Nanoencapsulated curcumin and praziquantel treatment reduces periductal fibrosis and attenuates bile canalicular abnormalities in Opisthorchis viverrini-infected hamsters

    Nanoencapsulated curcumin and praziquantel treatment reduces periductal fibrosis and attenuates bile canalicular abnormalities in Opisthorchis viverrini-infected hamsters Lakhanawan Charoensuk, Porntip Pinlaor, Supason Wanichwecharungruang, Kitti Intuyod, Kulthida Vaeteewoottacharn, Apisit Chaidee, Puangrat Yongvanit, Chawalit Pairojkul, Natthakitta Suwannateep, Somchai Pinlaor PII: DOI: Reference:

S1549-9634(15)00195-1 doi: 10.1016/j.nano.2015.10.005 NANO 1194

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

1 June 2015 1 October 2015 6 October 2015

Please cite this article as: Charoensuk Lakhanawan, Pinlaor Porntip, Wanichwecharungruang Supason, Intuyod Kitti, Vaeteewoottacharn Kulthida, Chaidee Apisit, Yongvanit Puangrat, Pairojkul Chawalit, Suwannateep Natthakitta, Pinlaor Somchai, Nanoencapsulated curcumin and praziquantel treatment reduces periductal fibrosis and attenuates bile canalicular abnormalities in Opisthorchis viverrini-infected hamsters, Nanomedicine: Nanotechnology, Biology, and Medicine (2015), doi: 10.1016/j.nano.2015.10.005

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Word count for abstract: 149

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Word count for manuscript: 4941 Number of references: 37

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Number of figures: 8 Number of tables: 0

Nanoencapsulated curcumin and praziquantel treatment reduces periductal

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viverrini-infected hamsters

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fibrosis and attenuates bile canalicular abnormalities in Opisthorchis

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Lakhanawan Charoensuka,d, Porntip Pinlaord,e, Supason

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Wanichwecharungruangg, Kitti Intuyodd,f, Kulthida Vaeteewoottacharnb,d, Apisit Chaideea,d, Puangrat Yongvanitb,d, Chawalit Pairojkulc,d, Natthakitta Suwannateeph, Somchai Pinlaora,d,*

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Department of Parasitology,bDepartment of Biochemistry,cDepartment of

Pathology, and dLiver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

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e

Centre for Research and Development in Medical Diagnostic Laboratory,

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Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

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Biomedical Science Program, Graduate School, Khon Kaen University, Khon

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f

Kaen, Thailand. g

Department of Chemistry, Faculty of Science, Chulalongkorn University,

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Bangkok, Thailand. h

Faculty of Science and Technology, Suan Dusit Rajabhat University, Bangkok,

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Keywords:

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Thailand.

Opisthorchis viverrini; periductal fibrosis; nanoencapsulated curcumin; praziquantel; bile canalicular abnormalities

Conflicts of interest: No interest of conflicts.

Abbreviations: OV, Opisthorchis viverrini; CCA, cholangiocarcinoma; NEC, nanoencapsulated curcumin; PZQ, praziquantel; PDF, periductal fibrosis; BC, bile canaliculi; Cur, curcumin; ALT, alanine transaminase; MMP, matrix

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metalloproteinases; TIMP, tissue inhibitors of MMPs; CTGF, connective tissue

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growth factor; α-SMA, alpha-smooth muscle actin; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor alpha; abcb11, ATP-binding cassette

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sub-family B member 11; abcc2, ATP-binding cassette sub-family C member 2; cyp7a1, 7α-hydroxylation of cholesterol; shp-1, small heterodimer partner

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This work was supported by The Higher Education Research Promotion Group and National Research University Project of Thailand, Office of the Higher

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Education Commission, through the Health Cluster (SHeP-GMS, Ph.D.54203,

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1165), Thailand.

* Corresponding author at: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. Tel.: +66 43 348 387; fax: +66 43 202 475. E-mail address: [email protected] (S. Pinlaor).

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Abstract

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This study investigated the effects of nanoencapsulated curcumin (NEC) and praziquantel (PZQ) treatment on the resolution of periductal fibrosis (PDF)

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and bile canalicular abnormalities in Opisthorchis viverrini infected hamsters. Chronic O. viverrini infection (OV) was initially treated with PZQ (OP) and subsequently treated with NEC (OP+NEC), curcumin (OP+Cur) or unloaded

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carriers (OP+carrier) daily for one month. OP+NEC treatment reduced the PDF by suppression of fibrotic markers (hydroxyproline content, α-SMA, CTGF,

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fibronectin, collagen I and III), cytokines (TGF- and TNF-α) and TIMP-1, 2, 3

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expression and upregulation of MMP-7, 13 genes, whereas OP, OP+carrier

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and OP+Cur did not. The higher activity of NEC in reducing fibrosis compared to curcumin was also demonstrated in in vitro studies. Moreover, OP+NEC also prevented bile canalicular (BC) abnormalities and upregulated several genes involved in bile acid metabolism. These results demonstrate that NEC and PZQ treatment reduces PDF and attenuates BC defect in experimental opisthorchiasis.

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Background

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Periductal fibrosis (PDF) and inflammation-mediated oxidative/nitrative DNA damage are consequences of Opisthorchis viverrini infection, a major

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health problem in the Greater Mekong Subregion, especially in northeastern Thailand. Currently, 6 million people are infected with this carcinogenic parasite, which may lead to the development of cholangiocarcinoma (CCA).1-3

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Moreover, oxidative/nitrative stress and bile reflux due to mechanical obstruction of bile canaliculi (BC) during O. viverrini infection can induce BC

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and bile secretory defects.4 In chronic opisthorchiasis patients, the intensity

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of O. viverrini infection is correlated with higher frequency of advanced PDF 5

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and high incidence of CCA.6 The prominence of PDF after chronic infection in animal models7 is similar to that seen in opisthorchiasis patients.1, 6 Praziquantel (PZQ) is currently the drug of choice for O. viverrini treatment. However, PDF induced by chronic infection usually remains long after liver fluke eradication by PZQ.8 Moreover, PZQ treatment induces dispersion of parasitic antigens from the dead worm, leading to increased oxidative/nitrative stresses.2, 9 These adverse effects may increase the risk of CCA.6 Thus, preventing fibrotic lesions as well as oxidative/nitrative stresses

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after PZQ treatment is a promising strategy for the prevention of

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opisthorchiasis-associated CCA.10 Several lines of evidence indicate that curcumin, a widely-used yellow

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pigment derived from the rhizomes of turmeric (Curcuma longa L.), possesses

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anti-inflammatory, antioxidant and antitumor properties11 and is currently being tested in clinical trials for treatment of various disorders.12, 13 However,

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curcumin is poorly soluble in water and easily degraded by alkaline solution, resulting in the reduction of its oral bioavailability.14 Thus, long term and high

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dose treatment are required for the anti-fibrotic 15 and anti-CCA effects.16 To

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overcome these limitations, curcumin has been loaded into a self-emulsifying

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drug delivery system, liposomes, polymeric nanoparticles and solid lipid nanoparticles.17

Previous work has shown that polymeric nanocarriers based on the blend of ethylcellulose and methylcellulose has high curcumin loading capacity, in vitro free radical scavenging activity, in vitro cytotoxic effect towards MCF-7 human breast adenocarcinoma and HepG2 hepatoblastoma cells, and improved in vivo oral bioavailability.18 Here we investigated the effect of curcumin-loaded polymeric nanocarriers (denoted here as nanoencapsulated curcumin or NEC) and PZQ treatment on the PDF and bile

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canalicular defect induced by O. viverrini infection in hamsters and in vitro.

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The paper also included a possible mechanism by which NEC and PZQ treatment could reduce fibrosis and attenuate bile canalicular changes in O.

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viverrini infection.

Methods

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All materials used in this study were described in the support materials. Preparation of nanoencapsuled curcumin

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Nanoencapsulation of curcumin (NEC) using a blend of ethoxy content

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(EC) and methoxy content (MC) was carried out by solvent displacement

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through dialysis as described elsewhere.18 EC (0.20 g) and curcumin (0.40 g) were co-dissolved in ethanol to a final volume of 75 ml. This solution was mixed with MC solution (0.20 g MC in 25 ml water). The obtained solution was then transferred into the dialysis tube (CelluSep T4, MWCO 12,000-14,000, Membrane Filtration Products, USA) and dialyzed against distilled water (1000 ml × 6 times). The final volume of the obtained aqueous suspension of NEC was then adjusted to 150 ml with water. Morphology of the NEC was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The amounts of curcumin incorporated into the polymeric

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particles and in the dialysate water were determined using a UV 2500 UV–vis

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spectrophotometer (Shimadzu Corporation, Japan) at 425 nm, with the aid of a calibration curve. The encapsulation efficiency (EE) and loading capacity were

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calculated as follows:

EE (%) = (weight of encapsulated curcumin/weight of curcumin originally used) × 100

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Loading (%) = (weight of curcumin in particles/weight of curcumin-loaded

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particles) × 100

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Animals

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Male golden hamsters (Mesocricetus auratus) aged 4-6 weeks were obtained from the Animal Unit at the Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. All animals were housed under conventional conditions and given ad libitum access to a stock murine diet (CP-SWT, Thailand). The Animal Ethics Committee of the Faculty of Medicine at Khon Kaen University approved this study (AE003/54).

Isolation of O. viverrini metacercariae and hamster infection

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O. viverrini metacercariae were isolated from naturally infected

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Cyprinid fish using the sedimentation method.19 Each hamster was inoculated

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orally with fifty metacercariae via gastric tube.

Experimental design

Seventy-two hamsters were divided into two groups: acute and chronic

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infection. Each experimental group was divided into six sub-groups: (1) normal control (N); (2) O. viverrini-infected hamsters (OV); (3) OV treated

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with PZQ (OP); (4) OP treated with unloaded carriers (OP+carrier); (5) OP

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treated with intact curcumin 50 mg/kg body weight (OP+Cur) and (6) OP

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treated with NEC 50 mg/kg body weight (OP+NEC). Infected hamsters in group 3-6 were treated with PZQ (300 mg/kg body weight for two constitutive days) at 1 and 3 month(s) post-infection for acute and chronic infected groups, respectively. Hamster feces were examined for O. viverrini eggs using the formalin concentration technique to confirm the effective treatment. After PZQ-treatment, either curcumin, carrier or NEC were orally administered daily by gastric tube for 1 month. The curcumin powder was weighed, dissolved in corn oil and given to the hamsters by gastric tube. Hamsters were euthanized through over inhalation of diethyl ether at the end

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of treatment and blood and liver tissues were collected for further

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Isolation of peribiliary myofibroblast cells

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investigation.4

Peribiliary myofibroblast cells were isolated for four months postinfection from the hepatic bile duct of three O. viverrini-infected hamsters by

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the enzymatic method as previously described 20 with slight modification. Briefly, after hamsters were anesthetized with diethyl ether inhalation,

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abdominal skins were sterilized with 70% ethanol and opened with sterile

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scissors. Livers were perfused with a phosphate buffer saline solution

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containing 1 antibiotics-antimycotics. Bile ducts were separated from liver tissue, minced, and incubated with DMEM high glucose containing 15% Fetal bovine serum (FBS), 1% antibiotics-antimycotics, 1140 U/ml collagenase type I, 0.1 mg/ml DNase I and 30 U/ml hyaluronidase at 37 C under agitation for 2 h. After incubation, the suspension was sequentially filtered through a 100 and a 70 µm nylon mesh, respectively. Then, red blood cells (RBC) were lysed with RBC lysis buffer, and cells were washed and centrifuged at 1500 g. Cells were plated on 6-well culture plates precoated with collagen I, and cultured in DMEM high glucose containing 15% FBS, 1% antibiotic-antimycotics under

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5% CO2 at 37 C. After one to four passes, peribiliary myofibroblasts were

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Toxicity of NEC on peribiliary myofibroblasts

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used for further experiments.

The toxicity of NEC in peribiliary myofibroblast cells was tested by cytotoxicity assay. Cells were briefly seeded in 96-well plates at a density of

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104 cells/well. After 24 h, the cells were treated with 10, 20 and 40 µM of NEC for another 24 h, 48 h, and 72 h, respectively. In addition, 1% DMSO and an

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unloaded carrier were used as treatment controls. Thereafter, cells were

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incubated with MTT solution (MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-

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Diphenyltetrazolium Bromide) for 4 h and then lysed with DMSO. The optical density was measured using a microplate absorbance reader at a 540 nm wavelength.

Western blot analysis Twenty micrograms of either crude liver extract or cell lysate were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk, membranes were incubated with primary antibody solution at 4 C overnight. After washing, the membranes were incubated with HRP-

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conjugated secondary antibody at 4 C for 1 h. Immunoreactivity was

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Histopathological and immunohistochemical studies

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visualized and relative band intensity was measured as previously described.4

Livers were buffered and formalin-fixed with 10% buffered formalin for 48 h. Fixed livers were then embedded in paraffin and cut into 5 m sections.

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Collagen tissue in the PDF area was stained using a picrosirius red kit according

described elsewhere.7

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to manufacturer’s instructions. The PDF was graded into five stages as

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For immunostaining, antigens in liver sections were retrieved via

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autoclaving at 110 oC in a citrate buffer. Endogenous peroxidase was quenched with 3% H2O2. Tissue sections were then incubated with specific primary antibodies at room temperature overnight and then incubated with secondary antibodies at room temperature for 1 h. Appropriate detection methods were used to detect targeted molecules. The CD10, α-SMA, fibronectin and CTGF staining patterns were graded based on the positive/negative percentages from ten areas examined in each sample under a high powered field.4, 15, 19, 21 For immunocytochemistry staining, peribiliary myofibroblast cells were cultured overnight on culture slides (104 cells/well) and fixed with ice-cold 95%

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ethanol. After washing, cells were incubated with 0.3% H2O2 in ethanol and

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blocked with 5% FBS. Cells were then incubated overnight with rabbit anti-αSMA and CK19 at 4 °C. Cells were then incubated with an HRP-conjugated

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secondary antibody for 1 h at room temperature and visualized under a phase contrast microscope (Nikon Corporation, Tokyo, Japan).

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Quantitative reverse transcription-polymerase chain reaction The oligonucleotide-specific primer pairs of MMPs-2, 7, 9, 13, TIMPs-1,

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2, 3, TNF-α, TGF-, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and

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CD10, abcc2, abcb11, cyp7a1 and shp-1 were constructed and used for

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identification of hamster genes as previously described.7, 19 Primers for α-SMA were: (5′ACTGGTATTGTGCTGGACTC3′ and 5′ATCTCACGCTCAGCAGTAGT3′), and for CTGF were: (5′ GCGCCTGTTCTAAGACCTGT3′ and 5′ GCAGCCAGAAAGCTCAAACT 3′). Total RNA isolation from the liver sample and cDNA production were performed as previously described.4 Relative mRNA expression was analyzed by LightCycler® 480 II using LightCycler® 480 SYBR Green I Master. All data were analyzed using LightCycler® 480 software and then processed using the 2¯Ct method relative to GAPDH mRNA. The data

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are expressed as fold change over normal hamsters (mean ± SD of duplicate

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Hydroxyproline content and ALT activity

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independent experiments, n = 4).

Serum level of collagen was determined by estimating hydroxyproline content using the base hydrolysis approach and the serum ALT activity was

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determined as described elsewhere.22

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Statistics

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Data are represented as mean ± SD and were analyzed with SPSS version

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15.0 for Window (SPSS Inc., Chicago, IL). P values less than 0.05 were considered statistically significant. Statistical significance of relative gene expression, ALT level, hydroxyproline levels, intensity of bands protein expression levels and mRNA expression levels among experimental groups were determined using one-way ANOVA. The Mann-Whitney U test was used to determine the graded score expression levels.

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Results

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Characterization of NEC The process gave an encapsulation percentage of 84.4% and the

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obtained product possesses curcumin-loading percentages of 45.8%. Particles are spherical with the size estimated from SEM of 214 ± 15 nm (Supporting

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Information Figure S1).

Histological study

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The accumulation of collagen tissue in the PDF area induced by O.

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viverrini infection is shown in Figure 1, A. Increased thickness of PDF was

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observed with moderate fibrosis and fibrous portal expansion in acute infection as shown in Supporting Information Table S1. Compared to the OV group, the PDF was significantly decreased to mild fibrosis in the OP, OP+carrier, OP+Cur and OP+NEC groups. In chronically infected hamsters, PDF was persistent in OV, OP and OP+carrier groups, and severe fibrosis and few septa were evident. Interestingly, NEC+PZQ treatment significantly decreased the thickness of fibrosis when compared to OV, OP and OP+carrier groups, while the grading score of fibrotic lesions was slightly reduced in the OP+Cur group (Table S1).

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Collagen genes expression and hydroxyproline level The effect of NEC on collagen gene expression and hydroxyproline

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levels during the fibrous tissue resolution was also tested (Figure 1, B-G). In acute infection, the highest mRNA expression levels of collagens I, III and hydroxyproline content were observed in the OV group (Figures 1, B, D and F).

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However, these fibrotic markers were significantly reduced in the OP, OP+carrier, OP+Cur and OP+NEC groups when compared to the OV group.

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In chronic infection, fibrotic markers were persistent in the OP and

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OP+carrier groups (Figures 1, C, E and G). However, a significant decrease in

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the expression of collagens I and III genes was observed in the OP+Cur and OP+NEC groups. Notably, NEC+PZQ treatment decreased mRNA expression of collagens I and III and hydroxyproline content significantly more than treatment with curcumin+PZQ. In addition, acute O. viverrini infection significantly increased ALT activity (a liver-injury marker) compared to the normal group, whereas its activity in the OP, OP+carrier, OP+Cur and OP+NEC groups were significantly decreased when compared to the OV group (Figure 1, H). In chronic infection, ALT activity was increased in the OV, OP, OP+carrier and OP+Cur groups, but

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the effect was not statistically significant. In the OP+NEC group, ALT activity

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was similar to the normal control (Figure 1, I).

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α-SMA expression

The localization of α-SMA, a marker of collagen-secreting cells, was observed mainly in the cytoplasm of fibroblasts (spindle-shaped cells) located

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in the basement membrane of bile duct epithelial cells (Figure 2, A). In the OV group, an immunoreactivity of α-SMA expression at fibrotic lesions was

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observed at higher intensity in chronic infection than in acute infection. In

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acute infection, expression of α-SMA was similar in the OP, OP+carrier and

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OP+Cur groups compared to the OV group, while in the OP+NEC groups there was a significant decrease in the expression of α-SMA compared to the OV and OP groups. During chronic infection, expression of α-SMA was significantly decreased in the OP+NEC group compared to the OV group, while in the OP, OP+carrier and OP+Cur groups was slightly decreased (Figure 2, A and Table S1). The mRNA expression level of the α-SMA gene was similar to an immunostaining result. Notably, NEC+PZQ treatment suppressed α-SMA transcription more than treatment with curcumin+PZQ (Figure 2, B and C).

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Western blot analysis showed a significant increase in α-SMA

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expression in the OV group compared to the normal group (Figure 2, D and E). Unexpectedly, α-SMA expression during acute infection was significantly

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decreased in the OP group compared to the OV group, while its α-SMA expression was no different from that in curcumin+PZQ treatment (Figure 2, D). In chronic infection, the effect of NEC+PZQ treatment on α-SMA

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expression was similar to an immunostaining pattern. Moreover, NEC+PZQ exhibited higher inhibitory effect on α-SMA expression than in OP and

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OP+Cur groups (Figure 2, E).

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MMPs, TIMPs and cytokine genes expression Figure 3 shows the effect of NEC+PZQ treatment on the mRNA expression of MMPs (Figure 3, A-H), TIMPs (Figure 3, I-N) and cytokinerelated genes (Figure 3, O-R). In acute infection, O. viverrini infection increased the mRNA expression levels of MMPs -2, 7, 9, and 13 compared to the normal control (Figures 3, A, C, E and G). Particularly, the transcription of MMPs-9, and 13 were significantly decreased in the OP, OP+carrier, OP+Cur and OP+NEC groups compared to the OV group (Figure 3, E and G). In addition, MMP-2 tended to decrease in the OP, OP+carrier, OP+Cur and

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OP+NEC groups (Figure 3, A) and MMP-7 tended to decrease in the

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OP+carrier, OP+Cur and OP+NEC groups compared to the OV group (Figure 3, C). In chronic infection, mRNA expression levels of MMPs-2, 7, 9 and 13 were

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increased in the OV, OP, OP+carrier, OP+Cur and OP+NEC groups compared to the normal group (Figures 3, B, D, F and H). Notably, mRNA levels of MMP9 significantly decreased and MMP-2 tended to decrease in the OP+NEC

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group compared to the OP group. In contrast, the OP+NEC group exhibited significantly increased expression of MMP-7 and MMP-13 compared to the

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OV, OP, OP+carrier, and OP+Cur groups.

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In acute infection, mRNA expression of TIMPs (TIMPs-1, 2 and 3) and

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cytokines (TGF- and TNF-α) (Figure 3, I, K, M, O and Q) were significantly increased in the OV group compared to the normal control. Compared to the OV group, these mRNA levels were significantly decreased in the OP, OP+carrier, OP+Cur and OP+NEC groups. In chronic infection, mRNA expression of these TIMPs and cytokine genes was increased in the OV, OP, OP+carrier and OP+Cur groups but decreased in the OP+NEC group compared to the OP, OP+carrier and OP+Cur groups (Figure 3, J, L, N, P and R). Notably, expression of TNF-α was still higher than the normal control in the OP+NEC group (Figure 3, R).

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Connective tissue growth factor expression (CTGF) An immunofluorescence revealed that only the OP+NEC group showed

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a statistically significant reduction of CTGF expression in contrast to the other OP groups, particularly in the case of chronic infection (Figure 4, A and Table S1).

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Expression of CTGF was confirmed by Western blot and real-time RTPCR analysis. The results revealed that the CTGF expression was correlated

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with the immunostaining pattern. In particular, the expression of CTGF was

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significantly decreased in the OP+NEC group in the case of both acute and

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chronic infection (Figure 4, B-E).

Fibronectin expression

Expression of fibronectin was significantly increased during acute infection and also increased during chronic infection in proportion to the degree of PDF in the OV, OP and OP+carrier groups compared to the normal group (Figure 5, A and Table S1). In contrast, the expression of fibronectin was decreased in the OP+Cur and OP+NEC groups compared to the OV, OP,

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and OP+carrier groups. The lowest expression of fibronectin was found in the

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OP+NEC group in the case of both acute and chronic infection. In acute infection, Western blot analysis revealed a similar increase in

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fibronectin protein expression in the OV, OP, OP+Cur and OP+NEC groups compared to the normal group (Figure 5, B). In chronic infection, fibronectin expression was significantly increased in the OV, OP and OP+Cur groups

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compared to the normal control, while NEC+PZQ treatment significantly decreased fibronectin expression compared the OV, OP and OP+Cur groups

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(Figure 5, C).

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Bile canalicular (BC) changes and gene expression involved in bile acid metabolism

A significant decrease in the immunostaining pattern of CD10 (BC marker) was observed in the OV group at acute and chronic infection compared to the normal group (Figure 6, A and Table S1). Also, CD10 expression was significantly decreased in the OV, OP and OP+carrier groups compared to the normal control, and its immunostaining pattern was significantly increased in the OP+NEC group compared with the OV and OP groups (Figure 6, A and Table S1). Interestingly, strong immunoreactivity of

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CD10 similar to that of a normal liver was observed in the NEC+PZQ group,

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indicating NEC+PZQ had the highest efficacy in preventing BC abnormality. In acute infection, the mRNA expression level of CD10 was significantly

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increased in the OP, OP+carrier, OP+Cur and OP+NEC groups compared with the OV group (Figure 6, B). Notably, in chronic infection only the OP+NEC group showed significantly increased CD10 gene expression compared with

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the OV group (Figure 6, C).

Moreover, mRNA expression levels of bile acid-independent bile flow

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(abcc2, Figure 6, D and E) and bile acid biosynthesis (cyp7a1, Figure 6, H and

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I)-related genes were significantly increased in the OP+NEC group compared

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to both the OV and OP groups of acute and chronic infection. In acute infection, bile acid-dependent bile flow (abcb11) expression was significantly increased in OP, OP+carrier, OP+Cur and OP+NEC (Figure 6, F). In chronic infection, a significant increase of abcc11 expression was observed only in the OP+NEC group (Figure 6, G). Interestingly, expression levels of these genes were unchanged in the OP+Cur group compared to the OV and OP groups. Furthermore, in acute infection, mRNA expression level of bile acid regulatory gene (shp-1) was significantly decreased in the OP+NEC group as compared with the OP group (Figure 6, J) and significantly decreased in OP+Cur and

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OP+NEC with chronic infection compared to the OV and OP groups (Figure 6,

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K).

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In vitro study

To confirm the in vivo results, peribiliary myofibroblasts were isolated from bile duct of O. viverrini-infected hamsters and treated with NEC in vitro.

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More than 90% of isolated peribiliary myofibroblasts were positive for α-SMA and negative for cytokeratin 19 (CK19), indicating no contamination by bile

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duct epithelial cells (Figure S2). NEC did not exhibit cytotoxic effects on

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peribiliary myofibroblasts after 24 h of treatment, except at 48 h and 72 h

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(Figure S3). Thus, we used the 24 h NEC treatment for further experimentation.

A high expression of α-SMA, a marker for differentiation between fibroblasts to myofibroblasts,23 was observed in the untreated control and 1% DMSO- and in the carrier-treated groups. In contrast, NEC treatment decreased α-SMA expression in a dose-dependent manner (10, 20 and 40 µM) compared to the control group (Figure 7, A). Treatment with 40 µM of curcumin treatment resulted in high α-SMA expression, similar to 1% DMSOand carrier-treated groups. Western blot analysis showed that NEC treatment

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dose-dependently decreased the expression levels of α-SMA, fibronectin,

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CTGF and vimentin, especially at a dose of 40 µM, compared to the control

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group (Figure 7, B). Curcumin (40 µM) had no effect on these markers.

Discussion

Persistence of PDF due to an imbalance between the synthesis and

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breakdown of the extracellular matrix (ECM) induced by chronic O. viverrini infection and PZQ treatment increases the risk of CCA.6, 10 In previous studies,

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the accumulation of PDF in acute O. viverrini infection was reversible, while

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PDF in chronic infection was advanced and irreversible.5, 24 In order to

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demonstrate the effect of NEC on the reversible and irreversible stages of PDF, we performed experimentation at acute and chronic phases of opisthorchiasis. In the case of acute infection, PDF induced by O. vivierrini infection can be spontaneously resolved after worm elimination, which may possibly be due to the activity of MMPs, such as MMP2 and MMP9. Alternatively, it may due to increase apoptosis or transformation to a quiescent state of activated stellate cells. 25 In the acute phase, a similar fibrolytic effect of curcumin and encapsulated curcumin was observed, suggesting that early-stage of PDF is potentially reversible after worm elimination. 8

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In the chronic phase, advanced PDF usually persists and is irreversible8

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but can be prevented.15 However, NEC was shown to be more effective in prevention of advanced PDF as reflected by the decrease of various fibrotic

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markers and suppressed TNF-α and TGF- gene expression to a greater extent than free curcumin, suggesting it might be due to the higher bioavailability of NEC.18 However, NEC treatment did not completely prevent advanced PDF,

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indicating that using a high dosage and/or long-term supplementation of NEC are required. Alternatively, persistence of advanced PDF even after the worms

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are eliminated by PZQ (OP and OP+carrier groups) may be due to the

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colonization of Helicobacter pylori in the biliary tree of the O. viverrini-infected

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hamsters26 which might promote fibrogenesis via induction of TNF-α and TGF- gene expression. Notably, their transcription levels were decreased by NEC and curcumin treatments, suggesting that this may be due to the partial antimicrobial activity of curcumin against Helicobacter pylori. 27 In addition, the results in all of the experiments involving the OP and OP+carrier groups were similar, indicating that the carrier did not affect any investigated parameters. Liver and bile duct injuries caused by O. viverrini infection increase TGFβ expression during wound healing, leading to the upregulation of α-SMA and

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CTGF-dependent fibronectin expression and promote fibrogenesis21, 28 Our

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results show that NEC+PZQ treatment (but not curcumin+PZQ) suppresses the expression of TGF-, CTGF, α-SMA, collagen I and III and fibronectin,

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resulting in a reduction of PDF. Anti-fibrotic effect of NEC was confirmed by in vitro study. NEC showed a dose-dependent suppression of α-SMA, fibronectin, CTGF, and vimentin expression in primary peribiliary myofibroblast cells

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isolated from chronic O. viverrini-infected hamsters, while curcumin did not. This effect of NEC on decreasing of various fibrotic markers may be due to its

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suppression of TGF- -mediated CTGF expression, a well-known central

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mediator of tissue remodeling and fibrosis.29

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Resolution of PDF might be mediated by the expression of MMPs and TIMPs, which regulate ECM homeostasis and are involved in tissue degradation.30 In acute O. virerrini infection, accumulation of fibrosis was found in concomitance with the increase of MMPs-2 and 9 expression levels. The role of these MMPs in fibrogenesis in the O. viverrini infection model has been demonstrated by previous studies. 7, 22 However, the resolution of fibrosis by NEC+PZQ treatment was associated with decreased transcription of MMPs-2, and 9 and TNF-α and TGF- levels, suggesting that these molecules might be involved in fibrolysis. In chronic O. viverrini infection, NEC+PZQ treatment

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inhibited TGF- and TNF- expression and subsequently decreased TIMPs,

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MMP2 and MMP9 expression via suppression of oxidative stress (data not shown), whereas PZQ and OP+Cur treatment had no effect. The suppression of

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TGF-β31 and reactive oxygen species (ROS) production 32 might cause a reduction

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of MMP-2 and MMP-9 and, thereby, prevent collagen synthesis.33 Alternatively, downregulation of TIMPs might lead to a decrease in fibrosis by increasing

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fibrolytic MMP activity (e.g. MMP-7 and MMP-13), resulting in cleavage of ECM and basement membrane proteins and the resolution of fibrosis.34

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O. viverrini infection not only induces inflammation-mediated

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oxidative/nitrative DNA damage via iNOS expression,3 but also induces BC

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changes. This reduces expression of bile secretory-related genes and bile volume,3, 4 which may increase the risk and severity of CCA. Although curcumin treatment prevents the reduction of microvilli density, it does not reduce the dilation of bile canaliculi lumen.35 In this study, NEC+PZQ treatment not only induced fibrolysis but also ameliorated the BC pattern change and bile acid metabolism genes alteration. Moreover, NEC+PZQ treatment increased the expression of CD10, a marker of BC and bile acid dependent (abcb11) and independent (abcc2) bile flow, and bile acid biosynthesis (cyp7a1) genes and decreased bile acid regulatory (shp-1) gene expression. This effect was perhaps

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mediated by the suppression of the oxidative/nitrative stress effect36 or the

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prolonging of antioxidant activity.37 In conclusion, the present study indicates that NEC+PZQ has a higher

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efficacy than curcumin+PZQ in reducing PDF by enhancing of MMPs and

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suppressing TIMPs and pro-fibrotic cytokines (TGF- and TNF-α) expression. NEC+PZQ also had a higher efficiency than curcumin+PZQ in improving bile

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canaliculi morphology and preventing genes involved in bile acid metabolism alteration. A possible molecular mechanism of NEC+PZQ treatment to reduce

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fibrosis and to attenuate BC alteration in O. viverrini infection is summarized in

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Figure 8. Our study indicated that NEC is a promising chemopreventive agent in

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reducing CCA development via the reduction of PDF and prevention of bile calicular alteration in opisthorchiasis.

Acknowledgements

We would like to acknowledge Dr. Justin Reese and Mr. Dylan Southard for their advice during manuscript preparation.

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Figure legends

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Figure 1. Effect of NEC on periductal fibrosis in the hamster liver. O. viverriniinfected hamsters (OV) were treated with either PZQ (OP), PZQ and carrier

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(OP+carrier), PZQ and curcumin (OP+Cur) or PZQ and NEC (OP+NEC) at 1 month (acute phase, white bars) and 3 months (chronic phase, gray bars). Data are represented as fibrotic tissue (red area) (original magnification 200)

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(A), relative mRNA expression of collagens I and III (B-E), serum hydroxyproline levels (F & G) and alanine transaminase activity (H & I). Data

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are reported as mean  SD levels of 6 hamsters. *P < 0.05, compared to the

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normal group, †P < 0.05, compared to OV group, ‡P < 0.05, compared to OP

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group. BD = bile duct, Ov = O. viverrini, N = normal hamsters.

Figure 2. Expression of α-SMA in the liver of O. viverrini-infected hamsters and treated with PZQ and NEC. Data are represented as an immunohistochemical staining (original magnification 400) (A), mRNA (B & C) and protein levels of α-SMA expression (D & E). The relative intensity is expressed as fold change over the normal control (N=3). *P < 0.05, compared to the normal group, †P < 0.05, compared to the OV group, ‡P < 0.05,

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compared to the OP group, §P < 0.05, compared to the OP+Cur group.

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Abbreviations are the same as in Figure 1.

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Figure 3. mRNA levels observed in the liver of O. viverrini-infected hamsters and treated with PZQ and NEC. The levels of the following genes are reported: MMPs-2, 7, 9, 13 (A-H), TIMPs- 1, 2, 3 (I-N) and cytokines (TGF- and TNF-α)

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(O-R). Data are reported as meanSD of 4 hamsters in duplicate experiments. The statistics, experimental groups and abbreviations are the same as in

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Figure 1 and 2.

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Figure 4. Expression of CTGF in the liver of O. viverrini-infected hamsters and treated with PZQ and NEC. Data are represented as fluorescent immunohistochemical staining (original magnification 400) (A), mRNA (B & C) and protein levels of CTGF expression (D & E). The relative intensity is expressed as fold change over the normal control (N=3). The statistics, animal groups and abbreviations are the same as in Figure 1 and 2.

Figure 5. Expression of fibronectin in the liver of O. viverrini-infected hamsters and treated with PZQ and NEC. Data are represented as an

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immunohistochemical staining (original magnification 400) (A), and protein

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expression level of fibronectin and the relative intensity is expressed as fold change over the normal control (N=3) (B & C). The statistics, experimental

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groups and abbreviations are the same as in Figure 1 and 2.

Figure 6. Expression of CD10 and genes involved in bile acid metabolism in

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the liver of O. viverrini-infected hamsters and treated with PZQ and NEC. Data are represented as an immunohistochemical staining of CD10, a marker of

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bile canalicular pattern (original magnification 400) (A), and the level of the

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following genes: CD10 (B & C), abcc2 (D & E), abcb11 (F & G), cyp7a1 (H & I)

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shp-1 (J & K). The statistics, animal groups and abbreviations are the same as in Figure 1 and 2.

Figure 7. Effects of NEC on the expressions of α-SMA in the peribiliary myofibroblast cells isolated from the bile duct of O. viverrini-infected hamsters. Cells were treated with either 1% DMSO (1% DMSO), unloaded carriers (carrier), curcumin 40 µM (Cur 40 µM), or various concentrations of NEC (10, 20 and 40 µM; NEC 10, 20 and 40 μM) compared to an untreated group (Control). Data are represented as an immunocytochemical staining of

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α-SMA (original magnification 200) (A), and the expression of α-SMA,

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fibronectin, CTGF and vimentin proteins and the relative intensities are expressed as fold change over the control (N=3) (B). Data are reported in

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duplicate experiments. #P < 0.05 compared to control.

Figure 8. Possible mechanism by which NEC and PZQ treatment could reduce

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fibrosis and attenuate bile canalicular changes in O. viverrini infection.

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Graphic abstract

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 O. viverrini infection causes periductal fibrosis (PDF) and bile canalicular (BC) abnormality.

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 PDF and BC defect were persistence after worm eradication by PZQ treatment.

 NEC and PZQ treatment reduces PDF better than curcumin and PZQ.

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 NEC and PZQ treatment also attenuates BC defect better than

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curcumin and PZQ.

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Graphical Abstract