1. Introduction
The liver is an important metabolic organ responsible, for the clearance of numerous toxins, drugs and pathogens, but it can also be injured by these harmful substances [
1]. Nowadays, acute hepatic failure has become one of the major diseases affecting human health around the world. It can be attributed to various factors such as hepatitis virus infection, and induction by drugs and toxins [
2]. Acetaminophen (APAP), a widely used analgesic and antipyretic drug, is safe and effective when taken at therapeutic doses [
3]. However, acetaminophen overdosing can cause hepatotoxicity and nephrotoxicity [
4]. Widespread use of APAP in hundreds of prescription and over-the-counter drugs has increased the prevalence of APAP hepatotoxicity [
5,
6].
Generally, APAP is metabolized primarily in the liver via glucuronidation and sulfation [
7]. APAP is also oxidized by cytochrome P450 enzymes to its chemically reactive metabolite which can cause liver and kidney damages [
8].
N-Acetyl-
p-benzoquinone imine (NAPQI) is known as the putative reactive metabolite of APAP. When APAP is at a high level, the accumulation of NAPQI, will result in increased utilization of GSH and depletion of GSH stores [
9]. Once intracellular GSH stores are depleted, excess NAPQI that is not conjugated with GSH may react with cellular proteins, including mitochondrial proteins [
10]. This situation leads to the formation of reactive oxygen and nitrogen species, and initiates lipid peroxidation that eventually results in liver cell destruction, necrosis, or apoptosis [
11,
12]. Although in clinical and experimental studies, many compounds have been investigated for their ability to protect against APAP-induced hepatotoxicity [
13,
14], the need for new treatment options remains high.
The roots of
Panax ginseng C. A Meyer (ginseng), one of the best known Traditional Chinese Medicines, have been reported to display adaptogenic effects in the endocrine, immune, cardiovascular, and central nervous systems [
15,
16]. Generally, the ginsenosides, which possess many pharmacological effects including anti-diabetes, anti-tumor, and anti-inflammation activity are considered the major active constituents in ginseng [
17]. Previous studies have demonstrated that the pharmacological and biological activities of steamed-processed ginseng (red ginseng and black ginseng) are greater than those of non-steamed (white ginseng) [
18]. The steaming process could lead to extensive conversion of the ginsenosides in non-steamed ginseng into new less polar degradation products such as ginsenoside Rg3, Rg5, Rk1, Rz1, F4, and Rg6 [
19,
20]. Black ginseng (BG), generated from ginseng through steaming and drying, is confirmed to exert many pharmacological effects, such as anti-cancer, anti-inflammatory, and anti-antioxidant actions [
21]. Though fermented ginseng (which also contains many less polar ginsenosides) was reported to protect against APAP-induced hepatotoxicity in a rat model [
22], the protective effect of BG on APAP-induced liver injury had not been reported so far. Therefore, it was of great interest to investigate the effect of BG on APAP-induced liver injuries.
On the basis of the above facts and taking into account that APAP overdose frequently results in life-threatening hepatotoxicity, we decide to explore the potential ameliorative effect of BG in a mouse model of APAP-induced liver hepatotoxicity. Importantly, to the best of our knowledge, the potential mechanisms underlying such hepatoprotective effects of BG are revealed in this work for the first time.
3. Discussion
Acetaminophen (APAP) is the most widely used over-the-counter analgesic and antipyretic medication in the world, with few side effects when taken in therapeutic doses. However, long-term or overdose usage of APAP can result in inflammation and necrosis of hepatocytes or even acute liver failure [
26]. Generally, APAP administration has been recognized as one of the most widely used in vivo animal models to induce liver injury to evaluate the hepatoprotective effects of natural medicines [
27].
Ginseng, the roots of
Panax ginseng, C.A. Meyer., is a Traditional Chinese Medicine widely used in China and Korea due to its good pharmacological activity. It has been demonstrated that steamed ginseng (e.g., red ginseng and black ginseng) exhibit more remarkable pharmacological activities and therapeutic efficacy than non-steamed ginseng (e.g., white ginseng) [
28]. The differences in their biological effects can be attributed to significant changes in the ginsenosides profile during steaming [
29]. Black ginseng (BG), produced from ginseng through steaming nine times and drying, respectively, shows various pharmacological effects, such as anti-cancer, anti-inflammatory, anti-stress and antioxidant properties [
21,
30]. Although it was reported that fermented ginseng containing more rare ginsenosides and could alleviate APAP-induced liver injury in a rat model [
22], the protective effect and the possible molecular mechanisms of action of BG on APAP-induced liver injury were still not clear. Therefore, the aim of the present study was to investigate the hepatoprotective effect and underlying mechanisms of BG on APAP-induced liver injury in mice.
Serum ALT and AST are the most sensitive biomarker enzymes used in the evaluation of acute liver injury. In the present study, we have observed significant increases in serum ALT and AST activities in APAP-induced mice compared with the normal group. The results in the present study showed that BG inhibited the serum levels of ALT and AST, indicating a significant protective effect of BG against APAP-induced liver damage, but this effect was not dose-dependent, which may be ascribed to the toxicity of the saponin ingredients found in high doses of BG that has been reported in former studies [
31]. Additionally, during the processing of BG, Maillard reactions can occur and produce some toxic byproducts [
32], which might account for the lower efficacy of BG at a high dose.
Oxidative stress, acts as an important pathogenic factor in APAP-induced hepatotoxicity, as reported in numerous animal models [
33]. APAP is metabolically activated by cytochrome P450 to generate the toxic metabolite NAPQI, which is immediately conjugated with GSH [
34]. Therefore, a high overdose of APAP may cause dramatic GSH depletion in the liver, affecting liver functions and leading to massive hepatocyte necrosis, liver failure or death [
35]. The MDA level is widely used as a marker of free radical mediated lipid peroxidation (LPO) injury [
36]. Previous reports confirmed that an elevated MDA level in liver tissues indicated that enhanced LPO can lead to tissue damage and the breakdown of the natural antioxidant defense mechanisms [
37]. In addition, there is also evidence of APAP-induced liver injury via an oxidative stress mechanism caused by increased MDA and reduced GSH levels [
38]. Interestingly, the results clearly exhibited that the MDA level increased significantly and the GSH level decreased significantly after APAP challenge and pre-treatment with two doses of BG effectively alleviated these alterations and reversed the oxidative stress levels. Therefore, the increase of GSH, and decreased level of MDA in liver tissues treated with BG suggested a host-detoxification process of BG in APAP-induced liver injury. Drug-metabolizing enzyme CYP2E1, serving as a site for the generation of reactive oxygen species (ROS), plays an important role in APAP-induced hepatotoxicity [
39]. In the present investigation, overexpression of CYP2E1 in liver tissues treated with APAP could be reversed after treatment with BG. Consistent with the MDA result, although 4-HNE immunofluorescence staining exhibited high fluorescence intensity in liver tissues after APAP exposure, this process was significantly blocked by pretreatment with BG.
Nitration of tyrosine (i.e., formation of NT) has been shown to be an excellent biomarker of peroxynitrite formation, and it is generally accepted that NT occurs in the centrilobular cells of the liver in overdose APAP-challenge mice. Peroxynitrite formation is formed by a rapid reaction between nitricoxide and superoxide, and peroxynitrile production was increased under APAP-toxicity conditions [
40]. Peroxynitrite initiates necrotic cell death in APAP hepatotoxicity associated with DNA damage through mitochondrial dysfunction [
41]. Our study clearly showed overexpression of 3-NT in liver tissues of APAP-treated mice, which was in line with the above findings. However, BG pretreatment at two doses reversed the APAP-mediated increase in 3-NT expression.
Generally, apoptosis is characterized by a fundamental cellular activity to maintain the physiological balance of the organism [
42]. Accumulating evidence has indicated that APAP-induced acute liver injury was associated with cell apoptosis. Apoptosis is an especially important form of cell death, and more and more reports have demonstrated apoptosis of hepatocytes after APAP exposure [
43]. Two important members of the Bcl-2 family involved in apoptosis are the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 [
44]. The findings of a western blotting analysis of liver tissues obviously indicated that the protein expression of Bax and Bax/Bcl-2 ratio were significantly suppressed while the protein expression of Bcl-2 was relatively increased, clearly indicating that BG exhibits anti-apoptotic properties in the context of APAP hepatotoxicity. In addition, Hoechst 33258 staining was employed to observe the apoptotic liver cells in the APAP-induced liver injury model. The results clearly showed a large area and high density of blue apoptosis staining, indicating the apoptosis of liver cells. However, the apoptosis in the BG groups was obviously alleviated and the liver damage was effectively improved, which confirmed that BG treatment could protect against APAP-induced liver cell apoptosis.
Recent evidence has indicated that inflammation plays an important role in the pathogenesis of APAP-induced liver injury. Therefore, overproduction of inflammatory cytokines could be considered a precursor to liver diseases, and the down-regulation of these inflammatory molecules is considered a beneficial effect [
14]. COX-2 and iNOS are enzymes involved in the development of inflammatory process development [
45]. In addition, the induction of COX-2, an inducible form of COX, can occur during tissue damage or inflammation in response to cytokines. It can be speculated that COX-2 plays a pivotal role in APAP-induced acute liver injury. Our immunohistochemical results revealed that the expression of iNOS and COX-2 was significantly increased in the APAP-injured mice. However, treatment with BG could effectively inhibit the overexpression of iNOS and COX-2 in liver tissues.
4. Materials and Methods
4.1. Plant Materials
The black ginseng (BG) was generated from the roots of P. ginseng and identified by Professor Wei Li (College of Chinese Medicinal Material, Jilin Agricultural University, Changchun, China). After cutting the black ginseng into slices, the cut pieces were ground to obtain a relatively homogenous drug powder and then sieved through a 40-mesh screen. The powder was dried at 60 °C until its weight remained constant and was well blended before use.
4.2. Chemicals and Reagents
Acetaminophen (>99.0%, batch No. A105808-25 g) was purchased from Aladdin Industrial Corporation (Shanghai, China). The kits for detection of alanine aminotransferase (ALT), aspartate transaminase (AST), glutathione (GSH), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Rabbit monoclonal anti-mouse iNOS, COX-2, Bax, Bcl-2, cytochrome P450 E1 (CPY2E1), 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) were purchased from Cell Signaling Technology (Danvers, MA, USA). Hoechst 33258 staining kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). DyLight 488-SABC and SABC-Cy3 immunofluorescence Staining kit was purchased from BOSTER Biological Technology (Wuhan, China). All other reagents and chemicals were of analytical grade and purchased from Beijing Chemical Factory (Beijing, China).
4.3. Identification and Analysis of Ginsenosides in Black Ginseng
Sample extraction was performed as previously described with some modifications [
46]. About 1.0 g of powdered BG was extracted two times with 100% methanol by ultrasonic-assisted extraction for 30 min. The combined extracts were concentrated with an evaporator, then diluted with methanol to 5.0 mL in a volumetric flask, the supernatant was then filtered through a 0.45 μm nylon membrane and injected into the HPLC for analysis.
Samples were analyzed on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV detector. Liquid chromatographic separations were achieved using a Hypersil ODS2 column (4.6 cm× 25 cm, 5 μm). The column temperature was set at 30 °C and the detection wavelength was set at 203 nm. The mobile phase consisted of a mixture of acetonitrile (A) and water (B) with a flow rate of 1.0 mL/min. The gradient elution was programmed as follows: 0–20 min, 18.5–20.5% A; 20–30 min, 20.5–25.5% A; 30–40 min, 25.5–35% A; 40–60 min, 35–45% A; 60–70 min, 45–60% A; 70–80 min, 60–70% A; 80–90 min, 70–80% A. The 20 μL sample solutions were directly injected into the chromatographic column manually.
4.4. Animals
Thirty-two male ICR mice, weighing 22–25 g, were provided by Experimental Animal Holding of Yisi Experimental Animals with Certificate of Quality No. of SCXK (JI) 2016-0003 (Changchun, China). The animals were housed in plastic cages under standard laboratory conditions (12 h light/dark cycle, relative humidity 60% ± 5%, and 25 ± 2 °C) and adaptability raise at least one week before commencement of the experiment. All animals handing procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). All experimental procedures were approved by the Ethical Committee for Laboratory Animals of Jilin Agricultural University (Permit Number: 16-008)
4.5. Experimental Groups and Treatment
The animals were randomly divided into four groups (eight animals per group) as follows: normal, APAP (250 mg/kg), APAP + BG (300 mg/kg) and APAP + BG (600 mg/kg). The treatment groups were administered BG by gastric intubation for seven consecutive days at doses of 300 mg/kg and 600 mg/kg body weight per day, and the normal and APAP groups were treated with 0.9% saline in the same way. After final administration, animals in APAP and BG-treatment groups received a single intraperitoneal (i.p.) injection of APAP (250 mg/kg) to induce acute liver injury in mice. All the mice were fasted for at least 12 h before intraperitoneal injected with APAP and dissected, they were allowed free access to water. Then, all the mice were killed by means of cervical vertebra dislocation, and their blood was collected. The serum was separated by centrifugation (3500 rpm, 10 min, and 4 °C) and stored at −20 °C for the determination of measuring aminotransferases. All the mice were sacrificed and the segregated livers were washed twice with saline, blotted dry on a filter paper, weighed. At the same time, shapes, colors and sizes of the livers were observed. Livers were dissected quickly, a small piece of tissue was cut off from the same part of the left lobe of the liver in each mouse and fixed in 10% buffered formalin solution (m/v) for histopathological analysis. The remaining liver tissues were stored at −80 °C for hepatic homogenate preparation.
4.6. Assay for Hepatic Function Biochemical Markers
To assess hepatic function, serum was used for the spectrophotometric determination of ALT and AST using commercially available detection kits according to the manufacturers’ instructions. In brief, the samples were transferred into a new 96-well plate containing substrates or buffer solution. After incubation at 37 °C, 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 U/L.
4.7. Assay for GSH and MDA in Liver
The liver tissues were homogenated in 50 mM phosphate buffer (pH 7.4). The resulting suspension was then centrifuged at 3500 r for 15 min at 4 °C, and the supernatant was used for the detection of GSH and MDA. In brief, the levels of GSH and MDA in liver homogenates were measured by commercial kits according to the manufacturer’s instructions [
47]. The amount of protein was measured using the Bradford assay [
48].
4.8. Histopathological Examination
For histopathological analysis, the liver tissues (n = 8 per group) were fixed in 10% buffered formaldehyde for over 24 h, subsequently processed by routine paraffin embedding and sectioned for 5 μm thickness. After hematoxylin-eosin (H&E) staining, slides were observed for histopathological changes using a light microscope (Leica, Wetzlar, Germany). The degree of hepatocellular necrosis was analyzed by Ridit analysis [
49].
4.9. Hoechst 33258 Staining
Hoechst 33258 staining was performed as previously described with some modifications [
50]. Briefly, at the end of the experiments, the liver tissues were dissected out and fixed in 10% neutral buffered formalin solution. We randomly chose three from each group. Then, these samples were cut into 5 μm sections and stained by Hoechst 33258 (10 μg/mL). After being washed with PBS for three times, stained nuclei were visualized under UV excitation and photographed under a fluorescence microscope (Leica TCS SP8). Image-Pro plus 6.0 was used to quantify the Hoechst 33258 staining.
4.10. Immunohistochemistry (IHC) and Immunofluorescence Analysis
Immunohistochemical staining was performed as previously described [
51]. Briefly, the 5 μm thick paraffin sections were deparaffinized and rehydrated with a series of xylene and aqueous alcohol solutions, respectively. After antigen retrieval in citrate buffer solution (0.01 M, pH 6.0) for 20 min, the slides were washed with TBS (0.01 M, pH 7.4) for three times and incubated with 1% bovine serum albumin for 1 h. The blocking serum was tapped off, and the sections were incubated in a humidified chamber at 4 °C overnight with primary antibodies including mouse polyclonal anti-iNOS (1:200) and anti-COX-2 (1:200), followed by secondary antibody for 30 min. Substrate was added to the sections for 30 min followed by DAB staining and hematoxylin counter-staining. The positive staining was determined mainly by a brownish-yellow color in the cytoplasm or nucleus of the cells. An image was taken by light microscopy (Olympus BX-60, Tokyo, Japan).
To assess the expression of CYP2E1, 4-HNE, and 3-NT in APAP-induced hepatotoxicity, immunofluorescence staining was exerted in liver tissues sections of APAP-induced groups and normal group as described for IHC analysis. In brief, the liver sections were taken microwave antigen repaired after deparaffinized and rehydrated, added serum sealing fluid for 20 min when sections reach room temperature, dilute primary mouse polyclonal anti- CYP2E1 (1:200), anti-4-HNE (1:100) and anti-3-NT (1:200) were added and keep 4 °C overnight. The sections washed with TBS (0.01 M, pH 7.4) three times, 2 min each and subsequently marked with fluorescence secondary antibody for 30 min at room temperature, then exposed by dilute DyLight 488-SABC (1:400) or CY3 (1:100) before 30 min incubation at 37 °C. The sections were washed with TBS (0.01 M, pH 7.4) for four times, 5 min every time. Nuclear staining was performed using 4, 6 diamidino-2-phenylindole (DAPI). Immunofluorescence staining was visualized using a Leica microscope (Leica TCS SP8), and immunofluorescence intensity were analyzed by Image-Pro Plus 6.0 software.
4.11. Western Blotting Analysis
Western blot analysis was performed as described previously [
52]. Briefly, the liver tissues were lysed in RIPA buffer and protein concentrations were measured by BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein (50 µg/lane) were analyzed by 12% SDS-PAGE and transferred onto PVDF membrane, then blocked with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20. After incubating with primary antibodies against Bax (1:2000) and Bcl-2 (1:2000) at 4 °C overnight, the membrane was then incubated with the secondary antibodies for 1 h at room temperature. Signals were detected using ECL substrate (Pierce Chemical Co., Rockford, IL, USA). Expression in the protein levels were measured by western blotting using Bio-Lad Laboratories-Segrate lately (Bio-Rad Laboratories, Hercules, CA, USA). The bands intensities were quantified by densitometry.
4.12. Statistical Analysis
All the results were expressed as means ± standard deviation (S.D). The data were analyzed using two-tailed test or one-way analysis of variance (ANOVA). GraphPad Prism 6.0 (ISI®, Philadelphia, PA, USA) was employed to make resulting data chart. The Nonparametric Test (Ridit analysis) was used for the histological examination comparison. p < 0.05 or p < 0.01 was considered to be significant.
