Protective effect of low molecular fraction of MGN-3, a modified arabinoxylan from rice bran, on acute liver injury by inhibition of NF-κB and JNK/MAPK expression

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Abstract

d-Galactosamine (GalN) induces acute hepatitis in experimental animals; this hepatitis has been shown to be suppressed by oral or intraperitoneal administration of modified arabinoxylan from rice bran (MGN-3), and active low molecular fraction isolated from MGN-3 (LMW). We previously reported that this protective mechanism is mediated in part by downregulation of interleukin-18 (IL-18). The present study shows for the first time that nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) and CD14 are involved in the suppressive action of LMW on GalN-induced hepatitis. Wistar rats (aged 4 weeks, SLC) were intraperitoneally treated with either MGN-3 or LMW. Then, rats were given GalN at 400 mg/kg at 1 h after the initial treatment. The serum activity of transaminases (ALT and AST) was significantly higher after GalN treatment; these changes were attenuated by MGN-3 and LMW. Furthermore, LMW abrogated inhibitor of κB kinase (IκB) degradation induced by GalN, and this was associated with the inhibition of NF-κB activation. Moreover, phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (JNK) protein expression in the liver after GalN treatment was significantly higher, and LMW reduced this increase. We also found that GalN treatment induced TLR4 and CD14 mRNA expression, and LMW significantly inhibited CD14 mRNA expression. These results suggest that the suppressive effects of LMW on GalN-induced hepatitis are possibly related to inhibition of NF-κB, JNK phosphorylation and CD14 expression.

Highlights

► Protective mechanism of LMW against GalN hepatitis is associated with NF-κB. ► GalN-induced JNK/MAPK was significantly suppressed by LMW. ► LMW suppressed GalN-induced CD14 mRNA expression but not TLR4.

Introduction

Hepatitis is a serious health problem worldwide associated with significant morbidity and mortality. A better knowledge of the basic mechanisms governing immune response in the pathogenesis of liver disease has allowed the development of targeted therapies for the management and treatment of hepatitis [1], [2], [3]. d-Galactosamine (GalN)-induced hepatitis has been used as an animal model for acute liver injury, since its morphological and pathophysiological characteristics are similar to those of human hepatitis B [4], [5]. Hepatitis induced by GalN in rats is considered to be mediated by inhibited macromolecular glycoprotein and RNA biosynthesis through depletion in cellular UTP concentration [6] and elevation of blood levels of tumor necrosis factor-α (TNF-α) caused by increasing absorption of lipopolysaccharide (LPS) endotoxin, from the intestine to the bloodstream [7], [8]. However, the precise mechanism for GalN-induced hepatitis has not yet been elucidated.

MGN-3, a modified water-soluble hemicellulose from rice bran has a variety of immune functions. It has been reported that NK cell, T cell, and B cell functions are augmented by MGN-3 both in vitro and in vivo [9], [10], [11]. In addition, when MGN-3 is administered in conjunction with conventional chemotherapeutic agents, it has been highly effective in inducing cancer remission in animal models [4]. In our previous study, we showed that GalN-induced hepatitis was suppressed in part by IL-18 reduction following ingestion of BioBran (MGN-3), a modified arabinoxylan from rice bran, or its active fraction (LMW). MGN-3 was hydrolyzed with HCl at 100 °C, and then the hydrolysate treated with cation or anion exchange resin was fractionated by molecular weight (high molecular weight fraction (≥ 2,000,000 Da), medium molecular weight fraction (2,000,000–400 Da), and low molecular weight fraction (LMW; ≤ 400 Da)). We concluded that LMW has a stronger hepato-protective effect than MGN-3 [12]. The molecular weight of LMW was measured by ESIMS, and an intense peak at m/z 409 was observed [12]. LMW is a mixture of monosaccharide and oligosaccharides, constituted by glucose as the main component (glucose, 22.8%; mannose, 1.5%; galactose, 0.5%; arabinose, 0.3%; protein, 2.85%) [12]. In our previous study, the results indicate that neutral oligosaccharides and monosaccharides in the LMW seem to be candidates for the effective ingredients for treating GalN-induced liver injury.

IL-18 is a unique activating cytokine belonging to a novel family of inflammatory cytokines that function in the immune response [13], [14], [15], [16]. In an animal experimental model, IL-18 is released from murine macrophages or Kupffer cells through Toll-like receptor 4 (TLR4)/CD14-dependent signaling pathways [17].

TLRs, members of the pattern recognition receptor family, sense pathogen-associated molecular patterns (PAMPs) for host defense; however, endogenous components from necrotic cells, referred to as damage to associate molecular patterns (DAMPs), were recently shown to activate TLR-mediated signals associated with innate immune responses [18]. In mammals, 12 members of the TLR family have been identified [19]. TLR4 is a transmembrane protein, mainly existing in macrophages, which recognize LPS or LPS-CD14 complexes, and mediates macrophage activation and pro-inflammatory cytokine release [12], [20]. CD14, a key gene of the innate immune system, functions as a receptor for LPS, a constitutive element of the bacterial cell wall. As a consequence of the CD14-LPS interaction at the level of the membrane, TLR-4 becomes activated. TLR-4 plays an important role in signal transduction in the innate immune response. Importantly, TLR-4 activates a transcription factor known as nuclear factor-κB (NFκB), and members of the mitogen-activated protein kinases (MAPKs) family including p38 kinase (p38), extracellular stress-related kinase 1/2 (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK) [21]. Activated CD14/TLR4 is associated with hepatic ischemia/reperfusion, regeneration, and alcoholic liver disease [19].

In an animal model, endotoxin stimulation induces activation of procaspase-1 in Kupffer cells, which cleaves preformed IL-18 (proIL-18) into IL-18, resulting in the release of IL-18 [22], [23]. IL-18 synergizes with IL-12 for IFN-γ production from lymphocytes, which fully activates the Kupffer cells to produce large amounts of TNF-α, a potent hepatotoxic cytokine [17]. However, the currently available data for the role of TLR4 and TLR4-signaling pathways fulminant hepatic failure are not sufficient.

In the present study, to study the mechanisms underpinning the protective effects of LMW against GalN-induced acute liver injury, we investigated the TLR4/CD14 pathway, NFκB and MAPKs.

Section snippets

Reagents

d-Galactosamine hydrochloride (GalN) was obtained from Sigma Chemicals (St. Louis, MO, USA); the SV Total RNA isolation system from Promega Corporation (Madison, WI, USA); the first-Strand cDNA synthesis kit for RT-PCR (AMV) from Roche Diagnostics (Mannheim, Germany); the SYBR® Premix Ex Taq™ II kit (perfect real-time PCR) from Takara Bio Inc. (Otsu, Japan); and the reaction mixture for PCR, AbsoluteTM QPCR SYBR Green mixes from Abgene (Epson, UK). MGN-3 was provided by Daiwa Pharmaceutical Co.

Hepato-protective effect of MGN-3 and LMW

Rats treated with GalN alone developed hepatocellular damage, as evident from a significant elevation in serum transaminase assays. Pre-treatment with MGN-3 by intraperitoneal injection afforded significant protection against GalN-induced liver injury. Pretreatment with LMW showed the same effect as MGN-3 (Fig. 1A).

LMW was administered to the rats at three different doses to quantitatively study the protective effects of LMW against GalN-induced liver injury. The AST and ALT activities of the

Discussion

Acute liver diseases constitute a global concern, and medical treatments for these diseases are often difficult to manage and have limited efficacy. Therefore, there has been considerable interest in the role of complementary and alternative medicines for treatment of liver diseases [25].

GalN is used as an experimental model of severe hepatic damage that closely resembles human viral hepatitis, due to its ability to inhibit RNA and protein synthesis in the liver, resulting in the subsequent

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