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Biliary intervention aggravates cholestatic liver injury, and induces hepatic inflammation, proliferation and fibrogenesis in BDL mice
Experimental and Toxicologic Pathology 63 (2011) 277–284
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Biliary intervention aggravates cholestatic liver injury, and induces hepatic inflammation, proliferation and fibrogenesis in BDL mice Yang-An Wen a,1, Ding Liu b,1, Qian-Yun Zhou a, Shi-Feng Huang a, Peng Luo a, Yu Xiang a, Shan Sun a, Dan Luo a, Yu-Fang Dong a, Li-Ping Zhang a,n a b
Department of Clinical Laboratory, the First Affiliated Hospital, Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing 400016, PR China Department of Laboratory Medicine, Affiliated Daping Hospital, Third Military Medical University, Chongqing 400042, PR China
a r t i c l e in fo
abstract
Article history: Received 7 September 2009 Accepted 21 January 2010
Obstructive cholestasis occurs in various clinical situations, whose pathological process is complex and not well known. The present study was initiated to display the complex and multifaceted pathological process caused by obstructive cholestasis in bile duct-ligated mice. Adult mice were bile-duct-ligated or sham-operated, and serum and liver tissues were collected at the indicated time points. Automatic biochemical analyzer was used to monitor serum biochemical index; TUNEL, HE staining, immunohistochemistry and Real-time PCR were employed to evaluate liver apoptosis, necrosis, inflammation, as well as proliferation and fibrosis. Our results demonstrated that obstructive cholestasis led to elevated serum biochemical indicators, with ALT peaking at day 3, indicative of acute hepatic dysfunction. Meanwhile, the number of TUNEL-positive cells increased significantly, and by 2 weeks, mild to moderate necrosis became apparent in BDL mouse livers, which consequently aggravated hepatic inflammatory responses as was demonstrated by increased expression of KC-1, MIP2, ICAM-1 and MPO in BDL mouse livers. Moreover, proliferative hepatocytes around periportal areas, manifested by enhanced cell mitosis and elevated expression of proliferative markers such as PCNA and Ki67, increased significantly after BDL, while increased CK-19-positive cells in bile ducts indicated bile duct hyperplasia. By 2 weeks, numerous a-SMA-positive cells and Sirius-stained collagen were observed, indicative of hepatic stellate cells (HSC) activation and fibrogenesis. In conclusion, biliary intervention led to a multifaceted hepatic pathological process characterized by aggravated liver injury and inflammatory reaction with enhanced cellular proliferation and fibrogenesis. Crown Copyright & 2010 Published by Elsevier GmbH. All rights reserved.
Keywords: Bile duct ligation Obstructive cholestasis Proliferation Inflammatory Fibrogenesis
Introduction Obstructive jaundice occurs due to bile duct obstruction in a variety of clinical settings, such as gallstone impaction, biliary atresia, and tumor compression (Hofmann, 2002). To date, surgery remains the primary option to release bile duct obstruction. However, pre-existing jaundice, frequently associated with liver cell injury and liver dysfunction, leads to significant morbidity and mortality when major operations are performed (Martignoni et al., 2001). Given this complex etiology and pathological progression, a comprehensive insight into the disease process is a pre-requisite for the exploitation of the effective and rational therapeutic strategies. Cholestatic liver injury involves the interplay among chemokines, toxic bile acids, inflammatory cells, endotoxemia, reactive
n
Corresponding author. Tel./fax: + 86 023 89012756. E-mail address: [email protected] (L.-P. Zhang). 1 Yang-An Wen and Ding Liu contributed equally to this work.
oxygen species and changes of the mitochondrial permeability transition (Roggin et al., 2001; Houdijk et al., 1998; Gores et al., 1998). Cholestasis results in the accumulation of hydrophobic bile acids, which may be responsible for hepatocellular apoptosis and necrosis, with apoptosis being a nearly ubiquitous response of the liver to injury (Galle et al., 1995). In addition to apoptosis, virtually all liver diseases are associated with an inflammatory response. The interplay between hepatocyte inflammation and apoptosis is complex. As a consequence of initial injury, hepatic inflammation develops, characterized by the activation of Kupffer cells, release of cytokines and chemokines, and, subsequently, neutrophil recruitment to the liver (Koeppel et al., 1997; Lin et al., 1997; Saito and Maher, 2000). Apoptosis was demonstrated to induce CXC chemokines, potent chemotactic agents for neutrophils, in the liver (Koeppel et al., 1997), and inhibition of hepatocyte apoptosis can block the transmigration of neutrophil into the liver (Lin et al., 1997; Parola et al., 1996). Deleterious effects of these chemokines in obstructive jaundice require further examination, particularly in the case of biliary intervention. Concomitant with apoptosis and necrosis,
0940-2993/$ - see front matter Crown Copyright & 2010 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.01.006
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liver cell proliferation, as a pathological compensation to injury, is common, and the pathogenesis is not well understood. Hence, a detailed description of these pathogenic changes is important for the interpretation of the available data as well as for the design of future studies. In this study, a bile duct ligation mouse model was established and utilized to gain comprehensive insights into the disease process of biliary intervention, and the multifaceted hepatic pathological process, including hepatic dysfunction, liver injury, inflammation responses, hepatocyte proliferation, as well as fibrogenesis were profiled in detail.
Materials and methods Animals and BDL model Female BALB/c mice, 6–8 weeks old, weighting 20–25 g, were fed on a standard laboratory diet with water and food. Obstructive cholestasis was induced by ligation of the common bile duct. Bile duct ligation was performed as previously described in detail (Saito and Maher, 2000). Sham-operated mice, used as controls, underwent a laparotomy with exposure, but no common bile duct ligation was performed. Blood and liver tissues were collected at indicated time points. All the animals were treated in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory animals and all the experimental procedures were approved by Animal Care and Use Committee of the Chongqing Medical University. Blood chemistry Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin (DBIL) and total bilirubin (TBIL) levels were measured in serum obtained from bile duct-ligated and sham-operated mice, by a standard autoanalyzer (OLYMPUS AU5400). Histology and TUNEL assay The liver tissues were diced into 5 5 mm2 sections, fixed in 4% paraformaldehyde for 48 h, and then embedded in paraffin. Tissue sections were prepared with a microtome and placed on glass slides. Liver necrosis was graded semiquantitatively on the tissue sections stained by hematoxylin and eosin. Grading criteria was represented as follows: 0, no necrosis; 1, very few necrotic cells; 2, less than one-third of the lobule displayed necrosis; 3, one-third to two-thirds of the lobule displayed necrosis; 4, more than two-thirds of the lobule displayed necrosis. TUNEL assay was performed with a commercially available kit (BOSTER, Wuhan, China), according to the manufacturer’s instructions. Hepatocyte apoptosis around the periportal areas and Glisson’s sheath was quantitated by counting the number of TUNNEL-positive cells in 30 random microscopic fields ( 200). Real-time polymerase chain reaction Total liver RNA was isolated using Qiagen RNA easy kit (Invitrogen, CA, USA), quantified using the RNA 6000 Nano Assay program of the Agilent 2100 Bioanalyzer (Agilent Technologies) and then reverse transcribed into cDNA using Oligo(dT) primer and Superscript II Reverse Transcriptase (Invitrogen, CA, USA), according to manufacturer’s manual. Real-time polymerase chain reaction was performed on an ABI 7000 Real-time PCR Instrument (Applied Biosystems, Foster City, CA) with the Brilliant SYBR
Green QPCR Master Mix (Stratagene, CA, USA) to quantify KC-1, MIP-2, ICAM-1 and TATA-box binding protein (TBP). The sequences of the primers were as follows: MIP-2 forward: 50 -TTTTCTGTAAGCCCCGAGAAG-30 ; MIP-2 reverse: 50 -CAATTGCTAAACGAGGCACTG-30 ; KC-1 forward: 50 -TGGGATTCACCTCAAGAACA-30 ; KC-1 reverse: 50 -TGGGGACACCTTAGCATC-30 ; ICAM-1 forward: 50 -CTAAGAGGACTCGGTGGATGG-30 ; ICAM-1 reverse: 50 -GGGGACAATGTCTCAGCTTTC-30 ; a-SMA forward: 50 -CCGCAAATGCTTCTAAGTCC-30 ; a-SMA reverse: 50 -GGGGGCCACCCTATAATAAA-30 ; TBP forward: 50 -AGGGATTCAGGAAGACCACAT-30 ; TBP reverse: 50 -AAGTAGCAGCACAGAGCAAGC-30 . Stock solutions of primer pairs were made in water that contained each primer at a final concentration of 10 mM. Each real-time PCR reaction contained 1 ml of cDNA, 10 ml of primer pair stock solution, and 9 ml of SYBR green reagent. Cycling parameters were 95 1C for 10 min, 40 cycles of 95 1C (30 s), 55 1C (1 min), 72 1C (1 min), followed by a melting curve analysis. All reactions were performed in triplicate. The median cycle threshold (CT) value was analyzed and normalized with the reference dye. The expression was then normalized against the expression of housekeeping gene TBP. The specificity of the PCR amplification was confirmed by the dissociation curve analysis.
Immunohistochemistry The liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then prepared on glass slides as sections. The sections were stained for MPO using a rabbit polyclonal antibody (NeoMarkers, RB-373-R7), ICAM-1, a goat polyclonal antibody (Santa Cruz Biotechnology, sc-1511), CK-19, a goat polyclonal antibody (Santa Cruz Biotechnology, sc-33119), PCNA, a rat monoclonal antibody (NeoMarkers), and Ki-67, a rat monoclonal antibody (Dako), respectively, which was followed by a second reaction with biotin-labeled anti-rabbit IgG for MPO, anti-goat IgG for ICAM-1 and CK-19, anti-rat IgG for PCNA and Ki-67, respectively (Zhongshan Goldenbrige Biotechnology Co., Ltd., Beijing, China). An avidin–biotin coupling reaction was performed on the sections, using SP kit (Zhongshan Goldenbrige Biotechnology Co., Ltd., Beijing, China).
Statistical analysis All data are presented as mean7SEM and analyzed by t-test or analysis of variance (ANOVA). A value of p o0.05 was considered to be significantly different.
Results Biliary intervention resulted in liver dysfunction in BDL mice To investigate liver dysfunction after BDL, serum levels for ALT, AST, ALP, and bilirubin in sham group and BDL group (day 1, 3, 5, 7 and 14 after surgeries) were determined (Fig. 1). There was a progressive increase of serum AST and ALT in BDL mice, with ALT reaching its peak at day 3 after BDL. The difference between ALT and AST levels in the BDL group and those of the Sham group was significant, indicating impaired liver function after BDL. Meanwhile, serum ALP, TBIL and DBIL were also significantly increased in BDL mice, indicating severe obstructive cholestasis happening in BDL mice model.
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Hepatocellular apoptosis and necrosis after biliary intervention To investigate hepatocellular injury after BDL, liver cell apoptosis and necrosis were detected by TUNEL and histopathological staining. After TUNEL staining, TUNEL-positive cells were
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observed and counted under microscope (Fig. 2). Result showed that the number of the apoptotic hepatocytes was significantly higher (p o0.05) in BDL mice (35.2 75.3 per field) than that in the sham-operated mice (5.871.6 per field). Liver necrosis was also observed in BDL mice, which was absent in sham mouse livers. After biliary intervention, necrosis developed progressively, and by 2 weeks, significant necrosis of the liver in BDL mice was observed, indicating mild to moderate parenchymal liver injury (Fig. 3). Biliary intervention augmented inflammation responses in BDL mice
Fig. 1. Changes of serum hepatic biochemical index in BDL and sham mice. Serum parameters were measured 1, 3, 5, 7 and 14 days after BDL surgery. By 2 weeks after operation, Serum total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in BDL and sham mice were detected. Each group consists of five animals. Serum AST, ALT, TBIL, DBIL and ALP levels were increased significantly in BDL mice, compared with sham mice (p o0.01 for all BDL groups versus sham).
Sham operated
To investigate the inflammation reactions in the livers of the BDL mice, expression of several inflammation-associated cytokines were evaluated in mouse livers. To investigate hepatic chemokines expression, mRNA was extracted from the mouse livers, and real-time PCR was employed to detect the expression of KC-1, MIP-2 and ICAM-1. As presented in Fig. 4; 2 weeks after biliary intervention, the transcriptional expressions of KC, MIP-2 and ICAM-1 gene were significantly elevated in BDL mice, while remaining in low levels in sham mice. To evaluate hepatic neutrophil infiltrations, liver specimens from mice were immunohistochemically stained for MPO, a specific neutrophil marker, and ICAM-1, an adhesion molecule that is also important for neutrophil extravasation and activation in the liver. Results showed that livers from sham-operated mice had very few
BDL-14d
Fig. 2. TUNEL staining of livers of BDL and sham-operated mice (200 ). Apoptotic cells in BDL mice (B) livers were significantly increased by 2 weeks after biliary intervention. However, few apoptotic cells were seen in sham mouse livers (A).
Sham operated
BDL-14d
Fig. 3. Liver necrosis after bile duct ligation (100 ). Two weeks after biliary intervention, liver necrosis in grade 1 or 2 and necrotic foci were observed in BDL mice (B) but was not present in sham mouse liver (A).
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MPO-positive cells in sinusoids and portal venules. On the other hand, 2 weeks after BDL, MPO-positive cells increased significantly, and accumulated in sinusoids and portal venules and then extravasated into the parenchymal tissue and the portal tracts, respectively (Fig. 5). Similarly, ICAM-1 positive cells were
Fig. 4. Quantitative RT-PCR analysis of the expression of KC-1, MIP-2 and ICAM-1 gene in BDL and sham mouse livers at 2 weeks after surgery. Expression of KC-1, MIP-2 and ICAM-1 were normalized to TBP as internal control. The expression of KC-1, MIP-2 and ICAM-1 in mRNA levels were significantly higher in BDL mice than in sham mice (p o 0.05).
Sham operated
significantly increased in BDL mouse livers at 2 weeks after operation (Fig. 6).
Liver cells proliferation after biliary intervention-mediated injury Cellular proliferation is a common compensatory reaction of the liver to cellular apoptosis and necrosis. To investigate hepatocyte proliferation after liver injury, cellular mitosis and expressions of proliferation-associated antigens were examined in the livers of the BDL mice. HE staining of the liver sections showed that liver cells in mitotic phase and cells with double nuclei increased significantly in BDL mouse livers 2 weeks after biliary intervention (Fig. 7), indicating hepatocytic proliferation after cholestasis. PCNA, an intranuclear polypeptide expressed in proliferating cells, was found to be highly expressed in liver cells after BDL injury (Fig. 8). Similarly, another proliferationassociated index highly associated with mitosis, Ki-67-positive cells also increased significantly in liver sections after biliary intervention (Fig. 8). Moreover, to compare the proliferation level between perivenular and periportal areas after BDL, Ki-67positive cells were counted under 30 random microscopic fields ( 200), and results showed that there was no difference of Ki-67positive cells number between perivenular (4276.3) and periportal (3975.8) areas. To investigate the biliary epithelial cellular proliferation, the expression of CK-19, a specific biliary marker, was evaluated in the mouse liver specimens. CK-19
BDL-14d
Fig. 5. Immunohistochemical staining of the mouse livers using anti-MPO (200 ). MPO-positive cells were significantly increased in BDL mice (B) after biliary intervention, and interestingly, MPO-positive cells are more often observed in the portal triad.
Sham operated
BDL-14d
Fig. 6. Immunohistochemical staining of mouse liver using anti-ICAM-1 (400 ). Two weeks after bile duct ligation, ICAM-1-positive cells were significantly increased in BDL mice (B).
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Sham operated
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BDL-14d
Fig. 7. Liver cells mitosis evaluation using HE staining (200 ). Two weeks after BDL, liver cells in mitotic phase (cells with double nuclei) increased significantly (B).
Fig. 8. Immunohistochemical staining of proliferative markers in mouse livers (200 ). PCNA and Ki-67, as proliferative markers of liver cell, were located in nucleus and brown-stained. Compared with sham group, PCNA (A and B)- and Ki-67 (C and D)-positive cell number were significantly higher in bile duct ligated mice. CK-19 was mainly expressed in ductal and ductular cells in the BDL mouse livers (F), but nearly no CK-19 expression was observed in sham mouse livers (E). (left lanes: sham group; right lanes: BDL group).
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expressions were clearly visible in ductal and ductular cells in the liver sections 2 weeks after BDL, but little CK-19 expression was observed in sham mouse livers (Fig. 8). These data strongly
suggest an increased cellular proliferation following apoptosis and necrosis after BDL injury. Activation of hepatic stellate cells and fibrogenesis after BDL Real-time PCR and immunohistochemistry were performed to detect the markers of liver fibrosis. Hepatic stellate cells (HSC) activation after biliary intervention was observed by the detection of a-SMA expression in livers. At 5 and 14 days after biliary intervention, a-SMA mRNA levels in BDL mice were significantly higher than those in sham-operated controls, as were shown by real-time reverse transcriptional PCR (Fig. 9). Immunohistochemically, at 14 days after BDL, numerous a-SMApositive cells were observed around the proliferating bile ducts and blood vessels in the portal areas (Fig. 10). Moreover, a-SMA-positive cells had infiltrated hepatic parenchyma. Similar to a-SMA-positive cells, collagen fibers, staining red with Sirius red, were observed to be deposited around the proliferating bile ducts and blood vessels in the portal areas and infiltrated the hepatic lobules in BDL mice (Fig. 10).
Discussion Fig. 9. Quantitative RT-PCR determination of a-SMA in mouse liver after BDL. aSMA increased continuously after BDL, and its level at day 5 and day 14 was significantly higher than in sham-operated mice (p o0.01). The a-SMA value was normalized to TBP as internal control.
Sham operated
In this study, we established BDL model in BALB/c mice and utilized it to profile detailed pathological changes involving liver injury, inflammation and cellular proliferation after bile duct
BDL-14d
Fig. 10. Representative Immunohistochemical staining of a-SMA (A and B) and Sirius red staining of collagen (C and D) in the mouse livers ( 200). Numerous a-SMApositive cells (stained dark brown) accumulated around proliferating bile ducts and blood vessels and infiltrated hepatic parenchyma in BDL mice, indicating significantly increased a-SMA expression 2 weeks after biliary intervention. A marked deposition of collagen around the proliferating bile ducts and blood vessels in the portal areas was evidenced by the Sirius red stain, which tinges the fibers of collagen in red. (left lane: sham group; right lane: BDL group). (For interpretation of the references to colour in this figure legend, the reader is referenced to the web version of this article.)
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ligation. Rat and mouse BDL liver injury models have been widely used to dissect the molecular mechanisms underlying acute and chronic liver injury (Seki et al., 2009; Patsenker et al., 2009). Mouse BDL model has the added advantage of being amenable to easier genetic manipulations and pharmacological interventions. 2 weeks after BDL, bile duct walls became thin and stiff, and hepatic gap junctions disappeared with the formation of large crater-like fenestrae in ductular epithelium and the development of focal epithelial necrosis (Lawson et al., 1998; Jaeschke, 2002), which led to the leakage of toxic substances such as hydrophobic bile acids and induction of liver injury. We demonstrated that soon after biliary intervention, liver dysfunction developed as evidenced by the increased serum biochemical index including ALT, AST, ALP, TBIL and DBIL, which lasted for more than 2 weeks, indicative of serious hepatic impairment by obstructive cholestasis. Concomitant with liver dysfunction, hepatic apoptosis and necrosis, important stimuli for HSC activation and inducers of fibrogenesis (Gujral et al., 2004), developed 2 weeks after BDL. The investigation of the underlying mechanisms are currently underway, and the interplay among toxic bile acids, reactive oxygen species and leukocyte activation/ infiltration (Gujral et al., 2003; Jaeschke and Lemasters, 2003; Gujral et al., 2004) may be involved. Cellular apoptosis is no longer viewed merely as a silent consequence of liver injury, but rather as an important inflammatory stimulus (Martignoni et al., 2001), indicating the association between apoptosis and inflammation, which, in turn, causes further liver injury. Following cholestatic injury in BDL mice, we showed that there was a significant increase in the hepatic expression of CXC chemokines including KC and MIP-2, which were shown to have played important roles in neutrophil recruitment through their chemoattractant activities (Jaeschke, 1997). Besides chemotaxis to inflammatory cells, KC and MIP-2 can stimulate the generation of reactive oxygen species which are cytotoxic to liver cells (Jaeschke, 2002; Kobayashi, 2008). Intercellular adhesion molecules (ICAMs), known to be involved in the adherence and transmigration of neutrophils from portal venules (Bautista, 1998), were found to be significantly increased in BDL mice in our study, which was concordant with the previously reported result (Sprenger et al., 1997). Myeloperoxidase (MPO) was previously demonstrated to be abundantly expressed in neutrophils and monocytes (Sprenger et al., 1997; Bautista, 1998) and associated with oxygen absorption during inflammation, reflecting its role in the evaluation of both the amount of neutrophils and the degree of neutrophil activation. Increased hepatic expression of MPO in BDL mice in our study indicated that the aggravated inflammation may further impair liver functions through an oxygenic pathway. Cellular proliferation is a compensatory pathological reaction to hepatic injury (apoptosis and necrosis), which can be evaluated by the detection of cell mitosis or proliferation-related markers (Colozza et al., 2005). So far, the report of hepatocellular proliferation in bile duct ligation model is still missing. In our present study, we observed numerous cells in the mitotic phase and double-nuclei cells in BDL mouse livers, indicating increased hepatocytic proliferation after cholestatic injury. On the other hand, the expression level of PCNA, a molecular marker highly associated with cell cycle and proliferation (Bhattacharyya et al., 2008), was found to be significantly increased in BDL mice. As another proliferation-associated index highly associated with mitosis (Vilar, 2007), Ki-67 was widely applied in the detection of cell proliferation (Bereford, 2006), and the significantly elevated number of Ki-67-positive cells in BDL mice in the present study was another evidence for increased hepatocytic proliferation. Besides hepatocytic proliferation, biliary epithelial cell proliferation after BDL, as a response to liver injuries, is obvious. CK-19, a marker specific for bile duct epithelial cells, was
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widely applied in the detection of biliary epithelial cell proliferation (Xu et al., 2004; Varma and Cohen, 2004). Immunohistochemically higher hepatic expression level of CK-19 in proliferated ductal and ductular cells presented another evidence for biliary epithelial cell proliferation after BDL. Hepatic stellate cell plays an important role in the pathogenesis of hepatic fibrosis and cirrhosis (Friedman, 2008). Consistent with this hypothesis, we observed that after BDL, hepatic stellate cells undergo activation and transdifferentiation to myofibroblasts as evidenced by the progressively elevated expression of a-SMA and increasingly enhanced deposition of collagen. Taken together, obstructive cholestasis caused by biliary intervention led to complex and multifaceted pathological changes in BDL mouse livers, involving liver dysfunction, hepatocytes apoptosis and necrosis, inflammation and cellular proliferation, which are interrelated. The comprehensive view of this disease process is useful for both the diagnosis and treatment of obstructive cholestasis.
Acknowledgements This work was supported by the National Science Foundation of China (Grant no. 30670965), the Key Subject of Ministry of Education (Grant no. 207099) and the Subject of Chongqing Science and Technology Commission (Grant no. CSTC, 2006BB5030). References Bautista AP. The role of Kupffer cells and reactive oxygen species during acute and chronic ethanol intoxication. Alcohol Clin Exp Res 1998;22:255–9. Bereford MJ. Measuring proliferation in breast cancer: practicalities and applications. Breast Cancer Res 2006;8:216–26. Bhattacharyya NK, Chatterjee U, Sarkar S, Kundu AK. A study of proliferative activity, angiogenesis and nuclear grading in renal cell carcinoma. Indian J Pathol Microbiol 2008;511(1):17–21. Colozza M, Azambuja E, Cardoso F, Sotiriou C, Larsimont D, Piccart MJ. Proliferative markers as prognostic and predictive tools in early breast cancer: where are we now? Ann Oncol 2005;16(11):1723–39. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008;134: 1655–69. Galle PR, et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 1995;182:1223–30. Gores GJ, Miyoshi H, Botla R, Aguilar HI, Bronk SF. Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases. Biochim Biophys Acta 1998;1366:167–75. Gujral JS, Farhood A, Jaeschke H. Oncotic necrosis and caspasedependent apoptosis during galactosamine-induced liver injury in rats. Toxicol Appl Pharmacol 2003;190:37–46. Gujral JS, Liu J, Farhood A, Hinson JA, Jaeschke H. Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice. Am J Physiol Gastrointest Liver Physiol 2004;286:G499–507. Hofmann AF. Cholestatic liver disease: pathophysiology and therapeutic options. Liver 2002;22(Suppl. 2):14–9. Houdijk AP, van Lambalgen AA, Thijs LG, van Leeuwen PA. Gut endotoxin restriction improves postoperative hemodynamics in the bile duct-ligated rat. Shock 1998;9:282–8. Jaeschke H. Cellular adhesion molecules: regulation and functional significance in the pathogenesis of liver diseases. Am J Physiol Gastrointest Liver Physiol 1997;273:G602–11. Jaeschke H. Inflammation in response to hepatocellular apoptosis. Hepatology 2002;35:964–6. Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia reperfusion injury. Gastroenterology 2003;125:1246–57. Kobayashi Y. The role of chemokines in neutrophil biology. Front Biosci 2008;13:2400–7. Koeppel TA, Trauner M, Bass JC, Thies JC, Schlosser SF, Post S, et al. Extrahepatic biliary obstruction impairs microvascular perfusion and increases leukocyte adhesion in rat liver. Hepatology 1997;26:1085–91. Lawson JA, Fisher MA, Simmons CA, Farhood A, Jaeschke H. Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and Fas-antibodyinduced liver injury. Hepatology 1998;28:761–7. Lin M, Rippe RA, Niemela O, Brittenham G, Tsukamoto H. Role of iron in NF-B activation and cytokine gene expression by rat hepatic
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Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Biliary intervention aggravates cholestatic liver injury, and induces hepatic inflammation, proliferation and fibrogenesis in BDL mice Yang-An Wen a,1, Ding Liu b,1, Qian-Yun Zhou a, Shi-Feng Huang a, Peng Luo a, Yu Xiang a, Shan Sun a, Dan Luo a, Yu-Fang Dong a, Li-Ping Zhang a,n a b
Department of Clinical Laboratory, the First Affiliated Hospital, Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing 400016, PR China Department of Laboratory Medicine, Affiliated Daping Hospital, Third Military Medical University, Chongqing 400042, PR China
a r t i c l e in fo
abstract
Article history: Received 7 September 2009 Accepted 21 January 2010
Obstructive cholestasis occurs in various clinical situations, whose pathological process is complex and not well known. The present study was initiated to display the complex and multifaceted pathological process caused by obstructive cholestasis in bile duct-ligated mice. Adult mice were bile-duct-ligated or sham-operated, and serum and liver tissues were collected at the indicated time points. Automatic biochemical analyzer was used to monitor serum biochemical index; TUNEL, HE staining, immunohistochemistry and Real-time PCR were employed to evaluate liver apoptosis, necrosis, inflammation, as well as proliferation and fibrosis. Our results demonstrated that obstructive cholestasis led to elevated serum biochemical indicators, with ALT peaking at day 3, indicative of acute hepatic dysfunction. Meanwhile, the number of TUNEL-positive cells increased significantly, and by 2 weeks, mild to moderate necrosis became apparent in BDL mouse livers, which consequently aggravated hepatic inflammatory responses as was demonstrated by increased expression of KC-1, MIP2, ICAM-1 and MPO in BDL mouse livers. Moreover, proliferative hepatocytes around periportal areas, manifested by enhanced cell mitosis and elevated expression of proliferative markers such as PCNA and Ki67, increased significantly after BDL, while increased CK-19-positive cells in bile ducts indicated bile duct hyperplasia. By 2 weeks, numerous a-SMA-positive cells and Sirius-stained collagen were observed, indicative of hepatic stellate cells (HSC) activation and fibrogenesis. In conclusion, biliary intervention led to a multifaceted hepatic pathological process characterized by aggravated liver injury and inflammatory reaction with enhanced cellular proliferation and fibrogenesis. Crown Copyright & 2010 Published by Elsevier GmbH. All rights reserved.
Keywords: Bile duct ligation Obstructive cholestasis Proliferation Inflammatory Fibrogenesis
Introduction Obstructive jaundice occurs due to bile duct obstruction in a variety of clinical settings, such as gallstone impaction, biliary atresia, and tumor compression (Hofmann, 2002). To date, surgery remains the primary option to release bile duct obstruction. However, pre-existing jaundice, frequently associated with liver cell injury and liver dysfunction, leads to significant morbidity and mortality when major operations are performed (Martignoni et al., 2001). Given this complex etiology and pathological progression, a comprehensive insight into the disease process is a pre-requisite for the exploitation of the effective and rational therapeutic strategies. Cholestatic liver injury involves the interplay among chemokines, toxic bile acids, inflammatory cells, endotoxemia, reactive
n
Corresponding author. Tel./fax: + 86 023 89012756. E-mail address: [email protected] (L.-P. Zhang). 1 Yang-An Wen and Ding Liu contributed equally to this work.
oxygen species and changes of the mitochondrial permeability transition (Roggin et al., 2001; Houdijk et al., 1998; Gores et al., 1998). Cholestasis results in the accumulation of hydrophobic bile acids, which may be responsible for hepatocellular apoptosis and necrosis, with apoptosis being a nearly ubiquitous response of the liver to injury (Galle et al., 1995). In addition to apoptosis, virtually all liver diseases are associated with an inflammatory response. The interplay between hepatocyte inflammation and apoptosis is complex. As a consequence of initial injury, hepatic inflammation develops, characterized by the activation of Kupffer cells, release of cytokines and chemokines, and, subsequently, neutrophil recruitment to the liver (Koeppel et al., 1997; Lin et al., 1997; Saito and Maher, 2000). Apoptosis was demonstrated to induce CXC chemokines, potent chemotactic agents for neutrophils, in the liver (Koeppel et al., 1997), and inhibition of hepatocyte apoptosis can block the transmigration of neutrophil into the liver (Lin et al., 1997; Parola et al., 1996). Deleterious effects of these chemokines in obstructive jaundice require further examination, particularly in the case of biliary intervention. Concomitant with apoptosis and necrosis,
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liver cell proliferation, as a pathological compensation to injury, is common, and the pathogenesis is not well understood. Hence, a detailed description of these pathogenic changes is important for the interpretation of the available data as well as for the design of future studies. In this study, a bile duct ligation mouse model was established and utilized to gain comprehensive insights into the disease process of biliary intervention, and the multifaceted hepatic pathological process, including hepatic dysfunction, liver injury, inflammation responses, hepatocyte proliferation, as well as fibrogenesis were profiled in detail.
Materials and methods Animals and BDL model Female BALB/c mice, 6–8 weeks old, weighting 20–25 g, were fed on a standard laboratory diet with water and food. Obstructive cholestasis was induced by ligation of the common bile duct. Bile duct ligation was performed as previously described in detail (Saito and Maher, 2000). Sham-operated mice, used as controls, underwent a laparotomy with exposure, but no common bile duct ligation was performed. Blood and liver tissues were collected at indicated time points. All the animals were treated in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory animals and all the experimental procedures were approved by Animal Care and Use Committee of the Chongqing Medical University. Blood chemistry Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin (DBIL) and total bilirubin (TBIL) levels were measured in serum obtained from bile duct-ligated and sham-operated mice, by a standard autoanalyzer (OLYMPUS AU5400). Histology and TUNEL assay The liver tissues were diced into 5 5 mm2 sections, fixed in 4% paraformaldehyde for 48 h, and then embedded in paraffin. Tissue sections were prepared with a microtome and placed on glass slides. Liver necrosis was graded semiquantitatively on the tissue sections stained by hematoxylin and eosin. Grading criteria was represented as follows: 0, no necrosis; 1, very few necrotic cells; 2, less than one-third of the lobule displayed necrosis; 3, one-third to two-thirds of the lobule displayed necrosis; 4, more than two-thirds of the lobule displayed necrosis. TUNEL assay was performed with a commercially available kit (BOSTER, Wuhan, China), according to the manufacturer’s instructions. Hepatocyte apoptosis around the periportal areas and Glisson’s sheath was quantitated by counting the number of TUNNEL-positive cells in 30 random microscopic fields ( 200). Real-time polymerase chain reaction Total liver RNA was isolated using Qiagen RNA easy kit (Invitrogen, CA, USA), quantified using the RNA 6000 Nano Assay program of the Agilent 2100 Bioanalyzer (Agilent Technologies) and then reverse transcribed into cDNA using Oligo(dT) primer and Superscript II Reverse Transcriptase (Invitrogen, CA, USA), according to manufacturer’s manual. Real-time polymerase chain reaction was performed on an ABI 7000 Real-time PCR Instrument (Applied Biosystems, Foster City, CA) with the Brilliant SYBR
Green QPCR Master Mix (Stratagene, CA, USA) to quantify KC-1, MIP-2, ICAM-1 and TATA-box binding protein (TBP). The sequences of the primers were as follows: MIP-2 forward: 50 -TTTTCTGTAAGCCCCGAGAAG-30 ; MIP-2 reverse: 50 -CAATTGCTAAACGAGGCACTG-30 ; KC-1 forward: 50 -TGGGATTCACCTCAAGAACA-30 ; KC-1 reverse: 50 -TGGGGACACCTTAGCATC-30 ; ICAM-1 forward: 50 -CTAAGAGGACTCGGTGGATGG-30 ; ICAM-1 reverse: 50 -GGGGACAATGTCTCAGCTTTC-30 ; a-SMA forward: 50 -CCGCAAATGCTTCTAAGTCC-30 ; a-SMA reverse: 50 -GGGGGCCACCCTATAATAAA-30 ; TBP forward: 50 -AGGGATTCAGGAAGACCACAT-30 ; TBP reverse: 50 -AAGTAGCAGCACAGAGCAAGC-30 . Stock solutions of primer pairs were made in water that contained each primer at a final concentration of 10 mM. Each real-time PCR reaction contained 1 ml of cDNA, 10 ml of primer pair stock solution, and 9 ml of SYBR green reagent. Cycling parameters were 95 1C for 10 min, 40 cycles of 95 1C (30 s), 55 1C (1 min), 72 1C (1 min), followed by a melting curve analysis. All reactions were performed in triplicate. The median cycle threshold (CT) value was analyzed and normalized with the reference dye. The expression was then normalized against the expression of housekeeping gene TBP. The specificity of the PCR amplification was confirmed by the dissociation curve analysis.
Immunohistochemistry The liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then prepared on glass slides as sections. The sections were stained for MPO using a rabbit polyclonal antibody (NeoMarkers, RB-373-R7), ICAM-1, a goat polyclonal antibody (Santa Cruz Biotechnology, sc-1511), CK-19, a goat polyclonal antibody (Santa Cruz Biotechnology, sc-33119), PCNA, a rat monoclonal antibody (NeoMarkers), and Ki-67, a rat monoclonal antibody (Dako), respectively, which was followed by a second reaction with biotin-labeled anti-rabbit IgG for MPO, anti-goat IgG for ICAM-1 and CK-19, anti-rat IgG for PCNA and Ki-67, respectively (Zhongshan Goldenbrige Biotechnology Co., Ltd., Beijing, China). An avidin–biotin coupling reaction was performed on the sections, using SP kit (Zhongshan Goldenbrige Biotechnology Co., Ltd., Beijing, China).
Statistical analysis All data are presented as mean7SEM and analyzed by t-test or analysis of variance (ANOVA). A value of p o0.05 was considered to be significantly different.
Results Biliary intervention resulted in liver dysfunction in BDL mice To investigate liver dysfunction after BDL, serum levels for ALT, AST, ALP, and bilirubin in sham group and BDL group (day 1, 3, 5, 7 and 14 after surgeries) were determined (Fig. 1). There was a progressive increase of serum AST and ALT in BDL mice, with ALT reaching its peak at day 3 after BDL. The difference between ALT and AST levels in the BDL group and those of the Sham group was significant, indicating impaired liver function after BDL. Meanwhile, serum ALP, TBIL and DBIL were also significantly increased in BDL mice, indicating severe obstructive cholestasis happening in BDL mice model.
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Hepatocellular apoptosis and necrosis after biliary intervention To investigate hepatocellular injury after BDL, liver cell apoptosis and necrosis were detected by TUNEL and histopathological staining. After TUNEL staining, TUNEL-positive cells were
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observed and counted under microscope (Fig. 2). Result showed that the number of the apoptotic hepatocytes was significantly higher (p o0.05) in BDL mice (35.2 75.3 per field) than that in the sham-operated mice (5.871.6 per field). Liver necrosis was also observed in BDL mice, which was absent in sham mouse livers. After biliary intervention, necrosis developed progressively, and by 2 weeks, significant necrosis of the liver in BDL mice was observed, indicating mild to moderate parenchymal liver injury (Fig. 3). Biliary intervention augmented inflammation responses in BDL mice
Fig. 1. Changes of serum hepatic biochemical index in BDL and sham mice. Serum parameters were measured 1, 3, 5, 7 and 14 days after BDL surgery. By 2 weeks after operation, Serum total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in BDL and sham mice were detected. Each group consists of five animals. Serum AST, ALT, TBIL, DBIL and ALP levels were increased significantly in BDL mice, compared with sham mice (p o0.01 for all BDL groups versus sham).
Sham operated
To investigate the inflammation reactions in the livers of the BDL mice, expression of several inflammation-associated cytokines were evaluated in mouse livers. To investigate hepatic chemokines expression, mRNA was extracted from the mouse livers, and real-time PCR was employed to detect the expression of KC-1, MIP-2 and ICAM-1. As presented in Fig. 4; 2 weeks after biliary intervention, the transcriptional expressions of KC, MIP-2 and ICAM-1 gene were significantly elevated in BDL mice, while remaining in low levels in sham mice. To evaluate hepatic neutrophil infiltrations, liver specimens from mice were immunohistochemically stained for MPO, a specific neutrophil marker, and ICAM-1, an adhesion molecule that is also important for neutrophil extravasation and activation in the liver. Results showed that livers from sham-operated mice had very few
BDL-14d
Fig. 2. TUNEL staining of livers of BDL and sham-operated mice (200 ). Apoptotic cells in BDL mice (B) livers were significantly increased by 2 weeks after biliary intervention. However, few apoptotic cells were seen in sham mouse livers (A).
Sham operated
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Fig. 3. Liver necrosis after bile duct ligation (100 ). Two weeks after biliary intervention, liver necrosis in grade 1 or 2 and necrotic foci were observed in BDL mice (B) but was not present in sham mouse liver (A).
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MPO-positive cells in sinusoids and portal venules. On the other hand, 2 weeks after BDL, MPO-positive cells increased significantly, and accumulated in sinusoids and portal venules and then extravasated into the parenchymal tissue and the portal tracts, respectively (Fig. 5). Similarly, ICAM-1 positive cells were
Fig. 4. Quantitative RT-PCR analysis of the expression of KC-1, MIP-2 and ICAM-1 gene in BDL and sham mouse livers at 2 weeks after surgery. Expression of KC-1, MIP-2 and ICAM-1 were normalized to TBP as internal control. The expression of KC-1, MIP-2 and ICAM-1 in mRNA levels were significantly higher in BDL mice than in sham mice (p o 0.05).
Sham operated
significantly increased in BDL mouse livers at 2 weeks after operation (Fig. 6).
Liver cells proliferation after biliary intervention-mediated injury Cellular proliferation is a common compensatory reaction of the liver to cellular apoptosis and necrosis. To investigate hepatocyte proliferation after liver injury, cellular mitosis and expressions of proliferation-associated antigens were examined in the livers of the BDL mice. HE staining of the liver sections showed that liver cells in mitotic phase and cells with double nuclei increased significantly in BDL mouse livers 2 weeks after biliary intervention (Fig. 7), indicating hepatocytic proliferation after cholestasis. PCNA, an intranuclear polypeptide expressed in proliferating cells, was found to be highly expressed in liver cells after BDL injury (Fig. 8). Similarly, another proliferationassociated index highly associated with mitosis, Ki-67-positive cells also increased significantly in liver sections after biliary intervention (Fig. 8). Moreover, to compare the proliferation level between perivenular and periportal areas after BDL, Ki-67positive cells were counted under 30 random microscopic fields ( 200), and results showed that there was no difference of Ki-67positive cells number between perivenular (4276.3) and periportal (3975.8) areas. To investigate the biliary epithelial cellular proliferation, the expression of CK-19, a specific biliary marker, was evaluated in the mouse liver specimens. CK-19
BDL-14d
Fig. 5. Immunohistochemical staining of the mouse livers using anti-MPO (200 ). MPO-positive cells were significantly increased in BDL mice (B) after biliary intervention, and interestingly, MPO-positive cells are more often observed in the portal triad.
Sham operated
BDL-14d
Fig. 6. Immunohistochemical staining of mouse liver using anti-ICAM-1 (400 ). Two weeks after bile duct ligation, ICAM-1-positive cells were significantly increased in BDL mice (B).
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Sham operated
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BDL-14d
Fig. 7. Liver cells mitosis evaluation using HE staining (200 ). Two weeks after BDL, liver cells in mitotic phase (cells with double nuclei) increased significantly (B).
Fig. 8. Immunohistochemical staining of proliferative markers in mouse livers (200 ). PCNA and Ki-67, as proliferative markers of liver cell, were located in nucleus and brown-stained. Compared with sham group, PCNA (A and B)- and Ki-67 (C and D)-positive cell number were significantly higher in bile duct ligated mice. CK-19 was mainly expressed in ductal and ductular cells in the BDL mouse livers (F), but nearly no CK-19 expression was observed in sham mouse livers (E). (left lanes: sham group; right lanes: BDL group).
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expressions were clearly visible in ductal and ductular cells in the liver sections 2 weeks after BDL, but little CK-19 expression was observed in sham mouse livers (Fig. 8). These data strongly
suggest an increased cellular proliferation following apoptosis and necrosis after BDL injury. Activation of hepatic stellate cells and fibrogenesis after BDL Real-time PCR and immunohistochemistry were performed to detect the markers of liver fibrosis. Hepatic stellate cells (HSC) activation after biliary intervention was observed by the detection of a-SMA expression in livers. At 5 and 14 days after biliary intervention, a-SMA mRNA levels in BDL mice were significantly higher than those in sham-operated controls, as were shown by real-time reverse transcriptional PCR (Fig. 9). Immunohistochemically, at 14 days after BDL, numerous a-SMApositive cells were observed around the proliferating bile ducts and blood vessels in the portal areas (Fig. 10). Moreover, a-SMA-positive cells had infiltrated hepatic parenchyma. Similar to a-SMA-positive cells, collagen fibers, staining red with Sirius red, were observed to be deposited around the proliferating bile ducts and blood vessels in the portal areas and infiltrated the hepatic lobules in BDL mice (Fig. 10).
Discussion Fig. 9. Quantitative RT-PCR determination of a-SMA in mouse liver after BDL. aSMA increased continuously after BDL, and its level at day 5 and day 14 was significantly higher than in sham-operated mice (p o0.01). The a-SMA value was normalized to TBP as internal control.
Sham operated
In this study, we established BDL model in BALB/c mice and utilized it to profile detailed pathological changes involving liver injury, inflammation and cellular proliferation after bile duct
BDL-14d
Fig. 10. Representative Immunohistochemical staining of a-SMA (A and B) and Sirius red staining of collagen (C and D) in the mouse livers ( 200). Numerous a-SMApositive cells (stained dark brown) accumulated around proliferating bile ducts and blood vessels and infiltrated hepatic parenchyma in BDL mice, indicating significantly increased a-SMA expression 2 weeks after biliary intervention. A marked deposition of collagen around the proliferating bile ducts and blood vessels in the portal areas was evidenced by the Sirius red stain, which tinges the fibers of collagen in red. (left lane: sham group; right lane: BDL group). (For interpretation of the references to colour in this figure legend, the reader is referenced to the web version of this article.)
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ligation. Rat and mouse BDL liver injury models have been widely used to dissect the molecular mechanisms underlying acute and chronic liver injury (Seki et al., 2009; Patsenker et al., 2009). Mouse BDL model has the added advantage of being amenable to easier genetic manipulations and pharmacological interventions. 2 weeks after BDL, bile duct walls became thin and stiff, and hepatic gap junctions disappeared with the formation of large crater-like fenestrae in ductular epithelium and the development of focal epithelial necrosis (Lawson et al., 1998; Jaeschke, 2002), which led to the leakage of toxic substances such as hydrophobic bile acids and induction of liver injury. We demonstrated that soon after biliary intervention, liver dysfunction developed as evidenced by the increased serum biochemical index including ALT, AST, ALP, TBIL and DBIL, which lasted for more than 2 weeks, indicative of serious hepatic impairment by obstructive cholestasis. Concomitant with liver dysfunction, hepatic apoptosis and necrosis, important stimuli for HSC activation and inducers of fibrogenesis (Gujral et al., 2004), developed 2 weeks after BDL. The investigation of the underlying mechanisms are currently underway, and the interplay among toxic bile acids, reactive oxygen species and leukocyte activation/ infiltration (Gujral et al., 2003; Jaeschke and Lemasters, 2003; Gujral et al., 2004) may be involved. Cellular apoptosis is no longer viewed merely as a silent consequence of liver injury, but rather as an important inflammatory stimulus (Martignoni et al., 2001), indicating the association between apoptosis and inflammation, which, in turn, causes further liver injury. Following cholestatic injury in BDL mice, we showed that there was a significant increase in the hepatic expression of CXC chemokines including KC and MIP-2, which were shown to have played important roles in neutrophil recruitment through their chemoattractant activities (Jaeschke, 1997). Besides chemotaxis to inflammatory cells, KC and MIP-2 can stimulate the generation of reactive oxygen species which are cytotoxic to liver cells (Jaeschke, 2002; Kobayashi, 2008). Intercellular adhesion molecules (ICAMs), known to be involved in the adherence and transmigration of neutrophils from portal venules (Bautista, 1998), were found to be significantly increased in BDL mice in our study, which was concordant with the previously reported result (Sprenger et al., 1997). Myeloperoxidase (MPO) was previously demonstrated to be abundantly expressed in neutrophils and monocytes (Sprenger et al., 1997; Bautista, 1998) and associated with oxygen absorption during inflammation, reflecting its role in the evaluation of both the amount of neutrophils and the degree of neutrophil activation. Increased hepatic expression of MPO in BDL mice in our study indicated that the aggravated inflammation may further impair liver functions through an oxygenic pathway. Cellular proliferation is a compensatory pathological reaction to hepatic injury (apoptosis and necrosis), which can be evaluated by the detection of cell mitosis or proliferation-related markers (Colozza et al., 2005). So far, the report of hepatocellular proliferation in bile duct ligation model is still missing. In our present study, we observed numerous cells in the mitotic phase and double-nuclei cells in BDL mouse livers, indicating increased hepatocytic proliferation after cholestatic injury. On the other hand, the expression level of PCNA, a molecular marker highly associated with cell cycle and proliferation (Bhattacharyya et al., 2008), was found to be significantly increased in BDL mice. As another proliferation-associated index highly associated with mitosis (Vilar, 2007), Ki-67 was widely applied in the detection of cell proliferation (Bereford, 2006), and the significantly elevated number of Ki-67-positive cells in BDL mice in the present study was another evidence for increased hepatocytic proliferation. Besides hepatocytic proliferation, biliary epithelial cell proliferation after BDL, as a response to liver injuries, is obvious. CK-19, a marker specific for bile duct epithelial cells, was
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widely applied in the detection of biliary epithelial cell proliferation (Xu et al., 2004; Varma and Cohen, 2004). Immunohistochemically higher hepatic expression level of CK-19 in proliferated ductal and ductular cells presented another evidence for biliary epithelial cell proliferation after BDL. Hepatic stellate cell plays an important role in the pathogenesis of hepatic fibrosis and cirrhosis (Friedman, 2008). Consistent with this hypothesis, we observed that after BDL, hepatic stellate cells undergo activation and transdifferentiation to myofibroblasts as evidenced by the progressively elevated expression of a-SMA and increasingly enhanced deposition of collagen. Taken together, obstructive cholestasis caused by biliary intervention led to complex and multifaceted pathological changes in BDL mouse livers, involving liver dysfunction, hepatocytes apoptosis and necrosis, inflammation and cellular proliferation, which are interrelated. The comprehensive view of this disease process is useful for both the diagnosis and treatment of obstructive cholestasis.
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