Application of miR-186-5p in preparation of medicine for preventing and / or treating medicine-induced liver injury

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of miR-186-5p in preparation of a medicine for preventing and / or treating drug-induced liver injury. According to the invention, the miR-186-5p, the miR-186-5p simulant or the miR-186-5p agonist is applied to the preparation of the medicine for preventing and / or treating the medicine-induced liver injury. The invention finds that miR-186-5p can be used as a target spot for treating DILI, and miR-186-5p, miR-186-5p simulants or miR-186-5p agonists can reduce neutrophil infiltration, relieve liver cell injury, improve liver dysfunction, inhibit neutrophil infiltration of liver tissues and change of pathological structures of the liver tissues, and also can reduce apoptosis in the liver tissues.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of miR-186-5p in preparing drugs for preventing and / or treating drug-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) is an important adverse drug reaction, which can lead to acute liver failure or even death in severe cases. Acetaminophen (APAP) is a widely used antipyretic and analgesic drug. APAP overdose is one of the most common causes of drug-induced acute liver failure (ALF) worldwide. When APAP is taken in excess, it leads to saturation of glucuronidation and sulfation, and is metabolized to the inactive compound N-acetyl-p-benzoquinone imine (NAPQI) through cytochrome C. Under physiological conditions, NAPQI can bind to reduced glutathione to prevent NAPOI from targeting mitochondria, but excessive NAPQI depletes glutathione in cells, then binds to biomacromolecules such as proteins, exerts toxic effects, induces mitochondrial damage, causes hepatocyte necrosis, activates inflammatory responses, and ultimately leads to acute liver injury (ALI). In addition, an important mechanism of APAP-induced hepatotoxicity is mitochondrial permeability transition (MPT). In addition to APAP-induced hepatotoxic effects, the occurrence of hepatocellular necrosis during drug-induced liver injury can further recruit neutrophils and activate inflammatory responses. More and more data indicate that activation of inflammatory responses in the liver is an important factor in the further development of hepatocellular injury. In particular, neutrophils can be rapidly recruited to the site of liver injury and mediate the intrahepatic cascade of inflammation. Studies have shown that neutrophils can mediate the deterioration of DILI, and neutrophils have been observed to have cytotoxic effects, which can aggravate inflammation under other sterile conditions, such as inflammation associated with hepatic ischemia-reperfusion injury and obstructive cholestasis. Another study showed that activated neutrophils tend to migrate to the site of injury and participate in the inflammatory process by forming neutrophil extracellular traps (NETs). Inhibition of neutrophil extracellular traps alleviated APAP-induced excessive ALI. Excessive neutrophil infiltration of liver tissue can cause severe inflammation and necrosis.

[0003] Currently, there are two treatment options for drug-induced liver injury. Early treatment is based on the antioxidant N-acetylcysteine ​​(NAC). NAC is a clinical standard treatment drug with a narrow treatment window. The best effect is achieved when NAC is used within 8-10 hours after APAP overdose. It can promote the synthesis of glutathione (GSH) in the body, consume NAPQI and reduce drug-induced liver injury. However, the clinical manifestations of acute DILI are mostly non-specific, and the incubation period varies greatly, ranging from 1 day to several months. Most patients have no obvious symptoms, so most patients miss the best treatment time. When liver damage develops into liver failure, the only effective treatment is liver transplantation. Although the proportion of liver transplantation has increased, most patients with life-threatening liver diseases cannot obtain liver transplantation, and liver transplantation has problems such as lack of liver donors, post-transplant rejection, and high costs. Therefore, research is urgently needed to find new treatment options. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a use of miR-186-5p in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention uses miR-186-5p in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0007] In a second aspect, the present invention uses miR-186-5p mimics in the preparation of drugs for preventing and / or treating drug-induced liver injury.

[0008] In a third aspect, the present invention uses a miR-186-5p agonist in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0009] The present invention finds that miR-186-5p can be used as a target for treating DILI. miR-186-5p, miR-186-5p mimics or miR-186-5p agonists can reduce neutrophil infiltration and alleviate liver cell damage, improve abnormal liver function, inhibit neutrophil infiltration in liver tissue and changes in liver tissue pathological structure, and reduce cell apoptosis in liver tissue.

[0010] As a preferred embodiment of the application of the present invention, the drug-induced liver injury includes drug-induced acute liver failure caused by antipyretic and analgesic drugs.

[0011] As a further preferred embodiment of the use of the present invention, the antipyretic and analgesic drug includes acetaminophen.

[0012] As a preferred embodiment of the application described in the present invention, the dosage of the miR-186-5p, miR-186-5p mimics or miR-186-5p agonist is 2 nmol / (2g-2.2g).

[0013] In a fourth aspect, the present invention provides a preparation for preventing and / or treating drug-induced liver injury, comprising at least one of miR-186-5p, a miR-186-5p mimic, and a miR-186-5p agonist.

[0014] As a preferred embodiment of the preparation of the present invention, it also includes at least one food or pharmaceutically acceptable auxiliary material.

[0015] As a preferred embodiment of the preparation of the present invention, the dosage form of the preparation is at least one of capsules, tablets, oral preparations, microcapsule preparations, injections, suppositories, sprays, ointments, gels, solutions, powders, lotions, tinctures, oils, creams, and aerosols.

[0016] As a preferred embodiment of the preparation of the present invention, the administration method of the preparation includes at least one of intravenous injection, intramuscular injection, subcutaneous injection, and intraperitoneal injection.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention verifies for the first time at the cellular level that transfection of miR-186-5p mimic can reduce acetaminophen (APAP)-induced hepatocyte apoptosis and inhibit the expression level of chemokine CXCL1. In mouse experiments, it is confirmed that intravenous infusion of agomir-186-5p, an agonist of miR-186-5p, can reduce the degree of APAP-induced drug-induced liver injury, reduce the area of ​​hepatocyte injury, and reduce the changes in the levels of ALT and AST in serum enzymes of liver function. agomir-186-5p reduces the expression level of CXCL1 in liver tissue and reduces the infiltration of neutrophils, thereby reducing drug-induced hepatocyte injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression levels of AST and ALT in the peripheral blood of the CTRL group and the DILI mouse model in Example 1.

[0020] Figure 2 The expression changes of CXCL1 detected in the DILI mouse model in Example 1; in the figure, A shows that the expression level of Cxcl1 mRNA in the liver tissue of DILI model mice is increased, B shows that the expression level of CXCL1 protein in the liver tissue of DILI model mice is increased by ELISA after liver tissue homogenization, and C shows that the expression level of CXCL1 protein in the liver tissue of DILI model mice is increased by immunohistochemical staining method.

[0021] Figure 3 The expression changes of CXCL1 in liver tissue sections of healthy subjects and patients with drug-induced liver injury in Example 1; in the figure, A is the immunohistochemical staining method showing that the expression of CXCL1 in liver tissue sections of patients with liver injury is increased, and B is the immunohistochemical staining method showing that the expression of myeloperoxidase (MPO) in liver tissue sections of patients with liver injury is increased.

[0022] Figure 4 This is a Venn diagram of the existing miRNAs and bioinformatics prediction data binding to CXCL1 in Example 2.

[0023] Figure 5 These are possible binding sites of miR-186-5p and human and mouse Cxcl1 mRNA in Example 2.

[0024] Figure 6 This is the result of the luciferase activity experiment in Example 2, which proves that miR-186-5p can bind to Cxcl1 mRNA.

[0025] Figure 7 The results of flow cytometry in Example 3 prove that miR-186-5p can reduce hepatocyte apoptosis in vitro; in the figure, A is the apoptosis of cells detected by flow cytometry, and B is the early apoptosis of AML12 cells in the corresponding indicator group (Annexin V + ) cell number, early apoptosis of AML12 cells after transfection with mimic-miR-186-5p (Annexin V + ) cell number decreased; C is the number of live AML12 cells in the corresponding indicated groups (Annexin V - PI - ) and the number of AML12 cells after transfection with mimic-miR-186-5p (Annexin V - PI - ) increased.

[0026] Figure 8 The following are the results of HE pathological sections and liver function index detection of mice in each experimental group after administration of agomir-186-5p in Example 4; in the figure, A is a schematic diagram of the construction of the model mice, B is HE staining indicating the presence of hepatocyte necrosis areas in the positive control tissue, and C is the increase in the levels of AST and ALT in the peripheral blood of mice in the positive control group, and the decrease in the levels of AST and ALT after administration of agomir-186-5p.

[0027] Fig. 9These are the results of flow cytometry detection of neutrophils in the liver and spleen tissues of mice in each experimental group after administration of agomir-186-5p in Example 4; in the figure, A is the change in the proportion of neutrophils in the liver tissue detected by the flow cytometer, B is the percentage of neutrophils in the liver after the administration of agomir-186-5p is decreased compared with the control group, and C is the percentage of neutrophils in the spleen tissue after the administration of agomir-186-5p is significantly reduced compared with the control group.

[0028] Fig.10 These are the results of Ly6G staining and CXCL1 staining of the liver of mice in each experimental group after administration of agomir-186-5p in Example 4; in the figure, A is an immunohistochemical staining method showing that the expression of Ly6G in the liver of mice is reduced after the administration of agomir-186-5p, and B is an immunohistochemical staining method showing that the expression of CXCL1 in the liver of mice is reduced after the administration of agomir-186-5p.

[0029] Fig.11 These are the results of the expression levels of inflammatory factors in the liver tissue of mice in each experimental group after administration of agomir-186-5p in Example 4.

[0030] Fig.12 It is a schematic diagram of the technical process of the present invention.

[0031] In the above figures, * indicates that there is a statistical difference between the two groups, * is p < 0.05, ** is p < 0.01, *** is p < 0.001, **** is p < 0.0001; the scale bar in the figure is 100μm or 200μm. DETAILED DESCRIPTION

[0032] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0034] In the following experimental examples, anti-CXCL1 was used as the CXCL1 neutralizing antibody, which was purchased from R&D Company with the catalog number MAB453-500.

[0035] C57BL / 6 male mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0036] Example 1: Analysis of CXCL1 expression in drug-induced liver injury

[0037] When a patient takes an overdose of APAP, the liver cells in the patient's body are damaged, and their structure and function are destroyed. If not treated in time, liver failure may develop.

[0038] CXCL1 (CXC chemokine ligand 1), also known as GROα (growth regulatory gene α), is a member of the CXC chemokine family. It mainly exerts its biological function by binding to its receptor CXCR2. CXCL1 is expressed in many immune cells and neurons. Its main function is to recruit immune cells, especially neutrophils, which can promote inflammatory responses, immunoregulation and angiogenesis. Numerous studies have shown that CXCL1 plays an important role in liver diseases. For example, in alcohol-induced liver injury, CXCL1 gene expression is significantly increased. In the extrahepatic bile duct injury (EHBD) model, the CXCL1 gene rose rapidly, and the CXCR2 antagonist was used to block the CXCL1-CXCR2 binding. The results showed that Ly6G + The number of neutrophils was significantly reduced, and the proliferation of extrahepatic bile duct epithelial cells was inhibited.

[0039] In order to explore the relationship between CXCL1 and drug-induced liver injury, the mRNA expression level and protein expression level of CXCL1 in mouse liver tissue were first detected. Several 8-week-old C57BL / 6 male mice were randomly selected to prepare the DILI mouse model. After weighing each mouse, warm APAP solution (dose of 400 mg / kg) was injected intraperitoneally. After 24 hours, the liver and peripheral blood of the mice were taken for testing. By detecting the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the peripheral blood of mice (AST and ALT are one of the commonly used indicators for detecting liver function, used to reflect the damage of liver cells), it was determined that the DILI mouse model was successfully prepared. Figure 1 As shown in Figure 2, the levels of AST and ALT in the serum of mice in the APAP group were significantly higher than those in the CTRL control group. Next, RT-qPCR, ELISA and immunohistochemistry were used to detect the expression level of CXCL1 in the liver tissues of normal mice and DILI model mice. The results are shown in Figure 2. Figure 2 As shown in AC, the mRNA expression level and protein expression level in the liver tissue of DILI model mice were increased. Figure 2 In the figure, the scale bar is 100 μm.

[0040] To further verify this result, liver tissue sections of healthy controls and patients with drug-induced liver injury were selected for immunohistochemical detection. Figure 3 As shown in AB, the expression of CXCL1 was significantly increased in the liver injury group, indicating that CXCL1 was related to drug-induced liver injury. Figure 3In the figure, the scale is 200 μm. Since drug-induced liver injury is an inevitable stage of drug-induced liver failure, blocking or reversing the development of liver injury is conducive to delaying or even reversing the progression of liver failure. Therefore, subsequent experiments consider whether inhibition of CXCL1 can be used to treat drug-induced liver failure.

[0041] Example 2: Bioinformatics prediction of miR186-5p targeting regulation of CXCL1 and luciferase assay verification

[0042] Bioinformatics websites (miRDB and ENCORI) were used to predict potential miRNAs that may interact with CXCL1, and these results were intersected with the microRNA data contained in extracellular vesicles secreted by human umbilical cord-derived MSCs searched from the GEO database (GSE215041). The candidate miRNA molecule obtained after the intersection of the three data was miR-186-5p ( Figure 4 ). The specific steps are:

[0043] Select the miRNA-mRNA interaction section in the ENCORI (https: / / rnasysu.com / encori / ) database website, select "CXCL1" as "target", and 11 potential miRNAs predicted to bind to CXCL1 are obtained from the database.

[0044] In the miRDB (https: / / www.mirdb.org / ) database website, "CXCL1" was selected as the "gene symbol", and 21 potential miRNAs predicted to bind to CXCL1 were obtained from the database.

[0045] The keyword "wharton's jelly MSC extracellular vesicles" was searched in the GEO (https: / / www.ncbi.nlm.nih.gov / geo) database to obtain the microRNA expression profile sequencing data of extracellular vesicles secreted by human umbilical cord-derived MSCs (GSE215041), of which 2588 microRNA genes were detected.

[0046] Finally, the above three sets of data were intersected through a Venn diagram, and the only candidate miRNA molecule was obtained as miR-186-5p.

[0047] In order to further study the specific function of miR-186-5p, the Targetscan website (https: / / www.targetscan.org / ) was used to predict the possible binding sites of miR-186-5p to the 3'-UTR region of human and mouse Cxcl1 mRNA sequences ( Figure 5 ) and detected by dual luciferase assay ( Figure 6 ), the results showed that miR-186-5p and Cxcl1mRNA 3'-UTR binding region can bind to and regulate the expression of Cxcl1, and this regulatory effect disappears after the 3'-UTR binding region site of CXCL1 is mutated.

[0048] Example 3: miR186-5p mimics alleviate drug-induced hepatocyte apoptosis

[0049] In vitro, according to the instructions provided by the reagent manufacturer (Ribo Bio), AML12 cells (hepatocyte line) were evenly plated in a 12-well plate, with 1×10 5 cells, and 30nM miR186-5p mimics (mimic-miR-186-5p) were transferred into the cells after 24 hours. Specifically: 24 hours after plating, 1.5μL mimic-miR-186-5p was diluted with 60μL riboFECTTMCP Buffer, gently mixed, and then 6μL riboFECTTMCP Reagent was added, pipetting and mixing, and standing at room temperature for 15 minutes to prepare a transfection complex. Finally, the complex was added dropwise to the complete medium without double antibodies, the old medium was discarded, and the new medium containing mimic-miR-186-5p was added to the AML12 cells. After 6 hours of transfection, the AML12 medium was replaced and cultured. After 24 hours of transfection, the AML12 cells were digested and collected with EDTA-free trypsin for standby use. The collected cells were stained according to the experimental method of the Annexin-V / PI apoptosis kit, and the apoptosis of the cells was detected by flow cytometry within 1 hour. The results showed that APAP-induced apoptosis (Annexin V + ) significantly decreased and increased the viable cells of AML12 cells (Annexin V - PI - ) Figure 7 ).

[0050] Example 4: Overexpression of miR186-5p in vivo alleviates drug-induced liver tissue damage

[0051] In order to further explore the therapeutic effect of miR-186-5p in the APAP-induced DILI mouse model, animal experiments were performed to evaluate the effect of agomir-186-5p in the treatment of drug-induced liver injury. The evaluation indicators included: 1) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the peripheral blood of mice (AST and ALT are one of the commonly used indicators for detecting liver function, which are used to reflect the damage of liver cells). 2) The apoptosis and necrosis of liver tissue structure were evaluated by hematoxylin-eosin staining (HE staining). 3) After the liver tissue was decellularized, the proportion of neutrophils in interstitial cells was detected by flow cytometry, and the positive levels of MPO and ly6G were evaluated by immunohistochemistry staining (IHC) to further verify the changes in neutrophils. 4) Cell apoptosis was detected by flow cytometry (Annexin V and PI staining reflect the level of cell damage).

[0052] (1) Preparation of reagents

[0053] APAP solution: Before mouse modeling, APAP powder was dissolved in PBS buffer heated at 60°C. The concentration of APAP solution was 20 mg / mL.

[0054] Agomir-186-5p / agomir-NC reagent: agomir-186-5p / agomir-NC powder was dissolved in PBS buffer to prepare a 100 nmol / mL solution.

[0055] (2) Preparation of mouse model of drug-induced liver injury

[0056] Eight-week-old C57BL / 6 male mice (20 g-22 g) were randomly selected, each mouse was weighed, and warm APAP solution (400 mg / kg) was intraperitoneally injected. Figure 8 BHE staining results showed that the positive control tissue had obvious hepatocyte necrosis areas. Figure 8 The results of C showed that the levels of AST and ALT in the peripheral blood of mice in the positive control group were significantly higher than those in the CTRL group, indicating that the DILI model was successfully prepared.

[0057] (3) Drug treatment

[0058] The animal experiment was divided into three groups: control group (CTRL), positive control group (APAP+agomir-NC) and treatment group (APAP+agomir-186-5p). The experimental process was as follows Figure 8A shows that the CTRL group did not receive any treatment, and the other two groups were all modeled with APAP solution. 4 hours after modeling, 200 μL of agomir-NC or agomir-186-5p totaling 20 nmol was injected into each mouse through the tail vein in the APAP+agomir-NC group and the APAP+agomir-186-5p group.

[0059] (4) Sampling and testing

[0060] After 24 hours of APAP administration, mice were anesthetized, peripheral blood was collected from the retinal venous plexus, placed in a 4°C refrigerator overnight, centrifuged at 2500 rpm and 4°C for 15 min, the upper serum was drawn, and the liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected after dilution with PBS buffer. The results are shown in Figure 8 B.

[0061] The abdominal cavity of the mouse was opened, and 10 mL of pre-cooled PBS buffer was slowly perfused through the portal vein of each mouse to wash away the remaining blood cells. The left lobe of the liver was then taken and fixed in 4% paraformaldehyde for 24 h, prepared into paraffin sections, and HE staining was performed. The results are shown in Figure 8 C. Another part of the liver was taken to extract RNA, which was used to detect the mRNA expression levels of inflammatory factors Il 6, TNFα and Cxcl 1 in mouse liver tissue. At the same time, part of the liver and spleen were taken and ground, and the changes in the proportion of neutrophils in mouse liver tissue were detected by flow cytometry. The results are shown in Fig. 9 .

[0062] The results showed that after agomir-186-5p administration, the apoptotic area of ​​the liver was reduced, and the serum AST and ALT levels were also reduced compared with the agomir-NC group ( Figure 8 BC). Fig. 9 As shown, agomir-186-5p administration significantly reduced the percentage of neutrophils in liver and spleen tissues. Fig.10 The results of IHC staining showed that the positive signals of Ly6G and CXCL1 proteins in the liver sections of the agomir-NC group were stronger, while the introduction of agomir-186-5p significantly reduced the expression of Ly6G and CXCL1 protein signals. Fig.11 The results showed that the introduction of agomir-186-5p could reduce the increasing trend of inflammatory factors in the liver tissue of mice, proving that agomir-186-5p could improve liver damage caused by APAP.

[0063] The present invention has been tested by cell experiments and animal experiments (see Fig.12) found that miR-186-5p can be used as a target for the treatment of DILI. The present invention verifies for the first time at the cellular level that transfection of miR-186-5p mimic can reduce acetaminophen (APAP)-induced hepatocyte apoptosis and inhibit the expression level of chemokine CXCL1. In mouse experiments, it is confirmed that intravenous infusion of the miR-186-5p agonist agomir-186-5p can reduce the degree of APAP-induced drug-induced liver injury, reduce the area of ​​hepatocyte injury, and reduce the changes in the levels of ALT and AST in serum enzymes of liver function. agomir-186-5p reduces the expression level of CXCL1 in liver tissue and reduces the infiltration of neutrophils, thereby reducing drug-induced hepatocyte injury.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of miR-186-5p in the preparation of drugs for preventing and / or treating drug-induced liver injury.

2. Application of miR-186-5p mimics in the preparation of drugs for preventing and / or treating drug-induced liver injury.

3. Use of miR-186-5p agonists in the preparation of drugs for preventing and / or treating drug-induced liver injury.

4. The use according to any one of claims 1 to 3, characterized in that: The drug-induced liver injury includes drug-induced acute liver failure caused by antipyretic and analgesic drugs.

5. The use according to claim 4, characterized in that: The antipyretic analgesic includes acetaminophen.

6. The use according to any one of claims 1 to 3, wherein the dosage of the miR-186-5p, miR-186-5p mimic or miR-186-5p agonist is 2 nmol / (2 g-2.2 g).

7. A preparation for preventing and / or treating drug-induced liver injury, characterized in that: Includes at least one of miR-186-5p, miR-186-5p mimics, and miR-186-5p agonists.

8. The preparation according to claim 7, characterized in that Also included is at least one food or pharmaceutically acceptable excipient.

9. The preparation according to claim 7, characterized in that The dosage form of the preparation is at least one of capsules, tablets, oral preparations, microcapsule preparations, injections, suppositories, sprays, ointments, gels, solutions, powders, lotions, tinctures, oils, creams, and aerosols.

10. The preparation according to claim 7, characterized in that The administration method of the preparation includes at least one of intravenous injection, intramuscular injection, subcutaneous injection, and intraperitoneal injection.

Images