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MicroRNA134 Mediated Upregulation of JNK and Downregulation of NFkB Signalings are Critically Involved in Dieckol Induced Antihepatic Fibrosis Sangyoon Lee, Jihyun Lee, Hyojung Lee, Bonglee Kim, Jaehwan Lew, Nam-In Baek, and Sung-Hoon Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01945 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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Journal of Agricultural and Food Chemistry

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MicroRNA134 Mediated Upregulation of JNK and Downregulation of NFkB

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Signalings are Critically Involved in Dieckol Induced Antihepatic Fibrosis

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Sang Yoon Lee+#, Jihyun Lee§#, HyoJung Lee§, Bonglee Kim§, Jaehwan Lew+,

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Namin Baek¤ and Sung-Hoon Kim§*

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§

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+

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Science and ¤Department of Oriental Medicine Biotechnology, Graduate School of

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College of Korean Medicine, Kyung Hee University, Seoul 131-701, South Korea Department of East West Medical Science, Graduate School of East West Medical

Biotechnology, Kyung Hee University, Yongin 446-701, South Korea

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ABSTRACT

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Though Dieckol, a phlorotannin of Ecklonia cava, was known to have anti-oxidant,

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anti-cancer, anti-diabetic and anti-inflammatory effects, the underlying anti-fibrotic

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mechanism of Dieckol still remains unclear until now. Thus, in the current study,

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inhibitory mechanism of Dieckol on liver fibrosis was elucidated mainly in hepatic

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stellate cells (HSCs). Dieckol exerted cytotoxicity in LX-2, HSC-T6 and HepG2

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cells with the reduced fibrosis features of large, spread out and flattened polygonal

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shapes in LX-2 cells compared to untreated control. Dieckol attenuated the

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expression of α-SMA and TGF-β1, increased sub-G1 phase population and induced

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caspase-3 activation and cleavages of PARP in HSCs. Furthermore, Dieckol

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decreased phosphorylation of ERK, p38, AKT, NF-kB and IkB and activated

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microRNA(miR)134 level and JNK phosphorylation in HSCs. Conversely, JNK

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inhbitor SP600125 reversed the effect of Dieckol on PARP, p-NF-kB, α -SMA and

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p-JNK in LX-2 cells. Likewise, miR134 overexpression mimic enhanced

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phosphorylation of JNK and NF-kB and reduced the expression of α -SMA and

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PARP cleavage, while miR134 inhibitor reversed the ability of Dieckol to cleave

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PARP and attenuate the expression of α -SMA in LX-2 cells. Overall, our findings

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suggest that Dieckol suppresses liver fibrosis via caspase activation and miR134

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mediated JNK activation and NF-kB inhibition.

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Keywords: Dieckol, apoptosis, JNK, NF-kB, miR134, liver fibrosis

o

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INTRODUCTION

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Liver fibrosis is a liver disease with excessive accumulation of extracellular matrix

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induced by chronic liver inflammation by alcohol abuse, metabolic diseases, viral

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hepatitis, cholestatic liver diseases and autoimmune diseases eventually leading to

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liver cirrhosis1-3. Also, it was well documented that the activation of hepatic stellate

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cells (HSCs) plays a pivotal role during the initiation and development of liver

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fibrosis4, 5. Activated HSCs by various kinds of pathologic factors are fibrogenic,

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and proliferative and subsequently accumulating ECM, while HSCs are normally

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quiescent6, 7. Thus, recently several researches were conducted targeting HSC

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activation with natural compounds such as oridonin8, galangin9, puerarin10,

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ligustrazine and paeoniflorin11 and herbal medicine such as Prunella vulgaris12,

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Sho-saiko-to13, since the HSC activation is an important event in fatty liver.

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Dieckol, a phlorotannin, derived from brown algae Ecklonia cava, Ecklonia

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stolonifera and Eisenia bicyclis was known to have neuroprotective14, antioxidant15,

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antidiabetic16

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antihyperlipidemic20 activity. Furthermore, Dieckol induced apoptosis in Hep3B

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cancer cells21, and also showed hepatoprotective22,

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tetrachloride or ethanol induced liver damage. Nevertheless, the underlying

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inhibitory mechanism of Dieckol on liver fibrosis is not clearly understood so far.

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Therefore, in the present project, inhibitory mechanism of Dieckol on liver fibrosis

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was elucidated in hepatic stellate cells (HSCs) in association with caspase related

and

anti-inflammatory17,

antithrombotic18,

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antibacterial19

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effect from carbon


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apoptosis and miR134 mediated JNK signaling, based on the report that miR134

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inhibits epithelial to mesenchymal transition (EMT)7 closely involved in liver

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fibrosis24-26.

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MATERIALS AND METHODS

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Cell cultures

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HepG2 carcinoma cells (ATCC® HB-8065™) and AML-12 normal murine

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hepatocyte cells(ATCC® CRL-2254™) were obtained from the American Type

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Culture Collection (ATCC, USA) and LX-2 human hepatic stellate cells, HSC-T6

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immotalized rat hapatic stellate cells were kindly supplied from prof. Friedman Scott,

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Johns Hopkins University. HepG2, HSC-T6 and AML-12 cells were cultured in

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DMEM supplemented with 10% FBS and 1% antibiotic (Welgene, Daegu, South

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Korea). LX-2 (immortalized human hepatic stellate cell line) cells were maintained

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in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 5% FBS and 1%

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antibiotic (Welgene, Daegu, South Korea).

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Isolation of a phlorotannin, Dieckol, from Ecklonia cava

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Ecklonia cava was havested in Jeju island in April spring, 2015 and identified by Dr.

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Namin Beak, a professor and pharmacognosist of Kyunghee University,. The

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samples were stored at CPMDRC deep freezer with the name of CPMDRC 1538.

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The dried E. cava (2.8 kg) was treated with n-hexane (10 L) in a sonication chamber

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and filtered. The residue was extracted in 80% MeOH including 0.3% ascrobic acid

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(10 L × 3) for 12 h on the shaker. The combined extracted solutions were filtered

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and evaporated to produce a MeOH extract (289 g). The MeOH extract was poured

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in H2O (4 L) and successively extracted with n-hexane (4 L × 2), EtOAc (4 L × 2),

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and n-BuOH(3.5 L × 2). Each layer was evaporated to obtain n-hexane fraction (12

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g), n-BuOH fraction (34 g), EtOAc fraction (63 g) and aqueous fraction (180 g). A

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part of EtOAc fraction (ECE, 55 g) was subjected to a celite column

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chromatography (c.c.) and eluted with CHCl3-MeOH (3:1) to yield 11 fractions

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(ECE1 to ECE11). ECE-3 fraction (6 g) was applied to a Sephadex LH-20 using

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80% MeOH as an eluting solvent to get 20 fractions (ECE3-1 to ECE3-20) along

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with a phlorotannin, dieckol (ECE3-19, 124 mg). Dieckol (dioxin-2-yl]oxy]-3,5-

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dihydroxyphenoxy]-dibenzo[b,e][1,4]dioxin-1,3,6,8-tetrol) was identified based on

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spectroscopic analyses such as NMR, MS, and IR in comparison with the data with

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those reported in the bibliography 27.

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Cell viability assay

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Cytotoxic effect of Dieckol (Fig. 1) derived from Ecklonia cava was assessed by

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using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

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Briefly, LX-2, HSC-T6, HepG2 and AML-12 cells (1×104 cells/well) were

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dispensed onto 96-well culture plate and treated by various concentrations of

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Dieckol for 24 h. The cells were incubated with MTT (1 mg/mL) (Sigma Chemical,

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St. Louis, MO, USA)

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overnight. Optical density (OD) was assessed using a microplate reader (Molecular

for 2 h and subsequently treated with MTT lysis solution

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Devices Co., Sunnyvale, CA, USA) at 570 nm. Cell viability was counted as a

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percentage of viable cells in Dieckol treated group vs untreated control.

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Sub-G1 accumulation assay

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Cell cycle analysis was conducted by propidium iodide (PI) staining. LX-2 cells

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exposed to Dieckol for 24 h were harvested and fixed in 70% ethanol. Thereafter,

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the cells were incubated at 37 °C with 0.1% ribonuclease (RNase) A in PBS for

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30 min and then suspended in PBS containing 30 µg/mL PI for 30 min at room

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temperature. Sub-G1 accumulation was evaluated from the stained cells by

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FACSCalibur (Becton Dickinson, USA) with the Cell Quest program (Becton

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Dickinson, Franklin Lakes, NJ, USA).

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DAPI staining and microscopic observation

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Nuclear morphological changes of the cells treated with Dieckol were observed by

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DAPI staining. Briefly, after LX-2 cells were exposed to 50 µM Dieckol, the cells

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were dispensed onto poly-L-lysine coated slides and then fixed using 4% methanol-

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free formaldehyde solution for 25 min at 4℃. After washing the slides with PBS,

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mounting medium with DAPI was poured over the entire section of slides and then

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visualized under Axio vision 4.0 fluorescence microscope (Carl Zeiss Inc., Weimar,

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Germany).

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Western blotting

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LX-2 and HSC-T6 cells exposed to Dieckol for 24 h were lysised in RIPA buffer

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(50 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl and 1% TritonX-100) containing

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protease inhibitors (Roche, Mannheim, Germany), and phosphatase inhibitors

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(Sigma-Aldrich, St. Louis, MO, USA). The protein extracts were separated on 8 to

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15% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes.

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The membranes were incubated with several primary antibodies. Pro-caspase-3

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(#9662, 1:1000), poly (ADP-ribose) polymerase (PARP#9542, 1:1000), p-AKT

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(#4060, 1:1000), AKT (#4691, 1:1000), NF-kB (#8242, 1:1000), p-NF-kB (#3033,

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1:1000), p-IkB (#9246, 1:1000), p-ERK (#9101, 1:1000), p38 (#9212, 1:1000), p-

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p38 (#9211, 1:1000), JNK (#9252, 1:1000), p-JNK (#9251, 1:1000) (Cell signaling,

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Beverly, MA, USA), α-SMA (#53015, 1:1000), TGF-β1 (#146, 1:1000) (Santa Cruz

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Biotechnologies, CA, USA) and β-actin antibody (#A5316,1:5000) (Sigma-Aldrich,

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St. Louis, MO, USA) were diluted in 3% BSA imixed in PBS-Tween20 (1:500-

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1:2000) overnight at 4°C, washed thrice with PBS-Tween20, and then incubated

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with HRP-conjugated secondary antibodies. The expression was detected by using

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ECL Western blotting detection reagent (GE Healthcare, Buckingham, England).

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Cells were exposed to 10 µM JNK inhibitor (SP600125) for 1 h and then exposed to.

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Dieckol (50 µM) for 24 h. Cell lysates were prepared and subjected to Western

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blotting with antibodies for p-JNK, α-SMA, TGF-β1, Pro-caspase-3, Pro-PARP,

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NF-kB, p-IkB and β-actin

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Cytoplasmic and nuclear fraction preparation for p-IkB and NF-kB

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LX-2 cells exposed to Dieckol for 24 h were fractionated into cytoplasmic and

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nuclear fractions using NE-PER nuclear and cytoplasmic extraction reagents

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(Thermo, Rockford, IL, USA) based on the manufacturer’s protocol for Western

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blotting with p-IkB and NF-kB.

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RT-qPCR analysis

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To evaluate the expression of miR134, total RNA from Dieckol treated LX-2 cells

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was isolated by QIAzol (Invitrogen, Carlsbad, CA, USA). To construct microRNA

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cDNA, GenoExplorer

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USA) was used based on the manufacture’s protocol. RT-qPCR was conducted with

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the LightCycler TM instrument (Roche Applied Sciences, Indianapolis, IN) using

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the following primers, miRNA134- forward: 5’- CAG GGT GTG TGA CTG GTT

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GA -3’; reverse- 5’-GAG GGT TGG TGA CTA GGT GG-3’ (Bioneer, Daejeon,

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Korea), hGAPDH-forward5’-CCA CTC CTC CAC CTT TGA CA-3’;reverse-5’-

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ACC CTG TTG CTG TAG CCA -3’ (Bioneer, Daejeon, Korea).

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miR134 mimic (sequence (5’-3’):UGUGACUGGUUGACCAGAGGGG) and

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miR134 inhibitor (sequence (5’-3’):UGUGACUGGUUGACCAGAGGGG) were

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ordered from Bioneer (Bioneer, Daejeon, Korea). miR Control (AccuTarget™

TM

miRNA cDNA kit (GenoSensor Corporation, Arizona,

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miRNA mimic Negative Control #1. Cat.No. SMC-2001) was purchased from

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Bioneer (Bioneer, Daejeon, Korea).

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MicroRNA (miR) transfection assay

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The miR134 mimic, miR134 inhibitor and miR control (200 nM) (Bioneer, Daejeon,

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Korea) were transfected into LX-2 cells using lipofetamine 2000 (Invitrogen,

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Carlsbad, CA, USA) reagent according to the manufacture’s protocol.

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Statistical analysis

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All Data were presented as means ± SD. Statistical significance was evaluated by

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using Student's t-test of SigmaPlot software (Systat Software Inc., Richmond, CA,

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USA).

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RESULTS

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Dieckol exibited cytotoxicity in LX-2, HSC-T6, and Hep G2 cells and effectively

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suppressed the expression of α-SMA and TGF-β1 with inactivated HSC

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morphology in LX-2 cells

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The chemical structure of Dieckol is shown in Fig. 1A. To evaluate the cytotoxic

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effect of Dieckol, MTT assay was performed in LX-2, HSC-T6, HepG2 and AML-

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12 cells. As shown in Fig. 1B, Dieckol at 25 and 50 µM significantly suppressed the

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viability of cells compared to untreated control. To test whether or not Dieckol

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suppresses the morphology of activated HSC, microscopic observation was

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performed in LX-2, HSC-T6, and HepG2 cells. As shown in Fig. 1C, the fibrosis

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features of large, spread out and flattened polygonal shapes were observed in LX-2,

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HSC-T6 and HepG2 cells, while elongated and round single cells were shown in

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Dieckol treated cells. To investigate the anti-fibrotic effect of Dieckol, the

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expression of α-SMA and TGF-β1, the liver fibrogenesis biomarkers, was evaluated

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by Western blotting. Dieckol attenuated the expression of α-SMA and TGF-β1 in

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LX-2 and HSC-T6 cells (Fig. 1D).

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Dieckol increased the number of apoptotic bodies and sub-G1 phase population

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of LX-2 cells.

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It was well documented that sub-G1 and apoptotic bodies are the features of

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apoptosis28. Thus, DAPI staining and cell cycle analysis were performed in LX-2

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cells. As shown in Fig. 2A, apoptotic bodies were shown in Dieckol treated LX-2,

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HSC-T6 and HepG2 cells compared to untreated control. Also, Dieckol treatment

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significantly increased sub-G1 phase population in LX-2 and HSC-T6 cells (Fig.

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2B).

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Dieckol regulated apoptotic proteins in LX-2, HSC-T6 and HepG2 cells.

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Caspases (cysteine-aspartic proteases) play a pivotal role in apoptosis induction. In

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general two types of apoptotic caspases are well known as initiator caspases

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(caspase-2, caspase-9, caspase-8 and caspase-10) and effector caspases (caspase-3,

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caspase-7 and caspase-6)29.

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of pro-caspase-3, and pro-PARP in LX-2, HSC-T6 and HepG2 cells (Fig. 3A).

Here Diekckol significantly attenuated the expression

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Dieckol modulated phosphorylation of MAPKs and NF-kB in LX-2 and HSC-

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T6 cells.

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There are accumulating evidences that MAPKs and NF-kB proteins play critical

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roles in the proliferation of several cells30. The roles of MAPKs and NF-kB were

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examined in Dieckol treated LX-2, HSC-T6 and HepG2 cells. As shown in Fig. 3B,

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Dieckol activated phosphorylation of JNK, but not that of ERK. In contrast,

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phosphorylation of p38 was attenuated by Dieckol in LX-2 cells, but activated in

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HSC-T6 cells. Also, Dieckol suppressed phosphorylation of AKT in LX-2 and HSC-

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T6 cells (data not shown). Likewise, phosphorylation of NF-kB and IkB (whole cell

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lysates) was significanlty reduced by Dieckol treatment in LX-2 and HSC-T6 cells

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(Fig. 3C).

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JNK inhibitor SP600125 reversed effects of Dieckol in LX-2 cells.

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To confirm the role of JNK in Dieckol induced anti-fibrotic and apoptotic effects in

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HSCs, the cells were treated with Dieckol with or without JNK inhibitor SP600125.

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As shown in Fig. 4A, 4B and 4C, JNK inhibitor SP600125 blocked the activation of

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caspase-3 and cleavage of PARP and inactivation of α-SMA and TGF-β1 induced

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by Dieckol in LX-2 cells. Consistently, JNK inhibitor SP600125 restored the

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decreased phosphorylation of IkB in cytoplasm and NF-kB in nucleus by Dieckol in

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LX-2 cells (Fig. 4D).

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miR134 plays a critical role in Dieckol induced antihepatofibrotic effect in LX-

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2 cells.

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The pivotal role of miR134 was examined in Dieckol induced antihepatofibrotic

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effect in LX-2 HSCs, because miR134 can be postulated as a potent molecular target

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for liver fibrosis, based on the previous evidences that miR134 inhibits proliferation

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and EMT that is closely involved in fibrosis process in cancer cells24, 31, 32. Here

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Dieckol upregulated the mRNA level of miR134 in LX-2 cells (Fig. 5A).

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Overexpression of miR134 using miR134 mimic enhanced the phosphorylation of

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JNK and cleavage of PARP, reduced phosphorylation of NF-kB and also attenuated

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the expression of α-SMA in LX-2 cells (Fig. 5B). In contrast, miR134 inhbitor

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reversed the ability of Dieckol to attenuate the expression of pro-PARP and α-SMA

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in LX-2 cells (Fig. 5C).

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DISCUSSION

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Liver fibrosis is known to be induced by various kinds of cytokines and extracellular

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matrix proteins (ECMs) produced by myofibroblasts from activated HSCs5, 33 due to

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several factors such as infectious diseases, metabolic derangements (non-alcoholic

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steatohepatitis), exposure to toxins and autoimmune diseases1, 3, 6, 34. Current study

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was undertaken to elucidate the molecular mechanism of Dieckol in HSCs, targeting

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liver fibrosis. Dieckol exerted cytotoxicty in LX-2, HSC-T6, and HepG2 cells,

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implying potential of apoptotic and antitumor effects of Dieckol.

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There are accumulating evidences that apoptosis induction in activated HSCs can

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exert antifibrotic activity35-37. Here Dieckol increased sub G1 phase population and

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cleaved PARP and activated caspase-3 in a mitichodiral dependent pathway in HSCs,

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indicating the cytotoxicity of Dieckol is exhibited via apoptosis induction in HSCs.

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It was well documented that α-SMA38, p-NF-kB39 and TGF-β140 play critical roles

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in EMT mediated liver fibrosis41 with the flattened polygonal shapes of HSCs.

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Consistently, the fibrotic features of large, spread out and flattened polygonal

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morphology were observed in LX-2 cells, whereas elongated and round single cells

265

were shown in Dieckol treated LX-2 cells. Furthermore, Dieckol attenuated the

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expression of α-SMA, p-NF-kB and TGF-β1 in LX-2 cells, demonstrating

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antihepatofibrotic potential of Deickol in HSCs.

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Previous evidences supported that MAPK and AKT signalings are involved in liver

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fibrosis42-45. Consistently, Dieckol suppressed phosphorylation of ERK, p38 and,

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AKT, but upregulated phosphorylation of JNK in HSCs. To confirm the critical role

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of JNK in apoptosis induction and antifibrotic effect, JNK inhbitor SP600125 was

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used in LX-2 cells. Here we found SP600125 disrupted the apoptotic and

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antihepatofibrotic effects of Dieckol to activate caspase-3 and suppress α-SMA, p-

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NF-kB and p-IkB in LX-2 cells, indicating the pivotal role of JNK in Dieckol

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induced antihepatofibrotic effect.

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It was well known that mammalian miRNAs are involved in various biological

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processes, such as proliferation, differentiation, oxidative stress resistance and tumor

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suppression46. Previous evidences revealed that miR29b47,

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miR133a50 prevent liver fibrosis and collagen maturation and miR101 suppresses

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liver fibrosis by targeting TGF-beta signaling pathway. However, in the current

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study, miR134 was targeted in activated HSCs, with the hypothesis that miR134 can

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be postulated as a potent molecular target for liver fibrosis, because miR134 inhibits

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proliferation and EMT in renal32 and non-small lung cancer cells24, 31.

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Through the current study to exaime the inhibitory mechanism of Dieckol on liver

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fibrosis, we found that Dieckol showed cytotoxicity, increased sub-G1 population

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and activated caspase-3 and PARP in HSCs. Furthermore, Dieckol attenuated the

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expression of α -SMA, TGF-β1 and p-NF-kB, and activated miR134 and p-JNK in

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, miR12249 and

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HSCs. Conversely, JNK inhbitor SP600125 blocked the apoptotic and antifibrotic

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effects of Dieckol and miR134 inhbitor reversed the ability of Dieckol to attenuate

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the expression of pro-PARP and α -SMA in LX-2 cells. Taken together, our findings

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support evidences that Dieckol suppresses liver fibrosis via caspase dependent

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apoptosis and miR134 mediated JNK and NF-kB signaling pathways.

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AUTHOR INFORATION

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Corresponding author

305

*

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Cancer Molecular Targeted Herbal Research Center, College of Korean Medicine,

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Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, South

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Korea. Tel: +82-2-961-9233; Fax: +82-2-964-1064; E-mail: [email protected]

309

#Equally contributed authors

Dr. Sung-Hoon Kim, M.D. (KMD), Ph.D.

310 311

ACKNOWLEDGMENTS

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This work was supported by the Korea Science and Engineering Foundation

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(KOSEF) grant funded by the Korea government (MEST) (2014R1A2A10052872).

314 315

Notes

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The authors declare no cometing financial interest with respect to the authorship

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and/or publication of this article.

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ABBREVIATIONS USED

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HSC, hepatic stellate cells;PARP, poly (ADP-ribose) polymerase;JNK, c-Jun N-

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terminal kinases; ERK, extracellular signal–regulated kinases ;α –SMA, alpha

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smooth muscle actin;PBS, Phosphate buffered saline;FBS, fetal bovine serum;

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NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells

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REFRENCES

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from Pueraria lobata (Willd.), protects against hepatotoxicity via specific inhibition

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FIGURE CAPTIONS

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Figure. 1. Effects of Dieckol on the cytotoxicity in LX-2, HSC-T6, HepG2 and

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AML-12 cells and hepatic anti-fibrosis in LX-2 and HSC-T6 cells. (A) Chemical

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structure of Dieckol. Molecular weight = 742.5. (B) Cytotoxic effect of Dieckol in

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LX-2, HSC-T6, HepG2 and AML-12 cells. LX-2, HSC-T6,HepG2 and AML-12

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cells were seeded onto 96-well microplates at a density of 1 X 104 cells/well and

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treated with various concentrations of Dieckol (0, 12.5, 25, 50, 80 and 100 µM) for

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24 h. Cell viability was determined by MTT assay. Data represent means ± SD. *

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P

MicroRNA134 Mediated Upregulation of JNK and ... - ACS Publications

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