Depletion of Apoptosis Signal-Regulating Kinase 1 Prevents Bile Duct Ligation–Induced Necroinflammation and Subsequent Peribiliary Fibrosis
2014; Elsevier BV; Volume: 184; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2013.11.030
ISSN1525-2191
AutoresHirotsugu Noguchi, Sohsuke Yamada, Atsunori Nabeshima, Xin Guo, Akihide Tanimoto, Ke‐Yong Wang, Shohei Kitada, Takashi Tasaki, Tatsuo Takama, Shohei Shimajiri, Hasita Horlad, Yoshihiro Komohara, Hiroto Izumi, Kimitoshi Kohno, Hidenori Ichijo, Yasuyuki Sasaguri,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoApoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K), is ubiquitously expressed and situated in an important upstream position of many signal transduction pathways. ASK1 plays a pivotal role in stressor-induced cell survival and inflammatory reactions. To ascertain the regulatory functions of ASK1 in bile duct ligation (BDL)–induced liver injury, we examined the net effects of ASK1 depletion on hepatic necroinflammation and/or fibrosis. We subjected C57BL/6 wild-type (WT) or ASK1-deficient (ASK1−/−) mice to sham or BDL surgery for 14 days. In day 3 BDL animals, ASK1−/− mice had significantly fewer bile infarcts along with more reduced interlobular or portal inflammatory infiltrate of various immune cells, including neutrophils, compared with WT mice in which ASK1 expression was markedly activated. Morphologically apoptotic hepatocytes or cholangiocytes were negligible in both the sham and BDL animals. In contrast, ASK1−/− mice had significantly less proliferating activity of not only hepatocytes but also large cholangiocytes than WT mice. Day 14 BDL ASK1−/− mice manifested potential antifibrogenic aspects of ASK1 deficiency, characterized by significantly fewer activated peribiliary fibrogenic cells and peribiliary fibrosis. These observations indicate that ASK1-mediated hepatic necroinflammation and proliferation, but not apoptosis, are closely linked to liver fibrosis and fibrogenesis. A specific ASK1 pathway blocker or inhibitor might offer a therapeutic strategy against human cholestatic diseases. Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K), is ubiquitously expressed and situated in an important upstream position of many signal transduction pathways. ASK1 plays a pivotal role in stressor-induced cell survival and inflammatory reactions. To ascertain the regulatory functions of ASK1 in bile duct ligation (BDL)–induced liver injury, we examined the net effects of ASK1 depletion on hepatic necroinflammation and/or fibrosis. We subjected C57BL/6 wild-type (WT) or ASK1-deficient (ASK1−/−) mice to sham or BDL surgery for 14 days. In day 3 BDL animals, ASK1−/− mice had significantly fewer bile infarcts along with more reduced interlobular or portal inflammatory infiltrate of various immune cells, including neutrophils, compared with WT mice in which ASK1 expression was markedly activated. Morphologically apoptotic hepatocytes or cholangiocytes were negligible in both the sham and BDL animals. In contrast, ASK1−/− mice had significantly less proliferating activity of not only hepatocytes but also large cholangiocytes than WT mice. Day 14 BDL ASK1−/− mice manifested potential antifibrogenic aspects of ASK1 deficiency, characterized by significantly fewer activated peribiliary fibrogenic cells and peribiliary fibrosis. These observations indicate that ASK1-mediated hepatic necroinflammation and proliferation, but not apoptosis, are closely linked to liver fibrosis and fibrogenesis. A specific ASK1 pathway blocker or inhibitor might offer a therapeutic strategy against human cholestatic diseases. It was previously proposed that hepatocytes undergoing apoptosis might provide a critical hit to drive progression from chronic inflammation to cirrhosis, especially in cholestatic injury, such as primary biliary cirrhosis, primary sclerosing cholangitis, biliary atresia, or chronic cholelithiasis, with reference to a single laboratory.1Miyoshi H. Rust C. Roberts P.J. Burgart L.J. Gores G.J. Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas.Gastroenterology. 1999; 117: 669-677Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 2Canbay A. Higuchi H. Bronk S.F. Taniai M. Sebo T.J. Gores G.J. Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis.Gastroenterology. 2002; 123: 1323-1330Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 3Yoon J.H. Gores G.J. Death receptor-mediated apoptosis and the liver.J Hepatol. 2002; 37: 400-410Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 4Canbay A. Taimr P. Torok N. Higuchi H. Friedman S. Gores G.J. Apoptotic body engulfment by a human stellate cell line is profibrogenic.Lab Invest. 2003; 83: 655-663Crossref PubMed Scopus (348) Google Scholar In striking contrast, several other groups5Schoemaker M.H. Gommans W.M. Conde de la Rosa L. Homan M. Klok P. Trautwein C. van Goor H. Poelstra K. Haisma H.J. Jansen P.L. Moshage H. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Gujral J.S. Liu J. Farhood A. Jaeschke H. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.Hepatology. 2004; 40: 998-1007Crossref PubMed Scopus (95) Google Scholar, 7Fickert P. Trauner M. Fuchsbichler A. Zollner G. Wagner M. Marschall H.U. Zatloukal K. Denk H. Oncosis represents the main type of cell death in mouse models of cholestasis.J Hepatol. 2005; 42: 378-385Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Nalapareddy P.D. Schüngel S. Hong J.Y. Manns M.P. Jaeschke H. Vogel A. The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.Am J Pathol. 2009; 175: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Mitchell C. Mahrouf-Yorgov M. Mayeuf A. Robin M.A. Mansouri A. Fromenty B. Gilgenkrantz H. Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.Lab Invest. 2011; 91: 273-282Crossref PubMed Scopus (25) Google Scholar have recently suggested that there is no evidence of hepatocellular apoptosis using morphologic criteria, but not terminal deoxynucleotidyl transferase end-labeling (TUNEL) staining, in or around necrotic foci (ie, bile infarcts) after bile duct ligation (BDL), which is used to induce cholestasis in rodents.1Miyoshi H. Rust C. Roberts P.J. Burgart L.J. Gores G.J. Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas.Gastroenterology. 1999; 117: 669-677Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 2Canbay A. Higuchi H. Bronk S.F. Taniai M. Sebo T.J. Gores G.J. Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis.Gastroenterology. 2002; 123: 1323-1330Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 3Yoon J.H. Gores G.J. Death receptor-mediated apoptosis and the liver.J Hepatol. 2002; 37: 400-410Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 5Schoemaker M.H. Gommans W.M. Conde de la Rosa L. Homan M. Klok P. Trautwein C. van Goor H. Poelstra K. Haisma H.J. Jansen P.L. Moshage H. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Gujral J.S. Liu J. Farhood A. Jaeschke H. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.Hepatology. 2004; 40: 998-1007Crossref PubMed Scopus (95) Google Scholar, 7Fickert P. Trauner M. Fuchsbichler A. Zollner G. Wagner M. Marschall H.U. Zatloukal K. Denk H. Oncosis represents the main type of cell death in mouse models of cholestasis.J Hepatol. 2005; 42: 378-385Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Nalapareddy P.D. Schüngel S. Hong J.Y. Manns M.P. Jaeschke H. Vogel A. The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.Am J Pathol. 2009; 175: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Mitchell C. Mahrouf-Yorgov M. Mayeuf A. Robin M.A. Mansouri A. Fromenty B. Gilgenkrantz H. Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.Lab Invest. 2011; 91: 273-282Crossref PubMed Scopus (25) Google Scholar They also concluded that BDL-induced oncotic necrosis but not apoptosis of hepatocytes is closely correlated with the severity of the inflammatory response, including high chemokine and/or cytokine expression.6Gujral J.S. Liu J. Farhood A. Jaeschke H. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.Hepatology. 2004; 40: 998-1007Crossref PubMed Scopus (95) Google Scholar, 8Nalapareddy P.D. Schüngel S. Hong J.Y. Manns M.P. Jaeschke H. Vogel A. The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.Am J Pathol. 2009; 175: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Mitchell C. Mahrouf-Yorgov M. Mayeuf A. Robin M.A. Mansouri A. Fromenty B. Gilgenkrantz H. Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.Lab Invest. 2011; 91: 273-282Crossref PubMed Scopus (25) Google Scholar Meanwhile, although cholangiocytes are a minor component of liver cells, comprising merely 3% to 4% of the rodent liver, cells lining the large bile ducts (large cholangiocytes) are the main target in animal models of cholestasis and intractable human cholestatic diseases.10Lazaridis K.N. Strazzabosco M. Larusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 11Yahagi K. Ishii M. Kobayashi K. Ueno Y. Mano Y. Niitsuma H. Igarashi T. Toyota T. 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Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) family member that is activated through distinct mechanisms in response to various cytotoxic stressors, including oxidative stress mediated by hydrogen peroxide, endoplasmic reticulum (ER) stress, and immune system mediators, such as tumor necrosis factor (TNF)-α, IL-1β, or Fas ligands.15Ichijo H. Nishida E. Irie K. Digike P.T. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Induction of apoptosis by ASK1, mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.Science. 1997; 275: 90-94Crossref PubMed Scopus (2062) Google Scholar, 16Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1031) Google Scholar ASK1 is situated in an important upstream position for many signal transduction pathways, such as the c-Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK), which subsequently induce inflammation and intrinsic apoptotic signaling through mitochondria-dependent caspase activation.15Ichijo H. Nishida E. Irie K. Digike P.T. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Induction of apoptosis by ASK1, mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.Science. 1997; 275: 90-94Crossref PubMed Scopus (2062) Google Scholar, 16Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1031) Google Scholar, 17Yamada S. Ding Y. 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ASK1 regulates cardiomyocyte death but not hypertrophy in transgenic mice.Circ Res. 2009; 105: 1110-1117Crossref PubMed Scopus (72) Google Scholar Moreover, we recently found that activation of ASK1 signaling enhances hyperlipidemia-induced necrotic lipid core formation by inducing macrophage apoptosis and accelerates mechanical injury–induced vascular remodeling via increased neovascularization and proinflammatory reaction and by stimulating apoptosis of smooth muscle cells and/or endothelial cells.17Yamada S. Ding Y. Tanimoto A. Wang K.Y. Guo X. Li Z. Tasaki T. Nabesima A. Murata Y. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.Arterioscler Thromb Vasc Biol. 2011; 31: 1555-1564Crossref PubMed Scopus (40) Google Scholar, 20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.Am J Pathol. 2013; 182: 597-609Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar ASK1 plays a significant role in the regulation of vascular cell apoptosis and inflammatory signaling in vivo and is correlated with plaque vulnerability in atherosclerosis.20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.Am J Pathol. 2013; 182: 597-609Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Other studies report that ASK1 expression plays a critical role in vivo in the regulation of apoptosis in type 2 pneumocytes in lung injury, with both antiapoptotic and anti-inflammatory properties.21Makena P.S. Gorantla V.K. Ghosh M.C. Bezawada L. Kandasamy K. Balazs L. Luellen C.L. Thompson K.E. Parthasarathi K. Ichijo H. Waters C.M. Sinclair S.E. Deletion of apoptosis signal-regulating kinase-1 prevents ventilator-induced lung injury in mice.Am J Respir Cell Mol Biol. 2012; 46: 461-469Crossref PubMed Scopus (16) Google Scholar However, few studies have investigated the relationship between ASK1 signaling pathway and cholestasis-induced injury, even though ASK1 is ubiquitously expressed. In the current study, we examined the roles of ASK1 in BDL-induced cholestatic liver injury using mice genetically deficient for ASK1 (ASK1−/−). Furthermore, we aimed to determine the net effects and key factors of ASK1 in liver necroinflammation or subsequent fibrosis. Experiments were performed using 6- to 8-week-old, male, C57BL/6 wild-type (WT) and ASK1−/− mice,17Yamada S. Ding Y. Tanimoto A. Wang K.Y. Guo X. Li Z. Tasaki T. Nabesima A. Murata Y. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.Arterioscler Thromb Vasc Biol. 2011; 31: 1555-1564Crossref PubMed Scopus (40) Google Scholar, 20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.Am J Pathol. 2013; 182: 597-609Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar weighing 18 to 22 g, maintained in a temperature and light-controlled facility with free access to standard rodent chow diet and water. ASK1−/− mice were developed on a C57BL/6 background.17Yamada S. Ding Y. Tanimoto A. Wang K.Y. Guo X. Li Z. Tasaki T. Nabesima A. Murata Y. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.Arterioscler Thromb Vasc Biol. 2011; 31: 1555-1564Crossref PubMed Scopus (40) Google Scholar, 20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.Am J Pathol. 2013; 182: 597-609Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar To produce a ligation-induced cholestatic liver injury (BDL) model, the peritoneal cavity was opened after a midline upper-abdominal incision, and the common bile duct was double-ligated with sterile surgical 7-0 silk sutures (Alfresa Pharma Corp., Tokyo, Japan) and cut between the ligatures in two groups of mice at 6 to 8 weeks of age under anesthesia [intraperitoneal injection of ketamine (100 mg/kg) (Daiichi Sankyo Co., Tokyo, Japan) and medetamidine (2 mg/kg) (Meiji Yakuhin Co., Tokyo, Japan)], as previously described.20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.Am J Pathol. 2013; 182: 597-609Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Sham-operated mice, as controls, underwent laparotomy with exposure but no ligation of the common bile duct. The fascia and skin of the midline abdominal incision were closed with sterile surgical 6-0 silk sutures (Alfresa Pharma Corp.). After a defined period of BDL or sham operation at 3 or 14 days, animals were euthanized by exsanguination under reanesthetization with an i.p. injection of ketamine-medetamidine, as follows: the peritoneal cavity was reopened, and blood samples were taken from the inferior vena cava, followed by immediate cannulation of the suprahepatic vena cava. In all animals, after blood was flushed out of the liver via the suprahepatic vena cava catheter, livers were excised and cut into small pieces and used for various experiments as described below. The 24-hour urine of day 3 BDL mice was collected using mouse metabolic cages (Sugiyama-Gen Co., Ltd., Tokyo, Japan). The Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health (Japan), approved the protocols. They were performed according to the Institutional Guidelines for Animal Experiments and the Law (no. 105) and Notification (no. 6) of the Japanese government. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Liver specimens were stained with H&E, Masson's trichrome, or picrosirius red stain, or immunohistochemistry (IHC) preparations in sequential sections, after fixation in 10% neutral buffered formalin for 24 hours.17Yamada S. Ding Y. Tanimoto A. Wang K.Y. Guo X. Li Z. Tasaki T. Nabesima A. Murata Y. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.Arterioscler Thromb Vasc Biol. 2011; 31: 1555-1564Crossref PubMed Scopus (40) Google Scholar, 20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.Am J Pathol. 2013; 182: 597-609Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 22Yamada S. Wang K.Y. Tanimoto A. Fan J. Shimajiri S. Kitajima S. Morimoto M. Tsutui M. Watanabe T. Yasumoto K. Sasaguri Y. Matrix metalloproteinase 12 accelerates the initiation of atherosclerosis and stimulates the progression of fatty streaks to fibrous plaques in transgenic rabbits.Am J Pathol. 2008; 172: 1419-1429Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Ding Y. Yamada S. Wang K.Y. Shimajiri S. Guo X. Tanimoto A. Murata Y. Kitajima S. Watanabe T. Izumi H. Kohno K. Sasaguri Y. Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.Antioxid Redox Signal. 2010; 13: 1477-1490Crossref PubMed Scopus (73) Google Scholar, 24Guo X. Yamada S. Tanimoto A. Ding Y. Wang K.Y. Shimajiri S. Murata Y. Kimura S. Tasaki T. Nabeshima A. Watanabe T. Kohno K. Sasaguri Y. Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.Antioxid Redox Signal. 2012; 17: 1362-1375Crossref PubMed Scopus (54) Google Scholar, 25Nabeshima A. Yamada S. Guo X. Tanimoto A. Wang K.Y. Shimajiri S. Kimura S. Tasaki T. Noguchi H. Kitada S. Watanabe T. Fujii J. Kohno K. Sasaguri Y. Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.Antioxid Redox Signal. 2013; 19: 1983-1998Crossref PubMed Scopus (76) Google Scholar Analyses were performed in BDL-induced cholestatic livers in all experiments, whereas the sham-operated livers served as controls. Livers embedded in paraffin for histologic examination were cut systematically in sequential sections of 4-μm thickness using a sliding microtome (Leica SM2010R; Leica Microsystems, Wetzler, Germany). For histologic analyses of the liver, images of H&E and special stained sections or IHC sections were captured and quantified using NanoZoomer Digital Pathology Virtual Slide Viewer software version 2.0 (Hamamatsu Photonics Corp., Hamamatsu, Japan). H&E-stained liver sections were used to measure the areas of bile infarcts (hepatic necrosis) and calculate the percentage of necrotic area divided by total area. In addition, apoptotic cells were identified in 10 randomly selected fields of interlobular and portal tracts per these H&E sections (original magnification, ×400), using morphologic criteria (cell shrinkage, chromatin condensation and margination, and apoptotic bodies).5Schoemaker M.H. Gommans W.M. Conde de la Rosa L. Homan M. Klok P. Trautwein C. van Goor H. Poelstra K. Haisma H.J. Jansen P.L. Moshage H. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Gujral J.S. Liu J. Farhood A. Jaeschke H. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.Hepatology. 2004; 40: 998-1007Crossref PubMed Scopus (95) Google Scholar, 7Fickert P. Trauner M. Fuchsbichler A. Zollner G. Wagner M. Marschall H.U. Zatloukal K. Denk H. Oncosis represents the main type of cell death in mouse models of cholestasis.J Hepatol. 2005; 42: 378-385Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Nalapareddy P.D. Schüngel S. Hong J.Y. Manns M.P. Jaeschke H. Vogel A. The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.Am J Pathol. 2009; 175: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Mitchell C. Mahrouf-Yorgov M. Mayeuf A. Robin M.A. Mansouri A. Fromenty B. Gilgenkrantz H. Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.Lab Invest. 2011; 91: 273-282Crossref PubMed Scopus (25) Google Scholar Liver fibrosis was quantified by Masson's trichrome and picrosirius red staining (Picrosirius Red Stain Kit; Polysciences, Inc., Warrington, PA) in 10 randomly selected fields of portal and periportal tracts per section (original magnification, ×400). In periportal tracts, fibers stained red with yellow birefringence under polarized light were defined as expressing collagen type I.25Nabeshima A. Yamada S. Guo X. Tanimoto A. Wang K.Y. Shimajiri S. Kimura S. Tasaki T. Noguchi H. Kitada S. Watanabe T. Fujii J. Kohno K. Sasaguri Y. Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.Antioxid Redox Signal. 2013; 19: 1983-1998Crossref PubMed Scopus (76) Google Scholar The proportions of collagen content were calculated for individual images in cholestatic BDL livers, excluding large bile ducts (>15-μm diameter) and vessels. Serum levels of total bilirubin and hepatic injury–related enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and total bile acids, were measured using commercial assay kits (Wako Pure Chemical Co., Osaka, Japan). To examine the hepatic bile acids, snap frozen liver tissue (30 mg) was homogenized and extracted with chloroform-methanol (2/1 v/v) solution. The organic phase was dried and resolubilized in 2-propanol. Then the hepatic contents of bile acids were determined using commercial assay kits (Wako Pure Chemical Co.). Urinary bilirubin concentrations were normalized with creatinine levels. One representative sequential section per mouse was prepared for IHC staining and was captured and evaluated by a NanoZoomer Digital Pathology Virtual Slide Viewer (Hamamatsu Photonics Corp.) to avoid potential bias.17Yamada S. Ding Y. Tanimoto A. Wang K.Y. Guo X. Li Z. Tasaki T. Nabesima A. Murata Y. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.Arterioscler Thromb Vasc Biol. 2011; 31: 1555-1564Crossref PubMed Scopus (40) Google Scholar, 20Tasaki T. Yamada S. Guo X. Tanimoto A. Wang K.Y. Nabeshima A. Kitada S. Noguchi H. Kimura S. Shimajiri S. Kohno K. Ichijo H. Sasaguri Y. 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