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    Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression

    2017-02-10 08:08:20

    Brussels, Belgium

    Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression

    Devaraj Ezhilarasan, Jonathan Evraerts, Brice Sid, Pedro Buc Calderon, Sivanesan Karthikeyan, Etienne Sokal and Mustapha Najimi

    Brussels, Belgium

    BACKGROUND: Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fbrosis consequent to chronic liver injury. Silibinin, a favonoid compound, has been shown to possess anti-fbrogenic effects in animal models of liver fbrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fbrogenic effect.

    METHODS: The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 μmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay.

    RESULTS: Silibinin inhibits LX-2 cell proliferation in doseand time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regula-tion was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinininhibited proliferation of LX-2 cells.

    CONCLUSION: The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

    (Hepatobiliary Pancreat Dis Int 2017;16:80-87)

    silibinin;

    hepatic stellate cells;

    in vitro;

    cell cycle arrest;

    proliferation

    Introduction

    Liver disease is one of the major causes of morbidity and mortality worldwide, affecting humans of all ages. According to WHO, liver fbrosis is a signifcant health problem with a worldwide mortality attributable to cirrhosis and primary liver cancer of around 1.5 millions death per year.[1]Liver fbrosis is the common fnal pathway of virtually all chronic infammatory liver injuries. Hepatic stellate cells (HSCs) are known as the primary source of the extracellular matrix (ECM) components in liver fbrosis.[2]Following liver injury of any etiology, HSCs undergo a response known as “activation”, which is the transition of quiescent cells into proliferative, fbrogenic, and contractile myofbroblasts.[3]

    HSC activation is a remarkably pleiotropic tightly programmed response occurring in a reproducible sequence. This sequence consists of “initiation” and “perpetuation” steps.[4]Initiation encompasses rapid changes in gene expression and phenotype that render the cells response to cytokines and other local stimuli. Perpetu-ation involves key phenotypic responses mediated by increased cytokine effects and remodeling of ECM. Discrete phenotype responses of stellate cell perpetuation include proliferation, chemotaxis, fbrogenesis, contractility, matrix degradation, retinoid loss, and cytokine release.[4]Proliferation of activated HSCs and their increased synthesis of ECM are essential steps in the initiation and progression of liver fbrosis in chronic liver diseases. Hence, targeting the anti-proliferative effect of HSCs is clearly an appropriate therapeutic approach for liver fbrosis.

    Plant derived antioxidant substances have been evaluated as a potential therapeutic approach for liver fbrosis. Curcumin,[5]caffeine,[6]and salvianolic acid B[7]have been found to suppress the proliferation of cultured HSCs. Silibinin (SBN), a standard extract isolated from the medicinal plantSilybum marianum(L.) (milk thistle), is the major bioactive component of silymarin. The most abundant favonolignans present in SBN are the diastereoisomers silybin A and silybin B.[8]Silymarin and SBN have been used for decades as a herbal remedy and as a hepatoprotectant in acute and chronic liver disease settings.[9]Severalin vitro,in vivoand clinical studies have demonstrated the antioxidant and hepatoprotective effects of silymarin and its major active constituent SBN in alcoholic or nonalcoholic chronic liver disease models.[10-15]Moreover, SBN has been found to be virtually nontoxic in both chronic and acute toxicity studies, and no LD50has been reported for these favonolignansin vivo, which further underscores its promise in chronic liver diseases and cancer prevention.[16]Several studies were also emphasized regarding the manifold inhibitory effects of SBN on cell proliferation, cell cycle regulation, and apoptosis induction in several cancer cell lines of different origins.[17-22]Although these studies imply that SBN has a suppressive effect on cell cycle, its infuence on the mechanisms of cell cycle arrest in HSC remains unclear.

    Our previous study has demonstrated that SBN is not cytotoxic to LX-2 cells, inhibits proliferation and does not induce apoptosis and cellular senescence.[23]Hence, in the present study, we examined the possible reasons for anti-proliferative effects of SBN on human hepatic stellate LX-2 cell line and explored the related signaling pathways. The potential effects of SBN on cyclin dependant kinase inhibitors (CDKIs) proteins i.e., Cip1/p21, Kip1/p27, p53, Akt and their downstream targets signaling pathways were particularly investigated.

    Methods

    Reagents and drugs

    Silibinin (C25H22O10; CAS No. 22888-70-6) was purchased from Sigma Chemical Co. (Belgium). It is a mixture of two diastereisomers (silybin A and silybin B).

    Cell culture

    LX-2 cells were kindly provided by Dr. S. L. Friedman, Mount Sinai School of Medicine, New York, USA. These cells are derived from normal human HSC that are spontaneously immortalized. LX-2 cells exhibit the typical features of HSCs in primary culture as for instance the expression of desmin, glial acidic fbrillary protein, and the response to platelet-derived growth factor BB and TGF-β1. LX-2 cells express α-smooth muscle actin (α-SMA) under all culture conditions.[24]The cells were cultured in Dulbecco's modifed Eagle's medium (DMEM) supplemented with 1% of fetal bovine serum (Gibco, Belgium) and 1% penicillin-streptomycin (Life Technologies, Belgium). Cell cultures were maintained at 37 ℃ in a fully humidifed atmosphere containing 5% CO2. Those used in the present study were from passages 6-10 after thawing.

    Cell treatment

    SBN was dissolved in 0.1% DMSO (v/v) according to Agarwal et al.[25]LX-2 cells were plated at 10 000 cells/cm2. Twenty-four hours later, cells were fed with fresh expansion culture medium supplemented with different fnal concentrations of SBN (10, 50 and 100 μmol/L) or the corresponding volumes of the vehicle. After 24 and 96 hours of incubation, cells were collected after 0.05% trypsin (Life Technologies) application. Total cell number was determined by counting each sample in triplicate using a KOVA Glasstic? Slide 10 under Leica DMIL inverted microscope. Viability was also evaluated by the trypan blue dye exclusion assay.

    Intracellular ATP level measurement

    LX-2 cells were seeded at density of 6×105cells in 6-well plates. After 24 hours, plated LX-2 cells were fed with fresh expansion culture medium supplemented or not with different fnal concentrations of SBN. After 24 and 96 hours of SBN incubation, cells were detached/ collected using 0.05% trypsin for intracellular ATP analysis. The assay was performed using Perkin Elmer luminescence kit by luminescence method. This method was based on the luciferase and luciferin reaction, which determines the number of viable cells present in the well, based on ATP levels. Briefy, 50 μL of lysis buffer was added to the plate containing 1×105LX-2 cells suspended in 100 μL of Iscove's medium without phenol red. The plate was shaken for 5 minutes on ice and 50 μL of luciferase substrate was added and incubated for 15minutes on ice. The emitted luminescence was recorded using Perkin Elmer 2030 multi label reader.

    Immunocytochemistry of Ki-67

    LX-2 cells (10 000/cm2) were plated on glass cover slips in 24-well culture dishes. After 24 hours postplating, cells were incubated in the presence of increasing doses of SBN for 96 hours. At the end of exposure, the cells were washed twice with D-PBS and then fxed using 4% formalin in PBS for 20 minutes at room temperature. After fxation, cells were washed twice with PBS and incubated with hydrogen peroxide (3.3%) for 3 minutes to inactivate the endogenous peroxidase activity. Thereafter, fxed cells were permeabilized after 10 minutes incubation with D-PBS containing 1% Triton X-100 at room temperature. Permeabilized cells were washed twice with D-PBS and incubated with blocking solution (1% BSA in PBS) for one hour at room temperature. Primary antibody of monoclonal mouse Ki-67 (Dako, Denmark) diluted at 1:150 in 0.1% BSA was incubated with the cells for one hour at room temperature. After three washes with D-PBS, cells were incubated with 200 μL of HRP labeled anti-mouse secondary antibody (Dako, Carpintaria, CA) for 45 minutes at room temperature. After three washes with D-PBS, detection was performed after 5 minutes incubation with liquid DAB and substrate chromogen (Dako, Denmark). Counterstaining was performed using Mayer' s hematoxylin for 10 minutes. Preparations were then mounted for microscopic analysis (DMIL, Leica, Belgium).

    Western blotting analysis

    LX-2 cells were lysed in RIPA buffer (20 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1% NP-40, 0.1% SDS, 1% deoxycholate sodium and 0.1% protease and phosphatase inhibitor cocktails, Sigma-Aldrich, Belgium). After extraction, protein concentration was estimated by the Bradford method (Biorad Laboratories) with BSA as standard. Total protein extracts were subjected to SDSPAGE and electroblotted onto nitrocellulose membranes (Hybond?-ECL, Amersham, CA). The membranes were blocked with 5% BSA, probed overnight at 4 ℃ with primary antibodies and 2 hours at room temperature with corresponding secondary antibodies. Immunoreactive bands were detected by enhanced chemiluminescence with protein A-horseradish peroxidase and the SuperSignal chemiluminescent system (Pierce, Rockford, IL). Details of primary and corresponding secondary antibodies used for this study are given in the Table.

    Sirtuin activity

    Mammalian sirtuins are involved in several cell biological functions including survival, resistance to stress and apoptosis. Sirtuin activity was analyzed using the SIRT-Glo? luminescent assay (Ref: G6450) according to the manufacturer's instructions. Control and treated LX-2 cells were trypsinized, counted and seeded at 105cells/100 μL of SIRT-Glo buffer per well of a 96-well plate (in duplicate). One hundred μL of SIRT-Glo reagent were added and the plate was shaken for 2 minutes at room temperature. The cell suspensions were incubated for 45 minutes at room temperature during the sirtuin activities deacetylates the luminogenic peptide substrate. Such deacetylation is measured using a coupled enzymatic system in which a protease, contained in the Developer Reagent, cleaves the peptide from aminoluciferin. The produced luminescence, which is proportional to SIRT-deacetylase activity, was measured using a Victor3 luminometer (Perkin Elmer). Substrate without cells and cells with no enzyme were used as negative controls.

    Statistical analysis

    The data were subjected to one-way analysis of variance (ANOVA) and Newman Keuls' multiple compari-son tests were performed to assess the signifcance of differences in the means of various treatment groups, using Graph pad prism software. The values were tabulated and presented as mean±SEM. APvalue <0.05 was considered statistically signifcant.

    Table. List of primary and secondary antibodies used for the study

    Results

    SBN inhibited LX-2 cell proliferation without affecting the cell viability

    Fig. 1. Effect of SBN treatment on LX-2 cells morphology (A), cell proliferation (B), intracellular ATP levels (C). Cells were incubated with vehicle or SBN at 10, 50 and 100 μmol/L in DMEM containing 1% FBS for 24 and 96 hours. Data were presented as mean±SEM of three individual experiments withn=3. *:P<0.05, #:P<0.001, compared with control.

    SBN treated LX-2 cells showed no difference in cell viability in comparison with both na?ve and vehicle-treated LX-2 cells either at different time points or variable concentrations. More than 90% of the LX-2 cells remained viable and they were observed as culture activated and acquired the initial myofbroblast-like characteristic features (Fig. 1A). SBN treatment induced a dose- and timedependent drop in the number of LX-2 cells as compared to both na?ve and vehicle-treated cells (Fig. 1B). The counted cells in SBN 10, 50 and 100 μmol/L for 24 hours treated groups represent 77%±7%, 67%±5%, and 59% ± 7% of the control groups, respectively (Fig. 1B). These percentages after 96 hour incubation were 79%±13%, 70%±14%, and 54%±12% of the control groups, respectively (Fig. 1B). Intracellular ATP assay showed that SBN did not affect intracellular ATP level in comparison with controls (Fig. 1C).

    The effect of SBN on cell cycle proliferation and apoptosis related proteins

    Western blotting demonstrated that SBN treated LX-2 cells signifcantly increased p53 and p27 expression at 24 hours in comparison with controls (Fig. 2). The maximum effect of SBN on protein induction was observed in cells treated with 100 μmol/L of SBN (Fig. 2). Conversely, no signifcant changes were observed in the levels of both p21 and survivin (inhibitor of apoptosis) protein levels at the times analyzed. No signifcant changes of these proteins were noticed after 96 hours. This is in line with the earlier activation of the abovementioned proteins and its impact on the progression of cell cycle arrest.

    An immunocytochemical analysis on Ki-67, a good marker of cell cycle arrest in proliferating cells, showed dose-dependent decrease in Ki-67 in 96 hours of SBN treated cells as compared to na?ve and vehicle treated groups (Fig. 3A). Quantifcation of Ki-67 protein nuclear expression confrmed the signifcant concomitant decrease in the percentage of Ki-67 positively immunostained cells and an increase of the negatively immunostained cells (Fig. 3B). It should be underlined that a maximum effect was observed at 100 μmol/L SBN. Theseresults pointed out the ability of SBN to induce a cell cycle arrest in proliferating LX-2 cells. There was no signifcant change in the Ki-67 expression after 24 hours of SBN treatment in LX-2 cells.

    Fig. 2. Effect of SBN on the protein expression of p53, p27, p21 and survivin on LX-2 cells. Cells were incubated with vehicle or SBN at 10, 50 and 100 μmol/L in DMEM containing 1% FBS for 24 hours. Protein expression of p53, p27, p21 and survivin were evaluated by Western blotting analysis. β-actin used as an internal control for optimization.

    Fig. 3. SBN treatment decreases Ki-67 expression in LX-2 cells. Cells were incubated with vehicle or SBN at 10, 50 and 100 μmol/L in DMEM containing 1% FBS for 96 hours. After 96 hours of treatment, cells were fxed in 4% formalin and immunocytochemical expression of Ki-67 was performed (A). Quantitative analysis was performed by counting Ki-67 positive and negative cells (B). Data were presented as mean±SEM of three individual experiments withn=3. *:P<0.05, #:P<0.001, compared with vehicle.

    Modulation of early G1 phase signaling molecules and transcription factors is correlated to the antiproliferative effect of SBN on LX-2 cells

    The protein expression of Akt (a G1 phase related protein) was a dose-dependent decrease in LX-2 cells exposed to SBN. The dose-dependent effect was more pronounced after 96 hours as compared to vehicle treated cells (Fig. 4). Akt phosphorylation (phospho-Thr308, phospho-Ser473) was not detectable in 24 hours. The phosphorylated Akt was positive at 96 hours in all studied groups. SBN at 100 μmol/L signifcantly decreased Akt and phosphorylated Akt (Fig. 4).

    Foxo3A, the downstream target of Akt, was increased by SBN (50 μmol/L) at 24 hours only (Fig. 4). SBN (100 μmol/L) signifcantly decreased pSer-Foxo3A after 96 hours treatment (Fig. 4).

    The activity of sirtuin modulated by SBN may also trigger the anti-proliferative effect on LX-2 treated cells

    Sirtuin activity was signifcantly increased in LX-2 cells treated with SBN for 96 hours as compared to controls (Fig. 5). This effect was more prominent at the highest concentration of SBN used. The sirtuin activity was not signifcantly changed after 24 hours of SBN treatment in LX-2 cells.

    Fig. 4. Effect of SBN on the protein expression of Akt, Foxo and their phosporylated proteins were analyzed in LX-2 cells. Cells were incubated with vehicle or SBN at 10, 50 and 100 μmol/L in DMEM containing 1% FBS for 24 and 96 hours. Expression of Akt, Foxo and their phosporylated proteins were evaluated by Western blotting analysis. β-actin used as an internal control for optimization.

    Fig. 5. SBN treatment increases sirtuin activity in LX-2 cells. Cells were incubated with vehicle or SBN at 10, 50 and 100 μmol/L in DMEM containing 1% FBS for 96 hours. Data were presented as mean±SEM of three individual experiments withn=3. *:P<0.05, #:P<0.01, compared with vehicle.

    Discussion

    The inhibition of HSC proliferation is one of the promising therapeutic strategies to prevent the progression of liver fbrosis in chronic liver diseases.[26]Several studies were documented the anti-proliferative effect of SBN through cell cycle arrest.[19,25,27]In liver context, it has been previously reported an ameliorative effect of SBN on repeated dimethylnitrosamine-induced liver fbrosis in rats.[10]Therefore, we hypothesized that SBN might modulate liver fbrosis development via an inhibition of the activated HSCs proliferation. Hence, in this study, we demonstrated the anti-proliferative effect of SBN in dosedependant manner in immortalized human LX-2 HSCs with no alteration of cell viability. We also decipheredthe pathways targeted by SBN to block LX-2 cell cycle.

    Our results clearly show a retard active proliferation of LX-2 cells by SBN which is in line with previously reported effects of this compound. Indeed, SBN has been proposed to exhibit anti-proliferative effect by different mechanisms including stimulation of cell cycle arrest, apoptosis and cellular senescence.[28,29]These effects were mainly observed in proliferating cancer cell lines. In our study, we mainly investigated the mechanisms involved in inhibiting the proliferation of the human non-tumor LX-2 cells.

    Promoting the clearance of activated HSCs by enhancing their cell death by apoptosis is considered as one of the effective strategy towards the mitigation of liver fbrosis.[30]SBN exposure has been reported to induce cytotoxicity in various cell lines.[31,32]In contrast to these reports, we did not observe a marked cytotoxicity by SBN in LX-2 cells. In addition, in SBN treated LX-2 cells we did not notice any signifcant change in survivin expression whatever the investigated SBN concentrations. Survivin has been reported to inhibit apoptotic cell death in severalin vitroandin vivostudies.[33]These effects are in agreement with the absence of cell death as determined by fow cytometry using annexin V/PI staining even after 96 hours treatment (data not shown). Altogether, these data clearly indicate that SBN treatment does not have any effect on inducing apoptosis of LX-2 cells. Moreover, the discrepancy in cytotoxic response to SBN exposure in the current study as compared to previous reports could be due to different SBN doses and cell types as well.[28]

    We thereafter analyzed if the observed SBN effect is due to an inhibition of LX-2 cell proliferation. To further delineate whether the anti-proliferative effect of LX-2 cells upon SBN treatment was mainly through cell cycle arrest, the effect of SBN on proteins which are pertinent to G1 phase of cell cycle was investigated. In cell cycle progression, activation of Akt downstream signaling pathway plays a pivotal role in stimulation of HSCs proliferation, survival and migration.[4,34,35]It is also reported that Akt plays an important role in proliferation of rat primary HSCs.[36]In the present investigation, we revealed that SBN exposure caused down-regulation of Akt (Akt, Akt-Thr308 and Akt-Ser473) protein expressions at 96 hours of SBN treatment. This suggests that SBN could inhibit cell proliferation even in the earlier downstream process of cell cycle progression by altering the Akt signaling. Our data are in line with other studies showing that inhibition of Akt expression occurs following SBN treatment in variousin vitromodels other than HSCs.[37,38]

    Activation of Foxo transcription factors has been shown to regulate HSC cell cycle via an activation of Akt pathway.[39,40]The activation of Foxo transcription factors also enhanced the expression of p21 protein, which causes cell cycle arrest in the G1 phase.[41,42]Adachi et al[39]has shown that p27 protein is also a crucial downstream target of the Foxo transcription factor, which control both HSC proliferation and differentiation. Our data demonstrated that SBN caused an increase in the expression of Foxo3A and its phosphorylated protein pSer-Foxo3A only at 24 hours treatment with highest concentration (50 μmol/L) used in this study. This discrepancy indicate that SBN treatment, especially at high dose levels induced cell cycle arrest and block the progression of proliferating LX-2 cells.

    Similarly, SBN increased p27 protein expression in dose-dependent manner in LX-2 cells. These results show that the inhibition of Akt expression by SBN is positively correlated to an up-regulation of the transcription factor Foxo3A as well as CDK1 protein p27 in LX-2 cells. The cell cycle progression is basically controlled by activation of several CDKs which is critically regulated by p21 and p27 proteins as well as by the tumor suppressor proteins i.e., p53.[43]Experiment in rat primary HSCs demonstrated that an increase in the expression of p53 causes an enhancement of p21 protein expression.[44]In the present investigation, SBN treatment caused a dose-dependent increase in the expression of p53 protein. However, the expression of p21 remains unchanged. The increased expression of p53 could have also contributed towards the inhibition of cyclin E binding with CDK2 leading to cell cycle arrest. The mechanism of SBN induced p53 protein up-regulation is unknown and studies on these lines are warranted. These results demonstrated that inhibition of Akt could possibly inhibit its downstream signaling and LX-2 cell proliferation via cell cycle arrest involving up regulation of p53 and p27.

    Data from above experiments suggest that SBN 24-hour treatment of LX-2 cells inhibit the early G1 phase of Akt downstream signaling thereby it could have enhanced the Foxo3A transcription factors. This enhanced transcription factor expression might have induced the p27 expression which might have caused cell cycle arrest. The Ki-67 protein is a proliferation index and is normally expressed at its peak during all the phases of cell cycle except G0 and early G1 phases.[45]The absence of Ki-67 protein in quiescent cells has created great interest because it serves as a marker of cell proliferation.[46]The dose-dependent decrease in the expression of Ki-67 as well as the number of negatively stained LX-2 cells exposed to SBN is a clear indication to show that these cells have not achieved their proliferative state and they probably remain quiescent.

    Fig. 6. Schematic picture of pathways modulated by SBN that potentially govern the G1 cell cycle phase of HSC. G1: growth phase 1; S: synthetis phase; G2: growth phase 2; M: mitosis phase; Rb: retinoblastoma protein; Rb-pi: phosporylated retinoblastoma protein; CDK2: cyclin-dependent kinase 2; Akt: protein kinase-B; Foxo1/3: Forkhead box 1/3.

    Recently, role of sirtuin has been implicated in liver fbrosis. A recent study reported that sirtuin ameliorates liver fbrosis by inducing the reversion of the activated stellate cell and apoptosis in LX-2 cells.[47]In the present study, SBN increased the activity of sirtuin in LX-2 cells. Further, studies also indicate that sirtuin can act as a tumor suppressor. Sirtuin suppressed intestinal tumorigenesis and colon cancer growth in a β-catenin-driven mouse model of colon cancer.[48]In view of the above scenarios, it is suggested that the observed anti-proliferative effect of SBN in LX-2 cells could be via the increase of sirtuin expression in this study. However, it has been apparent that the effects of sirtuin on various cell lines are not entirely clear and still a subject of controversy.

    In conclusion, our present study on the potential effect of SBN on LX-2 cell cycle arrest have shown that SBN inhibits Akt and it phosphorylated protein expression through an induced earlier expression of the transcription factor Foxo3A and its phosphorylation as well as an enhanced protein expression of p27 and p53 and activity of sirtuin. This refects the complex regulatory pathways that SBN may provoke LX-2 cell cycle arrest (Fig. 6).

    Contributors:ED and NM designed the research, analyzed the data and wrote the paper. ED, EJ and SB performed the research. CPB, KS, SE and NM contributed analytic tools and scientifc discussion. NM is the guarantor.

    Funding:None.

    Ethical approval:The protocol and experiments were approved by the ethical committees of the St-Luc Hospital and faculty of Medicine of Université Catholique de Louvain, Brussels, Belgium.

    Competing interest:No benefts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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    Received December 16, 2015

    Accepted after revision August 29, 2016

    Author Affliations: Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy (Ezhilarasan D, Evraerts J, Sokal E and Najimi M), and Louvain Drug Research Institute, Toxicology and Cancer Biology Research Group, PMNT Unit (Sid B and Calderon PB), Université Catholique de Louvain, 1200, Brussels, Belgium; Facultad de Ciencias de la Salud, Universidad Arturo Prat, Iquique, Chile (Calderon PB); Department of Pharmacology and Environmental Toxicology, Food and Hepatotoxicology Laboratory, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600 113, India (Karthikeyan S)

    Mustapha Najimi, PhD, Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Université Catholique de Louvain, Avenue Mounier, 52, Box B1.52.03, 1200, Brussels, Belgium (Tel: +3227645283; Fax: +3227645258; Email: mustapha.najimi@uclouvain.be)

    ? 2017, Hepatobiliary Pancreat Dis Int. All rights reserved.

    10.1016/S1499-3872(16)60166-2

    Published online December 28, 2016.

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