Background
The liver performs important functions such as lipid metabolism, protein synthesis, and circulatory detoxification [
1]. Acute or chronic hepatic injury can be caused by viral infections, non-alcoholic steatohepatitis, toxins, and alcoholism [
2,
3]. The pathological progression of hepatic cell damage can result in fibrosis, cirrhosis, and ultimately hepatocellular carcinoma [
4]. Hepatic fibrosis, a gradually progressing chronic liver disease, is a wound-healing response of the liver to repeated injury. Following acute injury, hepatocytes regenerate and replace necrotic or apoptotic cells, a process associated with inflammatory response. Hepatic fibrosis results from excessive accumulation of scar tissue following inflammation and liver cell death that occurs in most types of chronic liver disease.
Chronic exposure to carbon tetrachloride (CCl
4) is used to establish an experimental model of chronic liver disease, resulting in oxidative stress and hepatic injury. The mechanism of CCl
4-induced hepatotoxicity is well characterized [
5] and is mediated by radicals generated by CYP2E1, such as trichloromethyl and trichloromethyl peroxy radicals. Increased oxidative stress mediated by reactive oxygen species (ROS) following CCl
4 exposure may play an essential role in progression of liver damage [
6]. ROS induce tissue injury via lipid peroxidation and enhance hepatic fibrosis by increasing tissue levels of metalloproteinases inhibitors, resulting in increased collagen synthesis and accumulation [
7]. Antioxidant compounds, such as polyphenols, are effective in the treatment of chronic hepatic damage and fibrosis [
8,
9]. Polyphenols derived from plant extracts have demonstrated hepatoprotective properties [
10,
11].
Sasa veitchii, of the family Gramineaehas, has been used in Asia as a health-promoting food and folk medicine. In Japan, its leaves are used as a food wrapping material or sushi sheet to prevent food from rotting.
S. veitchii extract (SE) exhibits antioxidant, antitumor, antiulcer, anti-inflammatory, antimicrobial, antiviral, and anti-allergic activities [
12‐
17]. These effects are attributed to C-glycoside flavonoids and phenolic acids, which are abundant in these extracts [
18,
19]. In our previous study, we showed that SE reduced high-fat diet-induced obesity by modulating adipose differentiation and preventing hepatic steatosis in mice [
20,
21]. In addition, SE confers protection against CCl
4- and acetaminophen-induced acute hepatic injury in mice [
22,
23]. Since obesity and CCl
4- or acetaminophen-induced acute hepatotoxicity are commonly associated with inflammatory response, SE may have protective effects against other diseases with inflammatory components, such as chronic hepatitis.
To further characterize the therapeutic benefits of SE, we examined whether hepatitis induced by chronic exposure to CCl4 is prevented by treatment with SE.
Methods
Preparation of SE
Commercial, non-prescription SE was kindly provided by Sunchlon Co., Ltd. (Nagano, Japan). One milliliter of SE was made from 2.82 g of
S. veitchii leaves according to the company data [
23]. Chemical components of SE have been previously reported [
22].
Apparatus and chromatographic conditions
Three-dimensional high-performance liquid chromatography (HPLC) analysis was performed using a JASCO series (JASCO Corporation, Tokyo, Japan) system consisting of a JASCO PU-2089 Plus pump and a JASCO MD-2010 Plus photodiode array detector. Chromatographic separation was performed using a YMC-Pack ODS-AL S-5 column (5 μm, 250 mm × 4.6 mm i.d., YMC Co., Ltd., Tokyo, Japan) controlled at 40 °C (Shimadzu CTO-20 AC, Shimadzu Corporation, Kyoto, Japan). The mobile phases consisted of acetonitrile (A) and 50 mM sodium dihydrogen phosphate monohydrate (B). Gradient elution was performed at a flow rate of 0.8 mL/min with the following parameters: 0–60 min A:B (10:90, v/v) to A:B (90:10, v/v), 60–75 min A:B (90:10, v/v), and 75–80 min A:B (10:90, v/v). The injection volume was 10 μL (0.2 mg/mL). Scan data were collected from 220 to 600 nm.
Experimental protocol
Twenty-four male C57BL/6J mice weighing 19–21 g and aged 6 weeks were purchased from CLEA Japan, Inc. (Tokyo, Japan). Mice were housed in a temperature- and humidity-controlled environment (24 ± 1 °C and 55 ± 5%, respectively) with a standard 12 h light/dark cycle (8:00/20:00) and provided with food and water ad libitum. This experiment was approved by the Institutional Animal Care and Experimentation Committee of Kinjo Gakuin University. After acclimatization to laboratory conditions for 1 week, the mice (7-week old) were randomly divided into 4 groups (6 mice each): (1) control group, (2) SE-treated group, (3) CCl4-treated group, and (4) SE + CCl4-treated group. In the CCl4 and SE + CCl4 groups, mice were intraperitoneally (i.p.) injected with CCl4 (1 g/kg (5 ml/kg) at 22:00 twice per week) for 8 weeks. The control and SE groups received i.p. injections of olive oil as placebo. Mice in the SE and SE + CCl4 groups were orally administered SE daily (0.1 mL at 10:00 every day) for the duration of the treatment course. Saline vehicle was orally administered to the control and CCl4 groups.
Body weight was measured weekly throughout the study. Mice were euthanized 72 h following the final CCl4 injection and bled by cardiocentesis to obtain plasma samples, which were stored at − 80 °C until analysis. The liver and kidneys were weighed. In addition, separate liver samples were snap frozen in liquid nitrogen and stored at − 80 °C or fixed in 15% neutral buffered formalin (pH 7.4).
Plasma biochemical analysis
Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using a Transaminase CII kit (Wako Pure Chemical Industries, Osaka, Japan), as previously described [
24‐
26]. Plasma levels of tumor necrosis factor alpha (TNFα) were measured using a commercially available ELISA kit (eBioscience, San Diego, CA, USA), as previously described [
22]. For relative quantification, calibration curves were prepared using standard solutions.
Measurement of malondialdehyde and glutathione levels in the liver
Total malondialdehyde (MDA) levels in the liver were measured using a colorimetric microplate assay kit for thiobarbituric acid reactive substances (Oxford Biochemical Research, Oxford, MI, USA), as previously described [
24]. Glutathione (GSH) levels in the liver were examined using a GSSG/GSH quantification kit (Dojindo Laboratories, Kumamoto, Japan), as previously described [
22].
Histopathological analysis
A portion of the left lobe of the liver from each animal was perfused with 15% phosphate-buffered neutral formalin (pH 7.4), dehydrated, and embedded in paraffin. Embedded tissues were sliced into 4-μm thick sections and stained with hematoxylin and eosin (H&E), as previously described [
27,
28]. Liver fibrosis was quantified by using Masson’s trichrome (MT) staining according to the manufacturer’s protocol (ScyTek Laboratories, Inc., Logan, UT, USA). Necrosis area and aniline blue-positive area were calculated using ImageJ software (NIH). For immunohistochemistry staining, paraffin-embedded sections were deparaffinized and rehydrated in a graded ethanol series. Following antigen retrieval using proteinase K (Wako Pure chemical) and blocking of endogenous peroxidase using hydrogen peroxide (Wako Pure chemical), sections were incubated with rat anti-p65 monoclonal antibody (Santa Cruz, CA, USA) (1:160 dilution) as a primary antibody at 4 °C overnight (16 h). The sections were then incubated with a secondary antibody, anti-mouse IgG-FITC (MBL, Aichi, Japan) (1:160 dilution). In addition, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining.
Western blot analysis
Liver sections (80 mg) were homogenized in 720 μL ice-cold phosphate-buffered saline containing a phosphatase inhibitor and protease inhibitor (Nacalai Tesque, Kyoto, Japan) and centrifuged at 20,000 ×
g for 15 min at 4 °C. The supernatant from each sample was collected, and protein was extracted using a BCA protein kit (Nacalai Tesque). Protein samples (30 μg) were subjected to gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BioRad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride membrane using Trans-Blot Turbo Transfer System (BioRad). Next, the membrane was incubated with mouse anti-α-smooth muscle actin (anti-αSMA) monoclonal antibody (Santa Cruz), mouse anti-β-actin monoclonal antibody (MBL, Aichi, Japan), rabbit anti-c-Jun N-terminal kinase (JNK) polyclonal antibody, rabbit anti-phospho-JNK monoclonal antibody, rabbit-anti extracellular signal-regulated kinase (ERK) 1/2 monoclonal antibody, rabbit anti-phospho-ERK1/2 monoclonal antibody, rabbit-anti p38 monoclonal antibody, rabbit anti-phospho-p38 monoclonal antibody, peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, Beverly, MA, USA), and peroxidase-conjugated anti-mouse IgG (BioRad) using previously described conditions [
29]. Immuno-reactive bands were visualized with an ECL system (BioRad). Band intensity was measured using ImageJ software (NIH).
Statistical analysis
Multiple comparisons were performed using one-way analysis of variance (ANOVA) with Tukey’s test. All statistical analyses were performed using SPSS Statistics for Windows software (version 24.0; IBM Corp., Armonk, NY, USA). Differences were considered statistically significant at p values < 0.05.
Discussion
Hepatic fibrosis is a consequence of chronic liver damage associated with an increased morbidity rate and is considered a worldwide health risk [
2,
35]. Therefore, prevention of chronic liver fibrosis is important to improve our quality of life. CCl
4 is the most commonly used toxin for inducing hepatic fibrosis in experimental models owing to the similarity of CCl
4-induced hepatic fibrosis to hepatic fibrosis in humans. Repeated exposure to CCl
4 is known to enhance fibrogenesis in hepatic tissues of mice [
36]. CCl
4 is converted to trichloromethyl free radicals in the liver by CYP2E1, resulting in lipid peroxidation and liver injury [
5]. In the current study, we established a hepatic fibrosis model through repeated exposure to CCl
4 and evaluated the protective effects of SE against hepatic fibrosis.
Oxidative stress is a major cause of hepatic fibrosis progression in experimental models [
37]. Lipid peroxidation, a marker of oxidative damage, is associated with a wide range of diseases [
38]. Moreover, oxidative stress promotes collagen synthesis by activated hepatic stellate cells (HSC) [
39]. Increased levels of MDA, a product of lipid peroxidation, in hepatic tissues reflect oxidative stress in hepatic cells [
32]. Therefore, many antioxidants have been investigated as preventive and therapeutic agents against CCl
4-induced hepatic fibrosis [
40‐
43]. The present study showed that SE treatment decreased CCl
4-induced increases in ALT and AST levels, collagen deposition, and MDA production. These findings suggested that SE attenuated hepatic fibrosis through antioxidant activity, consistent with our previous study that reported the antioxidant property of SE [
23].
HSCs are major fibrotic precursor cells that transdifferentiate to the extracellular matrix, producing myofibroblasts [
34]. The present study showed that αSMA, a marker of the initiation phase of HSC activation, was elevated in the liver and was accompanied by bridging fibrosis. In addition, CCl
4-induced increases in αSMA were attenuated in the livers of mice treated with SE. These results suggested that SE inhibited HSC activation during the pathogenesis of CCl
4-induced liver fibrosis. Increasing evidence indicates that inflammatory cytokines, such as TNFα, are critical to HSC activation in the pathogenesis of liver fibrosis [
44]. Moreover, the relationship between NF-κB and inflammatory response is well characterized [
45,
46]. NF-κB is sequestered in the cytoplasm by binding to IκB. In response to stress or pro-inflammatory stimuli, NF-κB translocates to the nucleus through translational inhibition of IκB [
47]. Our present study showed that plasma levels of TNFα were significantly elevated in mice administered CCl
4. CCl
4-induced hepatic NF-κB nuclear translocation was inhibited in mice administered SE, indicating that the protective effect of SE was associated with decreased TNFα production and subsequent improvement of liver histopathology. These results suggested that SE suppressed CCl
4-induced HSC activation by inhibiting NF-κB activation and subsequent inflammatory response.
The MAPK family regulates cellular proliferation in response to cellular stresses and inflammation [
48,
49]. Three distinct MAPK pathways have been reported in mammalian cells: the c-Jun N-terminal kinase (JNK) pathway, extracellular signal-regulated kinase (ERK) pathway, and p38 MAPK pathway. These three pathways are associated with HSC activation in the pathogenesis of hepatic fibrosis [
50,
51]. Moreover, CCl
4 activates MAPK signaling [
35,
42]. The present study showed that SE treatment attenuated CCl
4-induced phosphorylation of hepatic JNK, ERK1/2, and p38. These results suggest that SE inhibited CCl
4-induced hepatic oxidative stress, inflammatory response, and HSC activation by attenuating the activation of MAPK signaling.
Most studies, including ours, utilized an extract obtained from
S. veitchii leaves, which are rich in bioactive compounds. The SE used in this study contained an abundance of SCC (250 μg/mL) [
23], which exerts anti-inflammatory and antioxidant effects [
52,
53]. Prior to this study, the active ingredient of SE had not been characterized. As such, we performed HPLC analysis to confirm the components of SE. A broad peak and a sharp peak were observed. The sharp peak corresponded to SCC contained in SE. Because the activities of SE are different from those of SCC, we speculated that the active ingredient of SE described in the present study is represented by the broad peak. Several antioxidant and anti-inflammatory compounds, such as polyphenols and flavonoids, have also been identified in
S. veitchii [
18,
54], indicating that these components may mediate the anti-fibrotic effects of SE. Since our present study was conducted using crude SE extracts, further investigation is needed to elucidate the active components of SE.