Accepted Manuscript

Oral intake of chicoric acid reduces acute alcohol-induced hepatic steatosis in mice

Marianne Landmann, Giridhar Kanuri, Astrid Spruss, Carolin Stahl, Ina Bergheim

PII: S0899-9007(13)00543-1

DOI: 10.1016/j.nut.2013.11.015

Reference: NUT 9168

To appear in: Nutrition

Received Date: 14 May 2013

Revised Date: 25 October 2013

Accepted Date: 12 November 2013

Please cite this article as: Landmann M, Kanuri G, Spruss A, Stahl C, Bergheim I, Oral intake ofchicoric acid reduces acute alcohol-induced hepatic steatosis in mice, Nutrition (2014), doi: 10.1016/j.nut.2013.11.015.

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Oral intake of chicoric acid reduces acute alcohol-induced hepatic steatosis in mice

Marianne Landmanna, Dr. Giridhar Kanuria, Dr. Astrid Sprussb, Carolin Stahlb, Prof. Dr. Ina

Bergheima

aSD Model systems of Molecular Nutrition, Friedrich-Schiller University, Jena, D-07743 Jena, Germany bDepartment of Nutritional Medicine (180a), University of Hohenheim, D-70599 Stuttgart, Germany

Short title: Chicoric acid and alcohol-induced steatosis

Keywords: alcohol, steatosis, endotoxin, chicoric acid

Corresponding author: Prof. Dr. Ina Bergheim

Friedrich-Schiller University, Jena

SD Model systems of Molecular Nutrition

Dornburger Str. 29

D-07743 Jena

Germany

Phone: +49/3641/949-730

Fax: +49/3641/949-672

E-mail: ina.bergheim@uni-jena.de

Word Count main article: 4991

Number of Figures: 4

Number of Tables: 2

Supplemental Material: 2

Affiliations: I. B. designed the research; M. L., G. K., A. S. and C. S. conducted the research; M. L., G. K., A. S. and C. S. analysed the data; M. L. and I. B. wrote the paper; I. B. advised the work and had primary responsibility for the final content. Abbreviations used: 4-HNE, 4- hydroxynonenal; ADH1, alcohol dehydrogenase 1; ALD, alcoholic liver disease; b.w., bodyweight; IκB, inhibitor kappa B; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MyD88, myeloid differentiation primary response gene 88; NFκB, nuclear factor kappa B; PAI-1, plasminogen activator inhibitor 1; PPARγ, peroxisome proliferator activated receptor gamma; ROS, reactive oxygen spezies; TLR4, toll-like receptor 4; TNFα, tumor necrosis factor alpha Acknowledgements: The present project was supported by a grant from Institute Danone for Nutrition. We thank Birgit Huber for her contributions.

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Abstract

Objective Acute and chronic consumption of alcohol can alter intestinal barrier function thereby

increasing portal endotoxin levels subsequently leading to an activation of toll-like receptor 4 -

dependent signalling cascades, elevated levels of reactive oxygen species and induction of tumor

necrosis factor (TNF) α in the liver. Recent studies suggest that chicoric acid found in Echinacea

pupurea, chicory and other plants, may possess anti-oxidant and anti-inflammatory effects. The aim

of the present study was to determine if chicoric acid can reduce acute alcohol-induced liver

damage. Research Methods & Procedures Female mice were given chicoric acid orally (4 mg/kg

b.w.) for 4 days prior to acute ethanol administration (6 g/kg b.w.). Furthermore, the effect of

chicoric acid on the LPS-dependent activation in an in vitro model of Kupffer cells (RAW264.7

macrophages) was assessed. Results Acute alcohol ingestion caused a significant increase in hepatic

triglyceride accumulation, which was associated with increased protein levels of the inducible nitric

oxide synthase (iNOS), 4-hydroxynonenal protein adducts, and active plasminogen activator

inhibitor 1 protein in the liver. Pre-treatment of animals with chicoric acid significantly attenuated

these effects of alcohol on the liver. In lipopolysaccharide (LPS)-treated RAW264.7 macrophages

pre-treatment with chicoric acid significantly suppressed LPS-induced mRNA expression of iNOS

and TNFα. Conclusion These data suggest that chicoric acid may reduce acute alcohol-induced

steatosis in mice through interfering with the induction of iNOS and iNOS-dependent signalling

cascades in the liver.

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Introduction

One of the first characteristics of the onset of alcoholic liver disease (ALD) is fat

accumulation in the liver, long thought to be a benign, non-progressive histological state. However,

results of recent studies suggest that steatosis may be a critical factor not only in the onset of ALD

but also in the progression of the disease to later stages (e.g. fibrosis and cirrhosis, (1)). Therefore,

therapies protecting against the onset of ALD may have beneficial effects in regards to the

development of later stages of the disease. Administration of ethanol using a binge-drinking mouse

model has been shown to produce pathological changes in the liver that resemble early alterations

(e.g. steatosis) found in humans (2). Utilizing this model, a treatment regimen of potential

therapeutic value for humans can be tested.

Studies suggest that acute and chronic alcohol consumption can cause intestinal barrier

dysfunction associated with an increased translocation of bacterial endotoxin, which in turn can lead

to an activation of hepatic Kupffer cells, formation of reactive oxygen species (ROS) and increased

release of tumor necrosis factor (TNF) α in the liver (3-5). Indeed, studies using iNOS - knockout

mice suggest that an induction of the inducible nitric oxide synthase (iNOS) in the liver, probably in

Kupffer cells, is a major contributor in the pathogenesis of ALD damage (6;7). Furthermore, it has

been shown that the activation of Kupffer cells after chronic alcohol ingestion is associated with an

increase of markers associated with M1 polarization of these cells (8). The effects of TNFα after

acute and chronic alcohol ingestion on hepatocytes have been shown to be at least partly mediated

through an insulin-dependent induction of the expression of the plasminogen activator inhibitor

(PAI)-1 (2). Thus, therapies either protecting against a loss of intestinal barrier function or the

endotoxin-induced activation of Kupffer cells could be useful therapeutic approaches in humans to

prevent ALD.

Chicoric acid (dicaffeoyl-tartaric acid) found in a number of plants like chicory (Cichorium

intybus) and echinacea (Echinacea purpurea) (9;10) , belongs to the category of natural occurring

hydroxycinnamic acids being discussed to possess medicinal virtues such as antioxidant, anti-

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inflammatory, antiviral or anti-nociceptive activities (9;11;12). Besides being a potent HIV

integrase inhibitor (13) it has been shown that chicoric acid may also attenuate the

lipopolysaccharide (LPS) -dependent activation of RAW267.4 macrophages (14). Indeed, Park et

al. found a marked protection against the LPS-induced activation of cells (e.g. ROS-formation and

pro-inflammatory cytokines) exposing cells to chicoric acid (0-64 µM) in the presence of luteolin

(0-16 µM). However, in these studies exposure to chicoric acid alone only had limited effects on

markers of oxidative stress and inflammation when exposing cells to 1 µg LPS/ ml (14). Therefore,

the aim of the present study was to test the hypothesis that chicoric acid protects against early

alcohol-induced liver injury in a mouse model of acute alcoholic steatosis and if so to determine

potential mechanisms involved.

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Material and Methods

Animals and treatment

All procedures were approved by the local Institutional Animal Care and Use Committee

(IACUC). Mice were housed in a pathogen-free barrier facility accredited by the Association for

Assessment and Accreditation of Laboratory Animal Care (AAALAC) at the University of

Hohenheim. Six-week-old female C57BL/6J mice (n=5-6 per group; Janvier) were given ad libitum

access to food and tap water. In earlier studies and pilot experiments, 6-8 week old female mice

were shown to be more susceptible to acute-induced ALD than male mice (15). Chicoric acid used

in all experiments was a synthetic form with 95 % (HPLC) purity purchased from Sigma-Aldrich.

Chicoric acid was dissolved in tap water and was added to the drinking water so that mice received

4 mg/kg bodyweight (b.w.) for 4 days prior to ethanol administration. Dosage of chicoric acid was

adapted from Kour et al. (12) who used 2 mg/kg b.w.; however, as in a pilot experiment 4 mg/kg

b.w. chicoric acid was found to be more effective, this dose was chosen for the present study.

Liquid intake was assessed daily and chicoric acid concentration was adjusted accordingly. On day

5, mice received one bolus of ethanol (6 g/kg b.w. intragastric) or isocaloric maltodextrin solution

as previously described (16). Animals were anesthetized with 80 mg ketamine and 6 mg xylazine

per kg b.w. (i.p.) 12 h after ethanol administration. Liver tissue was frozen immediately in liquid

nitrogen, fixed in 4 % buffered formalin and frozen-fixed in Tissue-Tek® O.C.T.™ compound

media (Sakura) for later sectioning and mounting on microscope slides, respectively.

Cell culture and treatment

Murine RAW264.7 macrophage-like cells (American Type Culture Collection), grown in

DMEM media (PAN), were cultured as detailed before (16). Cells were treated with 160 µg/mL

chicoric acid in serum-free media. This concentration was shown in pilot experiments to be the

most effective and not to affect cell viability, assessed by trypan blue exclusion (Supplementary

Data 1). After 1 h, media was removed and replaced with fresh serum-free media containing

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50 ng LPS/mL serum-free media (Sigma-Aldrich, Germany) in the presence or absence of chicoric

acid. Cells were incubated for 18 h, rinsed with PBS and lysed with Trizol (Invitrogen) for later

RNA isolation. Medium was collected for the detection of nitrite levels using Griess reagent kit

(Promega) as detailed previously (16).

Oil Red O staining and triglyceride determination

Frozen sections of liver (10 µm) were stained with Oil Red O (Sigma-Aldrich) as described

previously (16). Hepatic triglycerides were isolated and measured as previously detailed (16).

Ethanol concentration

To determine blood alcohol levels the ‘alcohol dehydrogenase-enzymatic’ method described

by Bonnichsen and Lundgren (19) was used (detection limit: 0.0125 µmol/µl).

Immunoblots

To determine occludin protein levels in the duodenum, intestinal tissue was homogenized in

peqGOLD TriFast™ (PEQLAB) and protein was isolated according to the manufacturers

instructions. For the detection of the phosphorylation status of Akt and IκB (Cell Signaling

Technology) in liver tissue, cytosolic protein was isolated and Western blotting was done as

previously described (17). Levels of pAkt and pIκB were normalized to total Akt and total IκB,

respectively, whereas signals of occludin (Zymed) were normalized to β-actin. Protein bands were

analyzed densitometrically using Flurochem software (Alpha Innotech).

Immunohistochemical staining for 4-hydroxynonenal (4-HNE) protein adducts and iNOS

protein

Liver sections embedded in paraffin were cut (5 µm) and stained for 4-HNE (AG Scientific)

and iNOS (Affinity BioReagents), respectively, using a polyclonal antibody as described previously

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(18). Using an image acquisition and analysis system incorporated in the microscope (Axio

Vert200M, Zeiss), the extent of staining in liver sections was defined as percent of the field area

within the default colour range determined by the software. To determine means, data from 8 fields

(200 x) of each tissue section were used.

ELISAs

Concentration of active PAI-1 was determined in liver homogenate of mice using a mouse-

PAI-1 ELISA kit (Molecular Innovations, LOXO). Using a Trans AM-ELISA-based kit (Active

Motif) activity of PPARγ was measured in nuclear extract isolated from liver.

RNA isolation and real-time RT-PCR

RNA isolation and real-time RT-PCR has been previously detailed (16). SsoFast EvaGreen®

Supermix (Bio-Rad) was used to prepare the PCR mix. Primer concentration in the PCR mix was

0.15 pmol/µl. Sequences are shown in Supplementary Data 2. The comparative CT method was

used to determine the amount of target, normalized to an endogenous reference (18S) and relative to

a calibrator (2-∆∆Ct). The purity of the PCR products was verified by melting curves and gel

electrophoresis.

Statistical analyses

Results are reported as means ± SEM. One-way ANOVA with Tukey’s post-hoc test was

used for the determination of statistical significance among treatment groups. A p≤0.05 was

selected as the level of significance before the study. Grubbs test to identify outliers was used.

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Results

Effect of chicoric acid treatment on acute alcohol-induced hepatic steatosis and markers of

ethanol metabolism

Ethanol treatment caused a significant ~10-fold accumulation of triglycerides in mouse

livers. In contrast, in livers of animals treated with chicoric acid prior to ethanol ingestion,

triglyceride levels were only ~4-fold higher than in livers of controls and were significantly lower

in comparison to animals only exposed to ethanol (Figure 1B). Similar results were also found in

liver sections stained with Oil Red O (Figure 1A: representative pictures). 12 h after ethanol

administration plasma ethanol levels were still slightly higher than in the respective control groups

(+40 %, p=0.50). Furthermore, no differences were found comparing CYP2E1 and ADH1 mRNA

in livers of ethanol-treated groups. However, expression of these two genes was markedly lower in

livers of mice treated with ethanol compared to the respective controls (Table 1).

Effect of chicoric acid on the LPS-induced activation of TLR-4-dependent signalling cascades

in vitro

Eighteen hours after LPS exposure, MyD88 mRNA expression was significantly induced in

LPS-challenged RAW264.7 macrophages (+ ~30 % in comparison to untreated cells, Table 2). In

contrast, mRNA expression of MyD88 was almost at the level of naïve controls in LPS-challenged

cells pre-treated with chicoric acid. In LPS-challenged RAW264.7 cells, mRNA expression of

iNOS was induced by ~100-fold in comparison to untreated cells (Table 1). This effect of LPS was

significantly attenuated in cells pre-treated with chicoric acid. After LPS-treatment TNFα mRNA

expression was also markedly attenuated in RAW264.7 macrophages pre-exposed to chicoric acid.

Effect of chicoric acid and acute alcohol exposure on occludin protein levels in the duodenum

and MyD88 mRNA expression in the liver

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Twelve hours after the acute alcohol exposure, protein levels of the tight junction protein

occludin in the duodenum did not differ between groups (Figure 2A). In contrast, MyD88 mRNA

expression was significantly induced in livers of animals exposed to alcohol in comparison to the

respective controls, whereas a similar effect on MyD88 mRNA expression was not found in livers

of mice treated with chicoric acid prior to the alcohol challenge (Figure 2B).

Effect of chicoric acid and acute alcohol exposure on PAI-1 and iNOS protein levels, 4-HNE

protein adducts and phosphorylation status of Akt and IκB in the liver

Protein levels of the active PAI-1 were significantly induced in livers of mice exposed to

alcohol only, whereas in livers of mice pre-treated with chicoric acid this effect of alcohol was

almost completely attenuated (Figure 3A). Expression of iNOS protein in the liver did not differ

between controls; however, in livers of mice exposed to alcohol only, protein levels of iNOS were

significantly induced ( ~1.7-fold). In contrast, in livers of mice treated with chicoric acid before the

acute alcohol exposure, iNOS protein levels were almost at the level of controls (Figure 4A,B). In

line with these findings, levels of 4-HNE protein adducts were also only found to be significantly

elevated in livers of mice treated with alcohol (~1.8-fold, Figure 4C,D). Phosphorylation of both,

Akt and IκB did not differ between groups (Figure 2C,D).

Effect of chicoric acid and acute alcohol exposure on PPARγ activity and CD11c mRNA

expression in the liver

With the dose used, chicoric acid did not significantly alter PPARγ activity in the liver

(Figure 3B). Furthermore, whereas expression of CD11c mRNA in the liver did not differ between

controls and alcohol-treated mice, expression of CD11c mRNA was markedly lower in livers of

mice treated with chicoric acid and herein particularly in those additionally treated with alcohol

(Figure 3C).

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Discussion

Despite intense research efforts during the last decades therapies preventing the onset but

also progression of ALD are still limited. Animal models that resemble the early stages of ALD in

humans, like steatosis or even steatohepatitis have not only been used to study molecular effects of

alcohol on the liver but also to test potential new therapeutic approaches. Several studies suggest

that acute and chronic ALD share similar mechanisms (for overview see (5;20)). For instance,

results of Rivera et al. (21) suggest that similar to the findings in models of chronic alcohol

exposure, the acute ingestion of 5 g ethanol/kg bodyweight can lead to a marked increase in portal

endotoxin levels as early as 90 min after the alcohol ingestion in rats. Furthermore, Bergheim et al.

(2) showed that acute and chronic intake of alcohol is associated with an induction of hepatic PAI-

1, supporting the hypothesis that acute and chronic alcohol-induced liver damage may at least in

part originate from similar molecular alterations (e. g. increased translocation of bacterial endotoxin

from the gut, activation of TLR-4-dependent signalling cascades in the liver and induction of pro-

inflammatory cytokines like TNFα). In the present study, the hypothesis that chicoric acid protects

against alcohol-induced fat accumulation in the liver was tested in a model of acute alcohol

ingestion. Neither blood alcohol levels nor expression of enzymes involved in ethanol metabolism

(e.g. ADH1 and CYP2E1) in the liver differed between the two ethanol-treated groups suggesting

that chicoric acid had no effect on hepatic ethanol metabolism. Interestingly expression of both ,

ADH1 and CYP2E1 was markedly lower in livers of ethanol-treated mice compared to controls

12 h after alcohol exposure. These results are somewhat contrary to those reported by others (22).

However, differences found between the present study and those of others might have resulted from

differences in the experimental setup (one bolus dose versus chronic exposure or repeated acute

exposure) and time of measurement (12 h after alcohol exposure versus directly after chronic

exposure). Despite no apparent differences in alcohol metabolism, oral pre-treatment of mice with

chicoric acid not only markedly reduced hepatic lipid accumulation induced by acute alcohol

exposure but also attenuated the induction of active PAI-1 protein, an acute phase protein

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previously shown to be involved in alcohol-induced hepatic steatosis but also hepatic inflammation

(23).

To determine if the protective effects of chicoric acid on alcohol-induced steatosis resulted

from a protection against the impairments of the intestinal barrier function, protein levels of the

tight junction protein occludin in the duodenum were determined. For occludin it was shown before

that a loss of this tight junction protein in the duodenum is associated with an increased

translocation of bacterial endotoxin in other models of liver damage (e.g. non-alcoholic fatty liver

disease (24)). However, in the present study, occludin protein levels did not differ between groups

suggesting that either 12 hours after the acute exposure to alcohol effects on occludin protein levels

are no longer found, or the increased translocation of bacterial endotoxin described before by others

after acute ingestion of alcohol (4;21) are not associated with altered occludin protein levels. This

will have to be addressed in future studies.

Results of Juskiewicz et al. suggested that the polyphenol fraction of chicory leaves, which

contain considerable amounts of chicoric acid (e.g. 2.13 g/100 g dried leaves), may decelerate

pro-oxidative processes in vivo (10). In the present study, the protective effects of pre-treating mice

with chicoric acid in drinking water against acute alcohol-induced liver steatosis were associated

with a repression of the induction of the mRNA expression of toll-like receptor adaptor protein

MyD88 but even more so of iNOS protein and the formation of 4-HNE protein adducts, the latter

being a marker of lipid peroxidation. In line with the findings of the in vivo studies, LPS-induced

expression of MyD88 and even more of iNOS and TNFα in RAW264.7 macrophages was markedly

attenuated in cells concomitantly treated with chicoric acid. Indeed, we recently showed that iNOS,

probably through NFκB-dependent signalling cascades may regulate expression of MyD88 in

RAW267.4 cells (unpublished data). The molecular mechanisms involved in the induction of

MyD88, iNOS and TNFα found in cells only treated with chicoric acid and also of MyD88 in livers

of mice treated with chicoric acid only remain to be determined in future studies. To our knowledge

an interaction of chicoric acid with transcription factors involved in the regulation of those genes

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has not yet been described. It may have been that chicoric acid used was slightly contaminated with

endotoxins; however, as chicoric acid interferes with the LAL test used to determine endotoxin

levels (16) it was not possible for us to determine actual levels of endotoxin in the chicoric acid

used in our experiments (data non shown). Taken together, these results suggest that chicoric acid

protected the liver from acute alcohol-induced liver damage through mechanisms involving a

protection against the induction of iNOS and subsequently the enhanced formation of ROS. These

data by no means preclude that chicoric acid may also alter intestinal barrier function. Rather our

data suggest that in the present study other mechanisms may have been involved in the protective

effects found.

Results of Park et al. (14) using LPS-stimulated RAW264.7 cells suggest that chicoric acid

in the presence of luteolin may exert its antioxidative and anti-inflammatory effect through an

attenuation of Akt phosphorylation followed by an inactivation of NFκB; however, effects of

chicoric acid alone only showed limited effects on parameters determined. In the present study,

phosphorylation of Akt and IκB did not differ between groups suggesting that the protective effects

of chicoric acid found in present study may have resulted from different mechanisms. Differences

between the results of Park et al. (14) and our study may have resulted from differences in the

experimental setup (e.g. cell culture vs. in vivo experiments) and differences in the concentration of

chicoric acid and LPS used in these experiments (e.g. Park et al. 0-64 µM chicoric acid and 1µg/ml

LPS (14)). These results do not preclude that chicoric acid may also have an effect on Akt and IκB

or NFκB in vivo; however, maybe other concentrations then the ones used in the present study are

required to affect these proteins.

In recently published studies it was shown that PPARγ is an essential regulator of the

activation of Kupffer cell activation (25) and that PPARγ ligands may attenuate the LPS-dependent

induction of iNOS in vitro (26). In the present study, the acute exposure to alcohol resulted in no

marked alterations in the activity of PPARγ in the liver of alcohol-exposed mice. Furthermore,

expression of CD11c, a marker of M1 polarisation of macrophages (8) was not found to be

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markedly altered in livers of mice treated with alcohol either. Taken together, these data suggest

that the protective effects of chicoric acid found in the present study did at least not primarily result

from alterations of PPARγ activity in the liver. The lack of alteration of markers of macrophage

polarisation may be due to the fact that in the present study alcohol was only given once and

expression was only determined at one time point.

In summary, the results of the present study suggest that chicoric acid may protect mice

from acute alcohol-induced liver steatosis through attenuating the alcohol-dependent induction of

iNOS and the subsequent formation of ROS as well as induction of TNFα and PAI-1. Indeed, our

data add further weight to the concept that pharmacological or nutritive supplementation of

substances interfering with the activation of the TLR-4-dependent signalling cascade may have the

potential to protect the liver from alcohol-induced damage.

Limitations

Several limitations need to be considered when interpreting the data. First, we used a model

of acute alcohol exposure, challenging mice only once with a dose of ethanol. However, results of

Rivera and our own group (2;21) suggest that acute and chronic ethanol-induced liver damage may

partly result from similar mechanisms. However, it should be emphasized that models of acute

alcohol exposure by no means resemble all effects of chronic alcohol consumption on the liver.

Nevertheless, models using an acute alcohol exposure can be used to study the very early effects of

alcohol on the liver in the development of chronic alcohol-induced liver damage and may be useful

tools to test the efficacy of new potential therapeutic interventions. Second, it cannot be ruled out

that at least some of the protective effects found in the present study resulted from changes at the

level of the intestine (e.g. metabolites formed by bacteria) rather than the liver. Indeed, it was

recently shown that bacterial esterases metabolize chicoric acid thereby producing probably also

potent secondary metabolites (27). Our results therefore may not be generalized to effects only

found at the level of the liver. Third, we used a mouse model and only one dose of chicoric acid.

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Therefore it remains to be determined if chicoric acid also has protective effects in humans and if

other doses may exert adverse effects.

Conclusion

Although future studies will be needed to identify the mechanism(s) responsible for the

protective effects of chicoric acid and to further explore the effects of chicoric acid in humans, our

results suggest that chicoric acid may be useful to prevent diseases associated with an induction of

iNOS/-dependent signalling cascades.

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Figure legends

Fig.1 Effect of pre-treatment with chicoric acid on alcohol-induced hepatic steatosis. (A)

Representative photomicrographs of Oil Red O staining (400 x) of liver sections and (B)

quantification of the hepatic triglycerides accumulation. Data are means ± SEM. Means without a

common letter differ (p<0.05). CA=chicoric acid enriched drinking water (4 mg/kg b.w.) for 4 d

before treatment; EtOH=ethanol (6 g/kg b.w.).

Fig.2 Effect of chicoric acid and acute alcohol exposure on occludin protein levels in the

duodenum, MyD88 mRNA expression and phosphorylation status of IκB and Akt in the liver.

Representative pictures and analysis of Western blots. (A) Occludin protein is normalized to β-

actin. (B) Relative expression of MyD88 mRNA normalized to 18S mRNA expression. (C) pAkt

protein is normalizes to Akt and (D) pIκB to IκB, respectively. Means without a common letter

differ (p<0.05). CA=chicoric acid enriched drinking water (4 mg/kg b.w.) for 4 d before treatment;

EtOH=ethanol (6 g/kg b.w.).

Fig.3 Effect of chicoric acid and acute alcohol exposure on protein levels of PAI-1 and PPARγ

and CD11c mRNA expression in the liver. (A) Expression of PAI-1 and (B) PPARγ protein as

well as relative (C) CD11c mRNA expression normalized to 18S mRNA in the liver. Data are

means ± SEM. Means without a common letter differ (p<0.05). CA=chicoric acid enriched drinking

water (4 mg/kg b.w.) for 4 d before treatment; EtOH=ethanol (6 g/kg b.w.).

Fig.4 Effect of chicoric acid and acute alcohol exposure on iNOS protein levels and 4-HNE

protein adducts in the liver. (A) Representative pictures of immunohistochemical staining of

iNOS and (C) 4-HNE and densitometric analysis of the staining of iNOS (B) and 4-HNE (D) in the

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liver. Data are means ± SEM. Means without a common letter differ (p<0.05). CA=chicoric acid

enriched drinking water (4 mg/kg b.w.) for 4 d before treatment; EtOH=ethanol (6 g/kg b.w.).

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Table 1 Effects of chicoric acid on parameters related to alcohol metabolism1

Control Ethanol Chicoric Acid Ethanol + Chicoric Acid

Blood:

Ethanol Level 100 ± 19 a 140 ± 26 a 116 ± 23 a 143 ± 23 a

2Liver:

ADH1 100 ± 18 a 56.5 ± 10 a,b 80 ± 8.5 a,b 44 ± 7 b

CYP2E1 100 ± 6.5 a,c 61 ± 9 b,c 85 ± 13 c 47 ± 6 b

1Data are means ± SEM and are normalized to percent of control. Means in a row without a

common letter differ (p<0.05).

2ADH1 and CYP2E1 mRNA expression are normalized to 18S mRNA expression.

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Table 2 Effect of chicoric acid on MyD88, iNOS and TNFα mRNA expression in LPS-challenged

RAW264.7 macrophages1

Control LPS Chicoric Acid LPS + Chicoric Acid

MyD88 100a 131 ± 8b 120 ± 5a,b 101 ± 6a

iNOS 100a 10168 ± 2559b 632 ± 177a 1528 ± 226a

TNFα 100a 2077 ± 295b 789 ± 48a,b 1484 ± 307b,c

1Data are means ± SEM, (n=4) and are normalized to percent of control. Means in a row without a

common letter differ (p<0.05).

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