, characterized in that: The drug uses hepatocyte-derived serum-free exosomes as active ingredients.

5. The drug according to claim 4, characterized in that The particle concentration of serum-free exosomes in the drug is (1-8)×10 11 particles / mL.

6. The drug according to claim 4, characterized in that The particle concentration of serum-free exosomes in the drug is 4.6×10 11 particles / mL.

7. The drug according to claim 4, characterized in that The medicament may further include a pharmaceutically acceptable adjuvant component.

8. The drug according to claim 4, characterized in that The dosage form of the drug is solution, lyophilized powder injection, capsule, tablet, powder or suppository.

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