JP2005176804A - Method for screening hepatic disease-treating agent by using performance of activating stat3 as index - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening a treating agent of hepatic diseases mediated by Fas. <P>SOLUTION: This method for identifying a medicine for the treatment of the hepatic diseases induced by a Fas-agonist comprises (a) a process of culturing cells capable of expressing a Stat3 gene in the presence of a candidate medicine under a condition in which the cells can express the Stat3 in the absence of the candidate medicine, and (b) a process of measuring the potency of the activation of the Stat3 in the cells obtained in the process (a), and in the case that the activation of the Stat3 is potentiated by the candidate medicine, the candidate medicine is identified as the medicine for the treatment of the hepatic diseases induced by the Fas-agonist. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Background of the Invention

The present invention relates to a method for screening a liver disease therapeutic agent using STAT3 activation ability as an index, and a pharmaceutical composition for treating liver disease containing a STAT3 activator.

Background Art Transcriptional signaling / activator-3 (Stat3) is expressed in various places and is temporarily activated by a wide variety of ligands such as IL-6, leukemia inhibitory factor, EGF, and PDGF. It is also transiently activated by several cancer receptor-type tyrosine kinases and non-receptor-type (Src-like) tyrosine kinases (Darnell, JE, Jr. 1997. STATs and gene regulation. Science. 277: 1630- 1635; Schuringa, JJ, Jonk, LJ, Dokter, WH, Vellenga, E., and Kruijer, W. 2000. Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1 / MKK -4 as signal trans-duction components. Biochem. J. 347: 89-96; Leu, JI, Crissey, MA, Leu, JP, Ciliberto, G., and Taub, R. 2001. Interleukin-6-induced STAT3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury Mol. Cell. Biol. 21: 414-424; Schuringa, JJ, van der Schaaf, S., Vellenga, E., Eggen, BJ, and Kruijer, W. 2002. LIF-induced STAT3 signaling in murine versus human embryonal carcinoma (EC) cells. Exp. Cell Res. 274: 119-129; Levy, DE, and Lee, CK 2002. What does Stat3 do? J. Clin. Invest. 109: 1143-1148. doi: 10.1172 / JCI200215650) . Stat3 is a molecule with DNA binding activity that is found in IL-6 stimulated hepatocytes and can selectively interact with enhancer elements in promoters of acute phase genes known as acute phase response elements. First reported (Zhong, Z., Wen, Z., and Darnell, JE 1994.Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science. 264: 95 -98; Lutticken, C., et al. 1994. Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science. 263: 89-92). Stat3 is activated by all IL-6 type cytokines that signal through gp130 and its related receptors, as well as several growth factors, oncoproteins, and interferons (Hirano, T ., Ishihara, K., and Hibi, M. 2000. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene. 19: 2548-2556; Bowman, T. , Garcia, R., Turkson, J., and Jove, R. 2000. STATs in oncogenesis. Oncogene. 19: 2474-2488). Much research has been done on the physiological functions of Stat3, and Stat3 is known to play an important role in the development and cell proliferation of various organs (Levy, DE, and L
ee, CK 2002. What does Stat3 do? J. Clin. Invest. 109: 1433-1148. doi: 10.1172 / JCI200215650). If the Stat3 gene is targeted for gene disruption, early embryo death occurs (Takeda, K., et al. 1997. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc. Natl. Acad. Sci. USA 94: 3801-3804), studies using mice with conditional Stat3 knockout (liver, lymphocyte, etc.) have been conducted (Takeda, K., et al. 1997. Targeted disruption of the mouse Stat3 gene leads to Proc. Natl. Acad. Sci. USA 94: 3801-3804; Li, W., Liang, X., Kellendonk, C., Poli, V., and Taub, R. 2002. STAT3 contributes to the mitogenic response of hepatocytes during liver regenera-tion. J. Biol. Chem. 277: 28411-28417). In liver-specific Stat3 knockout mice (LS3-KO mice), no abnormalities in liver development and structure were observed, but the mitogenic response after partial hepatectomy was reduced (Li, W., Liang, X., Kellendonk, C., Poli, V., and Taub, R. 2002. STAT3 contributes to the mitogenic response of hepatocytes during liver regenera-tion. J. Biol. Chem. 277: 28411-28417). In addition, many other studies have been reported showing that Stat3 is involved in several important early response genes immediately following partial hepatectomy (Leu, JI,
Crissey, MA, Leu, JP, Ciliberto, G., and Taub, R. 2001. Interleukin-6-induced STAT3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury. Cell. Biol. 21: 414-424; Debonera, F., et al. 2001. Activation of interleukin-6 / STAT3 and liver regeneration following transplantation. J. Surg. Res. 96: 289-295).

  In addition to promoting proliferation, Stat3 has anti-apoptotic properties. In fibroblasts, Stat3 competes with the proapoptotic action of Stat1 by inducing the expression of Bcl-xL and Survivin when stimulated with serum supply cessation and UV irradiation (Non-Patent Document 1). : Shen, Y., Devgan, G., Darnell, JE, and Bromberg, JF 2001. Constitutively activated Stat3 protects fibroblasts from serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1. Proc. Natl. Acad. Sci USA 98: 1543-1548). Stat3 may cause cell proliferation in some cell types by preventing apoptosis (Non-patent document 1: Shen, Y., Devgan, G., Darnell, JE, and Bromberg, JF 2001. Constitutively activated). Stat3 protects fibroblasts from serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1. Proc. Natl. Acad. Sci. USA 98: 1543-1548; Non-patent document 2: Grandis, JR, et al. 2000. Constitutive Activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proc. Natl. Acad. Sci. USA 97: 4227-4232; Non-patent document 3: Takeda, K., et al. 1998. Stat3 activation is responsible for IL- 6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J. Immunol. 161: 4652-4660). However, the exact mechanism of action of the anti-apoptotic properties of Stat3 has not been elucidated.

  Apoptosis is mediated by a variety of biochemical processes. One of the most well-characterized apoptosis pathways is induced by the surface molecule Fas (Non-patent Document 4: Nagata, S. 1999. Fas ligand-induced apoptosis. Annu. Rev. Genet. 33: 29-55; Non-Patent Document 5: Matsumura, H., et al. 2000. Necrotic death pathway in Fas receptor signaling. J. Cell Biol. 151: 1247-1256). Fas agonists cause the trimerization of Fas and then collect several molecules to form the cell death-inducing signal complex (DISC). The formation of DISC begins by collecting FADD on the cell membrane by binding Fas and its adapter protein FADD in the cell death region of each other. The other end of FADD contains two cell death effector regions (DEDs), which are caspase-8 (FLICE), or a homologue without its enzymatic activity, Fas inhibitory FLICE. Inhibitory protein (FLIP) is collected. Procaspase-8 is cleaved and activated after binding to FADD (non-patent document 6: Siegmund, D., et al. 2001. Fas-associated death domain protein (FADD) and caspase-8 mediate up- J. Biol. Chem. 276: 32585-32590; Non-Patent Document 7: regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP) -regulated pathway. Kovalovich, K., et al. 2001. Interleukin-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2, and Bcl-xL.J. Biol. Chem. 276: 26605 -26613; Non-Patent Document 8: Budd, RC 2002. Death receptors couple to both cell proliferation and apoptosis. J. Clin. Invest. 109: 437-442. Doi: 10.1172 / JCI200215077). This initiates a subsequent cascade reaction leading to apoptosis. Interestingly, IL-6, known to play an important role in liver regeneration via Stat3, induces necessary amounts of anti-apoptotic proteins such as FLIP, Bcl-2, and Bcl-xL. Protects the liver from Fas-mediated apoptosis (Non-Patent Document 7: Kovalovich, K., et al. 2001. Interleukin-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2 , and Bcl-xL. J. Biol. Chem. 276: 26605-26613).

In the clinical field, Fas has been shown to be important in hepatitis and other liver diseases (Non-Patent Document 9: Pinkoski, MJ, Brunner, T., Green, DR, and Lin, T. 2000. Fas. Am. J. Physiol. Gastrointest. Liver Physiol. 278: G354-G366; Non-Patent Document 10: Galle, PR, et al. 1995. Involvement of the CD95 (APO-1 / Fas) J. Exp. Med. 182: 1223-1230; Non-Patent Document 11: Kanzler, S., and Galle, PR 2000. Apoptosis and the liver. Semin. Cancer Biol. 10: 173-184 ). Because the Fas antigen is constitutively expressed on hepatocytes, the liver is susceptible to Fas-mediated apoptosis. Mice administered with anti-Fas antibody die due to liver failure (Non-patent Document 7: Kovalovich, K., et al. 2001. Interleukin-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2, and Bcl-xL. J. Biol. Chem. 276: 26605-26613). Fas ^{− / −} mice show hepatic hyperplasia, suggesting that the Fas pathway is important in determining liver structure and size. The Fas / FasL system appears to be involved in many pathological conditions such as graft-versus-host disease, liver grafts, hepatitis B and C, acute alcoholic hepatitis, and several biliary diseases (Non-patent document 9: Pinkoski, MJ, Brunner, T., Green, DR, and Lin, T. 2000. Fas and Fas ligand in gut and liver. Am. J. Physiol. Gastroin
Test. Liver Physiol. 278: G354-G366; Non-patent document 11: Kanzler, S., and Galle, PR 2000. Apoptosis and the liver. Semin. Cancer Biol. 10: 173-184; Non-patent document 12: Faubion, WA, et al. 1999. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J. Clin. Invest. 103: 137-145).

Shen, Y., Devgan, G., Darnell, J.E., and Bromberg, J.F. 2001. Proc. Natl. Acad. Sci. U. S. A. 98: 1543-1548 Grandis, J.R., et al. 2000. Proc. Natl. Acad. Sci. U. S. A. 97: 4227-4232 Takeda, K., et al. 1998. J. Immunol. 161: 4652-4660 Nagata, S. 1999. Annu. Rev. Genet. 33: 29-55 Matsumura, H., et al. 2000. J. Cell Biol. 151: 1247-1256 Siegmund, D., et al. 2001. J. Biol. Chem. 276: 32585-32590 Kovalovich, K., et al. 2001. J. Biol. Chem. 276: 26605-26613 Budd, R.C. 2002. J. Clin. Invest. 109: 437-442.doi: 10.1172 / JCI200215077 Pinkoski, M.J., Brunner, T., Green, D.R., and Lin, T. 2000. Am. J. Physiol. Gastrointest. Liver Physiol. 278: G354-G366 Galle, P.R., et al. 1995. J. Exp. Med. 182: 1223-1230 Kanzler, S., and Galle, P.R. 2000. Apoptosis and the liver. Semin. Cancer Biol. 10: 173-184 Faubion, W.A., et al. 1999. J. Clin. Invest. 103: 137-145

Summary of the Invention

  The present inventors used mice to examine whether Stat3 provides protection against Fas-mediated liver injury. Activation of Stat3 is due to inhibition of caspase activity in a redox-dependent or independent mechanism. Found to provide protection against liver damage mediated by Fas. The present invention is based on this finding.

  Accordingly, an object of the present invention is to provide a method for screening a therapeutic agent for a liver disorder mediated by Fas based on the ability to modulate the action of Stat3, and a pharmaceutical composition and hepatocytes for treating the liver disorder. And

  The screening method according to the present invention is a method for identifying a drug for the treatment of a liver disorder induced by a Fas agonist, comprising: (a) a cell capable of expressing a Stat3 gene in the presence of a candidate drug. Culturing the cells under conditions that allow the cells to express the Stat3 gene in the absence of the candidate drug, and (b) measuring the activation intensity of Stat3 in the cells obtained by the step (a). A method wherein the candidate drug is identified as a drug for the treatment of a liver disorder induced by a Fas agonist when the activation of Stat3 is enhanced by the candidate drug.

  A screening method according to another aspect of the present invention is a method for identifying a drug for the treatment of a liver disorder induced by a Fas agonist, comprising (a) a Ref-1 gene promoter and a functionally operative form thereof. A cell comprising a reporter gene linked to a promoter and capable of expressing the Stat3 gene is cultured in the presence of the candidate drug in the absence of the candidate drug under conditions that allow the cell to express the Stat3 gene. And (b) measuring the expression level of the reporter gene in the cell obtained by step (a), and when the expression level of the reporter gene is increased by the candidate drug, the candidate drug Are identified as drugs for the treatment of liver disorders induced by Fas agonists.

  Furthermore, the pharmaceutical composition according to the present invention is a pharmaceutical composition for treating a liver disorder induced by a Fas agonist, comprising a Stat3 activity enhancer and a pharmaceutically acceptable carrier.

  Furthermore, the hepatocytes according to the present invention are hepatocytes for use in the treatment of liver damage induced by a Fas agonist, comprising an expression vector comprising DNA encoding Stat3.

  Furthermore, a hepatocyte according to another aspect of the present invention can be obtained by a Fas agonistic substance comprising an expression vector comprising a DNA encoding Stat3 modified so as to constitutively form dimerized Stat3 having activity. Hepatocytes for use in the treatment of induced liver damage.

  ADVANTAGE OF THE INVENTION According to this invention, the new drug discovery means and pharmaceutical aiming at the treatment of the liver disorder induced by a Fas agonist substance are provided.

DETAILED DESCRIPTION OF THE INVENTION

  Transcriptional signaling / activator-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and liver regeneration. In the present specification, it has been demonstrated that Stat3 provides protection against Fas-mediated liver injury by experiments using mice aimed at examining the hepatoprotective effect of Stat3. More specifically, the following experimental results have been obtained. First, constitutively active Stat3 (Stat3-C) is overexpressed in mouse liver by intravenous injection of adenovirus vector, and then less than lethal dose of Fas agonist (Jo2) is administered intraperitoneally to the mice (0.3 μg / g body weight). Stat3-C dramatically suppressed both apoptosis and cell necrosis induced by Jo2. In contrast, liver-specific Stat3 knockout mice died after administration of Jo2. Stat3-C increased the expression level of FLICE inhibitory protein (FLIP), Bcl-xL and Bcl-2, and thus reduced the activity of FLICE and caspase-3, which are redox-independent. Interestingly, Stat3-C also increases the expression level of redox-related protein redox factor-1 (Ref-1) and suppresses the activity of caspase-3 that is sensitive to oxidative stress and redox, after administration of Jo2 Reduces apoptosis in the liver. These findings indicate that Stat3 activation results in protection against Fas-mediated liver injury through inhibition of caspase activity in a redox-dependent or independent mechanism.

  The above findings provide a method for identifying drugs for the treatment of liver damage induced by Fas agonistic substances.

  As used herein, the term “liver injury induced by a Fas agonist” refers to a substance that can act as an agonist (agonist) against Fas antigens present on the surface of hepatocytes, such as Fas ligand (FasL or CD95L). Also referred to as liver damage caused by liver apoptosis (cell death) induced by. Here, the mechanism of action when a Fas agonist induces liver apoptosis is not only the one that includes the binding of the Fas agonist to the Fas antigen, but also reactive oxygen species (oxidative stress) by the Fas agonist. It may be via induction of Examples of such liver disorders include fulminant hepatitis, fatty liver, cirrhosis, and jaundice liver. According to a preferred embodiment of the present invention, the liver injury is caused by a reactive oxygen species induced by a Fas agonist. For example, hepatitis B, hepatitis C, fulminant hepatitis, transplantation One-to-one host disease, alcoholic hepatitis, etc.

  As used herein, the term “treatment” means not only alleviating an already established condition, but also preventing a condition that is expected to develop later.

The drug identification method according to the first aspect of the present invention uses the ability to enhance the activation of Stat3 as an index, and therefore the drug identified by the method is a Stat3 activity enhancer. Therefore, this method is supposed to include the following steps:
(A ₁ ) culturing cells capable of expressing the Stat3 gene in the presence of the candidate drug in the absence of the candidate drug under conditions that allow the cells to express the Stat3 gene; and (b ₁ ) measuring the intensity of the activation of Stat3 in a cell obtained by _{(a 1).}

  In the drug identification method according to the first aspect of the present invention, when the activation of Stat3 is enhanced by the candidate drug, the candidate drug is identified as a drug for the treatment of a liver disorder induced by a Fas agonist. Is done.

In the step (a ₁ ), a cell having an endogenous Stat3 gene can be used as it is, but a cell genetically engineered to express the Stat3 gene is used regardless of the presence or absence of the endogenous Stat3 gene. May be. The amino acid sequence of Stat3 and the nucleotide sequence that encodes it are well known in the art. For example, the sequence of human Stat3 shown in SEQ ID NO: 1 and SEQ ID NO: 2 (NCBI access number: BC000627), and SEQ ID NO: 3 and sequence An example is the mouse Stat3 sequence represented by No. 4 (NCBI access number: NM — 011486). Those skilled in the art can refer to these sequences by standard methods (eg, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ( 1989) can be used to properly transfect cells.

According to a preferred embodiment of the present invention, the cell used in step (a ₁ ) is a mammalian cell. Various such mammalian cells are known in the art. For example, COS-7 cells, C127 cells, NIH3T3 cells, CHO cells, HEK293 cells, HeLa cells, BHK cells, SOAS-2 Examples thereof include cells. Moreover, you may use the liver cell isolated from the liver of a mammal, such as a human and a mouse | mouth, or a liver biopsy sample. Furthermore, the expression vector used in mammalian cells suitably includes, for example, a replication origin, a promoter, an enhancer, a ribosome binding site, a polyadenylation site, a splice donor site, a splice acceptor site, a terminator, a 5 ′ untranslated region, and the like. Is.

The culture in the step (a ₁ ) can be performed by a standard method well known in the art, for example, by incubating the drug with a candidate drug. The conditions under which the cells can express the Stat3 gene in the absence of a candidate drug can be achieved by methods known to those skilled in the art, for example, by activating the promoter in the expression vector used for transfection. Can be achieved. A person skilled in the art can appropriately select the culture medium, temperature, time, amount of candidate drug, amount of cells, additive, and the like in culture.

In step (b ₁ ), the intensity of activation of Stat3 can be measured by standard methods well known in the art, for example, by quantifying activated Stat3 protein. For example, it is known that when activated, Stat3 is phosphorylated in its C-terminal region to form a homodimer, and then moves into the cell nucleus and binds to DNA (promoter). Accordingly, in the first preferred method, the intensity of Stat3 activation is measured by measuring the amount of dimerized Stat3 protein in the cell, and more specifically, the protein extract from the cell is electrophoresed. The dimerized Stat3 protein can be detected by anti-Stat3 (for example, commercially available from BD Transduction Laboratories, Lexington, Kentucky, USA). In this method, whether or not the detected Stat3 protein is a dimer can be determined by the electrophoretic mobility. In the second preferred method, the intensity of Stat3 activation is measured by measuring the amount of Stat3 protein in the nucleus of the cell. More specifically, the protein extract from the cell nucleus is electrophoresed. The Stat3 protein can be detected by detecting the above-described anti-Stat3 or the like. Furthermore, in a third preferred method, the intensity of Stat3 activation is measured by measuring the amount of Stat3 protein bound to DNA in the cell (ie, Stat3 DNA binding activity), more specifically, Can be measured by electrophoretic mobility transfer assay using SIE-m67 oligonucleotide labeled as (5′-actgGGATTTTTCCCGTAAATGGTC-3 ′: SEQ ID NO: 5).

According to a preferred embodiment of the present invention, the intensity of activation of Stat3 in the cells obtained by the step (a ₁ ) is compared with the intensity of activation of Stat3 in control cells cultured in the absence of the candidate drug. The candidate drug is identified as a drug for the treatment of liver damage induced by Fas agonists. The control cells can be obtained by the same method as in step (a ₁ ) except that no candidate drug is added.

In the present invention, the Stat3 activity enhancer is preferably one that enhances Ref-1 gene expression by Stat3. Therefore, the drug identification method according to the second aspect of the present invention uses the ability to enhance the activity of Stat3 that promotes the transcription of the Ref-1 gene as an index. Therefore, this method is supposed to include the following steps:
(A ₂ ) a cell comprising a Ref-1 gene promoter and a reporter gene operably linked to the promoter and capable of expressing a Stat3 gene, in the presence of the candidate drug, Culturing the cells under conditions that allow the cells to express the Stat3 gene, and
(B ₂ ) A step of measuring the expression level of the reporter gene in the cells obtained by the step (a ₂ ).

  In the drug identification method according to the second aspect of the present invention, when the expression level of the reporter gene is increased by the candidate drug, the candidate drug is used as a drug for the treatment of liver damage induced by a Fas agonist. Identified.

  In the present specification, the term “reporter gene” means a gene capable of quantitatively detecting the expression product, a gene whose nucleotide sequence and / or amino acid sequence is known, and intensity depending on the expression level. Genes that can emit different signals (such as fluorescence). Therefore, it is possible to use the Ref-1 gene as a reporter gene. According to a preferred embodiment of the present invention, the reporter gene is a luciferase gene, a CAT gene or a lacZ gene.

In the step (a ₂ ), cells having an endogenous Stat3 gene and an endogenous Ref-1 gene can be used as they are, but the Stat3 gene is expressed regardless of the presence or absence of these endogenous genes. And / or cells that have been genetically engineered to contain a Ref-1 gene promoter and a reporter gene operably linked to the promoter may be used. The amino acid sequence of Stat3 and the nucleotide sequence that encodes it are well known in the art. For example, the sequence of human Stat3 shown in SEQ ID NO: 1 and SEQ ID NO: 2 (NCBI access number: BC000627), and SEQ ID NO: 3 and sequence An example is the mouse Stat3 sequence represented by No. 4 (NCBI access number: NM — 011486). The nucleotide sequence of the Ref-1 gene promoter is well known in the art, and examples thereof include the human Ref-1 gene promoter sequence shown in SEQ ID NO: 6. Furthermore, the gene sequence and amino acid sequence of Ref-1 protein are well known in the art, for example, the sequences of human Ref-1 shown in SEQ ID NO: 7 and SEQ ID NO: 8, and shown in SEQ ID NO: 9 and SEQ ID NO: 10. Mouse Ref-1 sequence. Those skilled in the art can refer to these sequences by standard methods (eg, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ( 1989) can be used to properly transfect cells.

According to a preferred embodiment of the present invention, the cell used in step (a ₂ ) is a mammalian cell. Various such mammalian cells are known in the art. For example, COS-7 cells, C127 cells, NIH3T3 cells, CHO cells, HEK293 cells, HeLa cells, BHK cells, SOAS-2 Examples thereof include cells. Moreover, you may use the liver cell isolated from the liver of a mammal, such as a human and a mouse | mouth, or a liver biopsy sample. Furthermore, the expression vector used in mammalian cells suitably includes, for example, a replication origin, a promoter, an enhancer, a ribosome binding site, a polyadenylation site, a splice donor site, a splice acceptor site, a terminator, a 5 ′ untranslated region, and the like. Is.

The culture in the step (a ₂ ) can be performed by a standard method well known in the art, for example, by incubating the drug with the candidate drug. The conditions under which the cells can express the Stat3 gene in the absence of a candidate drug can be achieved by methods known to those skilled in the art, for example, by activating the promoter in the expression vector used for transfection. Can be achieved. A person skilled in the art can appropriately select the culture medium, temperature, time, amount of candidate drug, amount of cells, additive, and the like in culture.

In the step (b ₂ ), the expression level of the reporter gene can be measured by a standard method well known in the art, for example, by quantifying a protein or mRNA that is an expression product of the gene. The amount of mRNA from the reporter gene can be measured by a method such as electrophoresis after amplification by RT-PCR using a primer pair specific thereto. The amount of protein from the reporter gene can be measured by immunoblotting or the like in which a protein extract obtained from cells is electrophoresed and then detected using an antibody specific for the protein. As a reporter gene, when a gene capable of emitting a signal (fluorescence or the like) having a different intensity depending on its expression level, such as a luciferase gene, a CAT gene, or a lacZ gene, is used, the signal (fluorescence or the like) By measuring the strength, the expression level of the reporter gene can be measured.

According to a preferred embodiment of the present invention, the expression level of the reporter gene in the cells obtained by the step (a ₂ ) is compared with the expression level of the reporter gene in control cells cultured in the absence of the candidate drug. If so, the candidate drug is identified as a drug for the treatment of liver damage induced by Fas agonists. Control cells can be obtained by the same method as in step (a ₂ ) except that no candidate drug is added. Moreover, it can be confirmed that the increase in the expression level of the reporter gene is due to the activation of Stat3 by performing the steps (a ₂ ) and (b ₂ ) using cells that do not express Stat3. it can. That is, in a cell that does not express Stat3, when the expression level of the reporter gene is decreased compared to a cell that expresses Stat3, it is determined that the increase in the expression level of the reporter gene is due to activation of Stat3. can do. As such cells that do not express Stat3, cells that do not have endogenous Stat3 gene can be used, or cells that have endogenous Stat3 gene knocked out or knocked down can be used. . For gene knockout or knockdown, use a method known to those skilled in the art, for example, an antisense method, a siRNA method using RNA interference (Elbashir, SM et al., Nature 411, 494-498, 2001), etc. Can do.

  The Stat3 activity enhancer can be used for treatment of liver damage induced by a Fas agonist. Therefore, according to the present invention, there is provided use of a Stat3 activity enhancer for treating a liver disorder induced by a Fas agonist. Furthermore, according to the present invention, there is provided a method for treating or preventing a liver disorder induced by a Fas agonist, which comprises administering to a subject a therapeutically effective amount of a Stat3 activity enhancer. The Furthermore, the present invention provides the use of a Stat3 activity enhancer in the manufacture of a medicament for treating liver damage induced by a Fas agonist. According to a preferred embodiment of the present invention, the Stat3 activity enhancer is not a naturally occurring substance but a non-natural substance such as a synthetic substance.

  The Stat3 activity enhancer can be identified by the drug identification method according to the first aspect of the present invention. The Stat3 activity enhancer is preferably one that enhances the activity of Stat3 that promotes the transcription of the Ref-1 gene, and this can be identified by the drug identification method according to the second aspect of the present invention.

  According to a preferred embodiment of the present invention, the Stat3 activity enhancer is an expression vector containing DNA encoding Stat3. Such an expression vector can be produced by standard techniques well known in the art with reference to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Further, the expression vector can appropriately include, for example, a replication origin, a promoter, an enhancer, a ribosome binding site, a polyadenylation site, a splice donor site, a splice acceptor site, a terminator, a 5 'untranslated region, and the like. Furthermore, as the expression vector, a viral vector such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or the like can be used.

  According to another preferred embodiment, the Stat3 activity enhancer is an expression vector comprising DNA encoding Stat3 modified so as to constitutively form dimerized Stat3 having activity. Stat3 is usually activated by phosphorylation in its C-terminal region, forming a homodimer. The term “constitutively” means that the expressed Stat3 forms a homodimer and is activated without the need for such phosphorylation. Stat3 (mutant Stat3) modified in this way has two disulfides in the C-terminal region with reference to the amino acid sequence of human Stat3 shown in SEQ ID NO: 2, the amino acid sequence of mouse Stat3 shown in SEQ ID NO: 4, and the like. It can be designed such that a bond (SS bond) is formed. For example, the mutant Stat3 derived from mouse contains 661 residues of 661 alanine residues and 663 asparagine residues (corresponding to 662 residues and 664 residues in SEQ ID NO: 4, respectively). It can be designed by changing to the base. Similarly, the mutant Stat3 derived from human also has the 661th alanine residue and the 663rd asparagine residue (corresponding to the 662th residue and the 664th residue in SEQ ID NO: 2, respectively). It can be designed by changing to a cysteine residue (Bromberg J. and Darnell JE, 2000, The role of STATs in transcriptional control and their impact on cellular function., Oncogene 19, 2468-2473). Furthermore, an expression vector expressing such mutant Stat3 can be produced by standard techniques well known in the art with reference to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Further, the expression vector can appropriately include, for example, a replication origin, a promoter, an enhancer, a ribosome binding site, a polyadenylation site, a splice donor site, a splice acceptor site, a terminator, a 5 'untranslated region, and the like. Furthermore, as the expression vector, a viral vector such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or the like can be used.

  The Stat3 activity enhancer can be administered by an appropriate route according to the disease or disorder to be treated or prevented, such as topical, intravenous, subcutaneous, intramuscular, oral, rectal, mucosal and the like. These therapeutically effective amounts are appropriately determined by a doctor or veterinarian according to the severity of symptoms, the age of the subject, the effectiveness of the specific drug used, the route of administration, the frequency of administration, etc. . Generally, the therapeutically effective amount is about 0.001 to about 1000 mg / kg body weight, preferably about 0.01 to about 10 mg / kg body weight, more preferably about 0.01 to about 1 mg / kg per day. Weight is kg.

  The Stat3 activity enhancer can be administered together with a pharmaceutically acceptable carrier. Thus, according to the present invention, there is provided a pharmaceutical composition comprising a Stat3 activity enhancer and a pharmaceutically acceptable carrier, the pharmaceutical composition for treating liver injury induced by a Fas agonist. Can be used. Pharmaceutically acceptable carriers such as vehicles, excipients, diluents and the like are appropriately selected by those skilled in the art depending on the route of administration, the properties of the specific drug used, and the like. The pharmaceutical composition according to the present invention preferably comprises a therapeutically effective amount of a Stat3 activity enhancer and a pharmaceutically acceptable carrier.

  Furthermore, according to the present invention, hepatocytes comprising an expression vector containing DNA encoding Stat3, and DNA encoding Stat3 modified so as to constitutively form dimerized Stat3 having activity Hepatocytes comprising an expression vector are provided, and these hepatocytes can be used for the treatment of liver damage induced by Fas agonistic substances. Such hepatocytes can be prepared by transfecting hepatocytes using the respective expression vectors. In addition, as a hepatocyte to be transfected, a liver cell or an established cell line isolated from a liver or a liver biopsy sample of a mammal such as a human or a mouse can be used. According to a preferred embodiment of the present invention, the hepatocytes are derived from an individual to be treated.

The hepatocytes according to the present invention can be administered in administration routes and therapeutically effective amounts generally used in the field of cell medicine. Therefore, the administration route is not particularly limited, but is preferably parenteral administration, more preferably intravenous administration, intradermal administration or subcutaneous administration, and further preferably intravenous administration. The therapeutically effective amount is also not particularly limited and is appropriately determined in consideration of the condition of the subject, for example, the patient's age, weight, sex, disease difference, symptom level, etc. In terms of conversion, it is preferably 5 × 10 ⁵ to 10 ⁹ , more preferably 0.5 to 60 × 10 ⁶ , and still more preferably 2 to 20 × 10 ⁶ . The timing of administration is not particularly limited, and may be any time before or after the onset of the disease.

  The present invention also provides the use of the hepatocytes according to the present invention for the manufacture of a medicament for the treatment of the above diseases. Furthermore, according to the present invention, there is provided a pharmaceutical composition comprising the hepatocytes according to the present invention, which can be used for the treatment of liver damage induced by a Fas agonistic substance. This pharmaceutical composition can be appropriately produced by those skilled in the art according to the administration route and dosage described above or according to well-known techniques in cell medicine.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the present invention.

Example 1: Protection of Fas-induced liver injury by Stat3 by a redox-dependent or independent mechanism The experiment in this example aims to investigate the protective effect of Stat3 against Fas-mediated liver injury. In this experiment, FLIP, Bcl-2, Bcl-xL and redox factor-1 (Ref-1) proteins are expressed by overexpressing constitutively active Stat3 (Stat3-C) using an adenovirus vector. It has been shown that concentrations are increased and Fas-mediated liver injury is dramatically suppressed.

1. Materials and methods
The adenovirus vector Stat3-C construct is obtained by substituting the 661th alanine residue and the 663rd asparagine residue with a cysteine residue in mouse Stat3 and further attaching a FLAG tag. This Stat3-C molecule can form a dimer even if the tyrosine residue at position 705 is not phosphorylated (Bromberg, J., and Darnell, JE 2000. The role of STATs in transcriptional control). and their impact on cellular function. Oncogene. 19: 2468-2473). A self-replication ability-deficient adenovirus vector (AxCAS3-C) encoding Stat3-C was constructed. Adenovirus (AdLacZ) encoding an inactive bacterial β-gal was used as a control vector. Adenovirus encoding full-length Ref-1 in the sense direction (AdRef) and adenovirus encoding in the antisense direction (AdFer) were constructed according to methods described in the literature (Ozaki, M., Suzuki, S., and Irani, K. 2002. Redox factor-1 / APE suppresses oxidative stress by inhibiting the rac1 GTPase. FASEB J. 16: 889-890).

Animal experiments C57BL / 6 male mice aged 6-8 weeks were used for the experiments. Adenoviral vectors were administered into the tail vein (3 × 10 ⁸ PFU / animal) 3 days before the experiment. In experiments using multiple types of viral vectors, the total amount of adenovirus administered was 3 × 10 ⁸ PFU / animal. After a 24-hour fast, mice were administered ip with Jo2 antibody (0.3 μg / g body weight; BD PharMingen, San Diego, California, USA). In some experiments, N-acetyl-L-cysteine (NAC) (200 mg / kg body weight) (Yamakawa, Y., et al. 2000. Interaction of platelet activating factor, reactive oxygen species generated) 1 hour before the experiment. by xanthine oxidase, and leukocytes in the generation of hepatic injury after shock / resuscitation. Ann. Surg. 231: 387-398), and a general apoptosis inhibitor Z-VAD-fmk (1 μg / body weight g; Enzyme Systems Products Inc., Livermore, California, USA) (Daemen, MARC, et al. 1999. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J. Clin. Invest. 104: 541-549).

Experiment using primary cultured hepatocytes Primary cultured hepatocytes were prepared from the liver of C57BL / 6 mice by collagenase perfusion method. Hepatocytes were seeded in 3 × 10 ⁶ cells / 10 cm culture dishes 24 hours prior to adenovirus infection. AdLacZ or AxCAS3-C was infected at 2MOI, and 2.5 μg / ml Jo2 was administered 48 hours later. Cells were harvested 48 hours after adenovirus infection for protein expression analysis, and an apoptosis assay was performed 8 hours after Jo2 administration.

Preparation of the LS3-KO mouse Stat3-flox mice (Takeda, K., et al 1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis:. Generation and characterization of T cell-specific Stat3-deficient mice J. Immunol. 161: 4652-4660) and albumin-Cre transgenic (Alb-Cre) mice (Yakar, S., et al. 1999. Normal) expressing Cre recombinase only in the liver under the control of the albumin promoter. LS3-KO mice were produced by mating with growth and development in the absence of hepatic insulin-like growth factor I. Proc. Natl. Acad. Sci. USA 96: 7324-7329). In order to increase the efficiency of Stat3 disruption, Stat3 hetero knockout (Stat3 ^+/− ) mice (Takeda, K., et al. 1997. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc. Natl. Acad. Sci. The Stat3 null allele was introduced onto the floxed allele by crossing with USA 94: 3801-3804). First, a Stat3 ^+/− mouse and an Alb-Cre mouse are mated to produce a Stat3 ^+/− , Alb-Cre mouse, and then this mouse and a mouse having a homozygous Stat3 floxed allele (Stat3 ^{flox / flox} ). The obtained Stat3 ^{flox / −} , Alb-Cre mouse was used as an LS3-KO mouse. Stat3 ^{flox / −} mice obtained by similar mating were used as control mice.

Western blot analysis The liver homogenate was centrifuged and the supernatant was used for the assay. 15% SDS-PAGE was performed using 50 μg of protein. Proteins transferred to the nitrocellulose membrane were treated with appropriate antibodies: anti-Fas, anti-Bcl-2, anti-Bcl-XL, anti-survivin (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), anti-FLIP-CT (Cell Signaling). Technology Inc., Beverly, Massachusetts, USA), anti-Ref-1, anti-manganese superoxide dismutase (anti-MnSOD; Transduction Laboratories, San Diego, California, USA), anti-thioredoxin (BD PharMingen), and anti-FLAG (Sigma-Aldrich) , St. Louis, Missouri, USA).

Electrophoretic mobility transfer assay Stat3 DNA binding activity was assayed using SIE-m67 oligonucleotide (5'-actgGGATTTTTCCCGTAAATGGTC-3 ': SEQ ID NO: 5) as a probe. The reaction mixture contains nucleoprotein extract (5 μg), DTT (2 mM), dI-dC (2 μg), ssDNA (10 μg / ml), and ³² P-labeled SIE-m67 probe (5 × 10 ⁵ cpm). did. This sample was electrophoresed on a 5% polyacrylamide native gel at 4 ° C. in 0.25 × Tris-Borate / EDTA (TBE) buffer.

Histological experiment Liver tissues were collected and fixed with 10% buffered formalin, and sections embedded in paraffin were stained with H & E. To immunohistochemically analyze the presence of Stat3 protein, samples were incubated overnight at 4 ° C. with 2.5 μg / ml primary antibody (anti-Stat3; BD Transduction Laboratories, Lexington, Kentucky, USA) Secondary antibody (biotin-conjugated anti-mouse IgG) was added.

Apoptosis assay
Cell death due to apoptosis of liver tissue was analyzed using an in situ Cell Death Detection Kit (Roche Diagnostics Corp., Basel, Switzerland). Liver samples embedded in paraffin were incubated with the TUNEL reaction mixture according to the instructions attached to the kit. In addition, apoptosis-induced cells were quantitatively evaluated using Cell Death Detection ELISA PLUS (Roche Diagnostics Corp.). In this assay, the lysate of liver tissue was used as it was (40 μg protein), and the assay was performed according to the instructions attached to the kit.

Caspase activity assay The activities of caspase-3, caspase-8 and caspase-9 were measured using CPP32 / Caspase-3 Colorimetric Protease Assay Kit, FLICE / Caspase-8 Colorimetric Protease Assay Kit, and Caspase-9 / Mch6 Colorimetric Protease Assay Kit, respectively. (Both were measured using Medical and Biological Laboratories Co., Tokyo, Japan). Liver homogenate supernatants were prepared (200 μg in 50 μl) and reacted with substrate at 37 ° C. for 1 hour prior to assay.

Liver Reactive Oxygen Species Assay in Liver Tissue Production of reactive oxygen species (ROS) in liver tissue was determined in two ways: lucigenin-enhanced chemiluminescence and hydrogen peroxide assay (Ozaki, M., et al. 2000). Inhibition of the Rac1 GTPase protects against nonlethal ischemia / reperfusion-induced necrosis and apoptosis in vivo. FASEB J. 14: 418-429). After the liver cells were isolated by perfusion method was added 100μl of cell suspension (10 ⁵ cells) lucigenin buffer 1 ml (75 mM lucigenin in HBSS). The chemiluminescent signal was monitored every 5 minutes for 30 minutes. The unit of synchrotron radiation (subtracting the measurement value of the blank sample and integrating over 15 minutes or more) was taken as the measurement value of superoxide production. Hydrogen peroxide production was measured using the Amplex Red Hydrogen Peroxide Assay Kit (Molecular Probes Inc., Eugene, Oregon, USA). Using the supernatant of the liver homogenate, the assay was performed according to the instructions attached to the kit. After subtracting the measured value of the blank sample, the red fluorescence signal was normalized by the protein concentration and the hydrogen peroxide concentration (expressed as mmol / mg protein) was calculated based on the standard curve.

mRNA analysis In order to conduct a multiprobe ribonuclease protection assay, the presence of the transcript of the gene of interest and L32 as an internal control were detected using a multiprobe template set (BD PharMingen). Probe synthesis, hybridization, and RNase treatment were performed according to the instructions attached to the kit. Protected transcripts were developed by electrophoresis on a denaturing 4.75% polyacrylamide gel and quantified using a PhosphorImager (Amersham Biosciences, Piscataway, New Jersey, USA).

For Northern blot analysis, total RNA was extracted from the cells and electrophoresed on a 1.4% agarose gel. RNA was transferred to a nylon membrane and 3 × 10 ⁷ cpm of ³² P-labeled single-stranded Ref-1 cDNA was hybridized. The membrane was washed and analyzed by autoradiography.

Transient transfection experiments and luciferase reporter assay The human Ref-1 promoter was cloned from genomic DNA of HeLa cells by PCR (Grosch, S., Fritz, G., and Kaina, B. 1998. Apurinic endonuclease (Ref- 1) is induced in mammalian cells by oxidative stress and involved in clastogenic adaptation. Cancer Res. 58: 4410-4416; Grosch, S., and Kaina, B. 1999. Transcriptional activation of apurinic / apyrimidinic endonuclease (Ape, Ref-1 ) by oxidative stress requires CREB. Biochem. Biophys. Res. Commun. 261: 859-863), which was cloned into pGL3 (Promega Corp., Madison, Wisconsin, USA). HeLa cells were seeded at a density of 1.5 × 10 ⁴ cells / cm ² , and transfection was performed using LipofectAMINE (Invitrogen Corp., Carlsbad, California, USA) as a transfection reagent according to the attached instructions. Forty-eight hours after transfection of the Ref-1 promoter gene, adenoviral vectors were infected at various predetermined MOIs. 36 hours after adenovirus infection, luciferase activity was evaluated using the Bright-Gloluciferase assay system (Promega Corp.) according to the attached instructions. The results of each reporter assay were confirmed to be reproducible in 3 independent experiments.

Statistical analysis Fisher's test was used to analyze the difference between multiple groups, and it was assumed that there was a significant difference in the 95% confidence interval. The number of animals (n) used in each experiment is shown in parentheses.

2. result
Expression of Stat3 in the livers of mice administered with AxCAS3-C and LS3-KO mice and an adenoviral vector (AxCAS3-C) encoding a constitutively active Stat3 gene were administered intravenously (1 × 10 ⁸ PFU / animal).

  Three days after adenovirus infection, the mouse liver was subjected to immunoblot analysis using anti-Stat3 and anti-FLAG, respectively, and immunohistochemical analysis using anti-Stat3. According to immunoblot analysis and immunohistochemical analysis using anti-Stat3, the total amount of Stat3 protein in the liver of mice inoculated with AxCAS3-C (Stat3-C mice) was the same as that of mice inoculated with saline (PS mice). ) And AdLacZ inoculated (LacZ mice). The expression of Stat3-C protein expressed from a foreign gene was confirmed by the binding of anti-FLAG in the liver of Stat3-C mice. According to immunohistochemical analysis, almost no Stat3 protein was detected in the livers of PS mice and LacZ mice, whereas Stat3-C protein was detected in both cytoplasm and nucleus in the livers of Stat3-C mice. It was. As is clear from these results, constitutively active Stat3 protein (Stat3-C) was well overexpressed in the mouse liver by administration of AxCAS3-C. Moreover, the abnormal structure of the liver by this Stat3-C was not seen.

  Three days after adenovirus infection, electrophoretic mobility transfer assay (EMSA) was performed on mouse liver. In addition, in order to confirm specific Stat3 DNA binding, competition assays and supershift assays using a chilled SIE-m67 probe and anti-Stat3 were performed. The DNA binding activity of Stat3 was not activated in quiescent livers in PS mice. AdLacZ infection only partially activated Stat3 DNA binding activity, whereas Stat3-C overexpression resulted in strong activation of its DNA binding activity.

Western blot analysis was performed on various tissues of control and LS3-KO mice. The amount of Stat3 protein in the liver of LS3-KO mice was less than 5% of Stat3 ^{flox / −} mice, whereas various other tissues, namely pancreas, kidney, heart, lung, adipose tissue, and skeleton In muscle, there was no difference in Stat3 expression level between control mice and LS3-KO mice.

Inhibition of liver damage induced by Fas agonists by Stat3-C The liver resulting from the expression of Stat3-C has a macroscopically normal size and color 3 days after virus administration, and is microscopic. A slight infiltration of inflammatory cells was observed. FIG. 1 is a diagram showing the results of intraperitoneal administration of 0.3 μg / g body weight of Jo2 to various mice and then examining the survival rate of the mice over 72 hours. Serious liver damage was induced by administration of the Fas agonist Jo2, but no individual died by 72 hours after administration in PS mice and Stat3-C mice. In LacZ mice, 3 out of 8 mice died by 12 hours after Jo2 administration, and in LS3-KO mice, all 5 mice died at 12 hours after Jo2 administration.

  At 6 hours after Jo2 administration, the color of the liver changed to dark red, but the mouse was alive. Histological analysis revealed extensive hepatic hemorrhage with necrosis at 6 and 24 hours after administration, and severe neutrophil infiltration at 72 hours after administration. The expression of Stat3-C markedly suppressed Fas-mediated liver injury. The liver was structurally preserved in appearance and changed to red in some places. In the liver, microscopically significantly less bleeding and necrosis were observed at 6 and 24 hours after Jo2 administration.

  FIG. 2 is a graph showing the concentrations of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in serum over 72 hours after Jo2 administration. In FIG. 2, “*” indicates a significant difference at P <0.01 with respect to the measured value of the LacZ mouse at 24 hours after administration of Jo2 (Fas agonist). Each data is represented as an average value ± standard error (n = 5). Serum concentrations of GOT and GPT were significantly increased in LacZ mice at 6 and 24 hours after Jo2 administration. On the other hand, the extent of such increase in GOT concentration and GPT concentration was dramatically reduced in Stat3-C mice. These experimental results demonstrate that Stat3-C provides resistance to Fas-mediated liver injury.

Inhibition of Fas agonist-induced apoptosis by Stat3-C In order to confirm the anti-apoptotic properties of Stat3, hepatic apoptosis induced by Jo2 was performed in three different ways: TUNEL staining of histological sections, DNA ladder assay And analyzed by ELISA.

  The results of TUNEL staining revealed that the widespread apoptotic cell death observed 6 hours after Jo2 administration in the livers of PS mice, LacZ mice and LS3-KO mice was dramatically reduced by the expression of Stat3-C. became. Moreover, in the DNA ladder assay 6 hours after Jo2 administration, samples from PS mice, LacZ mice and LS3-KO mice showed a typical DNA ladder pattern.

  FIG. 3 shows pretreatment with a general caspase inhibitor Z-VAD-fmk (1 μg / g) or an antioxidant NAC (200 mg / kg), and after 1 hour, apoptotic cell death was quantified by ELISA. It is a figure which shows the result. In FIG. 3, “*” indicates a significant difference at P <0.01 relative to the LacZ / Fas agonist-administered sample, and “**” indicates P <relative to the LacZ / Fas agonist-administered sample and the Stat3-C / Fas agonist-administered sample. A significant difference at 0.05 is indicated, and “***” indicates a significant difference at P <0.05 relative to a LacZ / Fas agonist and caspase inhibitor-administered sample and a LacZ / Fas agonist and NAC-administered sample. Each data is represented as an average value ± standard error (n = 5).

  FIG. 4 shows the dose-dependent effect of caspase inhibitor (Z-VAD-fmk) or antioxidant (NAC) on liver apoptosis after Fas agonist administration. In FIG. 4, “*” indicates a significant difference at P <0.01 relative to the untreated control sample, “**” indicates a significant difference at P <0.05 relative to the Fas agonist-administered sample, “*” “**” indicates a significant difference at P <0.05 relative to Fas agonist administration and caspase inhibitor administration (1 mg / kg and 2 mg / kg) samples. Each data is represented as an average value ± standard error (n = 5).

  According to FIGS. 3 and 4, when pretreated with Z-VAD-fmk (1 μg / g) or NAC (200 mg / kg), liver apoptosis induced by Jo2 is significantly inhibited. It was found that complete inhibition was not observed even when the drug dose was increased. However, according to FIG. 3, when these drugs were administered simultaneously, it was revealed that apoptosis was better suppressed, and the synergistic inhibitory effect was comparable to Stat3-C. These results indicate that activated Stat3 suppresses Fas-mediated liver injury by a caspase-dependent mechanism and / or a redox-dependent mechanism.

Induction of anti-apoptotic protein in liver expressing Stat3-C In order to elucidate the anti-apoptotic mechanism of Stat3 acting on Fas-mediated liver injury, the expression of apoptosis-related protein was first examined. Specifically, the expression levels of Fas, Bcl-xL, Bcl-2 and survivin in the liver tissue of PS mice, LacZ mice and Stat3-C mice were examined, and further, before administration of Jo2 (Fas agonist) and administration The expression level of these proteins was examined up to 72 hours later. Overexpressed Stat3-C did not suppress Fas expression in hepatocytes but increased its expression level. However, Fas was induced by AdLacZ infection to the same extent as Stat3-C mice. Stat3-C increased Bcl family proteins (Bcl-XL and Bcl-2) with anti-apoptotic properties, whereas adenovirus itself did not induce these proteins. In LacZ mice, Fa2 and Bcl-XL proteins were induced by Jo2 administration at 24 hours after administration. Stat3-C is a survivin protein that has been reported to be induced by Stat3 in several cell types (Shen, Y., Devgan, G., Darnell, JE, and Bromberg, JF 2001. Constitutively activated Stat3 protects fibroblasts From serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1. Proc. Natl. Acad. Sci. USA 98: 1543-1548).

Induction and activation of FLIP protein by Stat3-C and subsequent reduction of FLICE activity and caspase-3 activity FLICE and its active form, truncated FLICE, in the liver tissue of PS mice, LacZ mice and Stat3-C mice, The expression levels of caspase-3 and its active form, cleaved caspase-3, and FLIP and its active form, cleaved FLIP were examined, and further up to 72 hours before and after administration of Jo2 (Fas agonist). The expression levels of these proteins were examined. FIG. 5 is a diagram showing the results of an enzyme activity assay for caspase-3 and caspase-8 in the liver tissue of each mouse. In FIG. 5, “*” indicates a significant difference at P <0.05 with respect to a LacZ sample not administered with Fas agonist, and “**” indicates a significant difference at P <0.01 with respect to a LacZ / Fas agonist-administered sample. Show. Each data is represented as an average value ± standard error (n = 5).

  According to the comparison of Stat3-C mice with PS mice and LacZ mice, Stat3-C suppresses the expression of FLICE protein, and furthermore, the expression of caspase-3 is less than that of FLICE protein. It becomes clear to suppress. In LacZ mice, FLICE and caspase-3 were both processed and activated at 6 hours post-Jo2 whereas Stat3-C inhibited both expression and activation of these proteins by Fas agonists did. The FLIP protein was also induced by Stat3-C, which was markedly activated for 72 hours after Jo2 administration. FLIP protein was not induced by PS and LacZ. IL-6, one of the main ligands of Stat3, has been reported to increase the expression level of FLIP, Bcl-2 and Bcl-XL in the liver (Kovalovich, K., et al. 2001. Interleukin-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2, and Bcl-xL. J. Biol. Chem. 276: 26605-26613). Furthermore, Stat3-C suppressed caspase activity to the same extent as caspase inhibitors. These experimental results indicate that Stat3 plays an important role in IL-6-related anti-apoptotic effects in the liver.

Induction of redox-related protein Ref-1 by Stat3-C Pretreatment with NAC, an antioxidant, partially improved Fas-mediated liver apoptosis (FIG. 3), thus redox-related in liver overexpressing Stat3-C Protein expression was examined. Specifically, the expression levels of Ref-1, thioredoxin and MnSOD in the liver tissues of PS mice, LacZ mice and Stat3-C mice were examined, and further, up to 72 hours before and after administration of Jo2 (Fas agonist) The expression levels of these proteins were examined. The protein expression level of Ref-1 was significantly increased by Stat3-C before Jo2 administration, whereas this expression level was slightly decreased by LacZ. The amount of increased Ref-1 protein decreased after administration of Jo2. This result shows that Ref-1 transcripts were induced by Stat3-C at 48 to 72 hours after administration of AxCAS3-C (see FIG. 7 below), whereas mice administered PS or AdLacZ This is also supported by the experimental results that no Ref-1 transcript is induced in the liver. However, Stat3-C did not affect the amount of the other two redox-related proteins, thioredoxin and MnSOD. MnSOD is temporarily increased after Jo2 administration in the liver overexpressing Stat3-C, suggesting the existence of an oxidant-dependent mechanism of action in Fas-mediated liver injury. These experimental results indicate that activated Stat3 specifically increases the amount of Ref-1 in mouse liver.

Transcriptional promotion of Stat3-related proteins PS (untreated) mice and liver tissues of LacZ mice and Stat3-C mice 48 hours after gene transfer by adenovirus were analyzed for FLICE, FLIP, Fas, Ref-1, Bcl-xL. And a multiprobe ribonuclease protection assay targeting Bcl-2. FIG. 6 is a bar graph showing the magnification of the quantitative value when the quantitative value for the untreated mouse is 1. In FIG. 6, each data is represented as an average value ± standard error (n = 5).

  According to the results of the ribonuclease protection assay, 48 hours after gene transfer by adenovirus, the concentration of Fas mRNA was increased by LacZ and Stat3-C, and Bcl-xL, Bcl-2, FLIP and Ref-1 Was increased only by Stat3-C (FIG. 6). However, the concentration of FLICE mRNA was not changed by LacZ and Stat3-C.

  Ref-1 may play an important role in the suppression of liver damage due to its antioxidant and anti-apoptotic properties (Ozaki, M., Suzuki, S., and Irani, K. 2002. Redox factor) -1 / APE suppresses oxidative stress by inhibiting the rac1 GTPase. FASEB J. 16: 889-890), mRNA expression of Ref-1 was confirmed by Northern blot analysis. FIG. 7 is a graph showing the change over time in the Ref-1 mRNA concentration in the liver tissue of each mouse after adenovirus infection, and each plot shows the measured value when the measured value before infection for each mouse is 1. Represents magnification.

  According to the results of Northern blot analysis, Stat3-C increased Ref-1 mRNA concentration prior to Ref-1 protein expression 48-72 hours after AxCAS3-C infection (FIG. 7). No significant increase in Ref-1 mRNA concentration was seen in the livers of mice administered PS or AdLacZ.

  To examine whether Ref-1 transcription is promoted by Stat3-C, the activation of the Ref-1 promoter by Stat3-C was measured. Specifically, a luciferase reporter assay was performed for Ref-1 promoter activity stimulated by AxCAS3-C or AdLacZ. In this assay, pGL3 without the Ref-1 promoter was used as a control. FIG. 8 is a bar graph showing the results of this assay. Each measured value is expressed as a magnification when the measured value for a sample not infected with adenovirus is 1. In FIG. 8, “*” indicates a significant difference at P <0.01 relative to uninfected sample (0MOI) and LacZ (25MOI) sample, and “**” indicates uninfected sample (0MOI) and LacZ (125MOI) sample. The significant difference in P <0.01 with respect to is shown. Each data is represented as an average value ± standard error (n = 5). AdLacZ moderately stimulated Ref-1 promoter activity, whereas Stat3-C significantly stimulated Ref-1 promoter activity in a dose-dependent manner (FIG. 8).

Effect of Stat3 on primary cultured hepatocytes In order to examine whether Stat3-C directly targets hepatocytes, experiments were performed using primary cultured mouse hepatocytes.

  FIG. 9 is a bar graph showing quantitative measurement results of apoptotic cell death of primary cultured hepatocytes by ELISA. In FIG. 9, “*” indicates a significant difference at P <0.05 relative to the adenovirus-uninfected Fas agonist-untreated sample, and “**” indicates P <0.01 relative to the LacZ / Fas agonist-treated sample. Significant difference is shown. Each data is represented as an average value ± standard error (n = 5).

  Exogenous Stat3-C was well expressed by infection of AxCAS3-C with 2MOI in primary cultured hepatocytes and reduced Fas-induced apoptosis (FIG. 9). By treatment with Stat3-C, Ref-1, FLIP, Bcl-2 and Bcl-XL proteins were overexpressed in primary cultured hepatocytes and liver. This experimental result shows that Stat3 directly targets at least hepatocytes and has anti-apoptotic and antioxidant effects on hepatocytes.

Effect of Stat3-C on reduction of liver ROS production and apoptosis by Ref-1 To investigate the role of Ref-1 in liver ROS production and Fas-mediated liver injury, an adeno encoding the antisense strand of the full-length Ref-1 gene Viral vector (AdFer) was administered to mice. Specifically, mice were inoculated with AdLacZ alone, AxCAS3-C: AdLacZ (1: 1), and AxCAS3-C: AdFer (1: 1), respectively, and LacZ mice, Stat3-C: LacZ mice, and Stat3. -C: AdFer (Stat3-C: Fer) mice were used.

  According to the immunoblot analysis for Ref-1 protein and Stat3 protein in the liver, the amount of Ref-1 protein was increased in Stat3-C mice co-infected with AdLacZ, whereas Stat3- coinfected with AdFer. In C mice, the amount of Ref-1 protein was decreased. Ref-1 protein was expressed in the liver of LS3-KO mice.

  FIG. 10 is a bar graph showing the results of liver reactive oxygen species assay in various mice. In FIG. 10, “*” indicates a significant difference at P <0.05 relative to the LacZ sample, “**” indicates a significant difference at P <0.05 relative to the LacZ / Fas agonist-administered sample, and “** “*” Indicates a significant difference at P <0.05 relative to the Stat3-C: LacZ / Fas agonist-administered sample. “Casp inh” means that a caspase inhibitor Z-VAD-fmk was administered, and “RLU” means a relative light unit. Each data is represented as an average value ± standard error (n = 5). Liver ROS was produced 1 hour after Jo2 administration, which was reduced by overexpression of Stat3-C and Ref-1, and also by pretreatment with NAC. However, the amount of ROS produced was not changed only by the caspase inhibitor. The antioxidant effect of Stat3-C was partially reduced by co-infection with AdFer. Stat3-C was considered to have an antioxidant effect via induction of Ref-1 (FIG. 10).

  FIG. 11 is a bar graph showing the results of liver tissue apoptosis assay in various mice. In FIG. 11, “*” indicates a significant difference at P <0.01 relative to the LacZ sample, “**” indicates a significant difference at P <0.05 relative to the LacZ / Fas agonist-administered sample, and “** “*” Indicates a significant difference at P <0.05 relative to the Stat3-C: LacZ / Fas agonist-administered sample. Each data is represented as an average value ± standard error (n = 5). Liver apoptosis induced by Jo2 which is a Fas agonist was suppressed by pretreatment with NAC, and was also suppressed by Stat3-C. The inhibitory effect of apoptosis by Stat3-C was reduced by co-infection with AdFer (FIG. 11).

  FIG. 12 is a bar graph showing the results of enzyme activity assays for caspase-3, caspase-8 and caspase-9 in the livers of various mice. In FIG. 12, “*” indicates a significant difference at P <0.05 relative to a LacZ / Fas agonist-administered sample. Each data is represented as an average value ± standard error (n = 5). From the results of the caspase activity assay, Ref-1 and NAC reduced caspase-3 activity to the same extent and partially reduced caspase-9 activity induced by Jo2 administration, but not caspase-8 activity. It became clear (FIG. 12).

  Ref-1 is thought to reduce caspase activity (caspase-3 and caspase-9) by reducing oxidative stress. From these results, Stat3 increases the expression level of Ref-1, which inactivates caspase-3 and caspase-9, and as a result, suppresses apoptosis. Is shown.

FIG. 1 is a diagram showing the results of intraperitoneal administration of 0.3 μg / g body weight of Jo2 to various mice and then examining the survival rate of the mice over 72 hours. FIG. 2 is a graph showing the concentrations of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in serum over 72 hours after Jo2 administration. FIG. 3 shows pretreatment with a general caspase inhibitor Z-VAD-fmk (1 μg / g) or an antioxidant NAC (200 mg / kg), and after 1 hour, apoptotic cell death was quantified by ELISA. It is a figure which shows the result. FIG. 4 shows the dose-dependent effect of caspase inhibitor (Z-VAD-fmk) or antioxidant (NAC) on liver apoptosis after Fas agonist administration. FIG. 5 is a diagram showing the results of an enzyme activity assay for caspase-3 and caspase-8 in the liver tissue of each mouse. FIG. 6 is a bar graph showing the results of a multi-probe ribonuclease protection assay for FLICE, FLIP, Fas, Ref-1, Bcl-xL and Bcl-2 in various mice. FIG. 7 is a diagram showing the change over time in the Ref-1 mRNA concentration in the liver tissue of each mouse after adenovirus infection. FIG. 8 is a bar graph showing the results of a luciferase reporter assay for Ref-1 promoter activity stimulated by AxCAS3-C or AdLacZ. FIG. 9 is a bar graph showing quantitative measurement results of apoptotic cell death of primary cultured hepatocytes by ELISA. FIG. 10 is a bar graph showing the results of liver reactive oxygen species assay in various mice. FIG. 11 is a bar graph showing the results of liver tissue apoptosis assay in various mice. FIG. 12 is a bar graph showing the results of enzyme activity assays for caspase-3, caspase-8 and caspase-9 in the livers of various mice.

Claims (24)

A method for identifying a drug for the treatment of a liver disorder induced by a Fas agonist, comprising:
(A) culturing cells capable of expressing the Stat3 gene in the presence of the candidate drug in the absence of the candidate drug under conditions that allow the cell to express the Stat3 gene; and
(B) measuring the intensity of activation of Stat3 in the cells obtained by step (a),
A method wherein a candidate drug is identified as a drug for the treatment of a liver disorder induced by a Fas agonist when Stat3 activation is enhanced by said candidate drug.   2. The method of claim 1, wherein the liver injury is due to reactive oxygen species induced by a Fas agonist.   The method according to claim 1, wherein the cell used in the step (a) is a mammalian cell.   2. The method of claim 1, wherein step (b) comprises measuring the amount of dimerized Stat3 protein in the cell.   The method of claim 1, wherein step (b) comprises measuring the amount of Stat3 protein in the nucleus of the cell.   The method of claim 1, wherein step (b) comprises measuring the amount of Stat3 protein bound to DNA in the cell.   If the strength of Stat3 activation in the cells obtained by step (a) is enhanced in comparison with the strength of Stat3 activation in control cells cultured in the absence of the candidate drug, the candidate drug 2. The method of claim 1, wherein is identified as a drug for the treatment of liver damage induced by a Fas agonist. A method for identifying a drug for the treatment of a liver disorder induced by a Fas agonist, comprising:
(A) a cell comprising a Ref-1 gene promoter and a reporter gene operably linked to the promoter and capable of expressing a Stat3 gene in the presence of the candidate drug in the absence of the candidate drug Culturing the cells under conditions that allow the Stat3 gene to be expressed, and
(B) measuring the expression level of the reporter gene in the cell obtained by step (a),
When the expression level of the reporter gene is increased by the candidate drug, the candidate drug is identified as a drug for the treatment of a liver disorder induced by a Fas agonist.   9. The method of claim 8, wherein the liver injury is due to reactive oxygen species induced by a Fas agonist.   The method according to claim 8, wherein the cell used in the step (a) is a mammalian cell.   9. The method of claim 8, wherein step (b) comprises quantifying protein from the reporter gene in a cell.   9. The method of claim 8, wherein step (b) comprises quantifying mRNA from the reporter gene in a cell.   When the expression level of the reporter gene in the cell obtained by the step (a) is increased in comparison with the expression level of the reporter gene in a control cell cultured in the absence of the candidate drug, the candidate drug 9. The method of claim 8, wherein is identified as a drug for the treatment of liver damage induced by a Fas agonist.   A pharmaceutical composition for treating a liver disorder induced by a Fas agonist, comprising a Stat3 activity enhancer and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 14, wherein the liver injury is caused by reactive oxygen species induced by a Fas agonistic substance.   The pharmaceutical composition according to claim 14, wherein the Stat3 activity enhancer enhances the activity of Stat3 that promotes transcription of the Ref-1 gene.   The pharmaceutical composition according to claim 14, wherein the Stat3 activity enhancer is an expression vector comprising DNA encoding Stat3.   The pharmaceutical composition according to claim 14, wherein the Stat3 activity enhancer is an expression vector comprising DNA encoding Stat3 modified so as to constitutively form dimerized Stat3 having activity.   A hepatocyte for use in the treatment of a liver disorder induced by a Fas agonist, comprising an expression vector comprising a DNA encoding Stat3.   The hepatocyte according to claim 19, wherein the liver injury is caused by reactive oxygen species induced by a Fas agonist.   The hepatocyte according to claim 19, wherein the hepatocyte is derived from an individual to be treated.   Hepatocytes for use in the treatment of Fas agonist-induced liver injury comprising an expression vector comprising DNA encoding Stat3 modified to constitutively form dimerized Stat3 having activity .   The hepatocyte according to claim 22, wherein the liver injury is caused by reactive oxygen species induced by a Fas agonistic substance.   The hepatocyte according to claim 22, wherein the hepatocyte is derived from an individual to be treated.

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