ABSTRACT

Non-alcoholic steatohepatitis (NASH) can progress to cirrhosis or hepatocellular carcinoma. Oxidative stress is implicated in NASH progression. Fermented papaya preparation (FPP) has oxygen radical scavenging activity and is effective in oxidative stress-related diseases. We investigated the effects of FPP on NASH progression using a rat NASH model. Plasma biochemical parameters and lipid peroxidation in the liver were elevated in NASH rats. Histologically, the liver of NASH rats showed liver fibrosis, mitochondrial dysfunction and over-expression of microsomal CYP2E1. Myeloperoxidase activity and nuclear factor-kappaB activation were also markedly increased in NASH. Oral administration of FPP ameliorated these changes in NASH rats. These results suggest that FPP halts NASH progression through its anti-oxidative and antiinflammatory properties.

1. INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is the most frequent cause of liver damage, closely associated with obesity and the metabolic syndrome, and often progresses to non-alcoholic steatohepatitis (NASH). NASH has attracted attention in recent years based on its relation with cirrhosis and hepatocellular carcinoma. The pathological findings in NASH are similar to those found in alcoholic steatohepatitis; including liver inflammation, necrosis, apoptosis, and fibrosis, despite the lack of alcohol consumption [1]. There is no effective drug therapy for prevention or treatment of NASH. Therefore, development of novel supplements or drugs with preventive effect for NASH is desirable. The pathogenesis of NASH is not clear, but the “two hit theory” is the most accepted [2]. It is based on fat accumulation in hepatocytes as a first hit, followed by the second hit of oxidative stress (OS), inflammatory cytokines, and insulin resistance.

We previously established a clinically relevant rat model of NASH (Patent application No. PCT/JP2007/52477) [3]. This model is prepared as follows; rats are fed a choline-deficient high-fat diet for 4 weeks to induce steatosis, and are treated by intraperitoneal injections of nitrite for 6 weeks to induce hypoxia-related oxidative stress. Using this model, it has been demonstrated that OS causes extensive hepatic fibrosis, and that these effects can be ameliorated by anti-oxidants, such as fermented green tea extract [4], vitis coignetiae pulliat leaves [5,6], docosahexaenoic acid [7], and spirulina [8].

Fermented papaya preparation (FPP) is produced by fermentation of unripe papaya (Carica papaya Linn.) using a variety of enzymes. Nutritional analysis has demonstrated the presence of beneficial amino acids, some vitamins, and various trace metals, such as iron, calcium, magnesium, copper and zinc in FPP. Papaya is widely known as a traditional medicinal fruit with beneficial properties, such as the antioxidant activity [9,10], heaptoprotective activity against CCl₄ [11] and enhancement of phase II enzyme activity, such as glutathione S-transferase in hepatocytes [12]. Previous studies reported that FPP has radical scavenging activity [13,14] as well as protection against diseases related to oxidative stress, such as traumatic epilepsy [14-16], Alzheimer’s disease [17], contact hypersensitivity [18], and stress-induced gastric mucosal lesions [19].

The present study was designed to determine the effects of FPP on prevention of NASH progression, and to elucidate the mechanism of the action.

2. MATERIALS AND METHODS

2.1. Chemicals

FPP was obtained from SAIDO Corporation (Fukuoka, Japan). 5,5-Dimethyl-l-pyrroline N-oxide (DMPO) was purchased from Labotech Co. (Tokyo, Japan). Dodecyl maltoside (DM) was purchased from Dojin Laboratories (Kumamoto, Japan). Benzamidine hydrochloride and sodium nitrate were purchased from Nacalai Tesque Inc. (Kyoto, Japan). β-NADH was purchased from Oriental Yeast Co. (Osaka, Japan). Other reagents were obtained from Wako Pure Chemical Industries (Osaka). All chemicals used in this study were of the highest grade available.

2.2. Animals

Six-week-old male Wistar rats weighting 160 - 170 g were purchased from Shimizu Experimental Animals (Shizuoka, Japan). The rats were housed in filter-protected cages set at 23˚C and ambient light was controlled automatically to produce a light/dark 12 h cycle. The Animal Ethics Committee of Okayama University approved the study. All animal procedures described in this study were in strict accordance with the Guidelines for Animal Experiments at Okayama University Medical School.

2.3. Experimental Design

We induced fatty liver in rats by feeding them a choline-deficient high-fat (CDHF) diet (Oriental Yeast, Tokyo) for 4 weeks. The control group (n = 6) was fed the standard chow (Oriental Yeast Co.). The CDHF group (n = 6) was fed CDHF diet only through the experiment period of 10 weeks. NASH group (n = 6) was fed CDHF diet, followed by injection of sodium nitrite 30 mg/kg for 6 weeks from the 5th week [3]. In the NASH + PL (low dose of FPP: 1% solution) group (n = 6) and NASH + PH (high dose of FPP: 3% solution) group (n = 6) were orally administered FPP solution in drink in water for 6 weeks from 5th week. After the experiment period, the rats were fasted overnight and sacrificed by anesthetizing with diethyl ether. Tissue samples were prepared to determine oxidative injury and effects of FPP, by using biochemical and OS markers and assessment of histological changes. The liver samples were fixed in 20% formalin or snap-frozen in liquid nitrogen for histological examination and for analyses of lipid peroxidation and antioxidant activity.

2.4. Histological Examination

Paraffin-embedded tissue sections were cut (4-μm thick) and stained with Masson trichrome for fibrosis. Tissue morphology was assessed under an Olympus light microscope (Olympus, Tokyo).

2.5. Biochemical Analyses

The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma were determined by commercial enzyme assay kits (Wako Pure Chemical Industries, Osaka). The level of plasma alkaline phosphatase (ALP) was determined using a standard assay.

2.6. Reactive Oxygen Species (ROS) in Rat Liver Mitochondria

Liver mitochondria were prepared by differential centrifugation of the tissue homogenates. In brief, rats were transcardially perfused with ice-cooled 1.15% KCl buffer containing 5 mM benzamidine (pH 7.4) via the inferior vena cava [4]. The liver tissue was then removed and homogenized in 5 mM Tris-HCl buffer (pH 7.4) containing 0.25 mM sucrose and 100 mM KCl. The mixture was centrifuged at 2500 rpm for 10 min. The supernatant was collected and centrifuged at 9000 × g for 20 min. The resulting pellet containing the mitochondrial fraction was resuspended in the buffer and stored at −80℃ until analysis. The protein concentration was determined using a BCA kit (Pierce, Rockford, IL) with bovine serum albumin as a standard. Electron spin resonance (ESR) technique with DMPO was used to determine ROS generation in liver mitochondria. Rat liver mitochondria isolates (500 μg) were mixed in 0.1 ml potassium phosphate buffer (pH 7.4) containing 0.1% DM, 5 mM glutamate, 5 mM malate, 0.1 mM succinate, 0.92 mM DMPO, and 0.1 mM NADH, incubated for 5 min at 37℃ and then measured. Analyses were performed at room temperature under ambient atmosphere. Using a manganese signal as an internal standard, the relative peak height of the second signal obtained from the DMPO-OH adducts was evaluated. The conditions for ESR measurements were as follows; microwave power: 8 mW, modulation amplitude width: 0.1 mT, response time: 0.1 s, scanning time: 4 min, magnetic field: 336.0 ± 5 mT (JES-RE1X/HR, JEOL, Tokyo).

2.7. Western Blot Analysis

The levels of microsomal cytochrome P450 2E1 (CYP2E1) and nuclear factor kappa B (NF-κB) in the liver were analyzed by western blotting. The samples were solubilized in 0.5 M Tris-HCL (pH 6.8) with sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol and 0.05% bromophenol blue, then degenerated by incubation at 100˚C for 5 min. The samples were subjected to SDS-10% polyacrylamide gel electrophoresis. After electrophoresis, proteins are transferred to polyvinylidene fluoride membrane. Then, the non-specific binding proteins were blocked in 5% nonfat milk dissolved in TBS-T (Tween-20 in Tris-buffered saline, pH 7.4) for 1 hour and incubated with primary rabbit anti-human/rat CYP2E1 polyclonal antibodies (1:1500; Chemicon International, Temecula, CA) or mouse antihuman/rat NF-κB monoclonal for 1 hour, and the membranes were incubated with secondary anti-rabbit or mouse IgG antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour. β-actin or histon was used to assess equal protein loading. Protein bands were visualized using enhanced Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology).

2.8. Lipid Peroxidation Assay

The liver homogenate supernatants collected from centrifugation of 30 mg/ml liver homogenate in 120 mM KCl-30 mM phosphate buffer (pH 7.4), were mixed with Luminol (130 μg/ml) and analyzed in an Auto Lumat device (Tristar LB941, Berthold Technologies, Germany), at 37˚C for 5 min. Then, 0.33 mM tert-butyl hydroperoxide (t-BuOOH) was added, and the chemiluminescence (CL) intensity was detected for 120 min along with incubation at 37˚C. Data were expressed as mean ± SEM of the cumulative CL intensity for 120 min.

2.9. Myeloperoxidase Activity

Myeloperoxidase (MPO) activity was used as an index of neutrophil infiltration in the liver. The liver was homogenated in 0.3 M sucrose containing 0.22% cetyltrimethyl ammonium and 10 mM citirate (pH 5.0) and sonicated. The homogenates were centrifuged at 6500 × g for 20 min. The supernatants were reacted with substrate solution (3 mM tetramethylbenzidine, 120 μM resorcinol, and 2.2 mM H₂O₂) in shading and mixed with 4 N H₂SO₄, followed by reading absorbance at 450 nm.

2.10. Statistical Analysis

Values were expressed as mean ± standard error of the mean (SEM) of 4 - 6 rats. Differences among groups were examined by one-way analysis of variance (ANOVA) with Tukey test. A P value less than 0.05 was considered statistically significant.

3. RESULTS

3.1. Effects of FPP on Liver Histology

Figure 1 shows microphotographs of histological changes in a representative animal. Macrovesicular hepatic steatoses were evident in the liver of CDHF-treated rats, and marked fibril formations were observed in the liver of NASH rats by Masson-trichrome staining. However, these histological changes were attenuated in FPP-treated rats, and the effect was concentration-dependent.