Antrodia Cinnamomea Reduces Carbon Tetrachloride-induced Hepatotoxicity In Male Wister Rats

Materials and Methods

Reagents. CCl₄, olive oil and other reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Alanine aminotransferase (ALT) kit, aspartate aminotransferase (AST) kit and albumin were purchased from Roche Cobas Mira (Roche Diagnostic Systems, Montclair, New Jersey, USA).

Preparation of AC test solution. AC and 2 ml distilled water were mixed thoroughly to provide a final solution with a concentration of 350, 1,400 and 3,150 mg/kg. AC was obtained from Chang Gung Biotechnology Corporation, Ltd. (Taipei, Taiwan).

Experimental animals. Male Wistar rats, specific pathogen-free, 6 weeks old and weighing 250-300 g, were obtained from the Animal Medicine Center, College of Medicine, National Taiwan University and were used for the chronic CCl₄-induced liver injury model in all experiments. The rats were housed in plastic cages and maintained under standard conditions of controlled room temperature at 20±2°C and relative humidity of 75±15% with a 12 h light/night cycle with free access to standard laboratory diet and water, and filtered laminar air flow controlled room in the animal facility of the Animal Medicine Center, College of Medicine, National Taiwan University, Taipei, Taiwan. The use of experimental animals in this study was conducted under the guidance of the basic standards in the care and use of laboratory animals, which has been prepared and published by the National Institutes of Health. The study protocol has been approved by the Research Ethics Committee, the Institutional Animal Care and are in agreement with the Helsinki declaration.

Experimental design and protocol. After 1 week of acclimatization, a total of sixty rats were randomly divided into six groups (each group consisted of 10 rats) with healthy rats (Only H₂O, on-induction group; normal control), cirrhotic rat group with vehicle (olive oil) treatment only (CCl₄ + H₂O, negative control; non-treatment group), cirrhotic rat group with silymarin treatment (CCl₄ + silymarin, positive treatment control), and cirrhotic rat groups with three different doses of AC treatment (experiment groups). As a non-induction group, 10 rats without CCl₄ induction were fed a regular diet and double-distilled water. The other 50 rats treated with CCl₄ were further divided into non-treatment group (n=10), silymarin treatment group (n=10) and three AC treatment groups (n=30). In these 50 rats, animals were fed 20% CCl₄ in a 1:4 mixture with olive oil at a dose of 2 ml/kg, twice a week for 8 weeks to induce liver damage. During the 8-week induction period, negative control rats were treated only by vehicle and positive control rats were treated with silymarin. Rats in the three AC groups received AC at a low dose (350 mg/kg/day), medium dose (1400 mg/kg/day) or high dose (3150 mg/kg/day) daily for 8 weeks. The animals' weights were monitored prior to the start of experiment and then checked 2 times per week over the 8 week period. After the last dose, blood from all of the rats was drawn and collected by SST tube via caudal artery under mild ether anesthesia. Blood was allowed to clot at room temperature and the serum was separated by centrifuging at 4000 rpm for 15 min and kept at −20°C for further biochemical analysis. Rats were sacrificed, and the liver and spleen were dissected and used for histopathological (formalin fixed) and biochemical (frozen −80°C) studies.

Analysis of plasma transaminase activities and albumin level in serum. Serum samples were prepared at the end of 8 weeks and were assayed for GOT, GPT activity and albumin concentration according to the manufacturer's protocol for an estimation of liver function using a DxC 800 clinical chemistry analyzer with reagents (GOT lot number: M307050; GPT lot number: M312240; Albumin lot number: M310159) purchased from Beckman Coulter (Brea, CA, USA). These three tests were performed by the Animal Medicine Center, College of Medicine, National Taiwan University.

Assays for Superoxide Dismutase Assay (SOD), Glutathione Peroxidase (GSH-Px) and Catalase. Ten percent of homogenates of liver tissues were prepared separately in 100 mM KH₂PO₄ buffer containing 1 mM EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected and used for the following experiments as described below.

In this method NADH (RANSOD manufactured by RANDOX Ltd. UK) was used as the substrate (21). Reaction mixture of this method contained 0.1 mL of phenazine methosulphate (186 μM), 1.2 mL of sodium pyrophosphate buffer (0.052 mM; pH 7.0), and 0.3 mL of supernatant after centrifugation (1500 × g for 10 min followed by 10000 × g for 15 min) of tissue homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 mL of NADH (780 μM) and stopped after 1 min by adding 1 mL of glacial acetic acid. Amount of chromogen formed was measured by recording color intensity at 560 nm. Results are expressed in units/mg protein.

Glutathione peroxidase activity was assayed by the method of Mohandas et al. (22). Liver was homogenized with GSHPx cold buffer (50 mM Tris-HCl containing 5 mM EDTA and 1 mM dithiothreitol (DTT), pH 7.5). GSHPx activity was measured using a GSHPx assay kit. The reaction was initiated by the mixing glutathione, glutathione reductase, NADPH with cumene (isopropylbenzene) hydroperoxide, and then detecting conversion of NADPH to NADP with a spectrophotometer at 340 nm and 25°C for 5 min. The specific enzyme activity was measured as nanomoles of NADPH oxidized to NADP per minute per milligram protein (oxidized/min/mg protein).

CAT activities were determined according to manufacturer's protocols of corresponding kits. Using H₂O₂ as a substrate (23), 0.1 mL of the supernatant was mixed with 2.5 mL of 50 mM phosphate buffer (pH 5.0) and 0.4 mL of 5.9 mM H₂O₂, and change in absorbance was recorded at 240 nm after one min. One unit of CAT activity was defined as an absorbance change of 0.01 units/min.

Hepatic protein, malondialdehyde (lipid peroxidation) and hydroxyproline assays. Total liver protein concentration in each sample was measured using Coomassie blue assay reagent (KENLOR Industries Inc., CA, USA) based on an absorbance at 540 nm. Bovine serum albumin was used as a standard. The amount of total protein was expressed as mg/g tissue. Lipid peroxidation was measured using the method of Ohkawa et al. (24) and 2-thiobarbituric acid. Lipid peroxidation was expressed as the amount of malondialdehyde/mg protein. Hydroxyproline determination used procedures reported previously (25). After hydrolysis, dried liver tissue was oxidized by H₂O₂ and colored by p-dimethyl-aminobenzoaldehyde (Sigma Chemical Co. St Louis, Mo., USA). Absorbance was determined at 540 nm, and the amount of hydroxyproline was expressed as μg/g tissue.

Histopathology. For liver histology, liver tissue was immediately fixed in 10% buffered formaldehyde and the blocks were incubated twice with 10% neutral buffered formalin at room temperature for 0.5 h each, 75% alcohol at room temperature for 1 h, 85% alcohol at room temperature for 1 h, 95% alcohol at room temperature for 1 h, twice; 100% alcohol at 40°C for 1 h, twice; xylene at 40°C for 1 h, twice; and in molten wax at 60°C for 0.5 h and repeated 4 times. Blocks were then embedded in paraffin. Some paraffin sections were stained using Haematoxylin-Eosin stain solution (Muto Pure Chemicals, Co., Ltd., Tokyo, Japan). Other paraffin sections were stained using Sirius red stain (Direct Red 80) (Sigma Chemical Co. St Louis, Mo., USA). The stained sections were examined under a microscope for histopathological changes in liver architecture and photomicrographs taken.