Drinking alcohol has always been deemed essential in many areas, such as in social gatherings, status functions, personal interactions, and conformance. However, long-term excessive alcohol intake can result in alcoholic liver disease (ALD). ALD is the leading cause of cirrhosis and liver-related death worldwide for decades and is responsible for 4% of global mortality [1
]. ALD encompasses a histological spectrum of liver injury that ranges from steatosis (fatty liver) to alcoholic steatohepatitis (ASH), and in severe cases, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma [4
]. Thus, the control of ALD at an early stage, for example, at a stage prior to the occurrence of ASH, could be of great significance in preventing development of ALD.
Potential mechanisms of acute alcohol-induced liver injury are associated with oxidative stress, steaotosis, endotoxin, dysregulated immunity, and inflammation. However, in recent decades, studies have focused on the inflammatory pathway in ALD, and increasing evidence demonstrates that ASH is caused by the lipopolysaccharide (LPS) binding to toll-like receptor (TLR)-induced nuclear factor-kappa B (NF-κB) activation pathway [6
]. TLRs are pattern-recognition receptors that enable the innate immune system to react immediately to infections by recognizing both bacterial and viral constituents. Among the TLRs, TLR4 can initiate activation of NF-κB and cascade response further causing the accumulation of pro-inflammation cytokines, ultimately resulting in the aggravation of inflammatory progress [9
Although various treatments, such as nutritional therapy, pharmacological therapy, psychotherapy, and surgery, are currently available for the spectrums of ALD, no satisfactory therapy is available except for abstinence [13
]. Medications that act as anti-inflammatories and anti-oxidants, are frequently used as therapeutic drugs in ALD. For example, there is silymarin, which alleviates liver injury mainly by reducing free radical activity and lipid peroxidation, protecting the liver cell membrane, and promoting hepatocytes regeneration, and bifendate, which alleviates liver injury mainly by reducing serum level of alanine aminotransferase (ALT), inflammatory cell infiltration and liver histological changes. However, clinical applications are limited because of side effects among other reasons. For example, silymarin has poor oral-bioavailability, and bifendate may cause liver hypertrophy [16
]. Thus, finding convincingly effective treatment drugs with fewer side effects without compromising therapeutic effect continues to be an important goal.
Artichoke (Cynara scolymus
L.), an edible herbal medicine of the family Compositae, is a perennial herb widely studied because of its possible antioxidative and hepatoprotective effects [19
]. The extracts and derivatives from artichoke contain a variety of dicaffeoylquinic acids and many kinds of flavonoid functional compounds, such as cynarin (1,5-dicaffeoylquinic acids), chlorogenic acid (3-caffe-oylquinic acid), luteolin glucoside, and apigenin glucoside [23
], which exhibit anti-microbial, anti-allergic, anti-inflammatory, and anticancer effects. One study indicated that artichoke extract had potential in reducing hypercholesterolemia through preventing lipid peroxidation and ameliorating hepatic antioxidant status [25
]. Another report demonstrated that artichoke aqueous leaf extract reduced serum total cholesterol (TC), triglycerides (TG), very low density lipoprotein, glucose levels, and plasma malondialdehyde (MDA) levels in streptozotocin-treated diabetic rats [26
]. Reports also demonstrated that artichoke showed marked anti-inflammatory effects on tissue plasminogen activator-induced inflammation and antitumor activity in an in vivo two-stage carcinogenesis test in mice [27
]. Besides, it was reported that artichoke extract was very safe to the human body as no obvious side effects were observed after continuous medication for several months [28
]. Therefore, artichoke has a broad application prospects in ALD treatment due to its anti-oxidant and anti-inflammatory effects.
The purpose of this study was to investigate the prophylactic protective effects of ethanolic extract from artichoke on acute alcohol-induced injury in an acute ALD mice model. To date, the indicators of aspartate aminotransferase (AST), ALT, TG, TC, MDA, glutathione (GSH), and superoxide dismutase (SOD) were assessed. The possible mechanism for acute alcohol-induced liver injury was discussed using the signals of TLR4 and NF-κB.
2. Materials and Methods
Artichokes in freeze-dried powder (8.89% caffeic acid derivatives, 0.98% chlorogenic acid, 0.56% cynarin) were supplied by Huimei Agricultural Science and Technology Co., Ltd. (Hunan, China), and the artichokes were diluted into 0.04 g/mL, 0.08 g/mL, and 0.16 g/mL suspensions with distilled water, respectively. Edible alcohol was obtained from Beijing Red Star Co., Ltd. (Beijing, China), and edible alcohol was diluted with distilled water to a concentration of 40% (w/v). Regular chow diet (40–43% corn, 26% bran, 29% bean cake, 1% salt, 1% bone meal, 1% lysine) for mice was purchased from Huayueyang Biotechnology CO., Ltd. (Beijing, China). Bifendate was provided by Beijing Union Pharmaceutical Factory (Beijing, China), and bifendate was diluted as a 0.036 g/mL suspension with distilled water. Diagnostic kits for AST, ALT, TG, TC, SOD, MDA, and GSH were received from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Antibodies for TLR4 and NF-κB p50 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and Novus Biologicals, Inc. (Littleton, CO, USA), respectively. All other chemicals used were of analytical reagent and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Experimental Animals
Seven-week-old male Institute of Cancer Research (ICR) mice (25 ± 2 g) were supplied by the Shanghai Laboratory Animal Center (Shanghai, China) and acclimated for one week prior to use. These mice were kept under environmentally controlled conditions (12-h normal light/dark cycles, 22 ± 2 °C and 50 ± 10% relative humidity) with chow diet and water ad libitum. All animal experiments were approved by the Animal Care and Use Committee of the Huaqiao University (Approval No. SCXK (HU) 2012-0002) and followed the National Institutes of Health Guidelines for animal care (Approval No. HQ-ECLA-20160517).
The ICR mice were randomly divided into 6 groups of 10 mice per group:
Control group: mice were gavaged with same volume of 0.9% saline twice per day (interval time, one hour).
EtOH group (model group): mice were gavaged with same volume of 0.9% saline and with 12 mL/kg body weight (BW) alcohol one hour after saline administration per day.
Positive control group (EtOH + bifendate): mice were gavaged with 0.36 g/kg BW of bifendate and with 12 mL/kg BW alcohol one hour after bifendate pretreatment each day.
Low-dose artichoke group (EtOH + artichoke 0.4): mice were gavaged with 0.4 g/kg BW of artichoke and with 12 mL/kg BW alcohol one hour after artichoke pretreatment each day.
Middle-dose artichoke group (EtOH + artichoke 0.8): mice were gavaged with 0.8 g/kg BW of artichoke and with 12 mL/kg BW alcohol one hour after artichoke pretreatment each day.
High-dose artichoke group (EtOH + artichoke 1.6): mice were gaveged with 1.6 g/kg BW of artichoke and with 12 mL/kg BW alcohol one hour after artichoke pretreatment each day.
All groups were fed for 10 consecutive days. Then, all groups were fasted for 12 h and subsequently anesthetized by pentobarbital solution (60 mg/kg BW) before the experiment.
2.3. Serum Biochemical Assays
Blood samples were collected from the retrobulbar vessels of the mice. The samples were centrifuged at 1500 rpm for 10 min at 4 °C (GTR16-2, Beijing Era Beili Centrifuge Co., Ltd, Beijing, China) to separate the serum after standing for 1 h at room temperature. Serum ALT, AST, TG, and TC activities were subsequently subjected to diagnostic kit testing (Nanjing Jiancheng Institute of Biotechnology) according to the instructions provided using spectrophotometer determination (UV2550, Shimadzu Crop., Kyoto, Japan). Briefly, for assessment of ALT and AST, the samples were mixed with substrates or buffer solution. After incubation at room temperature for 5 min, the absorbance at 505 nm was measured. The final data of ALT and AST were represented as U/L. For assessment of TG and TC, the samples were transferred into a 96-well plate containing substrates or buffer solution. After incubation at 37 °C for 10 min, the plate was incubated for an additional time after adding color developing agent and the absorbance at 510 nm was measured. The final data are represented as μmol/L.
2.4. Hepatic Antioxidant and Oxidative Stress Marker Assays
The livers were weighed accurately. A total of 0.5 g liver tissue was cut and washed with distilled water, then excess water was dried up. Then the liver tissue was cut into slices and homogenated with nine volumes of phosphate buffer (4.5 mL) in an ice bath (pH 7.2–7.4). The resulting suspension was centrifuged at 12,000 rpm for 10 min at 4 °C (GTR16-2, Beijing Era Beili Centrifuge Co., Ltd, Beijing, China), and the supernatant was measured by diagnostic kits of SOD, MDA, and GSH (Nanjing Jiancheng Institute of Biotechnology) according to the manufacturer’s instructions. In brief, the concentrations of SOD, MDA and GSH were assayed by hydroxylamine method, thiobarbituric acid-reactive method and microplate method, respectively.
2.5. Histological Examination of Liver Tissue
Liver tissues were fixed in 10% neutral formalin buffer for 24 h, and 5-μm sections were cut and stained with hematoxylin and eosin (H&E), and then observed using a Nikon DS-Fi2 fluorescent microscope (Nikon, Tokyo, Japan). The magnification was 200×. At least 10 areas of each tissue slice were observed. Representative images were presented. Analyses of pathological changes were based on proportion of inflammation, necrosis (0 point, 0 foci; 1 point, <2 foci; 2 points, 2–4 foci; 3 points, >4 foci, per 200× field) and steatosis (0 point, <5%; 1 point, 6–33%; 2 points, 34–66%; 3 points, >66%), which were assessed by three examiners independently [29
2.6. Immunohistochemical Analysis of TLR4 and NF-κB
Liver tissues were fixed in 10% neutral formalin buffer and embedded in paraffin. Five-millimeter-thick paraffin sections were cut and were then heated in unmasking solution at 95 °C for 15 min after deparaffinization. Nonspecific binding sites were blocked with goat serum. Sections were then incubated overnight at 4 °C in a humidified chamber with the following primary antibodies: rabbit anti-TLR4 (1:50) and rabbit anti-NF-κB p50 (1:250). The examiners, blinded to the experimental groups, counted the cells labeled with TLR4 and NF-κB p50 throughout five random lesion regions in the stained areas under a 200× light microscope. Then, the expression levels of TLR4 and NF-κB p50 were analyzed by mean integrated optical density (IOD).
2.7. Statistical Analysis
All quantifications for assays were repeated for three times and a mean value was used by taking mean of the triplicate plus/minus standard deviation (mean ± SD) for each group. Statistically significant differences (p < 0.05) were evaluated by one-way analysis of variance using SPSS 18.0 (SPSS Inc., Chicago, IL, USA).