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. Author manuscript; available in PMC: 2012 Jun 6.
Published in final edited form as: Mol Pharm. 2011 May 6;8(3):958–968. doi: 10.1021/mp200115v

Cyclopamine attenuates acute warm ischemia reperfusion injury in cholestatic rat liver: Hope for marginal livers

Akshay Pratap 1, Ravi Pannakanti 2, Ningning Yang 2, Ramasubramanian Lakshmi 3, Kian A Modanlou 1, James D Eason 1, Ram I Mahato 2,*
PMCID: PMC3121156  NIHMSID: NIHMS289750  PMID: 21491930

Abstract

Cholestasis is a significant risk factor for immediate hepatic failure due to ischemia reperfusion (I/R) injury in patients undergoing liver surgery or transplantation. We recently demonstrated that inhibition of Hedgehog (Hh) signaling with cyclopamine (CYA) before I/R prevents liver injury. In this study we hypothesized that Hedgehog (Hh) signaling may modulate I/R injury in cholestatic rat liver. Cholestasis was induced by bile duct ligation (BDL). Seven days after BDL, rats were exposed to either CYA or vehicle for 7 d daily before subjecting them to 30 min of ischemia and 4 h of reperfusion. Expression of Hh ligands (sonic hedgehog, Patched-1 and Glioblastoma-1), assessment of liver injury, neutrophil infiltration, cytokines, lipid peroxidation, cell proliferation and apoptosis were determined. Significant upregulation of Hh ligands was seen in vehicle treated BDL rats. I/R injury superimposed on these animals resulted in markedly elevated serum alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin accompanied with increased neutrophil recruitment and lipid peroxidation. Preconditioning with CYA reduced the histological damage and serum liver injury markers. CYA also reduced neutrophil infiltration, proinflammatory cytokines such as TNF-α and IL-1β expression of α-smooth muscle actin and Type 1 collagen resulting in reduced fibrosis. Furthermore CYA treated animals showed reduced cholangiocyte proliferation, and apoptosis. Hepatoprotection by CYA was conferred by reduced activation of protein kinase B (Akt) and extracellular signal regulated kinase (ERK). Endogenous Hh signaling in cholestasis exacerbates inflammatory injury during liver I/R. Blockade of Hh pathway represents a clinically relevant novel approach to limit I/R injury in cholestatic marginal liver.

Keywords: Preconditioning, bile duct ligation, partial ischemia reperfusion, hepatocellular injury

INTRODUCTION

The number of cadaveric organ donors has remained static while the candidate waiting list for organ transplant has grown exponentially. This growing disparity in supply and demand has catalyzed the use of marginal grafts which are either steatotic or cholestatic.1,2 Marginal livers have an increased susceptibility to cold and warm ischemia reperfusion (I/R) injury resulting in poor graft outcomes.3–5 Cholestasis is a major risk factor for oxidative stress and complications of I/R injury. Recent studies have clearly established a critical role for neutrophils in the mechanism of hepatocyte injury during cholestasis.6 The mechanism by which cholestasis triggers the production of proinflammatory mediators that promote accumulation and activation of neutrophils in the liver, however, remains unknown. Elucidation of this pathway could provide clinically relevant insight into ways to prevent inflammation from occurring in cholestatic liver during I/R. One potential regulator of inflammation in the cholestatic liver may be the Hedgehog (Hh) signaling pathway. Hh ligands are soluble factors that interact with the plasma membrane-spanning receptor, Patched (Ptch), to de-repress the downstream intracellular signaling intermediate, Smoothened (Smo). Smo-initiated signals, in turn, activate Gli-family transcription factors to induce expression of Hh-responsive genes that regulate target cell proliferation, viability and differentiation.7 The mechanisms by which Gli genes are regulated in response to Hh signaling are still not completely understood. Unlike Gli1, which represents a direct transcriptional Hh target gene, Gli2 and also Gli3 are considered latent transcriptional regulators activated by Hh signaling. In fact, induction of Gli1 mRNA expression by Hh signaling is a reliable marker for pathway.8, 9 During embryogenesis, Hh ligands function as morphogens by modulating both mesendodermal fate and epithelial-mesenchymal transitions (EMT).10, 11 Consistent with these observations there is a growing evidence that Hh signaling regulates remodeling of various adult tissues, including the nervous system, skin, heart, lung, and gastrointestinal tract. Excessive Hh activity has also been noted in cancers arising in these tissues. 12–14 We have recently shown that Hh signaling is upregulated in normal liver after I/R injury and its inhibition confers hepatoprotection.15 Whether Hh is an essential regulator of inflammation during cholestasis, however, remains to be investigated. The studies presented herein tested the hypothesis that Hh is instrumental in hepatic injury during I/R of cholestatic liver. To this end, expression of Hh ligands and effect of inhibition Hh pathway by preconditioning with cyclopamine (CYA) was evaluated in the rat liver with cholestasis.

MATERIAL AND METHODS

Materials

CYA free base was purchased from LC Laboratories (Woburn, MA). Goat anti-rabbit SHH, Gli-1, Patch-1, TNF-α, collagen Type 1 and IL-1β primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho ERK, total ERK, phospho Akt, and total Akt primary antibodies were purchased from Cell Signaling (Danvers, MA). Goat anti-mouse CK-7, beta actin and α-smooth muscle actin were purchased from Abcam (Cambridge, MA). Mouse Proliferating cell nuclear antigen (PCNA) monoclonal antibody (Clone IPO-38) and TBARS assay kit was purchased from Cayman Chemical Company (Ann Arbor, MI). Goat anti-rabbit Alexa Fluor 488 and Alexa Fluor 594, rabbit anti-goat Alexa Fluor 488 and goat anti mouse 594 secondary antibodies and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA). SYBR Green real-time PCR master mix and reverse transcription reagents were purchases from Applied Biosystems (Foster city, CA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted.

Animals

Animal experiments were performed as per the NIH (http://grants1.nih.gov/grants/olaw/references/phspol.htm) and institutional animal care and use guidelines using the protocol approved by the University of Tennessee Animal Use and Care Committee. Animals were anesthetized with isoflurane.

Bile duct ligation

Bile duct ligation (BDL) or sham operation was performed as described previously16. After a midline laparotomy the bile duct was doubly ligated with 4-0 silk and transected. The sham operation was performed similarly, with the exception of ligating and transecting the bile duct.

Ischemia reperfusion

Ischemia reperfusion injury (I/R) injury was done 7 days after BDL as described earlier.17, 18 An atraumatic vascular clip was placed to interrupt the blood supply. The abdomen was closed with sterile staple sutures to prevent dehydration and possible contamination. The animals were kept in the recovery room under close supervision. After 30 min of partial hepatic ischemia, the clamp was removed and reperfusion was resumed. Sham operated mice underwent the same procedure without vascular clamping. The abdomen was closed in a double layer using 4-0 nylon. During the reperfusion, the mice were kept in clean cages. After 4h of reperfusion, animals were euthanized for blood and tissue collection.

Drug administration and sample collection

Rats were allocated randomly into the following groups, 5 rats per group: sham controls; I/R injury; BDL with I/R injury, underwent daily administration of beta cyclodextrin solution (10 mg/kg/day daily); BDL with I/R injury underwent daily administration of intraperitoneal CYA (10 mg/kg/day daily). Both vehicle and CYA treatment were started 3 days after BDL and continued once a day for 7 days. On the 14th day animals were subjected to I/R injury as described above and after 4 h of reperfusion were euthanized. Blood was collected via cardiac puncture and serum was collected immediately after euthanasia. Liver samples were immediately frozen in liquid nitrogen, or fixed in 10% neutral buffered formalin (NBF) prior to embedment in paraffin.

Assessment of Liver Injury

Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin were used as markers of liver injury. Their levels were measured using IDTox™ Alanine Transaminase color endpoint assay kit, IDTox™ Aspartate Transaminase (AST) Enzyme Assay Kit (ID Labs™ Inc, London, ON, Canada) and IDTox.™ Total bilirubin levels were measured using a MaxDiscovery™ Total bilirubin assay kit from Bio scientific (Austin, TX) and absorbance at 560 nm was measured using a UV spectrophotometer.

Hematoxylin-Eosin (H&E) Staining

Liver specimens were fixed in 10% NBF and embedded in paraffin. Liver sections (5μm) were stained with Hematoxylin/Eosin, and then analyzed blindly. The histological severity of I/R injury was graded using Suzuki’s criteria 19 Liver sections were also analyzed to evaluate biliary proliferation. Ten portal tracts were examined. A score of 0 was given if there were no proliferating ducts, a score of 1 for greater than 0 but less than 10%, a score of 2 for proliferation between 10% and 25%, a score of 3 for ducts greater than 25% but less than 50% and a score of 4 for proliferating ducts greater than 50%.

Hepatic Neutrophil infiltration

To assess neutrophil accumulation in the livers, tissue sections were stained for chloroacetate esterase present on neutrophils, using a Naphthol-ASD Chloroacetate Esterase Kit (Sigma Aldrich, St. Louis, MO). The numbers of neutrophils present in the sinusoids and extravasated into the parenchymal tissue were counted in 20 high-power fields. All cell counts were performed in a blinded fashion.

Lipid Peroxidation

Oxidative stress in the cellular environment results in the formation of highly reactive and unstable lipid hydroperoxides.20 Decomposition of the unstable peroxides derived from polyunsaturated fatty acids results in the formation of malondialdehyde (MDA), which can be quantified colorimetrically following its controlled reaction with thiobarbituric acid (TBARS).21 MDA assay from liver homogenates was performed using TBARS assay kit (Cayman Chemical Company, Ann Arbor, MI) using the manufacturer’s instructions.

Proliferating Cell Nuclear Antigen Immunohistochemistry

PCNA Immunoflorescence staining was performed on 5 μm fresh frozen sections using mouse monoclonal PCNA antibody.

TUNEL

Fresh frozen tissue sections (5 μm) were prepared, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed using in situ cell death detection kit (Promega, Madison, WI). Hepatocyte apoptosis in liver sections was quantitated by counting the number of TUNEL-positive cells in 10 random microscopic fields (X40), as previously described.22

Real Time Polymerase Chain Reaction

Shh, Patch-1, Gli-1, TNF-α, and IL-1β gene expression in rat livers was determined by real time PCR as described before, Briefly, total liver RNA was extracted and reverse transcribed to cDNA templates, followed by reverse transcription.. In all 100ng of cDNA was amplified by real-time PCR using SYBR Green dye universal master mix on an LightCycler®480 (LC 480) (Applied Biosystems, Inc., Foster City, CA) using the primers for Shh (NCBI Accession# NM_017221), Patched-1(NCBI Accession#; NM_053566), Gli-1 (NCBI Accession# XM_345832), TNF-α (NCBI Accession# NM_012675) and IL-1β (NCBI Accession# NM_031512). Following melting curve analysis, crossing point (Cp) was used for calculating the relative amount of mRNA compared to the house keeping gene, hypoxanthinephophoribosyltransferase (HPRT), and then scaled relative to controls, where control samples were set at a value of 1. Thus, results for all experimental samples were graphed as relative expression compared with the control.

Immunofluorescent Staining

Immunofluorescent staining was performed on snap frozen liver tissue. Briefly, 5μm cryosections were cut on lysine coated slides and fixed in 95% cold ethanol. Slides were air dried and stored at −80ºC till further use. The sections were blocked with 10% goat serum with 1% BSA in TBS for 2h at room temperature. Cryosections then incubated with the following primary antibodies overnight at 4°C: Shh, Ptch-1, Gli-1, CK- 7, α-smooth muscle actin, ICAM-1, and PCNA. The following secondary antibodies were used, anti-rabbit Alexa Fluor 488, anti-rabbit Alexa Fluor 594, anti-goat Alexa Fluor 488. Nuclear staining was performed using 4′, 6-diamidino-2-phenylindole (DAPI). Immunoflorescence was visualized on a Zeiss Apoplan Microscopy system.

Western blotting

Total protein was extracted by homogenizing liver tissues and lyzing in RIPA buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN). The protein concentration was determined using a Bio-Rad RC DC protein assay kit (Hercules, CA). Proteins were resolved on 4–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to Immobilon polyvinylidene fluoride (PVDF) membrane using iBlot™ Dry Blotting System (Invitrogen, Carlsbad, CA). After blocking with 5% nonfat dry milk in 1×PBST (PBS containing 0.05% Tween-20) for 1h at room temperature, the membranes were incubated with Shh, Ptch-11, Gli-1, TNF-α, IL-1β, α-sma, type 1 collagen, phospho and total ERK, phospho and total Akt, beta actin primary antibodies for 16 h at 4 °C as described.23 To correct for equal loading and blotting, all blots were re-probed with beta actin antibody. Membrane was then incubated with horseradish peroxidase (HRP)-conjugated anti-goat or anti-rabbit secondary antibody for 1 h at room temperature. Target proteins were detected by enhanced chemiluminescence (ECL) detection kit (GE Healthcare Life Sciences, Pittsburgh, PA). Quantitation of western blots was done using Gelscape software (http://www.gelscape.ualberta.ca:8080/htm/index.html).

Data Analysis

Data are presented as mean ± standard error of mean (SEM). Two sample comparisons were made and analyzed using two tailed unpaired t test. A P value <0.05 was considered statistically significant.

RESULTS

Bile duct ligation is associated with cholestasis and fibrosis

To confirm the establishment of cholestasis and fibrosis in our experimental group, 7 days after BDL three rats were euthanized and their liver harvested, fixed in 10% NBF and embedded in paraffin. In addition, livers from three sham operated animals were also harvested to serve as a control group. The livers of control animals showed normal gross morphology, histology and distribution of collagen (Figure 1A, upper panel). BDL animals showed enlarged pale yellow livers suggestive of cholestasis (Figure 1A, lower panel). Microscopic sections of BDL livers showed marked periportal ductal proliferation and inflammatory reaction. Picrosirrus red staining showed extensive collagen deposition around the ductular structures extending into the sinusoids and parenchyma (Figure 1A, lower panel).

Figure 1.

Figure 1

Figure 1

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Histological evaluation and expression of Hedgehog (Hh) ligands in BDL rats (A) Gross morphology, H&E and Picrosirrus red staining of liver specimens from sham controls and BDL rats are represented. Control livers were grossly normal, exhibited normal architecture and normal deposition of collagen supporting structures. In contrast, BDL livers were enlarged, cholestatic and microscopy showed bile duct proliferation, and extensive collagen deposition. (B–D) Shh, Ptch-1 and Gli-1 expression in normal and BDL rats quantified by real time polymerase chain reaction after correction for the housekeeping keeping gene HGPRT, **P < 0.01 vs. sham controls. (E) Total protein was isolated and Western blot analysis performed for Shh, Ptch-1 and Gli1 normalized to β actin. (F)Fresh frozen sections were immunostained using anti- Shh, Ptch-1 and Gli-1 and CK-7 (X 100 original magnifications). Hh ligands (green) and CK-7 (red) co localized in the biliary ductular epithelium (yellow)

Hepatic Expression of Hedgehog related molecules in cholestatic liver

To investigate the expression of Hh signaling in cholestatic liver, we performed real time PCR for mRNA expression of Sonic hedgehog (Shh), Patched 1 (Ptch-1) and Glioblastoma 1 (Gli-1) genes in sham operated and BDL rats. Weak expression of Shh was detected in healthy control rats. In contrast, significant upregulation of Shh, Ptch-1 and Gli-1 was seen at both mRNA (Figure 1B–D) and protein levels (Figure 1E). Dual immunoflorescence of Hh molecules with CK-7, a marker of mature and immature bile ductular cells, showed that Hh ligands co localized with CK-7 in ductular cells of bile ducts and sinusoids (Figures 1F).

Hedgehog pathway is abrogated by Cyclopamine

The major focus of this study was to inhibit Hh pathway by preconditioning BDL rats with CYA before subjecting them to I/R injury. Efficiency of Hh blockade was determined by mRNA expression of Shh, Ptch-1 and Gli-1 by real time PCR (Figure 2A–C) and protein expression of Ptch-1 (Figure 2D).

Figure 2.

Figure 2

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Hh pathway is efficiently inhibited by Cyclopamine. (A–C) real-time PCR showing downregulation of Hh signaling after treatment with CYA, *P<0.05. (D) Western blot for Ptch-1 used to confirm protein expression after CYA treatment.

Cyclopamine markedly blunted the increase of serum ALT, AST and Bilirubin

Thirty minutes of ischemia and 4 h of reperfusion significantly increased serum AST, ALT and bilirubin levels in BDL vehicle treated rats compared to control rats I/R rats (**P<0.01 and *P<0.05, Figure 3A–C). CYA resulted in a 28-fold, 4 and 10-fold decrease in serum ALT, AST and bilirubin levels. These results indicate that cholestatic livers are more susceptible to hepatocellular injury following ischemia reperfusion and inactivation of Hh signaling significantly attenuates this injury.

Figure 3.

Figure 3

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Cyclopamine markedly preserves liver functions and reduces histological liver injury. (A–C) Serum AST, ALT and Bilirubin in normal, I/R, vehicle treated BDL and CYA treated BDL rats after 30min ischemia and 4h reperfusion were quantified. Liver injury markers in CYA treated group were significantly lower than vehicle treated group, *P<0.05 and **P<0.01. (D) H& E staining of representative liver sections from rats as described above. Necrosis and infiltration of inflammatory cells were observed in vehicle BDL rats (arrow indicates vacuolization and sinusoidal congestion). These effects were attenuated in CYA treated group.

Preconditioning with Cyclopamine reduces histological liver injury

Histological architecture of control I/R rats after 4h of reperfusion showed minimal damage (Figure 3D). In contrast, vehicle treated BDL demonstrated confluent areas of necrosis, and intense inflammatory neutrophilic infiltrates (black arrow, Figure 3D). CYA treated animals, however, showed preservation of liver architecture. The degree of tissue injury (based on Suzuki criteria) was 1.1 ±0.02, 4.2 ±0.01 and 2.1 ±0.5 for I/R, BDL vehicle I/R and BDL CYA I/R treated rats respectively (P<0.05).

Cyclopamine blunts Neutrophil infiltration and lipid Peroxidation

To evaluate the role of Hh inhibition on the neutrophil infiltration, liver sections were stained with Naphthol AS-D chloroacetate esterase and intercellular adhesion molecule, ICAM-1 (Figure 4A). Control I/R rats subjected to 4 h of reperfusion showed minimal neutrophilic infiltration in the sinusoids (4±2 per 10HPF). The number of infiltrating neutrophils increased markedly in BDL vehicle treated animals (45±10 per 10HPF). CYA markedly reduced the neutrophilic infiltration by 33% (15±3 per 10HPF, **P<0.01, Figure 4B). Weak staining for ICAM-1 was seen along the endothelium of the portal vein in control I/R rats. Vehicle treated BDL rats showed more intense ICAM-1 expression along sinusoidal lining and endothelium of larger vessels. CYA treatment significantly attenuated the expression of ICAM-1. The rate of lipid peroxidation doubled in BDL vehicle treated group compared to control I/R rats (Figure 4C). In animals treated with CYA, tissue MDA level was markedly reduced.

Figure 4.

Figure 4

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Cyclopamine decreases neutrophilic infiltration, ICAM-1 expression and lipid peroxidation. (A) Naphthol D Acetate staining and ICAM-1 expression in liver sections of I/R, Vehicle and CYA treated BDL I/R rats. Marked reduction in neutrophils and decreased expression of ICAM-1 was seen in CYA treatment rats. (B) Quantification of neutrophils counted in 10HPF is shown,*P<0.01. (C) CYA treatment reduced MDA levels compared to vehicle treated group, **P<0.05.

Cyclopamine attenuates expression of proinflammatory cytokines

The production of TNF α and IL-1β in liver tissue was assessed by qRT-PCR and western blot analysis. Vehicle treated BDL rats showed marked upregulation of TNF-α and IL-1β mRNA compared to control I/R rats (Figure 5A and B). CYA downregulated mRNA expression of these cytokines. Real time PCR findings were confirmed by Western blot analysis of protein expression in liver homogenates. CYA treated rats showed a striking decline in TNF-α and IL-1β protein expression (Figure 5C).

Figure 5.

Figure 5

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Cyclopamine induces downregulation of proinflammatory cytokines. (A–B) Total RNA was isolated from I/R, Vehicle and CYA treated BDL I/R rats and expression of TNF-α and IL-1β was assessed by real time RT-PCR, *P<0.01. (C) Total protein from liver was extracted and subjected to western blotting for detection of TNF-α and IL-1β, *P<0.01.

Cyclopamine decreases HSC activation and bile duct proliferation

Next we explored the effect of CYA on early fibrosis. Extensive fibrosis was seen in rats BDL vehicle treated rats at 2 weeks. Histological evaluation for collagen depositon with Masson trichrome and Sirrus red staining demonstrated expanded areas of portal fibrosis (Figure 6A, upper and middle panel). Treatment with CYA significantly decreased the extracellular matrix deposition in BDL rats. The favorable changes mediated by CYA were also accompanied by marked reduction in expression of α-sma, a marker of activated hepatic stellate cells (Figure 6A, lower panel). These results were confirmed by immunoblotting for protein expression of α-SMA and Type I collagen (Figure 6B). Furthermore, there was markedly increased biliary duct proliferation in vehicle treated BDL animals as compared with CYA treatment, the latter showed mild to moderate biliary duct proliferation (Figure 6C). The bile duct proliferation score in vehicle treated and CYA treated BDL rats is shown in Figure 6D, in which it is evidently clear that CYA treatment resulted in reduced biliary proliferation compared to vehicle treated group. Taken together our results indicate that treatment with CYA reduces the activation of hepatic stellate cells and cholangiocyte proliferation after BDL. Concurrent with the above findings we further examined the effect of CYA on hepatocyte proliferation and apoptosis using anti-PCNA antibody and TUNEL respectively. BDL rats treated with saline showed increased proliferation compared to control I/R rats (Figure 6E, upper panel). The number of PCNA positive cells significantly declined in CYA treated group. Compared to control I/R rats, liver sections from vehicle treated BDL animals showed a large number of TUNEL positive cells with characteristic morphology for apoptosis (Figure 6E lower panel). Treatment with CYA decreased the number of TUNEL positive cells. The proliferation index and apoptotic index in CYA treated rats was reduced by 53.3% and 40% respectively when compared with vehicle treated group (Figure 6F).

Figure 6.

Figure 6

Figure 6

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Cyclopamine reduces liver fibrosis and bile duct proliferation. (A) Preconditioning with cyclopamine reduced collagen deposition (Masson trichrome and Sirrus red staining), and α-smooth muscle actin expression. Original magnification X40 (Zeiss AX10 microscope). (B) Total protein from liver was extracted and subjected to western blotting for detection of α-sma and Type 1 collagen, **P<0.05. (C) H&E and CK-7 expression in proliferating bile ducts in I/R, Vehicle and CYA treated BDL I/R rats. CK-7 staining in vehicle treated BDL I/R group showed florid and abundant biliary duct proliferation compared with CYA treated rats. (D) The degree of biliary proliferation was scored as in material and methods. (E) CYA treatment resulted in reduced number of PCNA and TUNEL positive cells. Original magnification X40 (Zeiss AX10 microscope). (F) Proliferation index and apoptosis index in each group was calculated by counting PCNA and TUNEL positive cells in 10 random (40X), **P<0.05.

Cyclopamine mediates anti-inflammatory effects by inhibiting the Akt and Mitogen protein kinase – ERK ½ pathway

We next investigated the association between Hh signaling and known cell survival pathways, i.e. the PI3K/Akt and MAPK signaling. The non-phosphorylated states of Akt and ERK1/2 remain unchanged after CYA treatment (Figure 7A and B). However, cyclopamine treatment induced a decrease in the phosphorylation state of Akt, and ERK1/2. Preventing activation of ERK and Akt indicates pivotal role of these signaling pathways in HSC activation and propagating inflammatory injury.

Figure 7.

Figure 7

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Cyclopamine reduces activation of PI-3K and MAPkinase signaling. (A and B) Total protein was extracted from liver tissue of I/R, Vehicle and CYA treated BDL I/R rats and phosphorylation of ERK1/2 and Akt was performed by Western blotting. Membranes were stripped and reprobed with total ERK and Akt antibodies to verify protein levels, *P<0.05.

DISCUSSION

Liver transplantation is now considered as a cure for end stage liver disease. However, as the number of patients on the liver transplant waiting list continues to grow, the demand for donor organs increases. Although expansion of donor criteria to include marginal livers such as those which are steatotic or cholestatic have become a significant source of transplantable organs their use is hampered by the higher risk of postoperative complications. Ischemia reperfusion is pivotal in causing hepatocellular damage in these marginal livers. We recently reported that Hh pathway is constitutively upregulated in healthy liver and renders it susceptible to I/R injury.15 Based on these observations we extended our hypothesis that Hh signaling may mediate inflammatory changes in cholestatic liver I/R injury. The principal findings of this study were 1) Hh pathway is upregulated in cholestatic liver, 2) Severity of ischemia reperfusion injury is more marked in cholestatic liver and parallels Hh expression, 3) Hh inhibition with CYA is hepatoprotective by reducing neutrophil infiltration, lipid peroxidation, and release of proinflammatory cytokines, 4) CYA reduces early fibrosis by downregulating activation of hepatic stellate cells and profibrogenic genes, and 5) CYA reduces the proliferation of biliary epithelium. Taken together our results provide new evidence for the role of endogenous Hh signaling in cholestatic liver subjected to warm IR injury.

Consistent with previously published reports, the present study demonstrates increased Hh expression in BDL rats by RT-PCR, immunoflorescence and western blot analysis.24–27 Hh pathway inhibiting experiments were performed by preconditioning BDL animals with CYA. Vehicle treated BDL rats subjected to 30 min of ischemia and 4 h of reperfusion showed marked increase in serum AST, ALT and bilirubin compared to CYA treated rats. Morphometric analysis of liver sections from vehicle treated BDL rats showed marked increase in inflammatory injury compared to CYA rats. Taken together, it is evidently clear that I/R elicited responses were greater when ischemia was superimposed upon cholestatic liver. These findings provide new evidence that endogenous Hh signaling promotes hepatocellular injury in cholestatic liver, which can be attenuated significantly by inhibiting this pathway.

One important feature of liver damage after I/R injury is inflammation.28, 29 The inflammatory response is more intense and exponentially amplified in cholestatic livers experiencing I/R injury.30 Most prominent among the inflammatory cells are neutrophils, which are recruited within hours of reperfusion.31 Neutrophil recruitment contributes to cytotoxic inflammatory injury by increased ICAM-1, and facilitating upregulation of proinflammatory cytokines such as TNF-α and interleukins. 32–34 Neutrophils can further aggravate I/R injury in cholestatic liver.35 Cumulatively, our data indicated extensive liver injury manifesting as markedly elevated liver enzymes, intense neutrophilic infiltration and ICAM-1 expression in vehicle treated BDL rats (Figure 4). This aggravated liver injury was substantially reduced by CYA. Recent studies have confirmed the role of proinflammatory cytokines like TNF-α and IL-1β in initiating and propagating inflammatory responses after I/R.36–39 These cytokines recruit neutrophils by up regulating vascular cell adhesion molecules and inducing neutrophil CXC cytokines.40 Genetic or pharmacological inhibition of TNF-α is an emerging strategy to reduce the burden of multiorgan I/R injury.37, 41 Our data in vehicle treated BDL rats showed increased expression of TNF-α and IL-1 β. CYA treatment was hepatoprotective by reduction in the expression of these proinflammatory cytokines (Figure 5). Lipid peroxidation was also reduced by CYA possibly secondary to overall decreased activation of neutrophils and inhibition of proinflammatory cytokines. There has been a recent report concluding that cholestasis protects mouse liver from I/R injury.42 On the contrary the results of our study and others indicate that cholestatic liver is more susceptible to I/R injury.43 Genetic differences in the animal models or other molecular mechanisms could affect the hepatocellular inflammatory responses to I/R injury in cholestatic livers.44

Cholestasis and I/R injury lead to significant alterations in both portal and arterial hemodynamics resulting in HSC activation, and cholangiocyte proliferation. The activated HSCs release proinflammatory cytokines and cell adhesion molecules thereby aggravating the existing inflammatory injury.45,46, 47 HSCs and cholangiocytes express target Hh genes and proliferate under Hh signaling resulting in increased ECM deposition and intrahepatic ductal mass.13 In our study, in vivo administration of CYA to BDL rats decreased expression of α-sma and Type-1 collagen resulting in decreased fibrosis. It also decreased the number of proliferating bile ducts. We also evaluated the effect of CYA on hepatocyte proliferation and apoptosis. In normal rat liver, the hepatocytes are mitotically quiescent with rare apoptosis.48,49 However, increased hepatocyte proliferation was noted under pathological conditions such as cholestasis. CYA treatment was associated with reduced hepatocyte proliferation and apoptosis. In support of our findings, studies have shown antiproliferative effects of CYA in other cell types.50, 51 The exact mechanism by which CYA exerts its antiproliferative effects is presently not clearly understood. However there is growing evidence showing regulation of proliferation and survival of HSC’s and expression of profibrogenic genes by both MAPkinase and PI3K-Akt signaling.52–54 In the present work we show that CYA treatment in BDL animals inhibited the activation of these signaling components suggesting a putative role of these well-characterized signaling molecules. However, more studies need to be carried out to completely delineate this signaling in our experimental system. Ductal proliferation seen after BDL is fundamentally crucial to sustain the enhanced functional and nutritional needs in addition to compensating for ongoing loss of ductal mass due to increased biliary pressure. One could argue that the inhibition of ductal proliferation by CYA would negatively affect liver regeneration and remodeling. Future studies in which the effect of CYA at different time points should determine whether CYA is detrimental in remodeling of I/R cholestatic liver.

In summary, cholestatic liver injury conclusion, the results of this study provide convincing evidence of hepatoprotection following warm ischemia reperfusion injury by Hh antagonist cyclopamine in a rat model of cholestasis.

Acknowledgments

FINANCIAL SUPPORT: This work was partly supported by a grant from the National Institute of Health (to R.I.M.; project number: EB003922)

ABBREVIATIONS

ALT

alanine transaminase

AST

serum aspartate transaminase

BDL

bile duct ligation

CK-7

cytokeratin-7

CYA

cyclopamine

DAPI

4′, 6-diamidino-2-phenylindole

ECL

enhanced chemiluminescence

EMT

epithelial mesenchymal transformation

ERK

extracellular signal regulated kinase

Gli-1

Glioblastoma-1

HBCD

2-hydroxypropyl-b-cyclodextrin

Hh

Hedgehog

HSC

hepatic stellate cells

ICAM-1

inter-cellular adhesion molecule

IL-1β

interleukin-1β I/R, ischemia reperfusion

MDA

Malondialdehyde

NBF

neutral buffered formalin

Ptch-1

Patched-1

PCNA

proliferating cell nuclear antigen

PVDF

polyvinylidene fluoride

α-SMA

alpha- smooth muscle actin

Shh

Sonic Hedgehog

TNF-α

tumor necrosis factor α

TUNEL

terminal deoxynucleotidyl transferase dUTP nick-end labeling

Footnotes

CONFLICT OF INTEREST: The authors disclose no conflicts.

References

  • 1.Scuderi V, Ceriello A, Maida P, Aragiusto G, Arenga G, Carfora T, Defez M, Giuliani A, Monti GN, Santaniello W, Sicoli F, Calise F. The Marginal Donor: A Single-Center Experience in Orthotopic Liver Transplantation. Transplantation Proceedings. 2006;38(4):1069–1073. doi: 10.1016/j.transproceed.2006.04.003. [DOI] [PubMed] [Google Scholar]
  • 2.Garcia CE, Bramhall S, Mirza DF. Use of marginal donors. Current Opinion in Organ Transplantation. 2000;5(2):50–56. [Google Scholar]
  • 3.Nadig SN, Periyasamy B, Shafizadeh SF, Polito C, Fiorini RN, Rodwell D, Evans Z, Cheng G, Dunkelberger D, Schmidt M, Self SE, Chavin KD. Hepatocellular ultrastructure after ischemia/reperfusion injury in human orthotopic liver transplantation. Journal of Gastrointestinal Surgery. 2004;8(6):695–700. doi: 10.1016/j.gassur.2004.04.002. [DOI] [PubMed] [Google Scholar]
  • 4.Verran D, Kusyk T, Painter D, Fisher J, Koorey D, Strasser S, Stewart G, McCaughan G. Clinical experience gained from the use of 120 steatotic donor livers for orthotopic liver transplantation. Liver Transplantation. 2003;9(5):500–505. doi: 10.1053/jlts.2003.50099. [DOI] [PubMed] [Google Scholar]
  • 5.Schaubel DE, Sima CS, Goodrich NP, Feng S, Merion RM. The Survival Benefit of Deceased Donor Liver Transplantation as a Function of Candidate Disease Severity and Donor Quality. American Journal of Transplantation. 2008;8(2):419–425. doi: 10.1111/j.1600-6143.2007.02086.x. [DOI] [PubMed] [Google Scholar]
  • 6.Jaeschke H, Farhood A, Smith C. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J. 1990;4(15):3355–3359. [PubMed] [Google Scholar]
  • 7.Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev Mol Cell Biol. 2005;6(4):306–317. doi: 10.1038/nrm1622. [DOI] [PubMed] [Google Scholar]
  • 8.Buttitta L, Mo R, Hui C-C, Fan C-M. Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction. Development. 2003;130(25):6233–6243. doi: 10.1242/dev.00851. [DOI] [PubMed] [Google Scholar]
  • 9.Ikram MS, Neill GW, Regl G, Eichberger T, Frischauf A-M, Aberger F, Quinn A, Philpott M. GLI2 Is Expressed in Normal Human Epidermis and BCC and Induces GLI1 Expression by Binding to its Promoter. J Investig Dermatol. 2004;122(6):1503–1509. doi: 10.1111/j.0022-202X.2004.22612.x. [DOI] [PubMed] [Google Scholar]
  • 10.Kimelman D, Griffin KJP. Vertebrate mesendoderm induction and patterning. Current Opinion in Genetics & Development. 2000;10(4):350–356. doi: 10.1016/s0959-437x(00)00095-2. [DOI] [PubMed] [Google Scholar]
  • 11.Li X, Deng W, Lobo-Ruppert SM, Ruppert JM. Gli1 acts through Snail and E-cadherin to promote nuclear signaling by [beta]-catenin. Oncogene. 2007;26(31):4489–4498. doi: 10.1038/sj.onc.1210241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sicklick JK, Li Y-X, Jayaraman A, Kannangai R, Qi Y, Vivekanandan P, Ludlow JW, Owzar K, Chen W, Torbenson MS, Diehl AM. Dysregulation of the Hedgehog pathway in human hepatocarcinogenesis. Carcinogenesis. 2006;27(4):748–757. doi: 10.1093/carcin/bgi292. [DOI] [PubMed] [Google Scholar]
  • 13.Omenetti A, Yang L, Li Y-X, McCall SJ, Jung Y, Sicklick JK, Huang J, Choi S, Suzuki A, Diehl AM. Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation. Lab Invest. 2007;87(5):499–514. doi: 10.1038/labinvest.3700537. [DOI] [PubMed] [Google Scholar]
  • 14.Toftgård R. Hedgehog signalling in cancer. Cellular and Molecular Life Sciences. 2000;57(12):1720–1731. doi: 10.1007/PL00000654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Pratap A, Panakanti R, Yang N, Eason J, Mahato R. Inhibition of Endogenous Hedgehog Signaling Protects Against Acute Liver Injury After Ischemia Reperfusion. Pharmaceutical Research. 2010:1–13. doi: 10.1007/s11095-010-0246-z. [DOI] [PubMed] [Google Scholar]
  • 16.Ezure T, Sakamoto T, Tsuji H, Lunz JG, III, Murase N, Fung JJ, Demetris AJ. The Development and Compensation of Biliary Cirrhosis in Interleukin-6-Deficient Mice. Am J Pathol. 2000;156(5):1627–1639. doi: 10.1016/s0002-9440(10)65034-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Liu P, Xu B, Quilley J, Wong PY-K. Peroxynitrite attenuates hepatic ischemia-reperfusion injury. American Journal of Physiology - Cell Physiology. 2000;279(6):C1970–C1977. doi: 10.1152/ajpcell.2000.279.6.C1970. [DOI] [PubMed] [Google Scholar]
  • 18.Rüdiger HA, Graf R, Clavien P-A. Sub-lethal oxidative stress triggers the protective effects of ischemic preconditioning in the mouse liver. Journal of hepatology. 2003;39(6):972–977. doi: 10.1016/s0168-8278(03)00415-x. [DOI] [PubMed] [Google Scholar]
  • 19.Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine. Transplantation. 1993;55(6):1265–72. doi: 10.1097/00007890-199306000-00011. [DOI] [PubMed] [Google Scholar]
  • 20.Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biology and Medicine. 1990;9(6):515–540. doi: 10.1016/0891-5849(90)90131-2. [DOI] [PubMed] [Google Scholar]
  • 21.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 1979;95(2):351–358. doi: 10.1016/0003-2697(79)90738-3. [DOI] [PubMed] [Google Scholar]
  • 22.Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, Gores GJ. Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology. 2003;125(2):437–443. doi: 10.1016/s0016-5085(03)00907-7. [DOI] [PubMed] [Google Scholar]
  • 23.Kepka-Lenhart D, Chen L, Morris S. Novel actions of aspirin and sodium salicylate: discordant effects on nitric oxide synthesis and induction of nitric oxide synthase mRNA in a murine macrophage cell line. Journal of Leukocyte Biology. 1996;59(6):840–846. doi: 10.1002/jlb.59.6.840. [DOI] [PubMed] [Google Scholar]
  • 24.Omenetti A, Popov Y, Jung Y, Choi SS, Witek RP, Yang L, Brown KD, Schuppan D, Diehl AM. The hedgehog pathway regulates remodelling responses to biliary obstruction in rats. Gut. 2008;57(9):1275–1282. doi: 10.1136/gut.2008.148619. [DOI] [PubMed] [Google Scholar]
  • 25.Greenbaum LE. Hedgehog signaling in biliary fibrosis. The Journal of Clinical Investigation. 2008;118(10):3263–3265. doi: 10.1172/JCI37189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hirose Y, Itoh T, Miyajima A. Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells. Experimental Cell Research. 2009;315(15):2648–2657. doi: 10.1016/j.yexcr.2009.06.018. [DOI] [PubMed] [Google Scholar]
  • 27.Syn W-K, Jung Y, Omenetti A, Abdelmalek M, Guy CD, Yang L, Wang J, Witek RP, Fearing CM, Pereira TA, Teaberry V, Choi SS, Conde-Vancells J, Karaca GF, Diehl AM. Hedgehog-Mediated Epithelial-to-Mesenchymal Transition and Fibrogenic Repair in Nonalcoholic Fatty Liver Disease. Gastroenterology. 2009;137(4):1478–1488.e8. doi: 10.1053/j.gastro.2009.06.051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kakkar AK, Lefer DJ. Leukocyte and endothelial adhesion molecule studies in knockout mice. Current Opinion in Pharmacology. 2004;4(2):154–158. doi: 10.1016/j.coph.2004.01.003. [DOI] [PubMed] [Google Scholar]
  • 29.Toledo-Pereyra LH, Lopez-Neblina F, Lentsch AB, Anaya-Prado R, Romano SJ, Ward PA. Selectin Inhibition Modulates NF-k B and AP-1 Signaling After Liver Ischemia/Reperfusion. Journal of Investigative Surgery. 2006;19(5):313–322. doi: 10.1080/08941930600889474. [DOI] [PubMed] [Google Scholar]
  • 30.Gujral JS, Farhood A, Bajt ML, Jaeschke H. Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology. 2003;38(2):355–363. doi: 10.1053/jhep.2003.50341. [DOI] [PubMed] [Google Scholar]
  • 31.Jaeschke H. Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol. 2006;290(6):G1083–1088. doi: 10.1152/ajpgi.00568.2005. [DOI] [PubMed] [Google Scholar]
  • 32.Jaeschke H, Farhood A, Smith C. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. The FASEB Journal. 1990;4(15):3355–3359. [PubMed] [Google Scholar]
  • 33.Jaeschke H, Farhood A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia- reperfusion injury in rat liver. American Journal of Physiology - Gastrointestinal and Liver Physiology. 1991;260(3):G355–G362. doi: 10.1152/ajpgi.1991.260.3.G355. [DOI] [PubMed] [Google Scholar]
  • 34.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. American Journal of Physiology - Gastrointestinal and Liver Physiology. 2004;286(3):G499–G507. doi: 10.1152/ajpgi.00318.2003. [DOI] [PubMed] [Google Scholar]
  • 35.Hiroyuki Y, Masaru M, Hiroaki S, Hiroshi I, Koji N, Satoshi A, Nobuyuki N, Michael JE, Alex BL. Obstructive jaundice impairs hepatic sinusoidal endothelial cell function and renders liver susceptible to hepatic ischemia/reperfusion. Journal of hepatology. 2000;33(1):59–67. doi: 10.1016/s0168-8278(00)80160-9. [DOI] [PubMed] [Google Scholar]
  • 36.Kei Y, Yuzo Y, Hidekazu Y, Yasuhide I, Hiroshi U, Kojiro T, Akio N, Yoshio Y. Suppression of tumor necrosis factor-α production and neutrophil infiltration during ischemia–reperfusion injury of the liver after heat shock preconditioning. Journal of hepatology. 2001;35(5):619–627. doi: 10.1016/s0168-8278(01)00191-x. [DOI] [PubMed] [Google Scholar]
  • 37.Esposito E, Mazzon E, Muià C, Meli R, Sessa E, Cuzzocrea S. Splanchnic ischemia and reperfusion injury is reduced by genetic or pharmacological inhibition of TNF-α. Journal of Leukocyte Biology. 2007;81(4):1032–1043. doi: 10.1189/jlb.0706480. [DOI] [PubMed] [Google Scholar]
  • 38.Camargo CA, Madden JF, Gao W, Selvan RS, Clavien P. Interleukin-6 protects liver against warm ischemia/reperfusion injury and promotes hepatocyte proliferation in the rodent. Hepatology. 1997;26(6):1513–1520. doi: 10.1002/hep.510260619. [DOI] [PubMed] [Google Scholar]
  • 39.Colletti LM, Kunkel SL, Walz A, Burdick MD, Kunkel RG, Wilke CA, Strieter RM. The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology. 1996;23(3):506–14. doi: 10.1002/hep.510230315. [DOI] [PubMed] [Google Scholar]
  • 40.Jaeschke H, Hasegawa T. Role of neutrophils in acute inflammatory liver injury. Liver International. 2006;26(8):912–919. doi: 10.1111/j.1478-3231.2006.01327.x. [DOI] [PubMed] [Google Scholar]
  • 41.Naoya M, Hisayasu W, Tsugiyasu K, Tamikazu N, Yasuhiro Y, Kuniaki S, Hisayoshi F, Kenji S, Mitsuru S. Improved myocardial ischemia/reperfusion injury in mice lacking tumor necrosis factor-α. Journal of the American College of Cardiology. 2002;39(7):1229–1235. doi: 10.1016/s0735-1097(02)01738-2. [DOI] [PubMed] [Google Scholar]
  • 42.Georgiev P, Navarini AA, Eloranta JJ, Lang KS, Kullak-Ublick GA, Nocito A, Dahm F, Jochum W, Graf R, Clavien P-A. Cholestasis protects the liver from ischaemic injury and post-ischaemic inflammation in the mouse. Gut. 2007;56(1):121–128. doi: 10.1136/gut.2006.097170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Kloek JJ, Levi M, Heger M, Van Der Loos CM, Gouma DJ, Van Gulik TM. Cholestasis enhances liver ischemia/reperfusion-induced coagulation activation in rats. Hepatology Research. 2010;40(2):204–215. doi: 10.1111/j.1872-034X.2009.00579.x. [DOI] [PubMed] [Google Scholar]
  • 44.Alaish SM, Torres M, Ferlito M, Sun C-C, De Maio A. The Severity of Cholestatic Injury Is Modulated By the Genetic Background. Shock. 2005;24(5):412–416. doi: 10.1097/01.shk.0000183392.83272.97. [DOI] [PubMed] [Google Scholar]
  • 45.Aleffi S, Petrai I, Bertolani C, Parola M, Colombatto S, Novo E, Vizzutti F, Anania FA, Milani S, Rombouts K, Laffi G, Pinzani M, Marra F. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology. 2005;42(6):1339–1348. doi: 10.1002/hep.20965. [DOI] [PubMed] [Google Scholar]
  • 46.Rubbia-Brandt L, Mentha G, Desmoulière A, Monte Alto Costa A, Giostra E, Molas G, Enzan H, Gabbiani G. Hepatic stellate cells reversibly express [alpha]-smooth muscle actin during acute hepatic ischemia. Transplantation Proceedings. 1997;29(5):2390–2395. doi: 10.1016/s0041-1345(97)00415-6. [DOI] [PubMed] [Google Scholar]
  • 47.Viñas O, Bataller R, Sancho-Bru P, Ginès P, Berenguer C, Enrich C, Nicolás JM, Ercilla G, Gallart T, Vives J, Arroyo V, Rodés J. Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation. Hepatology. 2003;38(4):919–929. doi: 10.1053/jhep.2003.50392. [DOI] [PubMed] [Google Scholar]
  • 48.Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri A, LeSage G, LaRusso NF. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. American Journal of Physiology - Gastrointestinal and Liver Physiology. 1998;274(4):G767–G775. doi: 10.1152/ajpgi.1998.274.4.G767. [DOI] [PubMed] [Google Scholar]
  • 49.Alpini G, Lenzi R, Zhai WR, Slott PA, Liu MH, Sarkozi L, Tavoloni N. Bile secretory function of intrahepatic biliary epithelium in the rat. American Journal of Physiology - Gastrointestinal and Liver Physiology. 1989;257(1):G124–G133. doi: 10.1152/ajpgi.1989.257.1.G124. [DOI] [PubMed] [Google Scholar]
  • 50.Mimeault M, Johansson SL, Henichart J-P, Depreux P, Batra SK. Cytotoxic Effects Induced by Docetaxel, Gefitinib, and Cyclopamine on Side Population and Nonside Population Cell Fractions from Human Invasive Prostate Cancer Cells. Molecular Cancer Therapeutics. 2010;9(3):617–630. doi: 10.1158/1535-7163.MCT-09-1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Hu W-g, Liu T, Xiong J-x, Wang C-y. Blockade of sonic hedgehog signal pathway enhances antiproliferative effect of EGFR inhibitor in pancreatic cancer cells1. Acta Pharmacologica Sinica. 2007;28(8):1224–1230. doi: 10.1111/j.1745-7254.2007.00620.x. [DOI] [PubMed] [Google Scholar]
  • 52.Gentilini A, Marra F, Gentilini P, Pinzani M. Phosphatidylinositol-3 kinase and extracellular signal-regulated kinase mediate the chemotactic and mitogenic effects of insulin-like growth factor-I in human hepatic stellate cells. Journal of hepatology. 2000;32(2):227–234. doi: 10.1016/s0168-8278(00)80067-7. [DOI] [PubMed] [Google Scholar]
  • 53.Son G, Hines IN, Lindquist J, Schrum LW, Rippe RA. Inhibition of phosphatidylinositol 3- kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis. Hepatology. 2009;50(5):1512–23. doi: 10.1002/hep.23186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Boaglio AC, Zucchetti AE, Sanchez Pozzi EJ, Pellegrino JM, Ochoa JE, Mottino AD, Vore M, Crocenzi FA, Roma MG. Phosphoinositide 3-kinase/protein kinase B signaling pathway is involved in estradiol 17beta-D-glucuronide-induced cholestasis: complementarity with classical protein kinase C. Hepatology. 2010;52(4):1465–76. doi: 10.1002/hep.23846. [DOI] [PubMed] [Google Scholar]

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