Application of IL-19 (interleukin-19) in preparation of medicine for preventing or treating medicine-induced liver injury

Abstract

The invention provides an application of IL-19 (interleukin-19) in preparation of a medicine for preventing or treating drug-induced liver injury. In the invention, the IL-19 protein, or the derivative or variant thereof, or the enhancer thereof, or the expression vector thereof can significantly improve the drug-induced liver injury, especially APAP-induced mouse liver injury, for example, the levels of ALT and AST are reduced, the liver necrosis area is reduced, the levels of inflammatory factors such as Tnfa, Il1b, Il6 and Ccr2 are reduced, the infiltration number of liver inflammatory cells is reduced, and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of IL-19 in the preparation of a drug for preventing or treating drug-induced liver injury, especially acetaminophen (APAP)-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) refers to liver injury induced by various prescription or over-the-counter chemical drugs, biological agents, traditional Chinese medicines, natural medicines, health products, dietary supplements and their metabolites and even excipients. Acetaminophen (APAP) is a common antipyretic and analgesic drug. Excessive use can lead to acute liver injury or even liver failure, endangering life (Jaeschke H, Adelusi OB, Akakpo JY, et al. Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls. Acta Pharm Sin B. 2021 Dec;11(12):3740-3755.). In recent years, APAP has become the most common cause of drug-induced liver injury and acute liver failure in European and American countries. So far, N-acetylcysteine is the only drug approved by the FDA for the treatment of drug-induced liver injury (Lee WM, Hynan LS, Rossaro L, et al. Acute Liver Failure Study Group. Intravenous N-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure. Gastroenterology. 2009 Sep;137(3):856-864.), but the treatment time window of N-acetylcysteine for drug-induced liver injury is short. Considering the actual situation, the time when patients with drug-induced liver injury seek medical treatment often exceeds the treatment time window of N-acetylcysteine. Therefore, there is an urgent clinical need for new drugs for the treatment of drug-induced liver injury.

[0003] Interleukin-19 (IL-19) is one of the cytokines of the IL-10 family. Its gene is located on human chromosome 1q32, containing 5 exons and 4 introns. Similar to IL-10, it is transcribed by two different promoters. IL-19 has 21% amino acid homology with IL-10. IL-19 exerts its functions by binding to receptors (IL-20R1 and IL-20R2) on the surface of target cells (He Y, Hwang S, Ahmed YA, et al. Immunopathobiology and therapeutic targets related to cytokines in liver diseases. Cell Mol Immunol. 2021 Jan;18(1):18-37.). Currently, there is little research on IL-19 in the field of liver diseases. In particular, there is no relevant report on the application of IL-19 in the treatment of drug-induced liver injury, especially APAP-induced liver injury. SUMMARY OF THE INVENTION

[0004] On the one hand, the present invention provides the use of interleukin-19 (IL-19) protein, or its derivatives or variants, or its enhancer, or its expression vector in the preparation of a medicament for preventing or treating drug-induced liver injury, especially acetaminophen (APAP)-induced liver injury.

[0005] In the present invention, the amino acid sequence of the IL-19 protein can be as shown in SEQ ID NO:1 (MKLQCVSLWLLGTILILCSVDNHGLRRCLISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTLETLQIIKPLDVCCVTKNLLAFYVDRVFKDHQEPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVIHDNYDQLEVHAAAIKSLGELDVFLAWINKNHEVMFSA) or SEQ ID NO:2 (MTNNLLTFYRDRVFQDHQERSLEVLRRISSIANSFLCVQKSLERCQVHRQCNCSQEATNATRIIHDNYNQLEVSSAALKSLGELNILLAWIDRNHLETPAA).

[0006] In the present invention, the amino acid sequence shown in SEQ ID NO:1 is the natural human IL-19 protein sequence, and the amino acid sequence shown in SEQ ID NO:2 is the natural mouse IL-19 protein sequence.

[0007] In the present invention, the derivative or variant of the IL-19 protein may refer to a molecule obtained by non-natural engineering modification of the IL-19 protein, which does not affect the function of the IL-19 protein and contains or consists of the IL-19 protein. In some embodiments, the derivative or variant may include an IL-19 protein containing one or more alterations, such as substitutions, insertions, and / or deletions, at one or more positions. In some embodiments, in the derivative or variant of the IL-19 protein, the IL-19 protein can be modified by chemical means, such as post-translational derivatization or modification of polypeptides, such as PEGylation and / or conjugation of a desired module (such as a therapeutic module) to a thiol group, such as a thiol group provided by an unpaired cysteine. In some embodiments, the derivative or variant may include a fusion protein thereof.

[0008] In some embodiments, the IL-19 protein may be a murine recombinant IL-19 protein, and its amino acid sequence is as shown in SEQ ID NO:3 (MLRRCLISVDMRLIEKSFHEIKRAMQTKDTFKNVTILSLENLRSIKPGDVCCMTNNLLTFYRDRVFQDHQERSLEVLRRISSIANSFLCVQKSLERCQVHRQCNCSQEATNATRIIHDNYNQLEVSSAALKSLGELNILLAWIDRNHLETPAA).

[0009] In some embodiments, the IL-19 protein may be a recombinant human IL-19 protein, and its amino acid sequence is as shown in SEQ ID NO:4 (LRRCLISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTLETLQIIKPLDVCCVTKNLLAFYVDRVFKDHQEPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVIHDNYDQLEVHAAAIKSLGELDVFLAWINKNHEVMSSA).

[0010] In the present invention, the enhancer of IL-19 may refer to any substance that can enhance the production of IL-19.

[0011] In the present invention, the expression vector of IL-19 may refer to an expression vector carrying a nucleotide sequence encoding the IL-19 protein, or its derivative or variant, which can enhance the expression of IL-19 in cells. The expression vector may include a viral vector or a non-viral vector, such as an IL-19 adenovirus, an Il-9 adeno-associated virus, an IL-19 overexpression plasmid. The method of enhancing IL-19 expression using an expression vector is well known in the art.

[0012] In some embodiments, the IL-19 protein, or its derivatives or variants, or its enhancer, or its expression vector can significantly improve drug-induced liver injury, especially APAP-induced liver injury in mice. For example, it can reduce the levels of ALT and AST, reduce the area of liver necrosis, reduce the levels of inflammatory factors such as Tnfa, Il1b, Il6, and Ccr2, and reduce the number of inflammatory cell infiltrations in the liver, etc. Description of the Drawings

[0013] Figure 1 A figure showing that IL-19 in Example 1 is involved in the pathological process of APAP-induced liver injury. Among them: A is a figure showing the measurement results of the serum IL-19 level in mice; B is a figure showing the measurement results of the mRNA levels of IL-19 and its receptor (IL-20R1 / R2) in the mouse liver, where *P≤0.05, **P≤0.01, ***P≤0.001; C is a figure showing the results of detecting the cellular distribution of IL-20R1 / IL-20R2 in the liver by immunohistochemistry; D is a figure showing the results of detecting the cellular distribution of IL-20R2 by flow cytometry; E is a figure showing the results of detecting the cellular distribution of IL-20R1 / IL-20R2 in the liver by immunofluorescence staining.

[0014] Figure 2 A figure showing that IL-19 gene knockout mice in Example 2 are more susceptible to APAP-induced liver injury and inflammation. Among them: A is a schematic diagram showing the administration method; B is a figure showing the results of detecting the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice using a kit; C is a figure showing the results of detecting the area of liver necrosis in mice by HE staining; D is a figure showing the results of detecting the mRNA levels of related inflammatory factors in the mouse liver by RT-qPCR; E is a figure showing the protein levels of related inflammatory factors in the serum of mice. Where *P≤0.05, **P≤0.01, ***P≤0.001.

[0015] Figure 3Figure showing that after APAP treatment in Example 2, more MoMFs infiltrated into the liver of IL-19 gene knockout mice. Among them: A is a figure showing the staining of liver immune cells by immunohistochemistry (CD45 - leukocytes, CD11b - myeloid leukocytes, IBA-1 - macrophages, Ly6g - neutrophils); B is a figure showing the statistical chart of the positive area of CD45 - leukocytes, CD11b - myeloid leukocytes, IBA-1 - macrophages, Ly6g - neutrophils; C is a figure showing the use of immunofluorescence multiplex staining method to distinguish resident Kupffer cells and monocyte - derived macrophages (MoMFs) in mouse liver; D is a figure showing the level of lipid peroxidation in mouse liver; E is a figure showing the mRNA levels of F4 / 80, Cx3cr1 and Ccr2 in mouse liver. Among them, *P≤0.05, **P≤0.01, ***P≤0.001.

[0016] Figure 4 Figure showing that in Example 3, rIL-19 can inhibit MoMFs from infiltrating into the liver and improve APAP - induced liver injury. Among them: A is a schematic diagram showing the application of rIL-19 in mice; B is a figure showing the comparison results of serum ALT and AST levels between control group mice and rIL-19 treatment group mice in an APAP - induced liver injury model; C is a figure showing the liver necrosis area of mice by HE staining; D is a statistical chart of the liver necrosis area of mice; E is a figure showing the IBA-1 immunohistochemical staining of mouse liver and the statistical chart of the positive area; F is a figure showing the detection results of the mRNA levels of Tnfa, Il1b, Il6, Ccr2 and Ccl4 in mouse liver. Among them, *P≤0.05, **P≤0.01, ***P≤0.001.

[0017] Figure 5 Figure showing the inhibitory effect of rIL-19 on macrophage activation in Example 4. Among them: A shows that IL-19 inhibits the activation of mouse bone marrow - derived macrophages (BMDM) by IL-1β and reduces the mRNA levels of Tnfa, Il1b, Ccl3, Ccl4; B shows that IL-19 reduces the phosphorylation degree of ERK and p38 proteins promoted by IL-1β in BMDM; C shows that IL-19 inhibits the activation of mouse macrophage cell line RAW264.7 cells by IL-1β and reduces the mRNA levels of Tnfa, Il1b, Ccl3, Ccl4. Among them, *P≤0.05, **P≤0.01, ***P≤0.001. Detailed implementation manners

[0018] Hereinafter, the present invention will be described in detail by examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0019] Materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0020] Antibody Information Table

[0021]

[0022]

[0023] Example 1: Observe whether IL-19 is involved in the pathogenesis of APAP-induced liver injury

[0024] Twenty-four male C57 mice were purchased from Shanghai Model Organisms Center, Inc. The animals were allowed to eat and drink freely, given standard pellet feed, and adaptively fed for 1 week in a standard light cycle (12 hours of light, 12 hours of darkness), at a room temperature of 22°C, and a constant humidity environment before the experiment. Acetaminophen (Sangon Biotech, Cat: 103-90-2) was dissolved in sterile PBS (Corning, Cat: 21-040-CV) (35 mg / ml, 55°C, heated for 10 min) to prepare an acetaminophen solution. The experimental mice were randomly divided into 4 groups (6 mice / group): control group, APAP 3h group, APAP 6h group, and APAP 24h group. The mice were starved overnight for 16 h before dosing; the dosing dose was 350 mg / kg; the dosing method was intraperitoneal injection. After the experiment, each mouse was weighed and anesthetized by inhaling isoflurane. After blood was collected from the orbital cavity, the abdominal cavity was opened to isolate the intact liver, which was then frozen and fixed.

[0025] Alanine aminotransferase test kit (Cat: C009-3-1) was used to measure alanine aminotransferase (ALT) in mouse serum, and aspartate aminotransferase test kit (Cat: C010-3-1) was used to measure aspartate aminotransferase (AST) in mouse serum. The specific operation was as follows: Mouse blood was centrifuged at 8000 rpm at 4°C for 5 min, and the supernatant was taken for standby. A 96-well plate was used to detect ALT and AST. Add 200 μL / well of R1 + 10 μL / well of diluted serum sample, gently tap and mix well, and incubate at 37°C for 5 minutes; after incubation, add 50 μL / well of R2, gently tap and mix well, and then quickly detect the absorbance at a wavelength of 340 nm using an enzyme-labeled instrument (Thermo Fisher Scientific). The absorbance was detected 5 times in total, once per minute for a total of 4 minutes. The ALT and AST values of each mouse were calculated by multiplying the calculated average rate × coefficient × serum dilution factor.

[0026] Determination of mouse serum IL-19 level: ELISA kit (Novus, Cat: NBP3-06800) was used to detect the content of IL-19 in mouse serum ( Figure 1In A), mouse blood was centrifuged at 8000 rpm for 5 min at 4°C, and the supernatant was taken for later use. The IL-19 level in mouse serum was measured according to the instructions of the IL-19 ELISA kit. The experimental results are as Figure 1 shown in A. After treatment with APAP for 3 h, 6 h, and 24 h, the IL-19 level in mouse serum was upregulated.

[0027] Determination of the mRNA levels of IL-19 and its receptors (IL-20R1 / R2) in mouse liver ( Figure 1 in B):

[0028] (1) Liver RNA extraction: Take about 30 - 40 mg of mouse liver (try to take similar parts from mice of the same batch) and put it into a grinding tube containing 1 ml of trizol and grinding beads. Grind it with a grinder at 60 HZ for 60 s to obtain a liver tissue homogenate. Incubate it in a 4°C refrigerator for 10 - 15 min for sufficient lysis. Add 200 μL of chloroform / 1 ml of Trizol (Nanjing Novoprotein Scientific Co., Ltd., Cat: R401-01-AA) (volume ratio 1:5), and shake it vigorously up and down. After standing for 3 min, centrifuge it at 12000 rmp at 4°C for 15 min. Take the supernatant and transfer it to a new EP tube. Generally, 100 - 200 μL is enough. Add an equal volume of cooled isopropanol (Sigma) and mix well. Incubate it at -20°C for 10 min or longer for precipitation. Centrifuge it at 12000 rmp at 4°C for 10 min. Discard the supernatant, add 1 ml of pre-cooled 75% ethanol (Sinopharm) (prepared with RNase-free water) (if 500 μL of trizol was used for lysis as above, add 500 μL of 75% ethanol). Centrifuge it at 7500 rmp at 4°C for 5 min. Discard the supernatant ethanol, centrifuge it at 7500 rmp at 4°C for 1 min. Carefully aspirate and discard the residual ethanol with a pipette tip. Add an appropriate amount of RNase-free water to dissolve the RNA (generally, 20 μL of water is used to dissolve cell samples, and 100 - 200 μL of water is used to dissolve tissue samples). Measure the RNA concentration.

[0029] (2) Reverse transcription: Reverse transcription system (Thermo Fisher Scientific, Cat: 1109566): RT buffer: 1 μL + RT primer: 1 μL + dNTP: 0.5 μL + Rtase: 0.5 μL + RNA sample: 7 μL (500 ng RNA + RNase-free water). Add them to an eight-well tube in the order of water + reverse transcription system + RNA, and place the eight-well tube in a PCR instrument (Bio-rad) for reverse transcription.

[0030] (3) Real-time fluorescence quantitative PCR: After the above reverse transcription reaction was completed, 100 μL of enzyme-free water was directly added to dissolve it, and the cDNA was aliquoted. PCR system configuration (10 μL system): 5 μL of SYBR green (Vazyme Biotech Co., Ltd., Cat: Q711-03-AA) + 0.5 μL of Primer (F+R) + 3 μL of Water + 1.5 μL of cDNA (the above dilution). The well plate added with the system was placed in an RT-qPCR instrument (Thermo Fisher Scientific) for RT-qPCR. The 2 —ΔΔCT -method was used to calculate the results.

[0031] RT-qPCR primer sequences

[0032]

[0033]

[0034] The experimental results are as Figure 1 shown in B below. The mRNA levels of liver IL-19 and its receptor (IL20R1 / R2) in mice treated with APAPA were significantly upregulated.

[0035] Flow cytometry was used to detect the cellular distribution of IL-20R2 ( Figure 1 shown in D below):

[0036] 1. Pre-cool the centrifuge in advance

[0037] 2. Prepare the mice. Anesthetize the mice with isoflurane. After the mice are anesthetized, collect blood by enucleating the eyeballs. Collect the blood in a 50 ml centrifuge tube containing 10 μL of EDTA (Invitrogen, Cat: CAM9260G) (for later use);

[0038] 3. Cut open the abdominal cavity of the mice, take out the liver, prepare a 6 cm dish, lay a nylon mesh on the dish, place the liver on the nylon mesh, and grind it with the piston of a 2 ml syringe;

[0039] 4. Transfer the ground cell suspension to a 50 ml centrifuge tube, supplement PBS to 50 ml, and centrifuge at 400 rpm (about 30 g) for 5 min (4 °C);

[0040] 5. Collect the supernatant and centrifuge at 1600 rpm for 5 min (4 °C);

[0041] 6. Discard the supernatant, collect the cell pellet, and add 15 ml of 40% percoll (Sigma, Cat: 17089101);

[0042] 7. After resuspending the cell pellet with 40% percoll, increase the speed of the centrifuge to 9 and decrease the deceleration to 1, then centrifuge at 2400 rpm for 5 min (4 °C);

[0043] 8. Discard the supernatant, add 2 ml of red blood cell lysis buffer (Thermo Fisher Scientific, Cat: 00-4300-54), gently pipette to mix well, and lyse on ice for 3 min;

[0044] 9. Add 10 ml of PBS to terminate the lysis, and centrifuge at 1600 rpm for 5 min;

[0045] 10. Discard the supernatant, collect the cell pellet and transfer it to a 1.5 mL Ep tube, and centrifuge at 400 xg for 5 min;

[0046] 11. For single-positive tubes, use a pipette to aspirate the supernatant. Add 200 μL of PBS to the pellet, pipette to mix well, then aspirate a small portion of the cells (30 μL) and supplement with 300 μL of MACS buffer to make a Blank (blank tube. When the sample types are the same, only take a portion of the cells from one of the samples as the blank tube. After preparing the blank tube, it can be placed at 4 °C). Then take out another portion of the cells for single-positive tube staining (single-positive tube: staining with a single antibody. Determine the amount of cells taken out according to the number of antibodies. When the sample types are the same, only take a portion of the cells from one of the samples for single antibody staining). Centrifuge the remaining cells at 400 xg for 5 min again. After discarding the supernatant, the cell pellet is used for subsequent staining;

[0047] 12. Stain cell viability with Zombie: Dilute Zombie (1:1000) with PBS (BioLegend, Cat: 423101), and resuspend the cell pellet with the diluted solution containing Zombie (add 100 μL for the cell pellet of each mouse), and stain at room temperature for 20 min (Zombie-low: live cells; Zombie-high: dead cells);

[0048] 13. Centrifuge at 400 g for 5 min;

[0049] 14. Discard the supernatant, add 200 μL of MACS buffer to resuspend the cells, take out a portion of the cells (30 μL) as the Zombie single-positive tube (when the sample types are the same, only take a portion of the cells from one of the samples as the single-positive tube), and the remaining cells are used as the sample tube for subsequent staining;

[0050] 15. Centrifuge the Zombie single-positive tube and the sample tube at 400 g for 5 min; (At this time, the antibody can be prepared: 100 μL of MACS buffer + 0.5 μL of antibody / sample)

[0051] 16. Antibody staining of single-positive tubes and sample tubes: Discard the supernatant, collect the cell pellet, resuspend the single-positive Zombie tube in 300 μL of MACS buffer, and add MACS buffer containing cell marker antibodies to the single-positive tube (added with a single antibody) and the sample tube. After resuspension, stain at room temperature or 4°C for 30 min.

[0052] 17. Discard the supernatant, add 300 μL of MACS buffer to resuspend the cells, centrifuge at 400 g for 5 min, collect the cell pellet, and add 300 μL of MACS buffer to resuspend the cells to obtain the sample tube, which can be used for instrument operation.

[0053] The experimental results are as Figure 1 shown in D below. IL-20R2 is mainly expressed in monocytes and neutrophils in the liver.

[0054] Immunohistochemistry and immunofluorescence staining were used to detect the cellular distribution of IL-20R1 / IL-20R2 in the liver ( Figure 1 shown in C and E below): Dewaxing: Place the slides containing mouse liver tissue sequentially in xylene (Sinopharm) (15 min), xylene: absolute ethanol (Sinopharm) 1:1 (3 min), absolute ethanol (3 min), absolute ethanol (3 min), 95% ethanol (3 min), 80% ethanol (3 min), 70% ethanol (3 min), and rinse with running water for 3 min. Antigen retrieval: Dilute 20× citrate (pH 6.0, Invitrogen, Cat: 005000) with distilled water to 1×. Place the slides rinsed with running water into the retrieval solution and put them in a microwave oven. First, heat at high power for 1 - 2 min until the liquid starts to boil, then switch to low power for 10 min to keep the liquid boiling intermittently for 10 min. Observe the liquid level at any time during this period to prevent the liquid from evaporating too fast and the tissue from being exposed. After antigen retrieval, place the slides in cold PBS and wash 2 times, 3 min each time. Block with 3% hydrogen peroxide for 15 min and wash with PBS. After blocking with 3% NGS (Abbexa, Cat: abs933 - 50 ml) for 1 h, incubate with the primary antibody overnight at 4°C. Take out the slides the next day and wash 3 times, 5 min each time. Incubate with the secondary antibody at room temperature for 1 h and wash 3 times, 5 min each time (immunofluorescence staining needs to be protected from light). Immunohistochemistry can be developed with DAB chromogenic solution (Vector, Cat: SK - 4105) (note that the development time should be the same for the same batch of sections). Observe under the microscope. When the target protein is stained dark brown and the background has no brown, stop the chromogenic reaction with water. After immunofluorescence DAPI staining, observe with a fluorescence microscope.

[0055] The experimental results are as Figure 1As shown in Figures DE, IL-20R1 and IL-20R2 are mainly expressed in monocyte-derived macrophages (MoMFs) and neutrophils in the liver.

[0056] The above results show that IL-19 and its receptor (IL-20R1 / R2) are significantly upregulated in the APAP model, and IL-19 is involved in the pathogenesis of the APAP model. IL-20R1 / R2 is highly expressed in monocyte-derived macrophages (MoMFs) and neutrophils in mouse liver.

[0057] Conclusion: IL-19 is involved in the pathological process of APAP-induced liver injury.

[0058] Example 2: Observation of the effect of IL-19 on APAP-induced liver injury-related indicators in mice

[0059] IL-19 knockout mice were purchased from Shanghai South Model Organisms Science Co., Ltd. IL-19 knockout mice were bred to obtain IL-19KO mice and their littermate control WT mice. The animals were allowed to eat and drink freely and given standard pellet feed. The experiment was started after 8-10 weeks of adaptive breeding in a standard light cycle (12 hours of light, 12 hours of darkness), room temperature of 22°C, and a constant humidity environment. Acetaminophen was dissolved in sterile PBS (35 mg / ml, 55°C, heated for 10 min) to prepare an acetaminophen solution. The experimental mice were divided into 2 groups (15 / group) according to the results of genotype identification.

[0060] Groups): WT group and IL-19KO group. Mice were starved for 16 hours before administration; dosage: 350 mg / kg; administration method: intraperitoneal injection ( Figure 2 Middle A). After the experiment, each mouse was weighed and anesthetized by inhalation of isoflurane. After blood was collected from the orbits, the abdominal cavity was opened to separate the complete liver, which was then frozen and fixed. The mouse blood was centrifuged at 8000 rpm for 5 min, and the supernatant was taken and frozen at -20°C for later use. The mouse serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a kit, the mouse liver necrosis area was detected using HE staining, and the mRNA level of mouse liver-related inflammatory factors was detected using RT-qPCR. The protein level of mouse serum inflammatory factors was detected using an ELISA kit. The number of inflammatory cell infiltration in the mouse liver was detected using immunohistochemistry and immunofluorescence methods.

[0061] The results are as follows Figure 2 As shown: In the APAP-induced liver injury model, the levels of ALT and AST in the serum of IL-19KO mice were significantly higher than those of WT mice ( Figure 2 Middle B). The liver necrosis area of ​​IL-19KO group mice was significantly higher than that of WT group mice ( Figure 2In C). The mRNA levels of Tnfa, Il1b, and Il6 in the livers of IL-19KO mice Figure 2 In D) and the protein levels of Tnfa, Il1b, and Il6 in the serum Figure 2 In E) were significantly higher than those in WT mice. TNF-α, IL-1β, and IL-6 are common inflammatory factors, and higher levels of them indicate more severe liver inflammation. The above results show that in the APAP-induced liver injury model, the liver injury and the level of liver inflammation in IL-19KO mice are more severe than those in WT mice.

[0062] By Figure 1 It can be seen that the receptors of IL-19 (IL-20R1 / R2) are mainly distributed in macrophages derived from monocytes. By immunohistochemical staining of liver immune cells (CD45 - leukocytes, CD11b - myeloid leukocytes, IBA-1 - macrophages, Ly6g - neutrophils), it was found that the number of IBA-1 positive macrophages in the livers of IL-19KO mice in the APAP-induced liver injury model was significantly increased compared with that in WT mice Figure 3 In A - B). By using immunofluorescence multiplex staining to distinguish resident Kupffer cells and monocytes-derived macrophages (MoMFs) in the mouse liver, the results showed that the infiltration number of MoMFs in IL-19KO mice was significantly higher than that in WT mice Figure 3 In C). The level of lipid peroxidation in the livers of IL-19KO mice was significantly higher than that in WT mice Figure 3 In D). The mRNA levels of F4 / 80, Cx3cr1, and Ccr2 in the livers of IL-19KO mice were also significantly higher than those in WT mice Figure 3 In E). CX3CR1 and CCR2 are markers of monocytes-derived macrophages. In the APAP-induced liver injury model, IL-19 inhibits the infiltration of monocytes-derived macrophages into the liver necrosis area.

[0063] Example 3: Inject the recombinant IL-19 protein (rIL-19) into mice via the tail vein

[0064] Twenty male C57 mice were purchased from Shanghai Model Organisms Center, Inc. The animals had free access to food and water, were given standard pellet feed, and were adaptively housed for 1 week in a standard light cycle (12 hours of light and 12 hours of darkness), at a room temperature of 22 °C, and in a constant humidity environment before the experiment. Acetaminophen was dissolved in sterile PBS (35 mg / ml, heated at 55 °C for 10 min) to prepare an acetaminophen solution. The experimental mice were randomly divided into 2 groups (10 mice / group): a control group and an rIL-19 administration group. The mice were fasted overnight for 16 h before drug administration; the dosing dose was 350 mg / kg; the dosing method was intraperitoneal injection. At 9 h after APAP administration, rIL-19 was applied to the mice ( Figure 4 in A). rIL-19 was purchased from MedChemExpress, with the catalog number HY-P7213. The rIl-19 protein was in dry powder form. rIL-19 was reconstituted in a 50 mM acetic acid solution in a laminar flow hood to prepare a mother liquor of rIL-19 with a concentration of 100 μg / ml. The dosing dose of rIL-19 was 0.1 μg / g, that is, 1 μL / g of the mother liquor per mouse body weight. To reduce injection error, the mother liquor was diluted 5-fold with sterile PBS in a laminar flow hood before using rIL-19 to prepare an rIL-19 dilution with a concentration of 0.02 μg / μL, and the dosing volume for mice was 5 μL / g of mouse body weight. The dosing method was tail vein injection. After the experiment, each mouse was weighed and anesthetized by inhaling isoflurane. After blood was collected from the orbital cavity, the abdominal cavity was opened to isolate the intact liver, which was then frozen and fixed. The mouse blood was centrifuged at 8000 revolutions per minute for 5 min, the supernatant was taken and stored at -20 °C for later use. A kit was used to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum, HE staining was used to detect the liver necrosis area in mice, and the RT-qPCR method was used to detect the mRNA levels of liver-related inflammatory factors in mice.

[0065] The experimental results are as Figure 4 shown: In the APAP-induced liver injury model, the levels of ALT and AST in the serum of mice in the rIL-19 treatment group at 24 h of APAP were lower than those of the control group mice ( Figure 4 in B). The liver necrosis area of mice in the rIL-19 group was significantly lower than that of the control group mice ( Figure 4 in C-D). rIL-19 inhibited the infiltration of IBA-1 positive cells into the liver necrosis area of mice ( Figure 4 in E). The mRNA levels of Tnfa, Il1b, Il6, Ccr2, and Ccl4 in the livers of mice in the rIL-19 treatment group were significantly lower than those of the control group mice ( Figure 4 in F). The above results indicate that rIL-19 can improve APAP-induced liver injury and liver inflammation in mice by inhibiting the infiltration of MoMFs into the liver necrosis area.

[0066] Example 4: Inhibitory effect of IL-19 on macrophages

[0067] Male C57 mice, 6 - 8 weeks old, were purchased from Shanghai Model Organisms Center, Inc. The animals had free access to food and water, were given standard pellet feed, and were adaptively fed for 1 week in a standard light cycle (12 hours of light, 12 hours of darkness), at a room temperature of 22°C, and a constant humidity environment before the experiment began. After anesthetizing the mice with isoflurane, the mice were sacrificed, and the mice and experimental equipment were thoroughly disinfected with 75% ethanol. The hind limbs of the mice were taken, and the intact hip joints and ankle joints were retained to isolate the bone marrow cells of the mice. The obtained mouse bone marrow cells were inoculated into a 24-well plate, and the cell suspension concentration was adjusted to 1×10 6 cells per well, and cultured in DMEM (Corning, Cat: 10 - 013 - CV) medium containing 10% fetal bovine serum and 1% antibiotics under the culture conditions of 37°C and 5% CO2. At the same time, M-CSF (10 ng / ml) was added to stimulate the mouse bone marrow cells to differentiate and mature into bone marrow-derived macrophages (BMDM). BMDM was activated in vitro with IL-1β. IL-1β was purchased from MedChemExpress, with the product number HY-P70437, and the administration concentration was 50 ng / ml. The cells were divided into 4 groups: control group, IL-1β (50 ng / ml) group, IL-1β (50 ng / ml) + IL-19 (10 ng / ml) group, and IL-19 (10 ng / ml) group. IL-19 was pretreated for 1 h, and IL-1β was treated for 6 h, with 5 replicate wells in each group. After the treatment, the cell plates for RT-qPCR analysis were lysed with ice-cold Trizol lysis buffer, lysed at 4°C for 15 - 20 min, and the cell lysates were collected. Chloroform, isopropanol, and 75% ethanol were added in sequence to extract the cell RNA. A reverse transcription system was prepared to reverse transcribe the obtained RNA. After the reverse transcription was completed, an RT-qPCR system was prepared and RT-qPCR was performed using an RT-qPCR instrument (Thermo Fisher Scientific) for data analysis and processing. For the cell plates for Western blot analysis, the cells were lysed with ice-cold RIPA lysis buffer containing protease inhibitors, lysed at 4°C for 15 - 20 min, and the cell lysates were collected. Centrifugation was carried out at 12000 rpm and 4°C for 15 min, and the supernatant was aspirated to measure the protein concentration by the BCA method. The protein loading amount was 40 μg, and 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) (Absin, Cat: abs9367) electrophoresis was performed, followed by NC membrane transfer, and non-specific blocking with 1% BSA (Absin, Cat: abs9157) for 1 h; the corresponding primary antibody was added and incubated overnight at 4°C, and then a horseradish peroxidase-labeled secondary antibody was added for hybridization. ECL luminescent solution was added, and imaging and analysis were performed using a gel imaging system (Tanon).

[0068] The experimental results showed that IL-19 inhibited IL-1β-induced macrophage activation by suppressing the activation of the ERK signaling pathway in macrophages( Figure 5 ).

[0069] Conclusion: IL-19 inhibits macrophage activation and infiltration into the liver necrosis area by suppressing the activation of the ERK signaling pathway in macrophages, thereby improving APAP-induced liver injury and liver inflammation. Treating APAP-induced liver injury using the method provided by the present invention can effectively improve the levels of liver injury and inflammation.

[0070] Summary: Intravenous injection of recombinant IL-19 protein through the tail vein can significantly improve APAP-induced liver injury. The previous experimental results showed that IL-19 alleviates APAP-induced liver injury and liver inflammation by inhibiting macrophage infiltration into the liver necrosis site. IL-19 is expected to become a new target for the treatment of drug-induced liver injury.

Claims

1. Use of interleukin-19 (IL-19) protein, or its derivative or variant, or its enhancer, or its expression vector in the preparation of a medicament for preventing or treating drug-induced liver injury.

2. The use according to claim 1, wherein, the amino acid sequence of the IL-19 protein is as shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. The use according to claim 1, wherein, the derivative or variant of the IL-19 protein refers to a molecule obtained by non-natural engineering modification of the IL-19 protein without affecting the function of the IL-19 protein.

4. The use according to claim 1, wherein, the amino acid sequence of the derivative or variant of the IL-19 protein is as shown in SEQ ID NO:

3.

5. The use according to claim 1, wherein, the amino acid sequence of the derivative or variant of the IL-19 protein is as shown in SEQ ID NO:

4.

6. The use according to claim 1, wherein, the enhancer of IL-19 refers to any substance that can enhance the production of IL-19.

7. The use according to claim 1, wherein, the expression vector of IL-19 refers to an expression vector carrying a nucleotide sequence encoding IL-19 protein, or its derivative or variant, which can enhance the expression of IL-19 in cells.

8. The use according to claim 7, wherein, the expression vector includes a viral vector or a non-viral vector.

9. The use according to claim 8, wherein, the expression vector includes IL-19 adenovirus, Il-9 adeno-associated virus, IL-19 overexpression plasmid.

10. The use according to any one of claims 1 to 9, wherein, the drug-induced liver injury is liver injury induced by acetaminophen (APAP).

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