CN120078878A

CN120078878A - Application of Tim-3 protein in the treatment of liver injury

Info

Publication number: CN120078878A
Application number: CN202510330042.XA
Authority: CN
Inventors: 黄晨璐; 方钟; 王丛; 袁正宏
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2025-03-20
Filing date: 2025-03-20
Publication date: 2025-06-03

Abstract

The present invention discloses an application of Tim‑3 protein in treating liver injury, belonging to the field of biomedical technology. The present invention finds that the soluble protein expressed by the Tim‑3 extracellular region has the effect of treating or alleviating liver injury, and the liver injury refers to acute or chronic liver injury induced by drugs, chemicals, and diet. The present invention confirms that Tim‑3 protein can effectively reduce the phenomenon of increased AST and ALT in the body, inhibit hepatocyte coagulative necrosis and/or hepatocyte watery degeneration, effectively alleviate the state of liver injury, have a definite therapeutic effect, and have a significant improvement effect, and have extremely important clinical transformation value.

Description

Application of Tim-3 protein in treating liver injury

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of Tim-3 protein in treating liver injury.

Background

Liver is one of the most important parenchymal organs in the body, and has important functions of secreting bile, synthesizing albumin, participating in glycolipid and vitamin metabolism, and detoxifying. Poor lifestyle, blindly administered drugs, viral infections, etc. can lead to chronic or acute damage to the liver.

Liver injury can cause liver function abnormality, affect metabolism and detoxification function of human body, and cause jaundice, ascites, hepatic encephalopathy and other symptoms. Liver injury to a severity that renders liver cells inoperable, with reduced regenerative capacity, with destruction of tissue structures, and causes liver dysfunction, known as liver failure. Liver failure has rapid disease progression, high treatment difficulty, high medical cost and poor overall prognosis.

At present, the main treatment means of acute liver failure is liver transplantation, but liver source is tense, so that the clinical application of the liver source is limited. As a replacement therapy for liver transplantation, bioartificial liver strategies have been developed clinically. Bioartificial liver is a liver replacement device constructed by bioengineering techniques. However, the research of bioartificial liver is still in the primary stage, the operation difficulty is far higher than that of the traditional liver transplantation, and the curative effect of the bioartificial liver is still not comparable with that of the real liver. Bioartificial livers are not only very costly to develop and produce, but also potentially trigger immune rejection in patients. It still cannot completely simulate the liver and cannot perform complex functions such as bile secretion, detoxification and the like. Thus, there is an urgent need in the art to develop drugs for liver damage including liver failure.

Tim-3 (also known as CD366 or HAVCR 2) is a transmembrane protein, an inhibitory molecule in adaptive immunity. Tim-3 consists of an extracellular region, a transmembrane region and an intracellular region. Tim-3 is expressed on CD4+ helper T cells and CD8+ cytotoxic T cells. Tim-3 can also be expressed on natural immune cells such as DC, NK cells, monocytes and macrophages. The inhibitory function of Tim-3 requires interaction with the adhesion protein CEACAM-1, and three other ligands are known to bind to Tim-3 and modulate antitumor immunity, galectin-9, ptdSer and HMGB15-8, and binding to these receptors can induce T cell apoptosis and impair Toll-like receptor and cytoplasmic receptor mediated recognition of foreign nucleic acids.

In addition, soluble Tim-3 protein can be detected in patient plasma. Plasma Tim-3 protein has been found to be associated with the efficacy, progression and prognosis of various diseases such as leishmaniasis, acute myeloid leukemia, non-small cell lung cancer, and the like. Patent document CN 110655566a discloses an application of a soluble Tim-3 recombinant protein and a mutant protein in preparing a medicament for regulating and controlling the function of monocytes, wherein the medicament for regulating and controlling the function of monocytes can be used for inhibiting excessive activation of monocytes of inflammatory patients such as liver failure patients, and in a specific cell experiment, the soluble Tim-3 recombinant protein can inhibit activation of monocytes to release inflammatory factors (such as TNF-alpha and HMGB 1).

In addition to the above list, other functions of the soluble Tim-3 protein have not yet been developed, and no research report has been made on the treatment of liver injury diseases other than liver failure using the Tim-3 protein. In view of this, the present invention particularly provides the following technical solutions.

Disclosure of Invention

The present invention unexpectedly found that a soluble protein expressed in the extracellular region of Tim-3 has an effect of treating or alleviating liver injury, which is a disease of liver injury other than liver failure. In a specific embodiment of the invention, the liver injury refers to acute or chronic liver injury induced by drugs, chemicals, diet. The invention aims to provide an application of Tim-3 protein in preparing a medicament for treating liver injury, and experiments prove that the Tim-3 protein can effectively reduce the phenomenon of AST and ALT elevation of organisms, inhibit the coagulation necrosis of liver cells and/or the water sample denaturation of liver cells, effectively relieve the liver injury state, has definite treatment effect and has potential clinical transformation value.

The aim of the invention is realized by the following technical scheme:

in a first aspect, the invention provides the use of a Tim-3 protein in the preparation of a product for the treatment and/or alleviation of liver damage, characterised in that the Tim-3 protein is selected from a natural protein or a recombinant protein, the Tim-3 protein comprising a human Tim-3 protein, a murine Tim-3 protein or a mammalian Tim-3 protein other than the human and murine Tim-3 proteins.

Preferably, the liver injury is selected from acute or chronic liver injury induced by drugs, chemicals, diet, alcohol.

In a specific embodiment of the present invention, the drug is selected from one or a combination of two or more of acetaminophen, methotrexate, e.g. cyclophosphamide.

In a specific embodiment of the present invention, the chemical substance is selected from one or a combination of two or more of carbon tetrachloride, concanavalin A (ConA), and polyinosinic-polycytidylic-acid (PolyI: C).

In a specific embodiment of the present invention, the diet is selected from one or a combination of two or more of a high fat diet, a high fructose diet, and a high cholesterol diet.

Further, the liver injury is selected from acetaminophen-induced acute liver injury, carbon tetrachloride-induced chronic liver injury or high-fat, high-fructose and high-cholesterol diet-induced liver injury.

The Tim-3 protein has at least one of the following functions:

(1) Reduces the AST and/or ALT level of the organism,

(2) Inhibiting hepatic cell coagulation necrosis;

(3) Inhibiting the degeneration of liver cell water sample.

In some embodiments of the invention, the Tim-3 protein is selected from a human Tim-3 protein or a murine Tim-3 protein.

In specific embodiments, the human Tim-3 protein is selected from any one of (hP 1) - (hP 3):

(hP 1) the protein has an amino acid sequence shown in SEQ ID NO. 1;

(hP 2) the protein has at least 80% identity with the amino acid sequence shown in SEQ ID NO.1 and the activity is the same;

(hP 3) the protein has the amino acid sequence shown in SEQ ID NO.1, and the protein has the same activity and is substituted by 1 or a plurality of amino acid residues, deleted or added.

In specific embodiments, the murine Tim-3 protein is selected from any one of (mP 1) - (mP 3):

(mP 1) the protein has the amino acid sequence shown in SEQ ID NO. 2;

(mP 2) the protein has at least 80% identity to the amino acid sequence shown in SEQ ID No.2 and the same activity;

(mP 3) the protein has the same activity and the amino acid sequence shown as SEQ ID NO.2 is replaced by 1 or a plurality of amino acid residues, deleted or added.

The term "sequence of at least 80% identity" refers to an amino acid sequence having a degree of 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

The term "several amino acid residues" means 2,3, 4, 5, 6, 7, 8, 9, 10.

In a specific embodiment of the invention, the human-derived amino acid sequence shown in SEQ ID NO.1 represents a soluble protein expressed in the extracellular domain of Tim-3, and the specific amino acid sequence is as follows:

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDV

NYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVT

PAPTLQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDS

GATIR

In a specific embodiment of the present invention, the murine amino acid sequence shown in SEQ ID NO.2 represents a soluble protein expressed in the extracellular domain of Tim-3, and the specific amino acid sequence is as follows:

FSGLTLNCVLLLLQLLLARSLENAYVFEVGKNAYLPCSYTLSTPGALVPMCWGKGF

CPWSQCTNELLRTDERNVTYQKSSRYQLKGDLNKGDVSLIIKNVTLDDHGTYCCRIQFP

GLMNDKKLELKLDIKAAKVTPAQTAHGDSTTASPRTLTTERNGSETQTLVTLHNNNGT

KISTWADEIKDS

In one embodiment of the invention, the Tim-3 protein is a recombinant protein prepared from a biological material comprising at least one of the following:

(a) A nucleic acid molecule encoding the Tim-3 protein;

(b) A recombinant vector comprising the nucleic acid molecule of (a);

(c) A transformed cell comprising (a) the nucleic acid molecule or (b) the recombinant vector;

(d) A recombinant virus comprising the nucleic acid molecule of (a) or the recombinant vector of (b).

In a specific embodiment of the invention, the original plasmid of the recombinant vector is pcdna3.4.

The host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells, preferably, the mammalian cells are selected from Chinese hamster ovary Cells (CHO), tumor cells, BHK cells or HEK293 cells.

The recombinant virus includes adenovirus, adeno-associated virus, vaccinia virus, herpesvirus or retrovirus vector.

In a specific embodiment of the invention, the Tim-3 protein is selected from a human Tim-3 protein or a murine Tim-3 protein.

In specific embodiments, the nucleic acid molecule encoding a human Tim-3 protein is selected from any one of (hDNA 1) - (hDNA):

(hDNA 1) a nucleotide sequence shown as SEQ ID NO. 3;

(hDNA 2) the nucleotide sequence shown as SEQ ID No.3 is substituted, deleted and/or added by 1 or several nucleotides, or the nucleotide sequence encoding the same active protein according to the degeneracy of the genetic code.

In specific embodiments, the nucleic acid molecule encoding a murine Tim-3 protein is selected from any one of (mDNA 1) - (mDNA 2):

(mDNA 1) a nucleotide sequence shown as SEQ ID NO. 4;

(mDNA 2) the nucleotide sequence shown as SEQ ID No.4 is substituted, deleted and/or added by 1 or several nucleotides or the nucleotide sequence encoding the same active protein according to the degeneracy of the genetic code.

The "several nucleotides" refers to 2,3,4, 5, 6, 7, 8, 9, 10.

In one embodiment of the invention, the Tim-3 protein is prepared by the following method:

(1) Directionally cloning the gene fragment shown in SEQ ID NO.3 or SEQ ID NO.4 into a pCDNA3.4 plasmid to form a recombinant vector;

(2) Transiently transfecting the recombinant vector into CHO cells to perform protein expression;

(3) Separating and purifying the protein to obtain Tim-3 protein.

The nucleotide sequence of the coded human Tim-3 protein shown in SEQ ID NO.3 is specifically as follows:

tctgaggtggagtacagggccgaggtgggtcagaacgcttatctgccttgcttttatacccccgccgcccctggtaacctggtgcctgtgtgttggggtaagggggcttgccctgtgtttgagtgtggaaatgtggtgctgaggacagacgagagggacgtgaattattggacctctaggtattggctgaatggcgattttaggaagggggacgtgagtctgaccatcgagaatgtgaccctggccgactctgggatctactgctgcaggattcaaatccccgggatcatgaacgacgagaagttcaacctgaagctggtgatcaagcccgccaaagtgacccccgctcctacactgcagagggatttcacagccgcctttcccagaatgctgaccaccaggggccacggacctgctgaaacacagaccctgggcagtctgcccgacatcaacctgacccagatctccaccctggccaacgaactgcgcgattcccgactggcaaacgacctgagggactccggagctaccattagg

the nucleotide sequence of the coded murine Tim-3 protein shown in SEQ ID NO.4 is specifically as follows:

tttagcggcctgaccctgaactgcgtgctgctgctgctgcagctgctgctggcgcgcagcctggaaaacgcgtatgtgtttgaagtgggcaaaaacgcgtatctgccgtgcagctataccctgagcaccccgggcgcgctggtgccgatgtgctggggcaaaggcttttgcccgtggagccagtgcaccaacgaactgctgcgcaccgatgaacgcaacgtgacctatcagaaaagcagccgctatcagctgaaaggcgatctgaacaaaggcgatgtgagcctgattattaaaaacgtgaccctggatgatcatggcacctattgctgccgcattcagtttccgggcctgatgaacgataaaaaactggaactgaaactggatattaaagcggcgaaagtgaccccggcgcagaccgcgcatggcgatagcaccaccgcgagcccgcgcaccctgaccaccgaacgcaacggcagcgaaacccagaccctggtgaccctgcataacaacaacggcaccaaaattagcacctgggcggatgaaattaaagatagc

the product for treating and/or relieving liver injury is a medicament, and the medicament comprises a therapeutically effective amount of Tim-3 protein and a pharmaceutically acceptable carrier or auxiliary material.

The "pharmaceutically acceptable carrier or adjuvant" includes any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. In a specific embodiment of the present invention, the pharmaceutically acceptable carrier or adjuvant includes at least one of a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, and a lubricant.

The pharmaceutical dosage forms of the invention include, but are not limited to, injection and powder injection.

The terms "treatment" or "improving" as used herein are used interchangeably to refer to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Here, therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, such that an improvement in symptoms is observed in the subject.

The technical proposal provided by the invention has the technical contribution to the field that:

The invention discovers that the soluble protein expressed by the extracellular region of Tim-3 has the effect of treating or relieving liver injury induced by drugs, chemicals or diet, and provides a new way for treating the liver injury. According to the experimental results provided by the invention, it can be proved that the effective dose of Tim-3 protein can be applied to a liver injury organism induced by medicines, chemical substances or diet to effectively reduce the phenomenon of AST and ALT elevation of the organism, inhibit the coagulation necrosis of liver cells and/or the water sample denaturation of liver cells and effectively relieve the liver injury state. The Tim-3 protein provided by the invention has definite therapeutic effect on treating liver injury, obvious improvement effect and extremely important clinical transformation value.

Drawings

FIG. 1 is the expression of the extracellular domain of Tim-3 protein.

FIG. 2 is a schematic representation of acetaminophen-induced acute liver failure protected by Tim-3 protein.

FIG. 3 is a graph showing Tim-3 protein protection against fatty liver damage induced by a high fat, high fructose and high cholesterol diet.

FIG. 4 is a schematic diagram of a Tim-3 protein protecting carbon tetrachloride-induced chronic liver injury.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 preparation of Tim-3 protein

The gene sequence of the coding human Tim-3 extracellular region (the amino acid sequence is shown as SEQ ID NO. 1) shown as SEQ ID NO.3 and the gene sequence of the coding mouse Tim-3 extracellular region (the amino acid sequence is shown as SEQ ID NO. 2) shown as SEQ ID NO.4 are respectively synthesized on a eukaryotic expression vector plasmid pCDNA3.4 by fusing 6XHIS tag, and the steps are completed by the Baiying biotechnology Co.

Taking out competent cells from a refrigerator at-80 ℃ and placing them in a refrigerating chamber of the refrigerator for thawing, adding plasmids carrying Tim-3 extracellular region genes into the corresponding competent cells, placing them in a refrigerator at 4 ℃ for ice bath for 8min, finishing the ice bath, placing the competent cells on a heating plate of a PCR instrument for heat shock for 90s at 42 ℃, taking out the competent cells after the heat shock is finished, closing the PCR instrument, placing the competent cells in the refrigerator at 4 ℃ again for ice bath for 4min, absorbing all the competent cell liquid, coating the competent cell liquid on a solid culture medium, placing the solid culture dish in an oven for culturing for 8 h, selecting single bacterial colonies on the solid culture dish, and shake culturing in a liquid culture medium.

100ML of bacterial liquid cultured overnight (12-16 h) is collected into a 50mL centrifuge tube with a mark, about 45mL of bacterial liquid is collected at one time, the bacterial liquid is collected by a centrifugal angle rotor machine at a speed of 8000rpm, a centrifugal angle of 4min or a centrifugal horizontal rotor machine at a speed of 4500rpm,15min, the supernatant is poured out to keep bacterial cells, the operation is repeated again, and the hooking mark and the bacterial collection are completed on each row of first centrifuge tube caps on a 36-hole acrylic frame. The plasmid of interest was extracted using the Qiagen plasmid large extraction kit (cat# 12362).

Expression and purification of Tim-3 protein. Diluting 30 mu g of the plasmid with 1ml of culture solution, mixing, diluting 60 mu l of transfection reagent with 1ml of culture solution, mixing, adding the solution 2 into the solution 1, mixing, incubating at 37 ℃ for 15 minutes, adding the mixed transfection solution into CHO cell liquid drop by drop, shaking and culturing in a shaking table for one week, collecting the supernatant, and centrifuging at 8000rpm for 5 minutes. The target protein in the supernatant was purified by nickel column affinity chromatography. 1xPBS, flow rate 1ml/min,20ml, equilibrium chromatography column, flow rate 1ml/min, adding collected cell supernatant, 1xPBS, flow rate 1ml/min,20ml, washing, eluting target protein with 300mM imidazole solution, 1ml/min, collecting by separating tubes, and 500 μl each. A total of 10 tubes were collected and absorbance values at 280nm were read using a NanoDrop instrument. And sucking the high-concentration protein into a dialysis bag, putting the dialysis bag into a beaker of 1XPBS, and dialyzing to remove imidazole to obtain purified human Tim-3 protein and murine Tim-3 protein.

The purity of human Tim-3 protein SDS-PAGE is >90% (FIG. 1A), the purity of HPLC is 82.471% (FIG. 1B), the purity of murine Tim-3 protein SDS-PAGE is >95% (FIG. 1C), and the purity of HPLC is 97.518% (FIG. 1D).

EXAMPLE 2Tim-3 protein protection against acetaminophen-induced acute liver injury

A model of acute liver injury in mice was constructed by intraperitoneal injection of acetaminophen (APAP) according to methods disclosed in the prior art by selecting 6 week old SPF grade male mice of variety C57BL/6, weighing 15-20g, standard feed feeding, free feeding and drinking, and room temperature 18-22 ℃. All experimental mice were fasted overnight prior to the experiment and were intraperitoneally injected with APAP aqueous solution at a dose of 300mg/kg the next day. Mice in the treatment group were intraperitoneally injected with the human Tim-3 protein or the murine Tim-3 protein prepared in example 1 at a dose of 400. Mu.g/mouse, respectively, one hour after APAP administration, and mice in the control group were injected with an equal volume of PBS solution. The orbit was sampled for 48 hours, mice were sacrificed, and livers were removed.

The serum of mice was submitted to the cymbidium and sanitation medical test for aspartic acid Aminotransferase (AST) and alanine Aminotransferase (ALT). The livers of the mice are placed in 4% paraformaldehyde for fixation for 24 hours, dehydrated by gradient ethanol, and subjected to HE staining after paraffin embedding and slicing, and placed in a 50 ℃ oven for 6 hours. Dewaxing xylene, staining gradient alcohol from high to low, hematoxylin, staining eosin, dehydrating gradient alcohol, and sealing with neutral resin for 15min each.

As shown in fig. 2A and 2B, treatment with human Tim-3 protein significantly reduced acute liver injury caused by APAP, and serum ALT and AST activity was significantly reduced in mice of the human Tim-3 treated group compared to PBS control group mice (p < 0.01).

Tim-3 of murine origin also significantly reduced liver injury in mice, including inhibition of elevation of ALT and AST (FIGS. 2C and D). And HE staining of the liver tissue sections of mice revealed extensive hepatic cell coagulation necrosis around the central veins of the liver lobules of PBS control mice (fig. 2E, 2F and 2G), the range of hepatic cell coagulation necrosis of murine Tim-3 treatment group was significantly lighter than that of PBS control mice (fig. 2H, 2I and 2J).

EXAMPLE 3Tim-3 protein protects against high-fat, high-fructose, high-cholesterol diet-induced liver injury

According to the method disclosed in the prior art, a fatty liver injury model of a mouse is constructed by freely feeding high-fat high-fructose high-cholesterol feed, namely, selecting a 6-week-old SPF-grade male mouse with a variety of C57BL/6, weighing 15-20g, freely feeding the high-fat high-fructose high-cholesterol feed (purchased from synergistic organism, product number XT 310), and continuously feeding for 16 weeks at room temperature of 18-22 ℃ to construct the fatty liver injury model. Mice in the treatment group were intraperitoneally injected with the Tim-3 protein prepared in example 1 at a dose of 400. Mu.g/mouse. The human Tim-3 protein is injected once, blood is taken after two days to detect ALT and AST in serum, the murine Tim-3 protein is injected once a week for three weeks continuously, and the control mice are injected with an equal volume of PBS solution. Blood was collected once per week from the orbit seven days after the third injection of Tim-3 protein. Serum ALT and AST detection protocols were as described above.

As shown in fig. 3A and 3B, treatment with human Tim-3 protein significantly reduced fatty liver injury induced by high fat, high fructose and high cholesterol diet, and significantly reduced ALT and AST levels in serum of mice in the treatment group compared to PBS control group mice. Also, tim-3 of murine origin can effectively relieve liver injury of mice with fatty liver model for a long period of time, and ALT and AST activities in serum are obviously reduced (figures 3C and 3D).

EXAMPLE 4Tim-3 protein protection of carbon tetrachloride-induced chronic liver injury

A model of chronic liver injury in mice was constructed by intraperitoneal injection of carbon tetrachloride (CCl ₄) according to methods disclosed in the prior art by selecting 6 week old SPF-grade male mice of the variety C57BL/6, weighing 15-20g, standard feed feeding, free feeding and drinking, and room temperature 18-22 ℃. A model of chronic liver injury was established by intraperitoneal injection of mice with 10% concentration (v/v) CCl ₄ prepared from edible corn oil, three times a week for 8 consecutive weeks, in an amount of 0.3mL/kg final concentration of CCl ₄. Mice in the treatment group were intraperitoneally injected with the murine Tim-3 protein prepared in example 1 at a dose of 400. Mu.g/mouse, and mice in the control group were injected with an equal volume of PBS solution. Three days after Tim-3 protein treatment, the orbit was sampled, mice were sacrificed, and livers were removed. Serum ALT and AST detection and liver tissue HE staining protocols were as described above.

As shown in fig. 4A and 4B, treatment with the murine Tim-3 protein significantly reduced chronic liver injury caused by chronic stimulation with CCl ₄, and significantly reduced ALT and AST activity in serum of mice in the treated group compared to PBS control group mice. HE staining was performed on liver tissue sections of mice, with significant liver cell water sample denaturation in PBS control mice (FIGS. 4C, 4D and 4E), and with significantly lower liver cell denaturation in treated mice than in control mice (FIGS. 4F, 4G and 4H).

The embodiments are only used to illustrate the technical scheme of the present invention, but not to limit the technical scheme, and although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical scheme described in the foregoing embodiments may be modified or some or all technical features may be equivalently replaced, and the modification or replacement does not deviate the essence of the corresponding technical scheme from the scope of the technical scheme of the embodiments of the present invention.

Claims

Use of a Tim-3 protein in the preparation of a product for the treatment and/or alleviation of liver damage, characterized in that said Tim-3 protein is selected from the group consisting of a natural protein or a recombinant protein, said Tim-3 protein comprising a human-derived Tim-3 protein, a murine-derived Tim-3 protein or a mammalian Tim-3 protein other than said human-derived Tim-3 protein and murine-derived Tim-3 protein.
2. The use according to claim 1, wherein the liver injury is selected from acute or chronic liver injury induced by drugs, chemicals, diet, alcohol.
3. The use according to claim 2, wherein the medicament is selected from one or a combination of two or more of acetaminophen, methotrexate, e.g. cyclophosphamide.
4. The use according to claim 2, wherein the chemical is selected from one or a combination of two or more of carbon tetrachloride, concanavalin a, polyinosinic cells.
5. The use according to claim 2, wherein the diet is selected from one or a combination of two or more of a high fat diet, a high fructose diet, a high cholesterol diet.
6. The use according to claim 1, wherein the Tim-3 protein has at least one of the following functions:

(1) Reduces the AST and/or ALT level of the organism,

(2) Inhibiting hepatic cell coagulation necrosis;

(3) Inhibiting the degeneration of liver cell water sample.
7. The use according to claim 1, wherein the Tim-3 protein is selected from the group consisting of human Tim-3 protein and murine Tim-3 protein,

The human Tim-3 protein is selected from any one of (hP 1) - (hP 3):

(hP 1) the protein has an amino acid sequence shown in SEQ ID NO. 1;

(hP 2) the protein has at least 80% identity with the amino acid sequence shown in SEQ ID NO.1 and the activity is the same;

(hP 3) the protein has the amino acid sequence shown in SEQ ID NO.1, is substituted by 1 or a plurality of amino acid residues, is deleted or added, and has the same activity;

the murine Tim-3 protein is selected from any one of (mP 1) - (mP 3):

(mP 1) the protein has the amino acid sequence shown in SEQ ID NO. 2;

(mP 2) the protein has at least 80% identity to the amino acid sequence shown in SEQ ID No.2 and the same activity;

(mP 3) the protein has the same activity and the amino acid sequence shown as SEQ ID NO.2 is replaced by 1 or a plurality of amino acid residues, deleted or added.
8. The use according to claim 1, wherein the Tim-3 protein is a recombinant protein, said recombinant protein being obtained from a biological material prepared by conventional techniques, said biological material comprising at least one of the following:

(a) A nucleic acid molecule encoding the Tim-3 protein;

(b) A recombinant vector comprising the nucleic acid molecule of (a);

(c) A transformed cell comprising (a) the nucleic acid molecule or (b) the recombinant vector;

(d) A recombinant virus comprising the nucleic acid molecule of (a) or the recombinant vector of (b).
9. The use according to claim 8, wherein the Tim-3 protein is selected from the group consisting of human Tim-3 protein and murine Tim-3 protein,

The nucleic acid molecule encoding human Tim-3 protein is selected from any one of (hDNA 1) - (hDNA):

(hDNA 1) a nucleotide sequence shown as SEQ ID NO. 3;

(hDNA 2) the nucleotide sequence shown as SEQ ID NO.3 is substituted, deleted and/or added by 1 or several nucleotides, or the nucleotide sequence of the same active protein is encoded according to degeneracy of genetic code;

the nucleic acid molecule encoding the murine Tim-3 protein is selected from any one of (mDNA 1) - (mDNA 2):

(mDNA 1) a nucleotide sequence shown as SEQ ID NO. 4;

(mDNA 2) the nucleotide sequence shown as SEQ ID No.4 is substituted, deleted and/or added by 1 or several nucleotides or the nucleotide sequence encoding the same active protein according to the degeneracy of the genetic code.
10. The use according to claim 9, wherein the Tim-3 protein is prepared by the following method:

(1) Directionally cloning the gene fragment shown in SEQ ID NO.3 or SEQ ID NO.4 into a pCDNA3.4 plasmid to form a recombinant vector;

(2) Transiently transfecting the recombinant vector into CHO cells to perform protein expression;

(3) Separating and purifying the protein to obtain Tim-3 protein.