Biochemical kits for the detection of AST (cat. no. C010-2-1), ALT (cat. no. C009-2-1), CHO (cat. no. A111-1-1), TG (cat. no. A110-1-1), nitric oxide (NO; cat. no. A013-2-1), H₂O₂ (cat. no. A064-1-1), lipid peroxide (LPO; cat. no. A106-1-3), total peroxide content (T-AOC; cat. no. A015-2-1), catalase (CAT; cat. no. A007-2-1), superoxide dismutase (SOD; cat. no. A001-3-2) and BCA (cat. no. A045-4-2) were purchased from the Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. ELISA kits for the detection of TNF-α (cat. no. 8066441), IFN-γ (cat. no. 8066449), IL-6 (cat. no. 8066455) and IL-33 (cat. no. 8063899) were purchased from Bossbio Biotech. RIPA buffer (cat. no. P0013B) was bought from Beyotime Institute of Biotechnology. Nrf2 (cat. no. sc-365949), NF-κB (cat. no. sc-8008) and β-actin (cat. no. sc-8008) antibodies were purchased from Santa Cruz Biotechnology, Inc. The chemiluminescence kit (cat. no. P0018S) was purchased from Beyotime Institute of Biotechnology. Anhydrous ethanol and other chemicals purchased were of analytical grade.

The whole-tree agarwood-inducing technique (patent no. ZL201010104119.5) was used to obtain artificially produced agarwood by the infusion agarwood-inducing technique (specimen no. JC2016112), wounding wild agarwood by causing damage via cuts to the trunk similar to wild agarwood formation (13,22) (specimen no. JC2016099) and burning agarwood via chisel-drilling using traditional or primitive fire drilling agarwood (13,22) (specimen no. JC2016076), which were purchased from the Guangdong Huzhou Guolin Agarwood Planting Cooperative. The aforementioned company was the experimental base of the agarwood-inducing production technology utilized in the present study. Agarwood was identified by Professor Jian-he Wei and stored in an air-conditioned sample room at 16°C from purchase. Agarwood alcohol extracts (AAEs) whole-tree agarwood-inducing technique alcohol extract (WTAAE), wild agarwood induced by ace wounds alcohol extract (WAAE) and burning-chisel-drilling agarwood alcohol extract (FBAAE) were extracted and prepared according to extraction methods reported in previous studies (20,22,23). Agarwood (1,000 g) was naturally dried at 30°C for a week, smashed and soaked in 95% ethanol (5 l) for 2 h. The resulting agarwood ethanol extract was extracted using reflux extraction for 1 h and filtered using 2-µm filter paper. The reflux extraction was repeated twice. The resulting ethanol solutions were combined, concentrated and dried in vacuo to obtain dark-brown WTAAE (140 g; 14%), WAAE (105 g; 10.5%) and FBAAE (140 g; 14%) which were stored in a freezer at −20°C.

Healthy, adult male C57 mice (n=80; weight, 20±2 g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Jing) 2017-022, Beijing, China). Animals were housed in a specific-pathogen-free laboratory animal room using a 12-h light/12-h dark cycle, with ad libitum access to food and water, and were housed for 3 days prior to experiments.

The 80 mice were randomly divided into eight groups: i) Normal; ii) model; iii) compound methionine choline tablets (CMCT); iv) WAAE; v) FBAAE; vi) 0.71 g/kg WTAAE; vii) 1.42 g/kg WTAAE; and viii) 2.84 g/kg WTAAE groups. The CMCT group was used as a positive control group. CMCT is a compound preparation of methionine choline used for the treatment of fatty liver and acute or chronic hepatitis. The dosage of WAAE (20,21), FBAAE (20,21) and WTAAEs (20,21) were determined according to the previously published conversion of human daily dosage of these compounds. The normal and model groups were orally gavaged with 20 ml/kg of distilled water. The CMCT group was orally garaged with 200 mg/kg CMCT daily. The WAAE and FBAAE groups were orally gavaged with 2.84 g/kg of the respective compounds daily. The WTAAE groups were orally gavaged with 0.71, 1.42 or 2.84 g/kg (equivalent to the mass of raw material for agarwood) (20,21) of treatment daily, respectively. All groups were fed a high-fat diet, except for the normal group, which was fed a normal maintenance diet. The composition of high fat feed included: 20% Sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate and the remaining food was base feed. All groups, except for the normal group, were orally gavaged with 30% ethanol, 20 ml/kg daily for 3 weeks (24). The corresponding test drugs were orally administered at 20 ml/kg daily from days 8–21. On the 21st day, the mice were fasted without water for 12 h, then blood and liver tissues were collected for the assessment of pharmacodynamic indexes. Animal care and experimental protocols were approved by the Animal Care and Use Committee at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (ethical approval no. SLXD-20180809012).

Mice were weighed 1 h after the final drug dose was administered. Mice were briefly anesthetized with cotton balls impregnated with ether. Mice were considered anesthetized when the animals lay on their back, their heart rate and breathing were even, muscles were relaxed, limbs were not moving, whiskers were not moving and their pedal reflex disappeared. Then, 0.5-1 ml of blood was collected from the retro-orbital sinus and centrifuged at 1,006.2 × g at 4°C for 15 min for serum analysis. Animals were sacrificed by cervical dislocation immediately following blood sample collection. The liver and adipose tissues were collected and weighed. The liver index and fat indexes were calculated as follows: Liver index (mg/g)=liver weight (mg)/animal weight (g); and fat index (mg/g)=fat weight (mg)/animal weight (g).

Histopathological changes in the liver of mice were analyzed using hematoxylin and eosin (H&E) staining (25). The liver tissues were fixed in 4% paraformaldehyde solution for 24 h at room temperature, embedded in paraffin, cut into 4-µm sections, and then stained with H&E for 25 min at room temperature. The specimens were imaged using a fluorescence microscope at ×400 magnification (Olympus Corporation). Histological damage was calculated using the following score system (PDI score): Score 0, absent (no destruction); score 0.5, few (0–20% destruction); score 1, mild (20–50% destruction); score 2, moderate (50–80% destruction); score 3, severe (80–100% destruction) (21,25).

The liver tissues were fully homogenized mechanically with saline (1:9 w/v) and were centrifuged at 1,006.2 × g at 4°C for 15 min, the supernatant was collected and used for measuring oxidative stress. The levels of NO, H₂O₂, LPO, CAT, SOD and T-AOC were measured using the respective biochemical kits according to the manufacturer's instructions.

Serum samples were collected to assess the levels of inflammatory cytokines. The levels of TNF-α, IL-6, IL-33 and IFN-γ were measured in serum using ELISA kits according to the manufacturer's instructions.

Total liver proteins were extracted from liver tissues using RIPA buffer. The protein concentration was measured using a BCA protein assay. The protein was loaded onto a 10% SDS-polyacrylamide gel (20 µg/lane), transferred onto a PVDF membrane and blocked with 5% skimmed milk at room temperature for 2 h. Membranes were incubated with primary antibodies for Nrf2 (1:500), NF-κB (1:1,000) and β-actin (1:2,000) at 4°C overnight. The membrane was then incubated with secondary antibodies (1:2,000; goat anti-rabbit secondary antibody; cat. no. A0208) at room temperature for 2 h. The membranes were washed three times using TBST (0.05% Tween) and visualized using an enhanced chemiluminescence kit. The membranes were imaged using a pro-gel imaging system and protein expression levels were semi-quantified using ImageJ Software V1.8 (National Institutes of Health).

Data were expressed as the mean ± standard deviation and were analyzed using SPSS (version 17.0; SPSS, Inc.). Differences between each group were analyzed by one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Liver and fat indexes were determined and were both significantly elevated in the model group compared with the normal group (Fig. 1). High liver and fat indexes are characterized by fat accumulation and liver edema, therefore, the high fat diet and alcohol consumption may lead to the accumulation of fat and the formation of fatty liver, which suggested that the model of AFL was successfully constructed in the present study. Pretreatment of mice with different AAEs significantly reduced the liver and adipose indexes compared with the model group for all AAEs, apart from pretreatment with 0.71 g/kg WTAAE which did not significantly impact the fat index. WTAAE pretreatment demonstrated a dose-dependent effect at reduced the liver and adipose indexes, which suggested that AAEs had a significant effect on relieving hepatic steatosis.

The levels of liver metabolic enzymes and lipids, such as AST, ALT, TG and CHO, are key markers used to evaluate liver function (2,6,7). The levels of AST, ALT, TG and CHO were significantly increased in mice in the model group compared with the normal group, which suggested the liver function was significantly impaired (Fig. 2). Pretreatment of mice with AAEs significantly reduced the levels of the aforementioned enzymes and lipids compared with the model group. In addition, WTAAE dose-dependently reduced the liver and adipose indexes, which suggested that AAE treatment demonstrated a marked effect on restoring the liver function in AFL mice.

The ethanol and high fat diet induced marked histopathological injuries, including lipid accumulation, edema, deformation, hemorrhagic injury, hepatic stellate cell necrosis and inflammatory cell infiltration (Fig. 3). AAEs markedly reduced this damage in liver tissues and protected against liver histopathological lesions. Mice treated with DBGTP, WAAE and FBAAE presented with less swelling and lower numbers of inflammatory cells. WTAAE pretreatment demonstrated dose-dependent protection against ALF. The pathological damage index (PDI) could show the results more directly (Fig. 3I). WTAAE and WAAE pretreatment had a similar effect at the 2.84 g/kg dose, which was more effective than FBAAE. These data suggested that AAEs demonstrated a significant protective effect on liver pathological injury in ALF mice.

Significantly increased levels of NO, LPO and H₂O₂ in the model group demonstrated the damage caused by oxidative stress compared with the normal group (Fig. 4A-C). AAE treatment significantly decreased the levels of lipid peroxidation. The levels of CAT, SOD and T-AOC were significantly lower in the model group compared with the normal group (Fig. 3D-F). The levels of CAT were significantly increased with AAE pretreatment compared with the model group, apart from in animals treated with 0.71 g/kg WTAAE. SOD levels were significantly increased in mice treated with WAAE and WTAAE at concentrations of 1.42 and 2.84 g/kg compared with the model group. Levels of T-AOC were significantly increased in mice pretreated with AAE compared with the model group. These data suggested that AAEs have an anti-oxidant effect and the effect of WTAAE was similar to WAAE and may be more effective than FBAAE at the highest dose tested (2.84 g/kg).

The levels of pro- and anti-inflammatory cytokines were analyzed to evaluate the potential anti-inflammatory activity of AAE treatment. The levels of TNF-α, IL-6 and IL-33 were significantly increased in the AFL mice compared with normal mice (Fig. 5A-C). AAEs significantly decreased the levels of IL-6 and IL-33 in all treatment groups compared with AFL mice. The levels of TNF-α were significantly decreased by FBAAE and all concentrations of WTAAE tested compared with the model group. WTAAE treatment at 2.84 g/kg appeared to be the most effective treatment regime tested to reduce levels of the aforementioned cytokines. In contrast, IFN-γ levels were significantly decreased in the model group compared with the normal group of mice (Fig. 5D). IFN-γ levels were significantly increased in the WAAE, FBAAE and WTAAE groups of mice and were highest with 2.84 g/kg WTAAE.

To further assess the underlying molecular mechanisms of AAEs, the protein expression levels of Nrf2 and NF-κB were assessed using western blotting (Fig. 6). The protein expression levels were semi-quantified and it was demonstrated that the protein expression levels of Nrf2 were significantly decreased in AFL mice compared with the normal group. WTAAE pretreatment significantly increased Nrf2 expression levels at each of the concentrations tested compared with the model group. The protein expression levels of NF-κB were significantly increased in the model group compared with the normal group; however, AEE pretreatment significantly decreased the protein expression levels of NF-κB compared with AFL mice. These results suggested that AAEs may exert anti-oxidative and anti-inflammatory effects.