Introduction

Clonorchiasis is food-borne trematodiasis caused by chronic infection with Clonorchis sinensis (C. sinensis) (Lv et al. 2012; Wang et al. 2012). C. sinensis has afflicted over 35 million people in eastern Asian countries, where clonorchiasis constitutes a significant public health concern. C. sinensis reside in the peripheral bile ducts and provoke pathological changes such as inflammation, dilatation, hyperplasia, fibrosis, and cholangiocarcinoma (Chen et al. 2014; Huang et al. 2012; Lim et al. 2008). However, molecular mechanism of clonorchiasis still remains to be elucidated. It was suggested that excretory/secretory products (ESPs) released by liver flukes may lead to pathologic changes in biliary epithelial cells or trigger cellular signals involved in hepatic fibrosis (Kim et al. 2009). ESPs are protein complex including proteases, antioxidant enzymes, and metabolic enzymes. It was reported that human cells exposed to ESPs from liver flukes (C. sinensis, Fasciola hepatica, and Opisthorchis viverrini) displayed diverse pathophysiological responses including proliferation (Lim et al. 2008), apoptosis (Serradell et al. 2007), and inflammation (Pinlaor et al. 2005). Our lab previously demonstrated that C. sinensis ESPs (CsESPs) could promote proliferation of human hepatic stellate cell (HSC) line LX-2 (a model cell line in hepatic fibrogenesis) and the effect of CsESPs was confirmed in animal model (Hu et al. 2009; Ma et al. 2007).

The transcription factor nuclear factor kappa B (NF-κB) has been shown to play a crucial role in HSC survival (Elsharkawy and Mann 2007). In addition, NF-κB has been reported to be upregulated in mouse RAW 264.7 macrophage cells treated with crude extracts from O. viverrini and in the nucleus of bile duct epithelial cells of O. viverrini-infected hamsters, suggesting the involvement of NF-κB activation in liver fluke infection (Pinlaor et al. 2006). NF-κB is a dimer of Rel family proteins, the most prevalent form of which consists of a heterodimer of RelA (p65) and p50. In the resting state, NF-κB is sequestered in the cytoplasm through covalent binding with inhibitory proteins called IκBs. Exposure of cells to external stimuli, including H2O2, ionizing radiation, cytokines, or bacterial endotoxins, results in activation of IκB and its subsequent proteolytic degradation (Morgan and Liu 2011).

Endogenous free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are produced through multiple sources including the electron transport chain in mitochondria and as by-products of enzymatic reactions involving NADPH oxidases (NOX), xanthine oxidase (XO), lipoxygenases (LO), cyclooxygenases (COX), and nitric oxide synthases (NOS). Excess intracellular free-radical generation can activate NF-κB and promotes serious cellular injuries and contributes to several human diseases (Morgan and Liu 2011). It was recently reported that treatment of human cholangiocarcinoma cells with CsESPs triggered increases in free radicals which participate in nuclear factor-κB-mediated inflammation in hepatobiliary diseases (Liang et al. 2013; Nam et al. 2012). However, the key components of CsESPs that play a major role in C. sinensis-induced activation of NF-κB are still under investigation.

In our previous study, using proteomic approaches, we identified the components of CsESPs, among which C. sinensis ferritin heavy chain (CsFHC) was suggested as a key component (Zheng et al. 2011). Ferritin heavy chain is a ferroxidase enzyme responsible for iron storage in prokaryotes and eukaryotes (Theil 1987). The ferritin molecule is comprised of 24 subunits of two structurally distinct subunit types: H-chain and l-chain. H-ferritin has been shown to regulate immune function, hematopoiesis, hepatocyte apoptosis, and cell differentiation (Recalcati et al. 2008). It has been shown that FHC regulates the intracellular iron which catalyzes the formation of toxic ROS (Cozzi et al. 2000). In this study, we investigated the role of CsFHC as a proinflammatory mediator in activating HSC through the NF-κB signaling pathway. We examined the activation of potential free radical-generating enzymes in LX2 cells treated with CsFHC and investigated the involvement of CsFHC-induced enzyme activation in NF-κB-mediated inflammatory processes. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

Materials and methods

Reagents

N-acetylcysteine (NAC), allopurinol (ALP; XO inhibitor), diphenyleneiodonium chloride (DPI; NOX inhibitor), and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). NG-nitro-l-arginine methyl ester (l-NAME; NOS inhibitor) and Bay 11-7082 (IκB-aphosphorylation inhibitor) were purchased from Beyotime Biotechnology (Beyotime Company, Shanghai, China). Polyclonal antibodies against the following proteins were obtained from the indicated sources: NF-κB p65, NF-κB p50, lamin B, calnexin, GAPDH, and horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech Company, Chicago, USA); IκB-α, p47phox, p67 Phox, and inducible NOS (iNOS) (Signalway Antibody LLC, Maryland, USA); All other chemicals were purchased from GBCBIO Technologies Inc. (Guangzhou, China).

Cloning, recombinant expression, and purification of His(6)-tagged CsFHC

The complete encoding sequence of CsFHC was searched in our draft genome of C. sinensis (Wang et al. 2011). The CsFHC full-length cDNA was directly cloned into vector pET-28a(+) (Novagen, USA), using forward primer 5′-GTAGGATCCATGTCCACTTCGTTG-3′ and reverse primer 5′-GCCAAGCTTTTAGTCATCTTCGCC-3′. After DNA sequencing, the recombinant plasmid was transformed into Escherichia coli (DE3) (Promega). The expression of recombinant protein was induced with 1 mM isopropyl-l-thio-β-galactopyranoside (IPTG) for 4 h at 37 °C in LB medium containing 50 mg/ml kanamycin. Cell lysate with pET-28a(+)-CsFHC was harvested after centrifugation at 4 °C for 15 min at 8,000×g and resuspended in native lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole, pH 8.0), sonicated on ice, and then centrifuged to separate the supernatant and sediment. Recombinant protein was purified by His-bind resin (Novagen) chromatography and dialyzed overnight in phosphate buffered saline (PBS) (pH 7.4). The purified rCsFHC was analyzed by 12 % SDS-PAGE stained with Coomassie brilliant blue G-250. Final recombinant soluble protein concentration was detected by the BCA protein assay kit (Beyotime, Guangzhou, China).

Preparation for antiserum

To prepare anti-CsFHC rat serum, recombinant CsFHC (rCsFHC) was emulsified with complete Freund’s adjuvant and subcutaneously immunized with 200 μg of protein for each rat at the first time. Subsequently, each rat was given 100 μg of protein (emulsified with equivalent incomplete Freund’s adjuvant) for three booster injections at 2-week intervals. Antiserum was collected at 8 weeks. The antibody titer was determined by enzyme-linked immune sorbent assay (ELISA).

Western blotting analysis

CsESPs were collected from adult worms according to previously described method (Zheng et al. 2011). Adult worms or metacercariae were crushed and cleaned by centrifugation at 10,000×g for 15 min at 4 °C to obtain soluble proteins. The recombinant CsFHC (3 μg/lane), CsESPs, and the total protein of adult worms and metacercariae (20 μg/lane, respectively) were resolved by 12 % SDS-PAGE and then immobilized onto PVDF membrane. The membranes were blocked with 5 % (w/v) skim milk at room temperature for 2 h, and then incubated with primary antibody (anti-rCsFHC rat serum, anti-His mouse serum, rat serum infected with C. sinensis, and naïve rat serum, respectively, 1:100 dilutions in 0.1 % BSA-PBS) overnight at 4 °C. After washing procedures, the membranes were incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rat IgG (1:3,000 dilutions in 0.1 % BSA-PBS, Proteintech Company) for 1 h at RT. The results were detected by ECL method.

RNA extraction and analysis of mRNA expression by quantitative RT-PCR (qRT-PCR)

The extraction of total RNA, complementary DNA synthesis, and PCR amplification were carried out as described elsewhere (Chen et al. 2014; Wang et al. 2011). Briefly, total RNAs from adult worms, excysted metacercariae, metacercariae, and eggs were extracted in Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentrations were detected by nucleic acid/protein analyzer (Beckman Coulter, USA). The preparation of the first-strand cDNA was carried out using RT-PCR kit (TaKaRa, Dalian, China) with the same quantity of total RNA as the template (1 μg). The mRNA expression level of CsFHC at different developmental stages was measured by qRT-PCR, and actin (GenBank accession no. EU109284) was used as the internal control (Wang et al. 2012). The primers were CsFHC: 5′-ATGTCCACTTCGTTGGCTCG-3′ and 5′-CTCGCTCTTCGTCAGACTGCT-3′, respectively, actin: 5′-ACCG TGAGAAGATGACGCAGA-3′ and 5′-GCCAAGTCCAAACGAAGAATT-3′, respectively. Quantitative RT-PCR reactions based on SYBR Green I fluorescence (TaKaRa) were performed by using Bio-Rad CFX96 instrument (Bio-Rad, USA). The PCR amplification program was 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s. The iQ5 software was used to analyze the relative quantification according to the 2−ΔΔCT method (Pfaffl 2001).

Immunolocalization of CsFHC in C. sinensis adult worm and metacercaria

Sectioned worms and metacercariae in paraffin wax were deparaffinized and incubated in the rat anti-CsFHC sera (1:200 dilutions). The collected metacercariae were immersed in PBS 0.3 % TritonX-100 for 1 h at RT before being fixed with cold acetone on a glass slide for immunohistochemical analysis. Preimmune rat serum was applied as a negative control. The sections were subsequently incubated with goat anti-rat IgG (1:400 dilutions with 0.1 % BSA in PBS, Alexa Fluor 594, Molecular Probe) for 1 h at RT in the dark, then imaged using a ZEISS Axio Imager Z1 fluorescent microscope.

Iron uptake assay and ferroxidase activity

To remove iron, the recombinant CsFHC protein was incubated for 18 h in a solution of 1 % thioglycolic acid and 0.1 M sodium acetate, pH 5.5. To chelate ferrous irons, excess 2, 2-bipyridine was added to the reaction and the solution was dialyzed exhaustively against 5 mM HEPES, pH 7.0 (Kim et al. 2002). The kinetics of iron uptake were quantified as described previously (Levi et al. 1988) with minor modifications. Briefly, CsFHC proteins or apo-horse ferritin (Sigma) were incubated in 100 mM HEPES (pH 6.0) containing 100 M ferrous ammonium sulfate. The ensuing amber color change was measured at 310 nm at room temperature. The ferro-oxidizing activity of recombinant CsFHC was assayed by employing transferrin as a ferric iron acceptor (Levi et al. 1988). The reaction mixture contained 0.1 M ferrous ammonium sulfate, 4 mg/ml apotransferrin (Sigma), and 0.2 M sodium acetate, pH 7.0, and the recombinant CsFHC at various concentrations. Increase of optical density was recorded at 470 nm for 10 min once the reactions started.

Cell culture and CsFHC treatment

The LX-2 human hepatic stellate cell line was cultured in DMEM (Thermo Hyclone, USA) containing 10 % fetal bovine serum (FBS, NQBB, Australia) and an antibiotic mixture at 37 °C in a humidified 5 % CO2 humidity. For CsFHC treatment, cells were seeded at ~70 % confluence on 35 or 100 mm culture dishes and grown for 24 h under culture conditions. Cells were gradually deprived of serum by incubation in 1 % FBS overnight, followed by incubation in serum-free medium for 3 h. These serum-starved cells were treated with 10 nM of CsFHC and incubated for indicated time.

Subcellular fractionation of cell lysates

Cells were homogenized with cold hypotonic lysis buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 5 mM MgCl2, add fresh protease inhibitor cocktail) on ice for 30 min, centrifuged at 600×g for 5 min, and further centrifuged at 13,000×g for 20 min. The supernatant was collected and ultracentrifuged at 55,000×g for 1.5 h. The resulting supernatant was saved as the cytosol fraction and the pellet was resuspended in lysis buffer (without sucrose) containing 1 % Triton X-100 for isolation of the total membrane fraction. Nuclear proteins were isolated using the nuclear and cytoplasmic extraction kit (Beyotime Biotechnology Inc., Guangzhou, China) according to manufacturer’s instructions. The protein concentration of extracts was measured and extracts were stored in aliquots at ~80 °C until use.

Enzymatic activity assays

XO activity was measured using the Amplex Red Xanthine/Xanthine Oxidase Assay Kit (Molecular Probes) according to the manufacturer’s instructions as previously described (Ribeiro et al. 2013). NO production was assessed by measuring nitrite/nitrate concentration in culture medium using a Total Nitric Oxide Assay Kit (Beyotime Biotechnology Inc.) according to manufacturer’s instructions as previously described (Xiong et al. 2009).

Immunoblot analysis

Total soluble proteins were extracted using RIPA buffer (GBCBIO) and centrifuged at 13,000×g for 20 min at 4 °C. The protein concentration in extracts was measured using BCA protein assay kit (Beyotime, China).

Thirty micrograms of total soluble proteins, cytosolic and membranous proteins, or 10 μg of nuclear proteins were separated by SDS-PAGE on 10 % gels, and transferred onto nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5 % skim milk in Tris-buffered saline containing Tween-20 and subsequently probed with antibodies specific for target proteins. After incubating with host-specific secondary antibodies, immune complexes were detected with enhanced chemiluminescence and quantitated by densitometric scanning of the X-ray film with a Image-pro plus 6.0 (Media Cybernetics). Blots were normalized against GAPDH (for cytosolic proteins), calnexin (for membranous proteins) or lamin B (for nuclear proteins).

IL-1β and IL-6 analysis by qRT-PCR

Total RNA was extracted from cells using Trizol reagent and reversely transcribed into cDNA using the reverse transcriptase kit. Equal volumes of single-stranded cDNA product were then used as templates for PCR amplification of the following targets using the indicated primer pairs: IL-1β, 5′-ATGATGGCTTATTACAGTGGCAA-3′ (sense) and 5′-GTCGGAGATTCGTAGCTGGA-3′ (antisense); IL-6, 5′-ACTCACCTCTTCAGAACGAATTG-3′ (sense) and 5′-CCATCTTTGGAAGGTTCAGGTTG-3′ (antisense); and GAPDH (internal control), 5′-GGAGCGAGATCCCTCCAAAAT-3′(sense) and 5′-GGCTGTTGTCATACTTCTCATGG-3′ (antisense). Quantitative real-time PCR based on SYBR Green I fluorescence (TakaRa) was performed as described above. The data were analyzed using the 2−ΔΔCT method (Pfaffl 2001).

Cytokine assay

Immunoreactive IL-1β and IL-6 were quantitated in untreated and CsFHC-treated culture supernatant using commercially available ELISA kits (NeoBioscience Technology Inc., China), according to the manufacturer’s instructions. A standard curve for each cytokine was generated by known concentration of human recombinant IL-1β or IL-6 protein. The absorbance at 450 nm was then used to calculate the level of IL-1β and IL-6 from the standard curve and adjusted by their dilution factor.

Statistical analysis

Data are expressed as means ± S.E.s of three or more independent experiments. Statistical significance was evaluated by one-way ANOVA, followed by a Student’s t test or Bonferroni’s test, as appropriate. Differences in mean values were considered statistically significant at P < 0.05.

Results

Expression, purification, and identification of rCsFHC

The soluble rCsFHC was expressed with His-tag in E. coli BL21 after being induced by 1 mM IPTG at 37 °C for 4 h. The molecular mass of purified protein was about 24 kDa (Fig. 1a), and the final protein concentration was 10 mg/l. Purified rCsFHC could be recognized by rat anti-rCsFHC serum, anti-His tag monoclonal antibody, serum from C. sinensis-infected rat and serum from CsESPs-immunized rat at 24 kDa, while not blotted with serum from naïve rat (Fig. 1b).

Fig. 1
figure 1

a Expression and purification of rCsFHC by 12 % SDS-PAGE. Protein molecular weight markers (lane M), lysate of E. coli with pET-28a (+) before induction (lane 1) and after induction (lane 2), lysate of E. coli with pET28a (+)-CsFHC before induction (lane 3) and after induction (lane 4), supernatant (lane 5), and precipitant (lane 6) of lysate of E. coli with pET28a (+)-CsFHC after induction (lane 7), and the purified recombinant CsFHC protein. b Western blotting of rCsFHC. rCsFHC reacted with sera from naive rats (lane 1), anti-His tag monoclonal antibody (lane 2), sera from C. sinensis-infected rats (lane 3), sera from rats immunized with the CsESP (lane 4), sera from rats immunized with rCsFHC (lane 5). CsESPs reacted with sera from rats immunized with rCsFHC (lane 6). Gene/protein expression analysis of CsFHC at different life cycle stages of C. sinensis. c Western blotting analysis. Thirty microgram of total proteins from each life cycle stage were subjected to SDS-PAGE and analyzed. Rat anti-rCsFHC serum was used as primary antibody at a dilution of 1:100. d Quantitative real-time PCR analysis. The transcription levels of CsFHC at life stages of adult worm, excysted metacercaria, egg, and metacercaria, and were analyzed by means of the 2−ΔΔCT ratio, with Csβ-actin serving as the internal standard. *P < 0.05

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Transcriptional level at different developmental stages

The result was calculated according to the previous methods. As shown in Fig. 1c, transcriptional level of CsFHC was detected at the life stages of adult worms, excysted metacercaria, metacercariae, and eggs of C. sinensis. The transcription level of CsFHC in egg was about 20 times higher than that in adult worm. The protein level of CsFHC was consistent with the transcriptional level. Egg has the highest expression level of CsFHC protein, followed by adult worm, metacercaria, and excysted metacercaria (Fig. 1d).

Immunohistochemical localization of CsFHC

Figure 2 showed that CsFHC was extensively distributed in metacercariae (Fig. 2e) and in vitellaria and cecum of adult worms (Fig. 2a). No specific fluorescence was detected either in adult worms or in metacercariae when treated with naïve mouse serum (Fig. 2c, g).

Fig. 2
figure 2

Immunolocalization of CsFHC in C. sinensis metacercariae and adult worms. In adult worms, CsFHC was localized in the vitellaria and cecum (a). CsFHC was extensively distributes in metacercariae (e). Negative controls were carried out with naïve serum (1:100 v/v) for adult worms (g) and metacercariae (c). b, d Corresponding bright fields for the adult worms. f, h Corresponding bright fields for metacercariae. The images were magnified at ×100 for adult worms and ×400 for metacercariae, respectively

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Iron incorporation and ferroxidase activity of recombinant CsFHC protein

In preparing the enzymatic assay, purified recombinant CsFHC protein was bleached of incorporated iron to the apo-form. The iron uptake of recombinant CsFHC protein was monitored by measuring optical absorbance of the amber product formed in the presence of ferrous iron and dioxygen. A dose-dependent increase of iron incorporation into recombinant CsFHC was observed (Fig. 3a), and optical absorbance of reaction mixture increased and reached a steady state in about 1 min after the reaction had started. The iron incorporation increased steadily from 1 min after the reaction started until the recombinant CsFHC had been completely saturated. To investigate the ferroxidase activity of recombinant CsFHC protein, the oxidation of ferrous to ferric iron was measured using apotransferrin as an acceptor molecule. The formation of the Fe(III)/transferrin complex was found to increase abruptly during the first 3 min after the reaction started and slowly approached to a steady state by 5 min (Fig. 3b). As was observed in iron incorporation assay, ferroxidase activity was dose-dependent to the amount of recombinant CsFHC added to the reaction mixture. After reaching an oxidation plateau, the second addition of transferrin caused a resumption of the same pattern of ferroxidase activity.

Fig. 3
figure 3

a The iron uptake activity of recombinant CsFHC protein. The reaction mixture contained recombinant CsFHC, 100 μM Fe++, 100 mM HEPES in a volume of 400 μl (n = 3). b Time course of ferroxidase activity of recombinant CsFHC. The formation of the Fe(III)/transferrin complex from ferrous ammonium sulfate and apotransferrin was measured in the presence of recombinant CsFHC. Mean results of triplicate measurements are shown

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Activation of NOX, XO, and iNOS by CsFHC exposure

We examined whether CsFHC triggered the activation of commonly known free radical-generating enzymes, namely NOX, XO, and NOS, in host cells. Activation of NOX was assessed by determining association of its subunits (p47phox and p67phox) with the cell membrane. As shown in Fig. 4a, b, increased amounts of immunoreactive p47phox and p67phox were detected in the membrane fraction following a 1-h treatment with CsFHC, with the most significant increases (P < 0.05) occurring between 9 and 15 h. This result indicates that CsFHC treatment promotes translocation of both p47phox and p67phox from the cytosol to the plasma membrane, suggesting NOX activation. We further investigated the possibility that the increased concentration of nitrite/nitrate reflected the conversion of NO produced by iNOS activation. Immunoblot analyses revealed a clear elevation in iNOS expression as early as 3 h after CsFHC treatment, with the most significant increase occurring at 15 h (Fig. 4d). We also examined the effect of CsFHC on NO generation by measuring nitrite/nitrate concentration in culture media. Nitrite/nitrate formation was significantly increased at 3 h, reaching a peak between 9 and 15 h, followed by a decrease at 24 h (P < 0.05, Fig. 4e). These time points coincide with the times of increased nitrite/nitrate concentration in culture media. XO activity was significantly increased in cells treated with CsFHC for 3 h, and reached a maximum level at 9 h before gradually declining between 15 and 24 h (P < 0.05); even at these later time points, XO activity was still higher than that in the untreated control (P < 0.05, Fig. 4g). These results demonstrate that NOX, XO, and iNOS enzymes in LX2 cells actively participated in the generation of free radicals triggered by CsFHC.

Fig. 4
figure 4

Effects of CsFHC treatment on the activity of free radical-generating enzymes and inhibition by specific inhibitors. Human hepatic stellate cells were treated with 10 nM of CsFHC for 0–24 h, or cells were preincubated with 5–20 μM of diphenyleneiodonium chloride (a), 50–200 μM of allopurinol (h), or 50–300 μM of l-NAME (f) for 1 h, followed by treatment with 10 nM of ferritin heavy chain for 9 h, and then harvested for immunoblot analysis and enzyme activity assays. a, b, c Accumulation of the NADPH oxidase subunits, p47phox and p67phox, in the membrane was assessed by immunoblotting. Calnexin was used as a loading control. Individual data were quantified as densitometric units and normalized with calnexin. Data in the graph are presented as the relative percentage of the untreated control. d, e Nitric oxide generation in culture media was measured using the Griess reaction after enzymatic conversion of nitrate to nitrite. Total soluble protein (30 μg) was immunoblotted using a polyclonal anti-inducible nitric oxide synthase antibody. GAPDH was used as a loading control. f The levels of nitrite/nitrate in culture media were measured in the absence or presence of different concentrations of l-NAME. g Xanthine oxidase activity was calculated as mU/mg after normalization to total protein content. h The inhibitory effect of allopurinol on excretory/secretory product-induced xanthine oxidase activity. (b, e, g: *P < 0.05, compared with the zero time point; c, f, h: # P < 0.05, compared with excretory/secretory product-untreated control, *P < 0.05, CsFHC only versus ferritin heavy chain plus each inhibitor)

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To confirm that the activation of NOX, XO, and iNOS induced by CsFHC is responsible for free-radical generation, we preincubated cells with a specific inhibitor of each enzyme for 1 h, followed by exposure to CsFHC for 9 h. Pretreatment with DPI, a flavoprotein inhibitor of NOX, significantly inhibited CsFHC-induced accumulation of both p47phox and p67phox in the membrane in a dose-dependent manner (P < 0.05, Fig. 4a, c). Similarly, pretreatment of cells with ALP (an XO inhibitor) or l-NAME (an NOS inhibitor) resulted in a dose-dependent inhibition of CsFHC-induced XO activation and elevation of nitrite/nitrate concentration (P < 0.05, Fig. 4f, h). Taken together, these results suggest that free radicals were generated in CsFHC-treated cells via the activation of NOX, XO, and iNOS.

Induction of NF-κB activation by CsFHC treatment

It has been reported that the activation of NF-κB can be controlled by ROS produced in response to various stimuli (Gloire et al. 2006). Moreover, increased levels of NF-κB and iNOS have been shown to colocalize in the bile duct epithelium of hamsters infected by O. viverrini, leading to oxidative DNA damage and elevated inflammatory cell infiltration (Pinlaor et al. 2006). These findings prompted us to examine whether CsFHC exposure was associated with NF-κB activation. The cytosolic and nuclear fractions of cells treated with 10 nM of CsFHC for 0–24 h were used to determine the degradation of IκBα and nuclear translocation of the NF-κB subunits, p65 and p50, by immunoblot analysis. As shown in Fig. 5a, b, cytosolic IκBα protein was almost completely degraded at 9 h, concomitant with the time-dependent nuclear accumulation of NF-κB p50 and p65 subunits (Fig. 5d, e). These results demonstrate that CsFHC treatment led to NF-κB activation in LX2 cells.

Fig. 5
figure 5

CsFHC-induced NF-κB activation and inhibitory effect of N-acetylcysteine. After treatment with 10 nM of CsFHC for the indicated times or cells were preincubated with different concentrations of N-acetylcysteine for 1 h before incubating with 10 nM of CsFHC for 9 h, degradation of cytosolic IκBα and nuclear accumulation of the NF-κB subunits, p50 and p65, were determined by immunoblotting using polyclonal antibodies against IκBα (a), p50 and p65 (d). Protein bands were quantified and the densities of IκB-α and p50/p65 bands were normalized to those of GAPDH and lamin B bands, respectively. The normalized IκB-α, p50 and p65 band densities from each group are presented as a percentage of those in ferritin heavy chain-untreated controls. Values are means ± S.E.s of three independent experiments (# P < 0.05, compared with ferritin heavy chain-untreated controls; c: *P < 0.05, compared with excretory/secretory product-untreated control; f *P < 0.05, CsFHC only versus CsFHC plus different concentrations of N-acetylcysteine)

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Involvement of CsFHC-triggered free radicals in NF-κB activation

To assess the role of intracellular free radicals generated in response to CsFHC exposure in NF-κB activation, we preincubated LX2 cells with the antioxidant NAC (1–10 mM) for 1 h before adding 10 nM of CsFHC, and then collected cytosolic and nuclear fractions after 9 h for immunoblot analysis. CsFHC-mediated IκBα degradation in the cytosol was significantly inhibited in a dose-dependent manner by NAC, with 10 mM of NAC reducing degradation by ~50 % (P < 0.05, Fig. 5a, c). Nuclear translocation of NF-κB p50 and p65 subunits was also suppressed to levels below those in CsFHC-treated controls (~260 % inhibition) by 5 mM of NAC (P < 0.05, Fig. 5d, f), demonstrating that NAC attenuated CsFHC-induced NF-κB activation by scavenging free radicals triggered by CsFHC. Because free radicals were produced by the activation of NOX, XO, and iNOS in CsFHC-treated cells, we explored the effects of each enzyme inhibitor on NF-κB activation. Preincubation of LX2 cells with 10 μM of DPI, 100 μM of ALP or 100 μM of l-NAME for 1 h prior to CsFHC treatment reduced IκBα degradation in the cytosol by ~67, ~24, and ~40 %, respectively, relative to that induced by CsFHC alone (P < 0.05, Fig. 6a, b). The relative effectiveness of these inhibitors suggests that ROS generated by NOX activity are the most effective inducers of NF-κB activation. Concomitant with this, the administration of inhibitors resulted in a significant disruption of CsFHC-induced NF-κB p50 and p65 nuclear accumulation (P < 0.05, Fig. 6c, d), indicating that the activities of these enzymes are closely associated with NF-κB activation.

Fig. 6
figure 6

NF-κB inactivation by NADPH oxidase, xanthine oxidase, and nitric oxide synthase inhibitors in CsFHC-treated cells. Cells were preincubated with 10 μM of DPI, 100 μM of ALP, or 100 μM of l-NAME for 1 h, followed by treatment with 10 nM of CsFHC for 9 h. Cytosolic and nuclear proteins were subjected to immunoblot analysis for IκB-α, NF-κB p50 and p65 expression levels. a Representative immunoblot of IκB-α expression. b Representative immunoblot of NF-κB p50 and p65 expression. Individual data were quantified and normalized to GAPDH (IκB-α) or lamin B (p50 and p65). Values in graphs are presented as a percentage of those in untreated controls, expressed as means ± S.E.s of three independent experiments (# P < 0.05, compared with CsFHC-untreated controls; *P < 0.05, CsFHC only versus CsFHC plus different concentrations of N-acetylcysteine)

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Proinflammatory cytokines IL-1β and IL-6 increased by CsFHC-induced NF-κB activation

It is generally accepted that helminth infection contributes to inflammatory responses in host cells. In particular, animal models of O. viverrini infection have shown accumulation of liver fluke-associated antigens in the intra- and extrahepatic bile duct epithelium, accompanied by marked local inflammatory responses (Vennervald and Polman 2009). Therefore, we analyzed the mRNA expression patterns of the most common proinflammatory cytokines, IL-1β and IL-6, during a 1–24 h CsFHC exposure. Quantitative real-time PCR analyses revealed a significant elevation in the expression of cytokines with 1 h of CsFHC exposure (P < 0.05). IL-1β expression reached a maximal level at 9 h, then declined afterwards, whereas IL-6 expression gradually increased up to 24 h (P < 0.05, Fig. 7a). Because NF-κB is a key transcription factor involved in initiating the inflammatory cascade and regulating inflammatory mediators, we investigated whether NF-κB inactivation attenuated CsFHC-induced upregulation of IL-1β and IL-6 mRNA expression. Cells were preincubated with different concentrations of the NF-κB inhibitor, Bay11-7082, for 1 h before treatment with 10 nM of CsFHC for 9 h. As shown in Fig. 7b, this irreversible inhibitor suppressed the CsFHC-induced increase in both IL-1β and IL-6 mRNA in a dose-dependent manner (P < 0.05), suggesting that NF-κB signaling pathways are involved in the inflammatory responses of LX2 cells during CsFHC exposure. To validate the correlation of mRNA expression with protein level, we measured the level of each cytokine in the culture supernatants of 9 h CsFHC-treated cells, as accessed by ELISA. The levels of both IL-1β and IL-6 secretion were significantly increased (P < 0.05), compared with that of the control (Figs. 7c, d). Taken together, these results indicated that the expressions of IL-1β and IL-6 were transcriptionally and translationally upregulated by CsFHC.

Fig. 7
figure 7

Expression of IL-1β and IL-6 in CsFHC-treated cells. a Cells were treated with 10 nM of CsFHC, harvested after 0 to 24 h, and subjected to Quantitative real-time PCR analysis. b Cells were preincubated with 5–20 μM of Bay11-7082 for 1 h, followed by treatment with 10 nm of ferritin heavy chain for 9 h. IL-1β and IL-6 mRNAs were detected by the reverse transcription-PCR. Each band was quantified and normalized to GAPDH band density. The ratios of IL-1β and IL-6 mRNAs to GAPDH from each group are presented as a percentage of that at zero time (a) or in ferritin heavy chain-untreated controls (b). c, d Cells were incubated with 10 nM of CsFHC or an equal volume of PBS (control; CON) for 9 h, and the production of IL-1β or IL-6 in culture supernatants was determined by ELISA. Data are presented as means ± S.E.s of three independent experiments. *P < 0.05 compared with the control. Values are means ± S.E.s of three independent experiments (*P < 0.05, compared with zero time (a) or ferritin heavy chain only versus ferritin heavy chain plus Bay11-7082 (Bay) (b); #P < 0.05, compared with ferritin heavy chain-untreated controls)

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Discussion

A major pathological consequence of liver fluke infection is inflammation of the hepatobiliary system of both naturally infected humans and experimentally infected animals (Lim et al. 2008; Rim 2005; Vennervald and Polman 2009; Yoon et al. 2001). In the present study, we identified and characterized a ferritin heavy chain protein of C. sinensis. We obtained soluble and stable rCsFHC, which could be recognized by both serum from C. sinensis-infected and serum from C. sinensis ESP-immunized mouse. qRT-PCR analysis demonstrated that CsFHC can be detected in different developmental stages of C. sinensis including adult worm, metacercaria, and egg stages, indicating that CsFHC was expressed across all stages of C. sinensis (Fig. 1). In this study, LX-2 cell line which retains features of HSCs was used as a model of HSC-induced liver fibrosis in vitro (Xu et al. 2005). We provided evidence that, LX2 cells exposed to C. sinensis CsFHC generate free radicals by the activation of NF-κB through NOX, XO, and iNOS, and proinflammatory molecules, IL-1β and IL-6, were subsequently upregulated. These results demonstrate that free radicals function as key triggering modulators of host NF-κB-mediated inflammation in response to CsFHC.

In hepatic fibrosis, HSCs become activated and trans-differentiate into proliferative myofibroblast cells upon chronic exposure to inflammatory environment (Friedman 2008). HSCs are the central cell type in the liver’s fibrogenic process and activated in many conditions that lead to liver fibrosis (Friedman 2004). Free radicals actively participate in diverse physiological processes, including cell growth, survival, induction and maintenance of the transformed state, cellular senescence, apoptosis, and even necrosis (Nam et al. 2012). We examined NF-κB activation in LX2 cells treated with C. sinensis CsFHC. Our results revealed that CsFHC promoted the degradation of cytosolic IκB-α, indicated by gradual increase in nuclear translocation of NF-κB p50 and p65 subunits. These inhibitory effects on enzyme activation were correlated with a reduction in the levels of intracellular ROS and NO, indicating the role of enzymatic free-radical generation in the response to CsFHC (Fig. 4).

CsFHC-induced activation of NF-κB was dose-dependently attenuated by the ROS scavenger, NAC (Fig. 5). Furthermore, pretreatment of cells with DPI, ALP, or l-NAME substantially antagonized CsFHC-induced activation of NF-κB (Fig. 6), demonstrating that free radicals generated by the activation of CsFHC played important roles in the activation of the NF-κB pathway. A number of studies also suggested that the redox status of cells participates in modulating NF-κB activation (Asehnoune et al. 2004; Gloire et al. 2006; Kim et al. 2008; Pantano et al. 2006). Consistent with our results, enzymatic free-radical generation has been reported in liver fluke-infected animals. For instance, ROS-induced apoptosis in rat eosinophils treated with F. hepatica ESPs is attenuated in the presence of DPI (Serradell et al. 2009). Finally, our experiments showed that CsFHC-induced NF-κB activation was associated with an increase in the levels of IL-1β and IL-6 mRNA and proteins (Fig. 7). IL-1β and IL-6 are pleiotropic cytokines that have diverse potentiating effects on proliferation, differentiation, and apoptosis, as well as on local and systemic inflammatory responses (Nam et al. 2012; Naugler and Karin 2008). IL-1β can cause inflammation and induce the expression of proinflammatory genes. In addition, IL-1β promotes the generation of ROS (Brigelius-Flohe et al. 2004). Our results suggested that proinflammatory cytokine production in host cells stimulated by CsFHC serves as a link to immune responses.

In conclusion, we have identified a new gene CsFHC from C. sinensis and investigated the role of CsFHC to trigger free radicals and mediate NF-κB inflammation signaling in human hepatic stellate cells LX2. We demonstrated that CsFHC was a component of CsESPs. The present study provides insight into the biochemical mechanism by which C. sinensis CsFHC induce inflammatory responses in host cells. Given the direct relationship between chronic inflammation and cancers, our findings implied that free radicals may play pivotal roles in the pathology that occurs in the liver tissues system during C. sinensis infection, and free radicals may contribute to the severe inflammation in clonorchiasis that has been associated with hepatic fibrosis. Further investigations on upstream and downstream elements in the NF-κB signaling pathway are required to elucidate the mechanisms underlying the progression of clonorchiasis.