INTRODUCTION

1-O-hexyl-2, 3, 5-trimethylhydroquinone (HTHQ), a synthesized vitamin E derivative, is a superoxide scavenger (Nihro et al., 1994; Hino et al., 1998; Liu et al., 2002). It retains the original property of vitamin E as a natural lipophilic chain-breaking antioxidant (Tappel, 1980; Sharma, 2007). Carbon tetrachloride (CCl₄)-induced liver injury model is widely used to investigate the mechanisms of liver damage and regeneration (Patrick-Iwuanyanwu et al., 2007; Samudram et al., 2008; Dahiru et al., 2010; Sonkusale et al., 2011). Several lines of evidence indicate that vitamin E derivatives prevent both enhancement of lipid peroxidation and synthesis of type I collagen caused by CCl₄-induced chronic liver damage and cirrhosis (Parola et al., 1992a; Houglum et al., 1991; Chojkier et al., 1998; Parola et al., 1992b). Recently, we demonstrated that HTHQ can inhibit development of CCl₄-induced hepatic cirrhosis in rats more potently than does vitamin E (An et al., 2010). It is well known that hepatocytes are destructed and regenerated when continuously exposed to toxic substances such as CCl₄ (Weber et al., 2003). Although CCl₄ has been shown to induce apoptosis in hepatocytes (Yang et al., 2010; Lee et al., 2008; Tirkey et al., 2005; Khalaf et al., 2009; Obidah et al., 2011), it remains unknown whether HTHQ can suppress CCl₄-induced apoptotic processes in hepatocytes.

Recently, we characterized naofen, a novel WD40 repeat domain-containing protein (An et al., 2008; Feng et al., 2008, 2010; Sato et al., 2010). Functional analyses revealed that naofen is a new mediator of spontaneous apoptosis via activation of caspase-3 in the HEK293 cell line (Feng et al., 2010) and in renal tubular epithelial cells in the streptozotocin-induced rat diabetic model (Sato et al., 2010). Although naofen is found in hepatocytes it is still largely unknown whether treatment with CCl₄ induces expression of naofen in these cells. Therefore, we used the rat CCl₄-induced cirrhosis model to test the effects of HTHQ on hepatocyte apoptosis and the in vivo expression of naofen.

MATERIALS AND METHODS

Animals, treatment and specimen collection: All rats received humane care with the prior approval of the Animal Care Committee of Aichi Medical University. The rats were kept in a climate-controlled room under a 12 h light-dark cycle with free access to food and tap water throughout the studies. They were divided into four groups: a solvent-treated group, a CCl₄-treated group, a CCl₄ plus HTHQ-treated group and a HTHQ-treated group. The method of administration and preparation of paraffin block of liver sample were performed essentially as described in a previous report (An et al., 2010). The consumption of drinking water was checked and there were no clear differences in the amounts of drinking water between the solvent-treated group and the HTHQ-treated groups.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay: Apoptosis was determined by the TUNEL assay using the ApopTag^® Plus in situ Apoptosis Detection Kit (Chemicon International, USA) according to the manufacturer’s instructions. The slides were counterstained with 0.5% methyl green for microscopic examination. Five (100x magnification) fields were randomly selected per slide and total hepatocytes counted per field. The mean percentage of apoptotic hepatocytes was calculated and compared between the different experimental groups.

In situ hybridization: Based upon the previous report (An et al., 2008), the sequence of the naofen probe was designed as follows: 5’-GCAGAAAGGATATATGCT CTGTGTACTTTA-3’. Hybridization was performed at 105°C for five min and 74°C for 45 min, with the probe diluted at 1:1500. Positive regions were visualized with DAB (Falma, Tokyo, Japan) and then counterstained with hematoxylin for microscopic examination. Five (100x magnification) fields were randomly selected per slide and total hepatocytes counted per field. The mean percentage of naofen-positive hepatocytes was calculated and compared between different experimental groups. Five independent experiments were performed.

Real-time polymerase chain reaction (PCR): Total RNA was extracted from frozen control and CCl₄-treated rat liver tissues using TRIzol^® reagent (Invitrogen, USA). Equal amounts of DNA-free total RNA from each sample were converted to cDNA using the ReverTra Plus kit (Toyobo, Osaka, Japan). Reverse transcription was performed at 65°C for five min, 42°C for 60 min and 85°C for five min. Quantitative real-time PCR was performed using TaqMan primers and probes for naofen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. PCR reactions and analyses were carried out using the Step One real-time PCR system and software (Applied Biosystems). The relative abundance of the target genes was obtained by normalizing to GAPDH expression according to the ΔΔC_T method as previously reported by An et al. (2010). Five independent experiments were performed.

Statistics: The significance of differences between the means of the total numbers of hepatocytes was determined by analysis of variance (ANOVA), evaluated by Scheffe’s method. The rates were analyzed with a non-parametric method, Kruskal-Wallis one-way ANOVA. Statistical significance was accepted at 0.05.

RESULTS

Eight weeks after starting injections of CCl₄, the liver architecture was completely distorted by fibrous septa and the formation of regenerative nodules (pseudo-lobules) (Fig. 1a). In order to evaluate the effects of agents on apoptosis, TUNEL staining was performed. The apoptosis of hepatocytes was observed around Glisson’s area in the CCl₄-treated group (Fig. 1b). The percentage of apoptotic cells in the liver of the CCl₄-treated group was 45.34±1.71, significantly greater than that in the solvent-only group (p<0.01) (Fig. 1e). In contrast, in the CCl₄ plus HTHQ-treated group, the formation of interlobular fibrosis and pseudo-lobules was clearly diminished, although the vacuoles and formation of enlarged portal veins were little affected by HTHQ (Fig. 1c, d). The percentage of apoptotic hepatocytes obtained in the CCl₄ plus HTHQ-treated group was 26.64±0.89, significantly smaller than that in the CCl₄-treated group (p<0.01).

In the next experiments, we examined whether treatment of CCl₄ induced expression of naofen in apoptotic hepatocytes. The expression of naofen mRNA in the HTHQ-treated group was almost identical to that in the solvent-treated group (Fig. 2a, d). In the CCl₄-treated group, expression of naofen mRNA increased robustly in the hepatocytes and its expression in the pseudo lobules were greater than that in other areas (Fig. 2b).

Fig. 1(a-e):

TUNEL expression in rat livers following carbon tetrachloride (CCl4)-induced cirrhosis, TUNEL staining. (a), Solvent-treated group, (b), CCl4-treated group, (c), CCl4 plus HTHQ-treated group, (d), HTHQ-treated group, Horizontal bar indicates 100 μm, (e) Percentage of apoptosis-positive hepatocytes in CCl4-treated rats, Con (-): Solvent-treated group, 8w (-): CCl4-treated group, 8w (+): CCl4+HTHQ-treated group, Con (+): HTHQ-treated group, **p<0.01 compared to the control group, †Hp<0.01 compared to the CCl4-treated group (n = 5, each)

The mean percentage of naofen-positive hepatocytes in the CCl₄-treated group was 78.40±4.16 (Fig. 2e), significantly greater than that obtained in the solvent-treated group (p<0.01). In contrast, in the CCl₄ plus HTHQ-treated group (Fig. 2c), the amount of naofen mRNA in the cytoplasm of hepatocytes around Glisson’s area was clearly diminished compared to the CCl₄-treated group. The mean percentage of naofen-positive hepatocytes in the CCl₄ plus HTHQ-treated group was 7.47±1.25 (Fig. 2e), significantly smaller than that in the CCl₄-treated group (p<0.01). Arrows show the same hepatocyte, indicating that among the abundant naofen-positive hepatocytes, some underwent apoptosis (Fig. 1b, Fig. 2b).

We also quantified the level of naofen mRNA expression using GAPDH mRNA as an internal standard. In the CCl₄-treated group, the normalized expression of naofen was 1.90±0.18 which was significantly greater than that in the solvent-treated group (p<0.01).

Fig. 2 (a-f):

Naofen mRNA expression in rat livers following CCl4-induced cirrhosis, (a) In situ hybridization assessment of naofen expression, (a) Solvent-treated group, (b) CCl4-treated group, (c) CCl4 plus HTHQ-treated group, (d) HTHQ-treated group, Horizontal bar indicates 100 μm, (e) Percentage of naofen-positive hepatocytes in CCl4-treated rats, **p<0.01 compared to the control group, ***p<0.01 compared to the CCl4-treated group (n = 5, each), (f) Ratios of naofen mRNA to GAPDH mRNA in liver tissues of CCl4-treated rats, ** p<0.01 compared to the control group, ***p<0.01 compared to the CCl4-treated group (n = 5)

By contrast, in the CCl₄ plus HTHQ-treated group, the normalized expression of naofen was 1.29±0.08 which was significantly smaller than that in the CCl₄-treated group (p<0.01) (Fig. 2f).

DISCUSSION

We have shown in the present study of rats that HTHQ effectively inhibits hepatocyte apoptosis caused by repeated hypodermic injections of CCl₄. The calculated doses of HTHQ from the consumed volumes of drinking water were in the range of 45.5-46.5 mg kg^-1 body weight/day, comparable with a previous report (An et al., 2010).

Shi et al. (1998) reported that apoptosis in rat livers could be observed histochemically after CCl₄ administration and Sun et al. (2003) reported that the activity of caspase-3 was increased in the rat liver and plasma after CCl₄ administration. Furthermore it is well known that the mechanism of hepatic injury by CCl₄ involves hepatocyte membrane lipid peroxidation, causing destruction of the cells and their intracellular organelles (Khalaf et al., 2009). In addition, we showed the apoptosis of hepatocytes by using TUNEL assay (Moorthy et al., 2009; Amin, 2009; Ali et al., 2011; Khorshid et al., 2011). Based on these observations it would be reasonable to assume that orally administered HTHQ mixed into the rats’ drinking water might reduce CCl₄-induced apoptotic changes by preventing enhancement of lipid peroxidation.

Naofen is a new intracellular component that may cause apoptosis by activating caspase-3 which mediates TNF-α-stimulated apoptosis (Feng et al., 2010). The present study revealed that the CCl₄-induced increase in expression of naofen mRNA correlated well with positive TUNEL staining in hepatocytes. In addition, HTHQ strongly suppressed the CCl₄-induced increase in naofen mRNA expression. It appears that HTHQ prevents fibrogenesis by inhibiting the naofen expression that mediates hepatocyte apoptosis.

The mechanism of induction of naofen mRNA in hepatocytes is currently obscure. When hepatocytes or cell lines were exposed to CCl₄, or to possible inflammatory mediators in in vitro studies, none of the examined mediators affected naofen expression (unpublished observation), implying that naofen expression may be induced by activation of non-parenchymal cells such as Kupffer cells. Further studies will determine whether non-parenchymal cells are involved.

In conclusion, we have demonstrated that HTHQ suppresses apoptosis during the development of CCl₄-induced liver cirrhosis in rats. In addition, the mode of action of HTHQ in preventing CCl₄-induced hepatic cirrhosis might include naofen-mediated apoptotic processes in rats.