JP2024067021A - Prevention and treatment of non-alcoholic fatty liver disease, liver cirrhosis, and liver cancer by controlling oncostatin M receptor signaling - Google Patents

Abstract

[Problem] To provide a novel drug for preventing and treating nonalcoholic steatohepatitis (NASH) that can inhibit the progression of nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and further to cirrhosis or liver cancer. [Solution] An agent for (1) preventing and/or treating nonalcoholic steatohepatitis (NASH), (2) inhibiting the progression of NASH to cirrhosis or liver cancer, or (3) treating cirrhosis or liver cancer, comprising a substance that enhances signaling mediated by oncostatin M (OSM) and its receptor. [Selected Figures] None

Description

The present invention relates to a preventive/therapeutic agent for non-alcoholic steatohepatitis, liver cirrhosis, or liver cancer. More specifically, the present invention relates to an agent for preventing the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis, improving the symptoms of non-alcoholic steatohepatitis, inhibiting the progression of non-alcoholic steatohepatitis to liver cirrhosis or liver cancer, or improving the symptoms of liver cirrhosis or liver cancer, which contains a substance that enhances signaling mediated by oncostatin M and its receptor.

Insulin resistance due to obesity leads to diabetes, hyperlipidemia, and hypertension. The accumulation of obesity, diabetes, hyperlipidemia, and hypertension is called metabolic syndrome, and is a serious risk factor for arteriosclerotic diseases. It is well known that arteriosclerotic diseases often lead to the death of diabetic patients, and about one-eighth of deaths in diabetic patients are due to liver diseases such as cirrhosis and liver cancer. In recent years, nonalcoholic fatty liver disease (NAAFLD), which causes these liver diseases, has been attracting attention. NAFLD develops due to the accumulation of fat in liver cells (a condition called nonalcoholic fatty liver (NAFL)) caused by insulin resistance and hyperlipidemia in metabolic syndrome (Figure 1). The pathology in which inflammation is added to NAFL is called nonalcoholic steatohepatitis (NASH), and if fibrosis progresses further, it progresses to liver cirrhosis and ultimately to liver cancer (Figure 1). NAFLD is a general term combining NAFL and NASH.

The prevalence of NAFLD in adults is high, at approximately 25%, in both developed and developing countries, and it is thought that 6-30% of NAFL patients progress to NASH. In Japan, the number of NAFLD patients is also estimated to be approximately 20 million, and the number of NASH patients is estimated to be 1-2 million, which is very large. Various treatments have been attempted for NASH, including drug therapy, phlebotomy, and liver transplantation, but there is currently no established treatment. In addition, there have been few research reports on the causes of liver cancer based on NAFL/NASH, and the development of an effective treatment is desired.

Oncostatin M (OSM) is a cytokine belonging to the IL-6 family that shares gp130 in its receptor complex. OSM was discovered as a factor that inhibits the proliferation of human tumor cells, but it has become clear that it is a molecule that has a variety of functions in addition to its antitumor effect. The inventors have previously reported that OSM improves fat accumulation in liver cells, inflammation in adipose tissue, and insulin resistance associated with metabolic syndrome through its receptor (Patent Document 1, Non-Patent Documents 1 and 2).

On the other hand, it has been reported that overexpression of OSM in the normal liver of wild-type mice significantly accelerates liver fibrosis, whereas conversely, liver fibrosis is significantly reduced when OSM knockout mice are used as a model for chronic hepatitis. It has also been reported that OSM may play a role in promoting fibrogenesis in chronic liver disease by regulating the response of hepatic stellate cells/myofibroblasts by increasing secretion of collagen I and TIMP-1 (Non-Patent Document 3). It has also been reported that OSM is overexpressed in animal models and patients with progressive NAFLD, and acts as a pro-fibrotic factor by stimulating the migration of myofibroblasts (Non-Patent Document 4).
Thus, it remains completely unknown whether promotion of OSM/OSMR signaling has a preventive or therapeutic effect on the progression of NAFL to NASH or on the development of NASH-based liver cancer.

JP 2013-67593 A

Komori, T. et al., J. Biol. Chem., 289(20): 13821-13837 (2014) Komori, T. et al., Diabetologia, 58: 1868-1876 (2015) Matsuda, M. et al., Hepatology, 67(1): 296-312 (2018) Foglia, B. et al., Cells, 9(28): (2020)

The object of the present invention is to provide a novel drug for preventing and treating NASH that can inhibit the progression of NAFL to NASH and further to liver cirrhosis and liver cancer.

In order to achieve the above-mentioned objective, the present inventors focused on OSM/OSMR signaling in the progression from NAFL to NASH, and further to liver cirrhosis and liver cancer, and investigated the role of OSM/OSMR in NASH and liver cancer based on NASH using NASH model mice and model mice that develop liver cancer through NASH. As a result, in NASH model mice, administration of OSM reduced inflammation of liver cells and improved liver function. Administration of OSM reduced inflammatory (M1 type) liver macrophages and inflammatory cytokine expression, inhibited liver cell death, and induced liver cell regeneration. In addition, administration of OSM increased the expression of endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) genes, which have anti-inflammatory effects. Next, the present inventors administered streptozotocin to NASH model mice to create a liver cancer model based on NASH, and compared the development of liver cancer between OSMRβ gene-deficient mice and wild-type mice. The incidence of liver cancer was higher in OSMRβ gene-deficient mice. When OSM was administered to these liver cancer model mice at the NASH stage, the development of liver cancer was significantly suppressed.
In addition, the inventors confirmed that the number of p21 positive cells, an indicator of senescent hepatocytes, was increased in the STAM model of OSMRβ gene-deficient mice compared to wild-type mice, and that NK cells, which act to remove senescent hepatocytes, were decreased.In addition, the inventors confirmed that intraperitoneal administration of OSM to the STAM model of OSMRβ gene-deficient mice increased the expression of CXCL10 in the liver after administration, and that stimulation of sinusoidal endothelial cells isolated from the liver of STAM model mice at the NASH stage with OSM increased the expression of CXCL10.
Based on these findings, the inventors have demonstrated that enhancing OSM/OSMR signaling can suppress the progression of NAFL to NASH, and further from NASH to cirrhosis or hepatocellular carcinoma. In addition, they have discovered that enhancing OSM/OSMR signaling can increase the expression of CXCL10 in sinusoidal endothelial cells, thereby promoting the accumulation of NK cells in the liver and promoting the removal of senescent hepatocytes by NK cells, thereby making it possible to treat NASH, cirrhosis, or hepatocellular carcinoma, and have thus completed the present invention.

That is, the present invention is as follows.
[1] A preventive and/or therapeutic agent for non-alcoholic steatohepatitis (NASH), comprising a substance that enhances signaling mediated by oncostatin M (OSM) and its receptor.
[2] The agent described in [1], wherein the substance is an agonist for the OSM receptor.
[3] The agent according to [2], wherein the agonist is OSM or a fragment thereof having liver damage ameliorating and/or liver regenerating activity.
[4] OSM is (a) or (b) of the following:
The agent described in [3], which is (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4; (b) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in which one to several amino acids have been substituted, deleted, inserted or added, and which has liver damage improving and/or liver regenerating activity.
[5] The agent according to any one of [1] to [4], which is for inhibiting the progression of NASH to liver cirrhosis or liver cancer.
[6] A method for screening a preventive and/or therapeutic agent for NASH, comprising:
A method comprising: (1) contacting a test substance with a cell expressing the OSM receptor; and (2) selecting a test substance that increases signaling via the OSM receptor compared to the absence of the test substance as a candidate drug for preventing and/or treating NASH.
Alternatively, the present invention is as follows.
[A1]
The present invention comprises a substance that enhances signaling via oncostatin M (OSM) and its receptor.
(1) For the prevention and/or treatment of nonalcoholic steatohepatitis (NASH),
(2) an agent for inhibiting the progression of NASH to cirrhosis or liver cancer, or (3) an agent for treating cirrhosis or liver cancer.
[A2]
The agent according to [A1], wherein the substance is an agonist for the OSM receptor.
[A3]
The agent according to [A2], wherein the agonist is OSM or a fragment thereof having liver damage ameliorating and/or liver regenerating activity.
[A4]
OSM determines that:
The agent described in [A3], which is (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4; (b) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in which one to several amino acids have been substituted, deleted, inserted or added, and which has liver damage improving and/or liver regenerating activity.
[A5]
A method for screening for a preventive and/or therapeutic agent for NASH, liver cirrhosis, or liver cancer, comprising:
A method comprising: (1) contacting a test substance with a cell expressing the OSM receptor; and (2) selecting a test substance that increases signaling via the OSM receptor compared to the absence of the test substance as a candidate drug for preventing and/or treating NASH, cirrhosis or liver cancer.

According to the present invention, by enhancing OSM/OSMR signaling, it is possible to inhibit the progression of NAFL to NASH, and further from NASH to cirrhosis or liver cancer, and by promoting the removal of senescent liver cells in liver cirrhosis or liver cancer, it is possible to improve the symptoms of these diseases. Furthermore, the OSM-OSMR signaling system can be used to discover new drugs for preventing and/or treating NASH, liver cirrhosis, and liver cancer.

FIG. 1 is a schematic diagram showing the progression of pathological conditions from metabolic syndrome through NAFL and NASH to the development of liver cancer, and showing the site of action of the effect of OSM discovered in the present invention. Figure showing the expression of OSM and OSMRβ in the livers of NASH model mice and model mice that develop hepatocellular carcinoma through NASH. As NASH model mice, C57BL/6J mice fed a methionine-choline deficient diet (MCD diet) (A, D; wild-type MCD) and ob/ob mice fed an MCD diet (B, E; ob/obMCD) were used. As a model mouse that develops hepatocellular carcinoma due to NASH, STAM mice (C, F) were used. The expression of OSM (A-C) and OSMRβ (D-F) in the livers of these mice was examined. In each graph, the wild-type mice are C57BL/6J mice fed a normal diet (MF). *p < 0.05 Localization of OSM and OSMRβ expression in the liver of NASH model mice. Expression of OSM and OSMRβ was examined in the liver of C57BL/6J mice fed an MCD diet. (A) Double immunostaining for OSM and F4/80. Arrowheads indicate double-positive cells. Counterstaining was performed with DAPI. Scale bar = 25 μm. (B) Immunostaining for OSMRβ. Counterstaining was performed with methyl green. OSMRβ was expressed in hepatocytes (arrowheads) and inflammatory cells (arrows). Scale bar = 50 μm. (C) Double immunostaining for OSMRβ and F4/80 (upper row) or Lyve-1 (lower row). Arrowheads indicate double-positive cells. Counterstaining was performed with DAPI. Scale bar = 10 μm. Figure 1. Expression of OSMRβ in the liver of STAM mice. Cells expressing OSMRβ were examined in the liver of STAM mice at the NASH stage. Double immunostaining of OSMRβ with albumin (A), F4/80 (B) or Lyve-1 (C) was performed. Counterstaining was performed with DAPI. OSMRβ was observed in hepatocytes (A), macrophages (B) and sinusoidal endothelial cells (C). Arrowheads indicate double-positive cells. Scale bar = 20 μm. Figure 1 shows progression of NASH in OSM-deficient mice. Wild-type (WT) and OSM-deficient (KO) mice were fed an MCD diet for 4 weeks. (A) Hematoxylin-eosin (HE) staining of the liver of an OSM-deficient mouse. Arrowheads indicate inflammatory cell foci. CV: central vein. Scale bar = 100 μm. (B) Quantification of the number of inflammatory cell foci in the liver of an OSM-deficient mouse. (C) Gene expression levels of inflammatory cytokines in the liver of an OSM-deficient mouse. (D) Measurement of blood AST/ALT levels in OSM-deficient mice. *p < 0.05 Mechanism of NASH progression in OSM-deficient mice. Wild-type mice (WT) and OSM-deficient mice (KO) were fed an MCD diet for 4 weeks to generate NASH models. (A) Immunostaining of F4/80 in the livers of wild-type mice (WT) and OSM-deficient mice (KO). Arrowheads indicate hepatic crown-like structures (hCLS). Counterstaining was performed with hematoxylin. CV, central vein. Scale bar = 100 μm. (B) Quantification of F4/80-positive areas in the livers of wild-type mice (WT) and OSM-deficient mice (KO). (C) Expression of macrophage M1 markers (iNOS, CCR2) and M2 markers (Arg1, CD206) in the livers of wild-type mice (WT) and OSM-deficient mice (KO). (D) Number of hCLS in the liver of wild-type mice (WT) and OSM gene-deficient mice (KO). (E) Images of TUNEL staining in the liver of wild-type mice (WT) and OSM gene-deficient mice (KO). Arrowheads indicate TUNEL-positive cells. CV: central vein. Scale bar = 100 μm. (F) Quantification of apoptotic cells by TUNEL staining in the liver of wild-type mice (WT) and OSM gene-deficient mice (KO). (G) Relative expression levels of GPX4 gene in the liver of wild-type mice (WT) and OSM gene-deficient mice (KO). *p < 0.05 Figure 2. Improvement of NASH by administration of OSM. OSM was administered intraperitoneally for one week (twice daily at 12.5 ng/g body weight per administration) to NASH model mice (ob/ob mice fed an MCD diet for 4 weeks). Veh: vehicle administration. (A) HE staining of the liver after OSM administration. Arrowheads indicate inflammatory cell foci. CV: central vein. Scale bar = 100 μm. (B) Quantification of the number of inflammatory cell foci in the liver after OSM administration. (C) Blood AST/ALT concentrations after OSM administration. (D) Immunostaining of F4/80 in the liver after OSM administration. Arrowheads indicate hCLS. Counterstaining was performed with hematoxylin. CV: central vein. Scale bar = 100 μm. (E) Quantification of the F4/80-positive area in the liver after OSM administration. (F) Quantification of the number of hCLS in the liver after OSM administration. (G) Gene expression levels of inflammatory cytokines in the liver after OSM administration. *p < 0.05 Figure 2 shows the mechanism of improvement of NASH by OSM administration. OSM was administered intraperitoneally for 1 week (twice daily at 12.5 ng/g body weight per administration) to NASH model mice (ob/ob mice fed an MCD diet for 4 weeks). Veh: vehicle administration. (A) TUNEL staining of liver after OSM administration. Arrowheads indicate TUNEL-positive cells. CV: central vein. Scale bar = 100 μm. (B) Quantification of apoptotic cells by TUNEL staining in liver after OSM administration. (C) Immunostaining of Ki67-positive cells in liver after OSM administration. Arrowheads indicate Ki67-positive cells. CV: central vein. Scale bar = 100 μm. (D) Quantification of Ki67-positive cells in liver after OSM administration. *p < 0.05 This figure shows changes in sinusoidal endothelial function and cell proliferation-related gene expression in NASH model mice following administration of OSM. OSM (12.5 ng/g body weight) was administered intraperitoneally to NASH model mice (ob/ob mice fed an MCD diet for 4 weeks), and changes in the expression of sinusoidal endothelial function-related genes (eNOS, VEGF) and hepatocyte proliferation-related genes (HGF, MET) in the liver were examined 1 and 2 hours after administration. Increased expression of eNOS and HGF was observed following administration of OSM compared to controls. *p < 0.05 This is a diagram showing the onset of liver cancer in the STAM model of OSMRβ gene-deficient mice. STAM models were created using OSMRβ gene-deficient mice (OSMRβ ^-/- ) and their control wild-type mice (WT), and the onset of liver cancer in the liver was examined. (A) A photograph of a liver resected from a STAM model mouse is shown. Both wild-type and OSMRβ gene-deficient mice developed liver cancer, but more liver cancers were observed in the OSMRβ gene-deficient mice. Scale bar = 1 cm. (B) The results of measuring the number of tumors are shown. * p < 0.05 Figure 2. Suppression of liver cancer development by administration of OSM. Eight-week-old STAM mice in the NASH stage were intraperitoneally administered OSM for one week (twice daily, 12.5 ng/g body weight per administration). Liver cancer development was assessed at 16 weeks of age. (A) Photograph of a liver resected from a STAM model mouse. While numerous liver cancers were observed in wild-type mice, administration of OSM suppressed the development of liver cancer. Scale bar = 1 cm. (B) Measured tumor numbers. * p < 0.05 Figure 12 shows the results of examining factors related to senescent hepatocytes in the STAM model of OSMRβ gene-deficient mice. Figure 12(A) shows that the livers of OSMRβ gene-deficient mice showed an increase in p21-positive cells, an indicator of senescent hepatocytes, compared to wild-type mice. Figure 12(B) shows that the livers of OSMRβ gene-deficient mice showed a decrease in NK cells. Figure 12(C) shows that the expression of CXCL10 was increased in the livers of the STAM model of OSMRβ gene-deficient mice after intraperitoneal administration of OSM. Figure 12(D) shows that the expression of CXCL10 was increased when sinusoidal endothelial cells isolated from the livers of the STAM model of NASH were stimulated with OSM. In the figure, "*" means p<0.05, and the scale bar means 50 μm. FIG. 1 is a schematic diagram showing the mechanism by which OSM suppresses NASH and the development of liver cancer based on NASH, and also treats liver cancer.

The present invention comprises a substance that enhances the signaling of oncostatin M (OSM) and its receptor.
(1) For the prevention and/or treatment of nonalcoholic steatohepatitis (NASH),
(2) to inhibit the progression of NASH to liver cirrhosis or liver cancer, or (3) to treat liver cirrhosis or liver cancer (hereinafter also referred to as "the agent of the present invention").

In this specification, "non-alcoholic steatohepatitis (NASH)" refers to a pathology in which fat accumulates in liver cells due to causes other than alcohol, such as insulin resistance and hyperlipidemia in metabolic syndrome, and which is accompanied by inflammation of liver cells. Non-alcoholic fatty liver (NAFL) and NASH are collectively referred to as NAFLD.

(Oncostatin M (OSM))
Oncostatin M (OSM) is a cytokine belonging to the IL-6 family, and in humans, it is translated into a precursor having an amino acid sequence of 252 amino acids as shown in SEQ ID NO: 2, and then the signal peptide at positions 1-25 and 31 amino acids at the C-terminus are removed to produce a mature form consisting of 196 amino acids at positions 26-221. The amino acid sequence of human mature OSM is shown in SEQ ID NO: 4. In this specification, proteins and peptides are described with the N-terminus (amino terminus) at the left and the C-terminus (carboxyl terminus) at the right, in accordance with the convention of peptide notation.

As used herein, "oncostatin M (OSM)" refers to
(a) the amino acid sequence of human mature OSM as set forth in SEQ ID NO:4;
(b) the amino acid sequence of an orthologue in another warm-blooded animal (e.g., monkey, cow, pig, rat, mouse, rabbit, dog, cat, sheep, chicken, etc.) of human mature OSM consisting of the amino acid sequence represented by SEQ ID NO: 4; or (c) the amino acid sequence of a natural allelic variant or genetic polymorphism of human mature OSM consisting of the amino acid sequence represented by SEQ ID NO: 4 or the orthologue of (b) above.
Preferably, the OSM is human mature OSM consisting of the amino acid sequence set forth in SEQ ID NO: 4, or a naturally occurring allelic variant or polymorphism thereof. Examples of such polymorphisms include, but are not limited to, SNPs registered in dbSNP (such as the SNPs not associated with disease (DISEASE ASSOCIATION: No) listed under "Variants" in UniProtKB P13725 (https://www.uniprot.org/uniprotkb/P13725/entry) in humans and the SNPs not associated with disease (DISEASE ASSOCIATION: No) listed under "Variants" in UniProtKB P53347 (https://www.uniprot.org/uniprotkb/P53347/entry) in mice).

(Oncostatin M Receptor (OSMR))
Oncostatin M receptor (OSMR) is a cell surface receptor that binds to OSM and transmits signals to downstream factors. It is a heterodimer of OSM-specific oncostatin M receptor β (OSMRβ) and gp130, which is common to IL-6 family receptors. OSM can bind to both OSMRβ and gp130 with low affinity, but cannot transmit signals into cells by binding alone. OSM binds with high affinity to the heterodimer of OSMRβ and gp130, activating the Ras-MAPK pathway and STAT3 and STAT5 via the JAK-STAT pathway, thereby transmitting intracellular signals.
OSMRβ is a single-pass transmembrane protein, and in humans, it has an amino acid sequence of 979 amino acids as shown in SEQ ID NO:6, of which positions 1-27 are a signal peptide, positions 28-740 are an extracellular domain, positions 741-761 are a transmembrane domain, and positions 762-979 are an intracellular domain. The nucleotide sequence including the portion encoding the amino acid sequence of human OSMRβ is shown in SEQ ID NO:5.

As used herein, "OSMRβ" refers to a protein that contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 28-979 in the amino acid sequence represented by SEQ ID NO: 6. "An amino acid sequence substantially identical to the amino acid sequence represented by amino acid numbers 28-979 in the amino acid sequence represented by SEQ ID NO: 6" means
(a) the amino acid sequence of an ortholog in another warm-blooded animal (e.g., monkey, cow, pig, rat, mouse, rabbit, dog, cat, sheep, chicken, etc.) of human mature OSMRβ consisting of the amino acid sequence represented by amino acid numbers 28-979 in the amino acid sequence represented by SEQ ID NO: 6; or (b) the amino acid sequence of a natural allelic variant or genetic polymorphism of human mature OSMRβ consisting of the amino acid sequence represented by amino acid numbers 28-979 in the amino acid sequence represented by SEQ ID NO: 6 or the ortholog of (a) above.
Preferably, OSMRβ is human mature OSMRβ consisting of the amino acid sequence represented by amino acid numbers 28-979 in the amino acid sequence represented by SEQ ID NO:6, or a naturally occurring allelic variant or genetic polymorphism thereof. Examples of the genetic polymorphism include, but are not limited to, SNPs registered in dbSNP.

The "substance that enhances signaling mediated by OSM and its receptor" used as an active ingredient of the agent of the present invention is not particularly limited as long as it can activate any of the above pathways through the interaction between OSM and OSMR, but it is preferably a substance that can bind to OSMR and activate signaling mediated by the receptor, i.e., an agonist for OSMR. Examples of agonists for OSMR include OSM itself or an equivalent thereof (hereinafter also referred to as "OSMs"). OSMs are proteins that contain an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO:4.

OSMs may be proteins isolated and purified from OSM-producing cells (e.g., activated macrophages, type I helper T (Th1) cells, etc.) or tissues containing them in humans or other warm-blooded animals (e.g., monkeys, cows, pigs, rats, mice, rabbits, dogs, cats, sheep, chickens, etc.). They may also be proteins chemically synthesized or biochemically synthesized in a cell-free translation system, or recombinant proteins produced from a transformant into which a nucleic acid having a base sequence encoding the above amino acid sequence has been introduced.

Examples of amino acid sequences substantially identical to the amino acid sequence represented by SEQ ID NO: 4 include amino acid sequences having an identity of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more with the amino acid sequence represented by SEQ ID NO: 4. Here, "identity" means the percentage (%) of identical amino acid residues relative to all overlapping amino acid residues in the optimal alignment (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. The identity of amino acid sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

OSMs are proteins that contain an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 and have the same quality of activity as a protein containing the amino acid sequence represented by SEQ ID NO: 4. Here, "activity" means any activity that suppresses the progression of NAFL to NASH and/or NASH to cirrhosis/hepatic cancer, such as, for example, receptor binding activity, activity to shift the phenotype of macrophages to non-inflammatory (M2 type), activity to suppress the expression of inflammatory cytokines (e.g., TNF-α, IL-1β, etc.), activity to suppress hepatocyte death, activity to promote liver regeneration, activity to induce the expression of eNOS and HGF, and anti-inflammatory activity in hepatocytes. Here, "quality of activity" means that the activities are qualitatively the same. Therefore, it is preferable that the activity of OSMs is equal to or greater than that of wild-type OSM, but the degree of these activities may be different.

Alternatively, the OSMs used in the present invention may be, for example, (i) an amino acid sequence in which one or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, and more preferably one to several (5, 4, 3, or 2)) have been deleted from the amino acid sequence represented by SEQ ID NO: 4; (ii) an amino acid sequence in which one or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, and more preferably one to several (5, 4, 3, or 2)) have been added to the amino acid sequence represented by SEQ ID NO: 4; (iii) an amino acid sequence in which one or more amino acids (e.g., about 1 to 30, preferably about 1 to 10, and more preferably one to several (5, 4, 3, or 2)) have been added to the amino acid sequence represented by SEQ ID NO: 4; These include (iv) an amino acid sequence in which one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, and more preferably one to several (5, 4, 3, or 2)) have been inserted into the amino acid sequence shown in SEQ ID NO: 4, or (v) a protein containing an amino acid sequence that is a combination of these.

When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited. For example, a high activity mutant in which part or all of the loop region between helix B and helix C of OSM is deleted (J. Biol. Chem. 287(39): 32848-32859) can be mentioned, but is not limited to this.

OSMs may also be fragments of the protein containing the amino acid sequence shown in SEQ ID NO:4 or a portion of the corresponding amino acid sequence of mature OSM, so long as they have the same activity as the protein containing the amino acid sequence shown in SEQ ID NO:4 (e.g., liver damage improving activity, liver regeneration activity, etc.).

The OSMs used in the present invention may be in the free form or in the form of a salt. Such salts include salts with physiologically acceptable acids (e.g., inorganic acids, organic acids) and bases (e.g., alkali metals, alkaline earth metals), and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

OSMs can be isolated from the extracellular matrix or culture supernatant of OSM-producing cells or tissues of humans or other warm-blooded animals, as described above, using known protein purification methods, such as chromatography, including reverse-phase chromatography, ion-exchange chromatography, and affinity chromatography. OSMs can also be produced according to known peptide synthesis methods. Peptide synthesis methods may be, for example, solid-phase synthesis or liquid-phase synthesis. That is, partial peptides or amino acids that can constitute OSMs can be condensed with the remaining portion, and if the product has a protecting group, the protecting group is removed to produce the OSMs.

In a preferred embodiment, OSMs can be produced by culturing a transformant containing nucleic acid encoding the OSMs, and isolating and purifying the nucleic acid from the resulting culture. Here, the nucleic acid may be either DNA or RNA. In the case of DNA, it is preferably double-stranded DNA. DNA encoding OSMs can be cloned, for example, by synthesizing an oligo DNA primer based on the cDNA sequence information of OSM, and amplifying it by RT-PCR using total RNA or an mRNA fraction prepared from cells that produce OSM as a template. Using the obtained cDNA as a template, various mutations can be introduced using site-directed mutagenesis methods known per se.

Examples of DNA encoding OSMs include DNA containing the nucleotide sequence shown in SEQ ID NO: 3, or DNA containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein having the same activity as the wild-type OSM described above. Examples of DNA that can hybridize under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 3 include DNA containing a nucleotide sequence that has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more with the nucleotide sequence shown in SEQ ID NO: 3.
The identity of the base sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3).
The DNA encoding OSMs is preferably DNA containing a region encoding wild-type human mature OSM having the nucleotide sequence shown in SEQ ID NO: 3, or DNA containing a region encoding various human mature OSM variant proteins. OSMs are produced by introducing the DNA into host cells and expressing and secreting them from the cells, so DNA encoding the human OSM precursor polypeptide consisting of the nucleotide sequence shown in SEQ ID NO: 1 can be used as long as the host cell has the ability to process the signal peptide and C-terminal prosequence.

It is also possible to construct DNA that codes for the entire length of OSMs by chemically synthesizing the DNA strand or by connecting synthesized overlapping oligo DNA strands using PCR or Gibson Assembly. The advantage of constructing the entire length of DNA by chemical synthesis or by combining PCR or Gibson Assembly is that the codons used can be designed over the entire length of the CDS to suit the host into which the DNA is introduced. When expressing heterologous DNA, it is expected that the amount of protein expressed will increase by converting the DNA sequence to codons that are frequently used in the host organism. Data on the codon usage frequency in the host can be obtained from, for example, the genetic code usage frequency database published on the website of the Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/index.html), or references that list the codon usage frequency in each host can be used.

Depending on the purpose, the cloned DNA can be used as is, or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have an ATG translation initiation codon at the 5' end, and a TAA, TGA, or TAG translation termination codon at the 3' end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.

An expression vector containing DNA encoding OSMs can be produced, for example, by excising a desired DNA fragment from the DNA encoding OSMs and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. Examples of expression vectors that can be used include plasmids derived from E. coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), yeast-derived plasmids (e.g., pSH19, pSH15), insect cell expression plasmids (e.g., pFast-Bac), animal cell expression plasmids (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo), bacteriophages such as λ phage, insect virus vectors such as baculovirus (e.g., BmNPV, AcNPV), and animal virus vectors such as retrovirus, lentivirus, vaccinia virus, adenovirus, adeno-associated virus, and herpes virus.
The promoter may be any promoter suitable for the host used to express the gene. For example, when the host is an animal cell, the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. may be used. For other hosts, a promoter known per se may be appropriately selected.

In addition to the above, expression vectors that contain enhancers, splicing signals, poly A addition signals, selection markers, SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori), etc., can be used as desired. Examples of selection markers include the dihydrofolate reductase gene, ampicillin resistance gene, and neomycin resistance gene.

OSMs can be produced by transforming a host with an expression vector containing DNA encoding the above-mentioned OSMs and culturing the resulting transformant. Examples of hosts that can be used include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, and animal cells. Examples of mammalian cells that can be used include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO(dhfr ^- ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, HeLa cells, HepG2 cells, and HEK293 cells. For other hosts, cells known per se can be appropriately selected.

Transformation can be carried out according to known methods depending on the type of host. Animal cells can be transformed, for example, according to the methods described in Cell Engineering Special Issue 8, New Cell Engineering Experimental Protocols, pp. 263-267 (1995) (published by Shujunsha) and Virology, vol. 52, pp. 456 (1973).

The transformant can be cultured according to a known method depending on the type of host.
For example, when a transformant whose host is an animal cell is cultured, a medium such as Minimum Essential Medium (MEM) containing about 5 to about 20% fetal bovine serum, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 medium, 199 medium, or Ham's F-12 medium can be used. The pH of the medium is preferably about 6 to about 8. The culture is usually carried out at about 30°C to about 40°C for about 15 to about 60 hours. Aeration or stirring may be performed as necessary.
In this manner, OSMs can be produced intracellularly or extracellularly in transformants.

OSMs can be separated and purified from the culture obtained by culturing the transformant by methods known per se. Such methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that utilize mainly differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. These methods can also be combined as appropriate.

If the OSMs thus obtained are in free form, the free form can be converted into a salt by a method known per se or a method similar thereto, and if the OSMs are obtained as a salt, the salt can be converted into the free form or another salt by a method known per se or a method similar thereto.

In another embodiment of the present invention, the agonist for OSMR may be a substance other than OSMs that can bind to OSMR and activate signaling via the receptor. The substance may be a low molecular weight compound, or a medium or high molecular weight compound such as a nucleic acid (e.g., an aptamer), a peptide, a protein, or an agonist antibody. These agonists for OSMR may be identified using the "screening method of the present invention" described below.

In a preferred embodiment, the agonist for OSMR may be an agonist antibody for the receptor. The agonist antibody may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. These antibodies can be produced according to publicly known methods for producing antibodies or antisera. The antibody isotype is not particularly limited, but is preferably IgG, IgM or IgA, and particularly preferably IgG. The Fc regions of IgG1 and IgG2 can have effector functions such as ADCC, ADCP, and CDC, so it may be preferable to use IgG4, which has low effector functions, but this is not limited thereto.

The agonist antibody against OSMR may bind to OSMR in any manner as long as it can activate or enhance signaling via the receptor (e.g., Ras-MAPK pathway, JAK-STAT pathway). For example, it may bind to either OSMRβ or gp130 and promote dimerization with the other subunit, or it may specifically bind to the site where both subunits of the OSMRβ/gp130 complex associate. Furthermore, it may be a bispecific antibody containing an antibody portion having a specific affinity for each subunit. The agonist antibody against OSMR may be a complete antibody molecule, or a fragment such as Fab, Fab', F(ab') ₂ , a genetically engineered conjugate molecule such as scFv, scFv-Fc, minibody, diabody, or a derivative thereof modified with a molecule having a protein stabilizing effect such as polyethylene glycol (PEG), as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen.

In a preferred embodiment, since the agonist antibody against OSMR is used as a pharmaceutical intended for administration to humans, the antibody (preferably a monoclonal antibody) is an antibody with a reduced risk of exhibiting antigenicity when administered to humans, specifically a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., and is particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering in accordance with standard methods. Although fully human antibodies can also be produced from human-human (or mouse) hybridomas, in order to provide large amounts of antibodies stably and at low cost, it is desirable to produce them using human antibody-producing mice or phage display methods.

In another embodiment, the agonist for OSMR may be, for example, a substance (e.g., a small molecule compound) identified by the screening method of the present invention described below.

In one embodiment of the present invention, a "substance that enhances signaling mediated by OSM and its receptor" may be a substance that is capable of inducing or enhancing expression of the OSM gene present in a warm-blooded animal to which the agent of the present invention is administered. Examples of such substances include epidermal growth factor (EGF)/interleukin (IL)-2/IL-3/erythropoietin (EPO)/granulocyte-colony stimulating factor (G-CSF) (EMBO J 15: 1055-1063, 1996), granulocyte-macrophage-colony stimulating factor (GM-CSF) (Am J Physiol Cell Physiol 301: C947-C953, 2011), prostaglandin _E2 ( _PGE2 ) (J Neurosci 22: 5334-5343, 2002), human gonadotropin (hCG)/β-hCG (Cytobios 92: 159-163, 1997), and cisplatin (Immunol Cell Biol 75:492- 496, 1997), phorbol 12-myristate 13-acetate (PMA) (Proc Natl Acad Sci USA 83:9739-9743, 1986), and the like.

(Medicines containing substances that enhance the signaling of OSM and its receptors)
Any of the above substances that enhance the signaling of OSM and its receptor enhances the signaling of OSM/OSMR and exhibits the activity of shifting the phenotype of liver macrophages to non-inflammatory (M2 type), the activity of suppressing the expression of inflammatory cytokines (e.g., TNF-α, IL-1β, etc.), the activity of suppressing liver damage (cell death), the activity of promoting liver regeneration, the activity of inducing the expression of eNOS and HGF, and the anti-inflammatory activity in liver cells, thereby suppressing the progression of NAFL to NASH and/or NASH to liver cirrhosis and liver cancer. Therefore, these substances can be used as a preventive and/or therapeutic drug for NASH. Furthermore, substances that enhance the signaling of OSM and its receptor enhance the expression of CXCL10 gene in sinusoidal endothelial cells and promote the accumulation of NK cells in the vicinity of sinusoidal endothelial cells, thereby promoting the removal of senescent liver cells in the vicinity of sinusoidal endothelial cells (i.e., the liver). Therefore, these substances can also be used as a therapeutic drug for NASH, liver cirrhosis, or liver cancer. Here, "prevention" includes not only preventing the onset of NASH (progression from NAFL to NASH) but also delaying its onset. "Treatment" includes not only improving the symptoms of NASH but also inhibiting the progression of NASH to liver cirrhosis and even liver cancer, and preventing recurrence.

The above-mentioned medicines containing substances that enhance signaling of OSM and its receptor can be administered orally or parenterally (e.g., intravascular administration, subcutaneous administration, etc.) to humans or other warm-blooded animals (e.g., monkeys, cows, pigs, rats, mice, rabbits, dogs, cats, sheep, chickens, etc.), preferably humans, as liquid preparations or as pharmaceutical compositions in an appropriate dosage form.

The above-mentioned substance that enhances the signaling of OSM and its receptor may be administered on its own or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the above-mentioned substance that enhances the signaling of OSM and its receptor and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

For compositions for parenteral administration, for example, injections, suppositories, intranasal preparations, etc. are used, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying the above-mentioned substance that enhances the signaling of OSM and its receptor in a sterile aqueous or oily liquid that is usually used for injections. As aqueous solutions for injection, for example, saline, isotonic solutions containing glucose and other adjuvants, etc. are used, and may be used in combination with appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As oily solutions, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with benzyl benzoate, benzyl alcohol, etc. as solubilizing agents. The prepared injection solution is preferably filled into a suitable ampoule. Suppositories for rectal administration may be prepared by mixing the above-mentioned substance that enhances the signaling of OSM and its receptor with a conventional suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

The above-mentioned parenteral or oral pharmaceutical compositions are conveniently prepared in a dosage unit form that matches the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. The substance that enhances signaling of OSM and its receptor is preferably contained in an amount of usually 0.1 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

The dosage of the above-mentioned medicine containing the substance that enhances the signaling of OSM and its receptor varies depending on the subject, symptoms, route of administration, etc., but for example, a single dose of the substance that enhances the signaling of OSM and its receptor is usually about 0.0001 to 20 mg/kg body weight, and it is convenient to administer a low molecular weight compound orally or parenterally about 1 to 5 times a day, or an antibody by intravenous injection once a day to once every few months. Similar amounts can be administered in other parenteral and oral administrations. If the symptoms are particularly severe, the dosage may be increased according to the symptoms.

The compositions described above may contain other active ingredients as long as no undesirable interactions occur when combined with the OSM and the substance that enhances the signaling of its receptor. For example, other active ingredients include, but are not limited to, drugs for treating underlying diseases (e.g., diabetes, dyslipidemia, hypertension, etc.) such as bioglitazone, statins, ezetimibe, and angiotensin II receptor antagonists, vitamin E, etc.

(Method of screening for preventive and/or therapeutic agent for NASH, liver cirrhosis, or liver cancer)
The present invention also provides a method for screening for a preventive and/or therapeutic agent for NASH, liver cirrhosis, or liver cancer, comprising:
The present invention provides a method (hereinafter also referred to as the "screening method of the present invention") comprising the steps of: (1) contacting a test substance with a cell expressing OSMR; and (2) selecting a test substance that increases OSMR-mediated signaling compared to the absence of the test substance as a candidate drug for preventing and/or treating NASH, cirrhosis, or liver cancer (hereinafter also referred to as "NASH, etc.").
Here, since signal transduction from OSM does not occur when OSMRβ or gp130 is used alone, a complex of OSMβ and gp130 is used as OSMR. Examples of cells that express OSMR include macrophages and Th1 cells.

For example, when macrophages expressing OSMR are used, the cells are incubated in a medium suitable for culturing the cells in the presence and absence of a test substance, and then, for example, activity to shift the phenotype of macrophages to non-inflammatory (M2 type), activity to suppress the expression of inflammatory cytokines (e.g., TNF-α, IL-1β, etc.), activity to suppress liver damage (cell death), activity to promote liver regeneration, activity to induce the expression of eNOS or HGF, anti-inflammatory activity in liver cells, etc. are used as indicators. If any of the above activities is significantly increased in the presence of the test substance, the test substance can be selected as a candidate for a preventive and/or therapeutic drug for NASH, etc. Alternatively, using cultured cells, a construct in which each subunit of a split enzyme is fused to each of the intracellular signaling domain of OSMRβ and the intracellular signaling domain of gp130 is introduced and expressed on the cell membrane, and a substrate for the enzyme is added to the medium to visualize the enzyme reaction. If the enzyme activity is significantly increased in the presence of the test substance, the test substance can be selected as a candidate for a preventive and/or therapeutic drug for NASH, etc. Alternatively, when a test substance is contacted with sinusoidal endothelial cells obtained from a subject with NASH, liver cirrhosis, or liver cancer, if expression of the CXCL10 gene in the sinusoidal endothelial cells is significantly increased in the presence of the test substance, the test substance can be selected as a candidate for a preventive and/or therapeutic drug for NASH, etc.

The preventive and/or therapeutic agents for NASH, etc. identified by the screening method of the present invention can be formulated in the same manner as pharmaceuticals containing substances that enhance the signaling of OSM and its receptor described above, and can be administered to the warm-blooded animal that is the recipient of the treatment.
The present invention will be described in more detail below by way of examples, but these are merely illustrative and do not limit the scope of the present invention in any way.

(Method)
Experimental animals
C57BL/6J mice used in this example were purchased from Japan SLC (Shizuoka, Japan). ob/ob mice, OSM gene-deficient mice, and OSMRβ gene-deficient mice were bred in-house. OSM gene-deficient mice (Minehata K et al., Int J Hematol 84: 319-327, 2006) and OSMRβ gene-deficient mice (Tanaka M et al., Blood 102: 3154-3162, 2003) were strains previously developed. All mice were kept in a room with a 12-hour light-dark cycle (light period: 8:00 a.m. to 8:00 p.m.), temperature of 22.0-25.0°C, humidity of 50-60%, and free access to food (MF; Oriental Yeast, Tokyo, Japan) and drinking water. All experiments were reviewed and approved by the Animal Experimentation Committee of Wakayama Medical University, and were performed in compliance with the guidelines for animal experiments of Wakayama Medical University. We also made efforts to reduce the burden on animals, in accordance with the Ministry of the Environment's standards for the care and storage of laboratory animals and the reduction of pain.

Generation of mouse models of NAFL, NASH, and NASH-mediated hepatocellular carcinoma
NAFL model mice were prepared from 12-week-old male ob/ob mice that had been fed MF as a normal diet. NASH model mice were prepared by feeding 8-week-old male ob/ob mice a methionine-choline deficient (MCD) diet (A02082002B; Research Diets, New Jersey, USA) for 4 weeks. NASH-mediated hepatocellular carcinoma model mice were prepared as previously reported (STAM mice; Fujii et al., Med Mol Morphol 46: 141-152, 2013). Specifically, 2-day-old male C57BL6/J mice were subcutaneously administered streptozotocin (200 μg; Wako Pure Chemical Industries, Osaka, Japan) and fed a high-fat diet (HFD32; CLEA Japan, Tokyo, Japan) from 4 weeks of age. Subsequently, experiments were conducted using STAM mice aged 6 weeks (NAFL stage), 8 weeks (NASH stage), 12 weeks (cirrhosis stage), and 16 weeks (liver cancer stage).

Establishment of a NASH model in OSM-deficient mice
Eight-week-old male OSM-deficient mice and their wild-type littermates were fed an MCD diet for four weeks.

Establishment of STAM model in OSMRβ gene-deficient mice
Two-day-old male OSMRβ knockout mice and their wild-type littermate mice were subcutaneously administered streptozotocin (200 μg; Wako Pure Chemical Industries, Ltd.) and fed a high-fat diet (HFD32) from 4 weeks of age. Hepatocellular carcinoma was then evaluated at 16 weeks of age.

Administration of OSM to NASH model mice and STAM mice Recombinant mouse OSM (R&D Systems, Minnesota, USA) was administered intraperitoneally at a dose of 12.5 ng/g body weight to the NASH model mice and 8-week-old STAM mice. The above doses were administered twice daily for one week to investigate the effect of OSM on the treatment of NASH and the development of hepatocellular carcinoma. To investigate the direct effect of OSM on the liver, experiments were performed 1 and 2 hours after administration of the above doses of OSM.

Blood collection and measurement of serum AST and ALT Blood was collected from the orbital venous plexus of the mice, and serum AST and ALT were measured at Nagahama Life Science Laboratory (Shiga, Japan) after separation.

Organ collection and tissue section preparation Mice were anesthetized with isoflurane, and blood was removed by transcardial injection of saline, followed by perfusion fixation with transcardial injection of 4% paraformaldehyde. Livers were collected and immersion-fixed in 4% paraformaldehyde for 3 hours at 4°C. After immersion fixation, the livers were dehydrated in 30% sucrose solution for TUNEL staining and immunostaining. They were then embedded in OCT compound (Sakura Finetech, Tokyo, Japan), solidified by immersion in n-hexane containing dry ice, and stored at −80°C. Liver sections 6 μm thick were prepared using a cryostat. For HE staining, paraffin blocks were prepared after immersion fixation, and liver sections 4 μm thick were prepared using a microtome.

TUNEL staining
TUNEL staining was performed using the TdT In Situ Apoptosis Detection Kit (R&D Systems). The sections were incubated with the labeling reaction mix (a mixture of biotinylated TdT dNTPMix, TdT enzyme, and manganese) at 37°C for 60 minutes, and then incubated with HRP-labeled streptavidin at room temperature for 1 hour. The sections were then stained with diaminobenzidine (DAB) and counterstained with methyl green.

Immunostaining Immunostaining was performed as previously described (Komori et al., J Biol Chem, 28.8: 21861-21875, 2013). Sections were incubated with 5% normal donkey serum (Jackson ImmunoResearch, PA, USA) for 1 h at room temperature. Sections were then incubated overnight at 4°C with anti-OSM (1:100; R&D Systems), anti-OSMRβ (1:200; R&D Systems), anti-albumin (1:100; Nordic Immunological Laboratories, Tilburg, The Netherlands), anti-Lyve-1 (1:100; Abcam, Cambridge, UK), anti-F4/80 (1:50; Serotec, Oxford, UK), anti-Ki67 (1:500; Novocastra Laboratories, Newcastle, UK), and anti-p21 (1:100; Abcam). For the fluorescent staining method, the sections were incubated with secondary antibodies (Cy3-labeled anti-goat IgG, Cy2-labeled anti-rabbit IgG, and Cy2-labeled anti-rat IgG; Jackson ImmunoResearch) for 1 h at room temperature and counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). For the enzyme immunoassay, the sections were incubated with secondary antibodies (NICHIREI-Histofine simple-stain MAX-PO, Nichirei, Tokyo, Japan) for 1 h at room temperature and stained with DAB, followed by counterstaining with methyl green. Stained images were photographed using an epifluorescence microscope (BX5O; Olympus) equipped with a digital CCD camera (DP71; Olympus, Tokyo, Japan).
For NK1.1 staining, a direct method was used: sections were incubated with anti-CD16/CD32 antibody (1:100; BD Biosciences, San Jose, USA) for 1 h at room temperature, and then incubated with APC-labeled anti-NK1.1 antibody (1:100; Thermo Fisher Scientific, Waltham, USA) overnight at 4°C. Sections were then counterstained with DAPI and photographed using a confocal microscope (LSM900; Carl Zeiss, Oberkochen, Germany).

RNA extraction, real-time PCR
RNA extraction and real-time PCR were performed as previously reported (Komori et al., J Biol Chem, 288: 21861-21875, 2013). RNA was extracted from the liver using TRI reagent (Molecular Research Center, Ohio, USA). The concentration of the extracted RNA was measured, and cDNA was produced using the High Capacity cDNA Reverse Transcription Kit from Applied Biosystems (California, USA). Real-time PCR was performed using the TaqMan method, with the Rotor-Gene Probe PCR Kit (Qiagen) as the master mix and the Rotor gene Q (Qiagen) as the real-time PCR device. The TaqMan probe was TaqMan Gene Expression Assays (Applied Biosystems) for OSM (Mm01193966_ml), OSMRβ (Mm00495424_ml), TNF-α (Mm00443258_ml), IL-1β (Mm00434228_m1), IL-10 (Mm00439616_ml), iNOS (Mm00440502_m1), and CCR2 (Mm00 438270_m1), Arg1 (Mm00475988_m1), CD206 (Mm01329362_m1), eNOS (Mm00435217_ml), VEGF (Mm00437306_ml), HGF (Mm01135184_ml), MET (Mm01156972_ml), GPX4 (Mm00515041_ml), CXCL10 (Mm00445235_m1), and 18S (Hs99999901_sl) were used. Ct values were calculated from the obtained data, and relative quantitative analysis was performed using the ΔΔCt method.

An experimental system for OSM stimulation of sinusoidal endothelial cells prepared from the liver of a STAM model of NASH
Eight-week-old STAM mice were anesthetized with isoflurane and their livers were harvested. Livers were isolated using collagenase IV (Sigma, St. Louis, USA), and sinusoidal endothelial cells were isolated using an autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) with magnetic bead-conjugated anti-CD146 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated cells were cultured and stimulated with OSM (50 ng/ml; R&D Systems) for 1 and 2 h before experiments.

Statistical analysis Data are expressed as mean ± standard error, and statistical analysis was performed by Student's t-test. Differences of p < 0.05 were considered statistically significant.

(result)
To investigate the role of OSM in the development of NASH and hepatocellular carcinoma, we used C57BL/6J mice fed an MCD diet and ob/ob mice fed an MCD diet as NASH model mice, and STAM mice as a model mouse that develops hepatocellular carcinoma due to NASH, and examined the expression of OSM and OSMRβ in the livers of these mice. OSM expression was significantly increased in the livers of both model mice compared to the livers of wild-type mice (C57BL/6J mice fed an MF diet) (Fig. 2A-C). OSM was also increased in the livers of ob/ob mice fed an MCD diet and STAM mice compared to the livers of NAFL (ob/ob mice fed an MF diet) (Fig. 2B, C). Furthermore, OSMRβ expression was significantly higher in the livers of NASH mice compared to the livers of wild-type mice (Fig. 2D-F). Furthermore, in STAM mice, OSM and OSMRβ were significantly higher in the livers of hepatocellular carcinoma (Fig. 2C, F). These results suggest that OSM may be involved in the development of NASH and hepatocellular carcinoma.

When we examined OSM-expressing cells in the livers of NASH model mice (C57BL/6J mice fed an MCD diet), we found that OSM was expressed in F4/80-positive macrophages (Fig. 3A). Next, to investigate the site of action of OSM in the livers of NASH patients, we examined OSMRβ-expressing cells in the livers of NASH model mice (C57BL/6J mice fed an MCD diet) and STAM mice during the NASH stage. In both model mice, OSMRβ was expressed in hepatocytes (Fig. 3B, Fig. 4A), macrophages (Fig. 3C, Fig. 4B), and sinusoidal endothelial cells (Fig. 3C, Fig. 4C). These results suggest that OSM may act on hepatocytes, sinusoidal endothelial cells, and macrophages in the livers of NASH patients.

To investigate the role of OSM in the development of NASH, OSM-deficient mice were fed an MCD diet and the severity of NASH was assessed. Compared to wild-type mice, the livers of OSM-deficient mice showed a significant increase in inflammatory cell foci (Fig. 5A, B) and a significant increase in gene expression of the inflammatory cytokines TNF-α and IL-1β (Fig. 5C). Gene expression of the anti-inflammatory cytokine IL-10 was significantly decreased in the livers of OSM-deficient mice (Fig. 5C). In addition, blood AST and ALT concentrations were measured to examine the severity of liver damage, and a significant increase was observed in the OSM-deficient mice (Fig. 5D). These results suggest that OSM deficiency may exacerbate NASH induced by the MCD diet.

To investigate the cause of NASH exacerbation in OSM-deficient mice, we investigated the number and phenotype of hepatic macrophages, which are important for the development of NASH. Macrophages are mainly classified into inflammatory M1 type and anti-inflammatory M2 type. In the livers of OSM-deficient mice, there was a significant increase in macrophages (Fig. 6A, B), a significant increase in gene expression of M1 type markers iNOS and CCR2, and a significant decrease in gene expression of M2 type markers Arginase-1 and CD206 (Fig. 6C), suggesting that there is a significant increase in inflammatory (M1 type) macrophages in the livers of OSM-deficient mice. In addition, there was a significant increase in hepatic crown-like structures (hCLS; structures in which macrophages surround and phagocytose hepatocytes undergoing cell death) in the livers of OSM-deficient mice (Fig. 6D). We then investigated cell death in the livers of OSM-deficient mice, and found a significant increase in apoptotic images by TUNEL staining (Fig. 6E, F). Furthermore, gene expression of glutathione peroxidase 4 (GPX4), an inhibitor of ferroptosis, an iron-dependent cell death, was significantly decreased (Fig. 6G). These results suggest that OSM deficiency may promote hepatocyte death and increase inflammatory macrophages, exacerbating NASH.

The above results suggest that OSM may suppress NASH. Therefore, to examine the therapeutic effect of OSM against NASH, OSM was administered intraperitoneally for one week to NASH model mice (ob/ob mice fed an MCD diet) and the severity of NASH was examined. In the control group, numerous inflammatory cell foci were observed in the liver, but these were significantly improved by OSM administration (Fig. 7A, B). Furthermore, a significant decrease in blood AST and ALT concentrations was observed in the OSM-administered group compared to the control group (Fig. 7C). These results suggest that OSM may have a therapeutic effect against NASH.

The mechanism by which OSM improves NASH was investigated. Administration of OSM significantly reduced hepatic macrophages (Fig. 7D, E) and gene expression of TNF-α and IL-1β (Fig. 7G). Furthermore, there was a significant reduction in hCLS (Fig. 7F) and apoptosis (Fig. 8A, B). Furthermore, there was a significant increase in Ki67-positive hepatocytes, a marker of cell proliferation (Fig. 8C, D). These results suggest that OSM may improve NASH by suppressing hepatocyte death and inducing hepatocyte regeneration.

To investigate the effect of OSM in the liver of NASH patients, OSM was administered intraperitoneally to NASH model mice (ob/ob mice fed an MCD diet) and genes induced by OSM were examined. One and two hours after OSM administration, the expression of eNOS, a gene associated with sinusoidal endothelial function that acts as an anti-inflammatory agent, and HGF, a gene associated with hepatocyte proliferation, were significantly increased (Figure 9).

To investigate the effect of OSM on the development of NASH-based liver cancer, we created STAM models in wild-type and OSMRβ gene-deficient mice and compared the development of liver cancer in the liver. Both wild-type and OSMRβ gene-deficient mice developed liver cancer, but the OSMRβ gene-deficient mice developed significantly more liver cancer (Fig. 10A, B). These results suggest that deficiency of OSM signaling may promote the development of NASH-based liver cancer.

To investigate the inhibitory effect of OSM on the development of liver cancer, we administered OSM intraperitoneally for one week to 8-week-old STAM mice in the NASH stage and examined the development of liver cancer. While the development of numerous liver cancers was observed in the control group, administration of OSM significantly suppressed the development of liver cancer (Fig. 11A, B). These results suggest that OSM may suppress the development of liver cancer caused by NASH.

Furthermore, we created a STAM model using OSMRβ gene-deficient mice (OSMRβ-/-) and their control wild-type mice (WT) to examine factors related to senescent cells in the liver. In the livers of OSMRβ gene-deficient mice, an increase in p21-positive cells, an indicator of senescent hepatocytes, was observed compared to wild-type mice (Fig. 12(A)). In addition, the number of NK cells (NK1.1-positive cells), which work to remove senescent hepatocytes, was decreased (Fig. 12(B)). Furthermore, when we examined the expression of CXCL10, a type of chemokine that induces NK cells, we found that the expression of CXCL10 in the liver was increased after intraperitoneal administration of OSM (Fig. 12(C)). Furthermore, when sinusoidal endothelial cells were isolated from the liver of the STAM model in the NASH stage and stimulated with OSM, an increase in the expression of CXCL10 was observed (Fig. 12(D)). In Fig. 12, "*" means p<0.05, and the scale bar is 50 μm.

The results shown in FIG. 12 reveal that administration of OSM leads to increased expression of the CXCL10 gene in sinusoidal endothelial cells. As described above, without wishing to be bound by theory, CXCL10 is a type of chemokine that induces NK cells (NK1.1-positive cells) that act to remove senescent hepatocytes. Therefore, it is expected that NK cells will accumulate near sinusoidal endothelial cells (in other words, in the liver) after administration of OSM. The accumulated NK cells will promote the removal of senescent hepatocytes in NASH, cirrhosis, or liver cancer, and as a result, it is expected that the condition of NASH, cirrhosis, or liver cancer will be improved. Therefore, in this aspect, the present invention can be considered as a therapeutic or preventive agent for NASH, cirrhosis, or liver cancer.

These results suggest that during the progression of NAFL to NASH, OSM produced by macrophages suppresses liver damage and inflammation via hepatocytes, macrophages, and sinusoidal endothelial cells, and further indicates that OSM enhances the expression of CXCL10 in sinusoidal endothelial cells, which in turn promotes the removal of senescent hepatocytes in liver tissue by NK cells (Figure 13). Through these actions, OSM is thought to have the effects of suppressing the progression of NAFL to NASH, improving NASH, and suppressing the onset of liver cirrhosis and liver cancer, as well as improving liver cirrhosis and liver cancer (Figure 13).

The agent of the present invention is extremely useful in that it enhances OSM/OSMR signaling, thereby making it possible to inhibit the progression of NAFL to NASH, and further from NASH to cirrhosis or liver cancer, and further making it possible to treat liver cirrhosis and liver cancer. Furthermore, the screening method of the present invention can use the OSM/OSMR signaling system to search for new preventive and/or therapeutic drugs for NASH, liver cirrhosis, or liver cancer, and is therefore useful as a drug discovery tool for developing curative drugs for NASH, liver cirrhosis, and liver cancer.

Claims (5)

The present invention comprises a substance that enhances signaling via oncostatin M (OSM) and its receptor.
(1) For the prevention and/or treatment of nonalcoholic steatohepatitis (NASH),
(2) an agent for inhibiting the progression of NASH to cirrhosis or liver cancer, or (3) an agent for treating cirrhosis or liver cancer. The agent according to claim 1, wherein the substance is an agonist for the OSM receptor. The agent according to claim 2, wherein the agonist is OSM or a fragment thereof having liver damage improving and/or liver regenerating activity. OSM determines that:
The agent described in claim 3, which is a polypeptide consisting of (a) the amino acid sequence represented by SEQ ID NO: 4; (b) the amino acid sequence represented by SEQ ID NO: 4 in which one to several amino acids have been substituted, deleted, inserted or added, and which has liver damage improving and/or liver regenerating activity. A method for screening for a preventive and/or therapeutic agent for NASH, liver cirrhosis, or liver cancer, comprising:
A method comprising: (1) contacting a test substance with a cell expressing the OSM receptor; and (2) selecting a test substance that increases signaling via the OSM receptor compared to the absence of the test substance as a candidate drug for preventing and/or treating NASH, cirrhosis or liver cancer.