CN114748506A - Application of bone marrow mesenchymal stem cell exosome - Google Patents

Abstract

The invention provides an application of a mesenchymal stem cell exosome in bone marrow. Liver function impairment was alleviated by injecting BMSCs-Exo into HIRI Kunming mice. Wherein miR-25-3p is remarkably reduced in HIRI liver, BMSCs and BMSCs-Exo both contain miR-25-3p, miR-25-3p expression is increased after miR-25-3p mimics (miR-25-3p agomir and miR-25-3p mimic) are respectively transferred into HIRI mice and AML12 liver cells with hypoxia/reoxygenation, the expression reduction of apoptosis and inflammation related factors is detected by qRT-PCR and Western blotting, and the liver injury is reduced by measuring the content of aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) in serum and hematoxylin-eosin (HE) staining. In addition, the expression reduction of genes and related proteins of MAPK/ERK signal channels and PI3K/AKT signal channels is detected by qRT-PCR and Western blotting. Therefore, miR-25-3p inhibits HIRI injury-induced hepatocyte apoptosis by inhibiting MAPK/ERK and PI3K/AKT signaling pathways, so that HIRI is improved, and a new idea is provided for treating HIRI.

Description

Application of bone marrow mesenchymal stem cell exosomes

technical field

The present invention relates to the fields of biology and medicine, in particular to the application of bone marrow mesenchymal stem cell exosomes.

Background technique

Hepatic ischemia-reperfusion injury (HIRI) refers to a pathological phenomenon in which the blood supply to the liver is blocked for a period of time during liver surgery, and the blood flow is restored. . HIRI is a common clinical problem in the management of liver trauma, hepatic lobectomy, and liver transplantation. It can lead to organ failure, increase the incidence of rejection, and have a great impact on the prognosis and survival of patients. So far, there is still a lack of clinically effective interventions. In the process of ischemia-reperfusion, cells are in a state of oxidative stress, endoplasmic reticulum stress and calcium overload, mitochondrial swelling and outer membrane rupture, cytochrome C release, thereby triggering apoptosis signaling pathway, leading to apoptosis occur.

Mesenchymal stem cells (MSCs) are one of the most widely used stem cell types in clinical practice for immunomodulation, organ reconstruction, and tissue repair. Exosomes (exosomes, Exo) are secreted under other stimuli. Exosomes are considered as a new therapeutic tool for the delivery of functional proteins, mRNAs, miRNAs and lncRNAs to transport bioactive molecules to recipient cells to modulate cellular functions, which have higher biological Safety and more stable signaling efficiency make it a strong candidate for cell-free therapy. Studies have shown that mesenchymal stem cell exosomes can regulate cell apoptosis and reduce hepatic ischemia-reperfusion injury. However, stem cell exosomes contain a variety of biologically active substances, which may play a role in reducing hepatic ischemia-reperfusion injury by stem cell exosomes. The mechanism needs to be studied in depth.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide an application of bone marrow mesenchymal stem cell exosomes.

One of the objectives of the present invention is achieved by the following technical solutions: the application of a bone marrow mesenchymal stem cell exosome in the preparation of a medicine for treating liver ischemia-reperfusion injury, the medicine being used to inhibit the MAPK/ERK signaling pathway and PI3K/AKT signaling pathway.

As one embodiment, the bone marrow mesenchymal stem cell exosomes comprise miR-25-3p.

The second purpose of the present invention is to provide an application of miRNA derived from bone marrow mesenchymal stem cells exosomes in the preparation of a drug for treating liver ischemia-reperfusion injury.

The second object of the present invention is achieved by the following technical solutions: the application of a miRNA derived from bone marrow mesenchymal stem cells exosomes in the preparation of a drug for the treatment of liver ischemia-reperfusion injury, and the drug is used for inhibiting MAPK/ ERK signaling pathway and PI3K/AKT signaling pathway.

As an embodiment, the miRNA is miR-25-3p.

The third object of the present invention is to provide a medicine for treating liver ischemia-reperfusion injury.

The third object of the present invention is achieved by the following technical scheme: a medicine for treating liver ischemia-reperfusion injury, the medicine contains components that inhibit the MAPK/ERK signaling pathway and the PI3K/AKT signaling pathway.

As one embodiment, the drug contains miR-25-3p.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention finds that the exosome-derived miRNAmiR-25-3p from human bone marrow mesenchymal stem cells inhibits HIRI injury-induced hepatocytes by inhibiting the MAPK/ERK and PI3K/AKT signaling pathways Apoptosis, thereby reducing liver ischemia-reperfusion injury. Therefore, the present invention provides a new idea for the treatment of HIRI.

Description of drawings

Fig. 1A is a result diagram of the diameter size of exosomes detected by ZetaView provided in the embodiment of the present invention;

1B is a morphological characteristic diagram of exosomes displayed by transmission electron microscope provided in the embodiment of the present invention;

Fig. 1C is a result diagram of the detection of exosome marker proteins by western blotting provided in the embodiment of the present invention;

Figure 2 is a graph showing the results of the content of ALT and AST in the serum after the BMSCs exosomes provided in the embodiment of the present invention were injected into HIRI mice and reperfused for 3h, 6h, and 9h respectively;

Figure 3 shows the detection of miR-25-3p in the liver of HIRI mice and its control CK, BMSCs, BMSCs-Exo, BMSCs-Exo-treated HIRI mice and its control PBS-treated HIRI mice by qRT-PCR provided in the example of the present invention The result graph of the content in the group;

Figure 4A and Figure 4B are graphs of the results of Tunel detecting the apoptosis of liver tissue cells provided in the embodiment of the present invention; wherein, the white cells in Figure 4A represent apoptotic cells, and the gray cells represent normal cells;

Figure 4C is a graph of the results of detecting the content of GSDMD and NLRP3 mRNA in liver tissue by qRT-PCR provided in the embodiment of the present invention;

Figure 4D and Figure 4E are the results of Western blot detection of the content of Caspase1 in liver tissue provided in the embodiment of the present invention; wherein, lanes 1 and 2 of Figure 4D are saline NS, lanes 3 and 4 are miR-25-3p agomir ;

Figure 4F is a result diagram of the detection of the content of inflammation-related genes in AML12 cells by qRT-PCR provided in the embodiment of the present invention;

Figure 5A is a graph showing the results of serum ALT and AST levels after the miR-25-3p mimic provided in the embodiment of the present invention treats mice with ischemia-reperfusion injury;

Fig. 5B is a graph showing the results of HE staining of liver tissue after miR-25-3p mimic provided in the embodiment of the present invention treats mice with hepatic ischemia and reperfusion;

Figure 6A and Figure 6B are the results of Western blot detection of the expression of p-AKT and p-ERK in liver tissue provided in the embodiment of the present invention; wherein, lanes 1 and 2 in Figure 6A represent NS, and lanes 3 and 4 represent agomir ;

FIG. 6C is a result diagram of the qRT-PCR detection of the mRNA content of PI3K, Raf, Ras, and MEK genes in liver tissue provided by the embodiment of the present invention;

FIG. 6D is a graph showing the results of detecting the mRNA levels of PI3K and MEK genes in AML12 hypoxia-reoxygenated hepatocytes by qRT-PCR according to the embodiment of the present invention.

Detailed ways

Hereinafter, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, on the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. . In the following examples, unless otherwise indicated, all reagents used are of analytical grade, and all reagents used can be obtained from commercial sources. The experimental methods for which specific conditions are not indicated in the text are usually in accordance with conventional conditions, such as those described in the book "Molecular Cloning Experiment Guide" published by Science Press, edited by J. Sambrook et al. in 2002, or as suggested by the manufacturer. conditions of. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the present invention.

1. Experimental method

1. Collection, purification and identification of BMSCs exosomes

Human bone marrow mesenchymal stem cells (BMSCs) were purchased from Wuhan Proceeds Life Science and Technology Co., Ltd. (CP-H166) and cultured in DMEM medium containing 10% fetal bovine serum. When the cells reached 70%-80% confluence, Rinse with PBS, inoculate 1:3 into serum-free medium and culture for 48h, collect the supernatant at 4°C, centrifuge at 300g for 10min, retain the supernatant; centrifuge again at 2000g for 10min, retain the supernatant; centrifuge at 10,000g for 30min, retain the supernatant After filtration with a 0.22 μm filter membrane, centrifuge at 100,000 g for 2 h to retain the precipitate. Add PBS and mix well, centrifuge again at 100,000g for 2h, and retain the precipitate, which is the purer exosomes. Add 100 μL of PBS to the exosomes and store them in a -80°C freezer. Particle analyzer to detect exosome diameter. The morphology of exosomes was observed by transmission electron microscopy. Western blot identification of exosome surface markers.

2. Experimental animals

SPF grade male Kunming mice, weighing 20-25 g, were purchased from Hunan Slike Jingda Laboratory Animal Co., Ltd. (production license: SCXK (Xiang) 2021-0002). Feed normal mouse chow with free access to filtered tap water. Room temperature 22℃±1℃, humidity 65%±5%, 12h light/dark alternation. The animal experimental protocol was reviewed and approved by the Animal Ethics Committee of Guilin Medical College. The experimental process was implemented in strict accordance with the National Standard of the People's Republic of China (GB/T 35892-2018) for the Guidelines for Ethical Review of Laboratory Animal Welfare.

3. Source of miR-25-3p mimics

miR-25-3pmimic and its control miR-25-3p mimic NC, and miR-25-3p mimic for animals, were purchased from Guangzhou Ribo Biotechnology Co., Ltd. miR-25-3pagomir. Normal saline (NS) was used as a control for miR-25-3p agomir. .

4. Preparation of mouse liver ischemia-reperfusion injury model

Mice were divided into experimental group (agomir) and control group (NS), with 3 mice in each group. Twenty-four hours before surgery, each mouse was injected with 100 μl normal saline diluted 13 nmol agomir or 100 μl normal saline through the tail vein. Fasting for 12 hours before surgery and free drinking water. 4% chloral hydrate was anesthetized by intraperitoneal injection of 0.1ml/10g, and the limbs were fixed with tape. Sterilize with 70% alcohol, open longitudinally from the middle of the abdomen, cut the skin layer and muscle layer respectively, up to the xiphoid process. The left and middle lobes of the liver were exposed, and the hepatic arteries and portal veins of the middle and left lobes were clipped with non-invasive vascular clips. During the clipping process, the incision was covered with wet gauze, and the mice were kept warm on a constant temperature heating pad at 37°C. After 60 minutes of continuous ischemia, the vascular clip was quickly removed to restore the ischemic liver blood flow, the abdominal muscles and skin were sutured layer by layer, and the incision was disinfected. Mice were harvested at 6 h after reperfusion.

5. Cell hypoxia and reoxygenation model

Mouse hepatocytes (AML12) were purchased from the Chinese Academy of Sciences Type Culture Collection Cell Bank (SCSP-550). Cells were cultured in DMEM medium containing 10% fetal bovine serum. Cells were plated in 24-well plates at a density of 1.5×105～2.5×105 cells/well 24h in advance. When the cell coverage reached about 60%-70%, miR-25-3p mimic and its control miR-25-3p mimic NC were transfected with 100nM. After 24 h, cells were incubated with PBS under hypoxia (5% CO2, 1% O2 and 94% N2, 37°C) for 6 h and then under normal conditions for 12 h.

6. Serological index detection

The mice were reperfused for 6 hours. The mouse eyeballs were blood collected, left standing at room temperature for 30 minutes, and then centrifuged at 1800g for 15 minutes at 4°C. The upper serum was taken, and the kit of Nanjing Jiancheng Bioengineering Institute was used to determine the aspartate aminotransferase (AST) in serum. ) and alanine aminotransferase (ALT) levels, the OD value of each well was measured by a microplate reader with a wavelength of 510 mm.

7. Tunel experiment

The liver tissue was fixed with 4% paraformaldehyde for 48 hours, dehydrated with graded alcohol, embedded in paraffin, and sectioned by a paraffin microtome with a thickness of 4 μm. Bake in a 60°C oven for 4.5 hours, dewax, and hydrate with gradient alcohol. Proteinase K was treated at 37°C for 20 min, 50 μl of TUNEL reaction mixture was added to each tissue, and the reaction was carried out at 37°C in a humid box for 1 h in the dark; 100 μl of DAPI staining solution was added dropwise, and the incubator was incubated at room temperature for 20 min in a constant temperature incubator; water-soluble mounting medium cover sheet. Using a fluorescence microscope, the green wavelength was 488 nm, and the red wavelength was 596 nm, observed and photographed, and the positive rate of TUNEL was calculated.

8. Hematoxylin-eosin (HE) staining experiment

The liver tissue was fixed with 4% paraformaldehyde for 48 hours, dehydrated with graded alcohol, embedded in paraffin, and sectioned by a paraffin microtome with a thickness of 4 μm. Bake in a 60°C oven for 4.5 hours, dewax, and hydrate with gradient alcohol. Hematoxylin staining for 5 min, hydrochloric acid-ethanol differentiation for 30 sec, and eosin staining for 5 min. Dehydrated with graded alcohol and mounted with neutral gum. Optical microscope observation.

9. Quantitative real-time PCR (qRT-PCR) experiment

mRNA and miRNA were extracted from AML12 cells or liver tissue using UNIQ-10 column Trizol total RNA extraction kit from Sangon Bioengineering (Shanghai) Co., Ltd. The OD260 value and OD260/280 ratio were determined by spectrophotometer, and the RNA concentration was calculated according to OD260. The integrity of total RNA was identified by 1% agarose gel electrophoresis. miRNA and mRNA were reverse transcribed to cDNA using Maxima ReverseTranscriptase (Thermo Scientific). 2X SG Fast qPCRMaster Mix (High Rox, B639273, BBI, ABI) was used for qRT-PCR for mRNA and RiboBio miRNA detection for miRNA according to the instruction manual. The relative expression levels of genes were determined by 2-DDCT method. Endogenous controls for mRNA and miRNA expression were GAPDH and U6, respectively. The primer sequences are as follows:

10. Western blot experiment

Liver tissue or cells were added to protease inhibitor and RIPA lysis buffer to extract total protein. Protein concentrations were determined using the BCA protein quantification kit (Solarbio). According to the protein molecular weight, different concentrations (10%-15%) of separating gel were selected for electrophoresis separation, the PVDF membrane was activated with methanol, a "sandwich" structure was installed, and the protein was transferred to the PVDF membrane by electrophoresis. The membrane was washed three times with PBST, the primary antibody was incubated at 4°C overnight, and the secondary antibody was incubated at 37°C for 1 h. The membrane was washed three times with PBST, and protein bands were detected with ECL kit (Millipore). The gel imaging system scans and quantifies the densitometric values of the bands.

11. Transmission electron microscope

Hepatocytes pre-fixed with 2% paraformaldehyde-2.5% glutaraldehyde, rinsed with cold buffer for 15 min × 3 times, fixed with 1% osmic acid at room temperature for 2 h, dehydrated with gradient ethanol, replaced twice with propylene oxide, propylene oxide and Epon 812 resin mixture was soaked overnight, embedded in Epon 812 resin, polymerized at 35°C for 24h, 45°C for 12h, 55°C for 12h and 60°C for 24h, after positioning the semi-thin section, ultrathin section at 50nm with a glass knife, uranyl acetate and citric acid Lead double electron staining, JEM-1230 transmission electron microscope observation. After the cells were digested, the cells were made into a suspension before embedding and slicing. The exosomes were dropped on a carrier copper grid with a pore size of 2 nm for electron microscopy. The exosomes were left standing for 2 minutes, negatively stained with 3% phosphotungstic acid for 5 minutes, and transmitted through JEM-1230. Electron microscope observation.

12. Statistical analysis

SPSS 25.0 statistical software was used for analysis, and P<0.05 was considered statistically significant.

2. Experimental results

1. Isolation and identification of BMSCs exosomes

The culture supernatant of P3 generation BMSCs cells was collected and subjected to differential centrifugation. The obtained exosomes were analyzed by particle analyzer for particle size of exosomes. Figure 1A shows the results of ZetaView detection of the diameter of exosomes; Figure 1B shows the morphological characteristics of exosomes using transmission electron microscopy; Figure 1C shows the results of Western blot detection of marker proteins. The specific results are as follows: The diameter of the particles in the purified exosomes detected by ZetaView is about 60-200nm. Transmission electron microscopy showed that the diameter of exosomes was about 60-200 nm, and low electron density components were seen in the cells, which were round or oval, and the cell membrane structure was complete. Westernblot showed positive for CD9 and CD63 and negative for Calnexin in exosomes.

2. BMSCs exosomes can improve liver injury caused by HIRI

In order to detect the effect of BMSCs exosomes on HIRI mice, 100 μg of BMSCs exosomes were dissolved in 100 μL of PBS, injected into HIRI mice through the tail vein, and reperfused for 3h, 6h, and 9h, respectively, and ALT in serum was detected by eyeball blood collection. , AST content. Please refer to Figure 2, the results show that at the three time points of reperfusion 3h, 6h, and 9h, the ALT and AST of the BMSCs exosome injection group were lower than those of the PBS injection group.

3. miR-25-3p participates in BMSCs exosomes to alleviate HIRI injury

In order to clarify the correlation between miR-25-3p and BMSCs exosomes in alleviating HIRI injury, miR-25-3p was detected by qRT-PCR in the liver of HIRI mice and its control CK, BMSCs, BMSCs-Exo, and BMSCs-Exo treatments. The content in HIRI mouse group and its control PBS-treated HIRI mouse group. Referring to Figure 3, it can be seen that the expression of miR-25-3p in HIRI liver is lower than that in CK group; both BMSCs and BMSCs-Exo contain miR-25-3p; when BMSCs-Exo is transferred into HIRI mice through the tail vein , the content of miR-25-3p in liver tissue increased. Therefore, miR-25-3p was present in BMSCs and their exosomes, and was involved in the mitigation of HIRI injury by BMSCs exosomes.

4. miR-25-3p inhibits apoptosis

The miR-25-3p mimic, agomir, can overexpress miR-25-3p in animals. It was injected into HIRI mice through the tail vein. After 6 hours of reperfusion, the liver was taken to make paraffin sections. The cell apoptosis was detected by Tunel test. Label the nuclei. Please refer to FIG. 4A and FIG. 4B , the results show that the rate of Tunel-positive cells in the experimental group (agomir group) is lower than that in the saline injection group (NS group). Please refer to Figure 4C, qRT-PCR detection showed that the content of GSDMD and NLRP3 mRNA in the liver tissue of the experimental group was also lower than that of the normal saline group. Please refer to Figure 4D and Figure 4E, the content of Caspase1 protein in the liver tissue of the experimental group was lower than that of the NS group. The miR-25-3p mimic mimic can overexpress miR-25-3p in cells. The miR-25-3p mimic and control NC were transferred into AML12 hypoxia-reoxygenated hepatocytes, and qRT-PCR was used to detect the contents of inflammation-related genes. , please refer to Figure 4F, it was found that the levels of TNF-α, IL-6, IL-1b and IL-18 were lower than those of the control group, and the TNF-α and IL-1b reached a significant difference.

5. miR-25-3p attenuates liver injury

Serum ALT and AST levels generally reflect the degree of hepatocyte damage. After transfecting HIRI mice with miR-25-3p agomir, blood was collected from the eyeball to detect the content of ALT and AST in serum. Please refer to 5A, the results showed that the ALT and AST contents of the miR-25-3p group were lower than those of the control NS group, and the ALT contents were significantly decreased. Please refer to 5B to observe the structure of liver tissue. It is found that the structure of the liver lobules in the experimental group is complete, the cords of liver cells are neatly arranged, and the cytoplasm is uniform, while the structure of the liver lobules in the control NS group is not clear, the arrangement of liver cells is disordered, enlarged to varying degrees, and vacuoles appear. Degeneration and inflammatory infiltration.

6. miR-25-3p regulates MAPK/ERK and PI3K/AKT signaling pathways

In order to elucidate the signaling pathway of miR-25-3p regulating cell apoptosis, the liver tissues of mice in the experimental group and the control group were collected for detection. Please refer to Figure 6A and Figure 6B, Western blot results showed that overexpression of miR-25-3p significantly inhibited the expression of p-AKT and p-ERK. Referring to Figure 6C, the qRT-PCR results showed that the levels of PI3K, Raf, Ras and MEK were also lower than those of the control group. When AML12 cells were transfected with miR-25-3p mimic, see Figure 6D, qRT-PCR also showed decreased PI3K and MEK mRNA levels. Among them, Raf, Ras, MEK and ERK are related factors of MAPK/ERK signaling pathway, and PI3K and AKT are related factors of PI3K/AKT signaling pathway. This shows that miR-25-3p inhibits MAPK/ERK signaling pathway and PI3K/AKT signaling pathway.

7. Summary

In the examples of the present invention, through the above experiments and analysis, it can be seen that the expression of miR-25-3p is down-regulated in the liver of HIRI mice, and the injection of BMSCs exosomes into HIRI mice can alleviate HIRI, and the experimental verification miR-25-3p was present in BMSCs and their exosomes, and was involved in BMSCs exosomes attenuating HIRI injury.

After transfecting miR-25-3p into mouse hepatocyte AML12 hypoxia-reoxygenation model, qRT-PCR showed that the expressions of inflammatory factors TNF-α, IL-6, IL-1β and IL-18 were decreased. miR-25-3p also reduced the expression of apoptosis-related factors Caspase-1, NLRP3, and GSDMD in the liver tissue of HIRI mice. This indicates that miR-25-3p plays a role in inhibiting inflammation and apoptosis in HIRI, and it can be concluded that miR-25-3p can slow down liver injury by detecting the content of ALT and AST in serum and observing the structure of liver tissue. .

Some studies have found that down-regulation of cellular communication network factor 1 (CCN1) can reduce ALT, AST levels, MPO activity, IL-6 and TNF-α levels, inhibit MEK/ERK signaling pathway, and reduce inflammation in C57BL/6HIRI mice reaction and liver damage. Some studies have also found that cerebroproteinhydrolysate-I (CH-I) plays a neuroprotective role by inhibiting the expression of MEK-ERK-CREB and increasing the expression of BDNF after cerebral ischemia/reperfusion injury. Neurobehavioral function in arterial embolized rats. Thus, it indicates that inhibition of MEK/ERK signaling pathway can reduce the inflammatory response and protect the organs of ischemia/reperfusion. Another study found that octreotide (OCT) can inhibit the expression of p62 by inhibiting the PI3K/AKT/mTOR/S757-ULK1 signaling pathway, and increase the expression of Beclin-1, ATG7 and LC3, leading to liver autophagy and maintaining a series of antioxidants. and anti-inflammatory cascade, protect the liver from ischemia-reperfusion injury. At the same time, the PI3K/AKT pathway is involved in the regulation of apoptosis. And some studies have found that TLR4 is activated after spinal cord injury, which promotes the expression of lncRNA-F630028O10Rik. This lncRNA acts as a ceRNA of the miR-1231-5p/Col1a1 axis and enhances the apoptosis of microglia after spinal cord injury by activating the PI3K/AKT pathway. Thus, it indicates that inhibiting the PI3K/AKT signaling pathway can reduce the inflammatory response and protect the organs of ischemia/reperfusion. Therefore, inhibition of MEK/ERK signaling pathway and PI3K/AKT signaling pathway can reduce the inflammatory response and protect the organs of ischemia/reperfusion.

The present application found through experiments that miR-25-3p reduced the content of Raf, Ras, MEK and ERK in the MAPK/ERK signaling pathway, and also decreased the content of PI3K and AKT in the PI3K/AKT signaling pathway. Therefore, miR-25-3p inhibits MAPK/ERK signaling pathway and PI3K/AKT signaling pathway.

In conclusion, miR-25-3p inhibits HIRI injury-induced hepatocyte apoptosis by inhibiting MAPK/ERK and PI3K/AKT signaling pathways, thereby improving HIRI and providing a new idea for the treatment of HIRI.

The above-mentioned embodiments are only preferred embodiments of the present invention, and cannot be used to limit the scope of protection of the present invention. Any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of the present invention. Scope of protection claimed.

Claims (6)

1. Use of a mesenchymal stem cell exosome for the preparation of a medicament for treating hepatic ischemia reperfusion injury, the medicament for inhibiting the MAPK/ERK signalling pathway and the PI3K/AKT signalling pathway.

2. The use of claim 1, wherein said bone marrow mesenchymal stem cell exosomes comprise miR-25-3 p.

3. The application of miRNA derived from mesenchymal stem cell exosomes in preparation of a medicine for treating liver ischemia-reperfusion injury is characterized in that the medicine is used for inhibiting a MAPK/ERK signaling pathway and a PI3K/AKT signaling pathway.

4. The use of claim 3, wherein the miRNA is miR-25-3 p.

5. A medicament for the treatment of hepatic ischemia reperfusion injury comprising a component which inhibits the MAPK/ERK signaling pathway and the PI3K/AKT signaling pathway.

6. The medicament for treating hepatic ischemia-reperfusion injury according to claim 5, wherein the medicament comprises miR-25-3 p.

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