Background
Bamboo leaves have been extensively used in folk medicine as an antifebrile and antihypertensive medication for centuries [
1,
2]. In addition, in Japan, due to their antimicrobial activity, bamboo leaves have been used to wrap sushi sheets to protect against bacterial spoilage. Recently, extracts of bamboo leaves were reported to have multiple biological activities including antioxidant activity and cancer prevention [
3,
4]. Furthermore, previous studies have demonstrated the antitumor [
5], antioxidant [
1,
6], antiviral [
7], anti-inflammatory [
8], and anti-allergic [
9] activities of the extract.
The liver is most vulnerable to attack by chemical toxic agents since it is one of the most internal organs with multiple functions such as detoxification and protein synthesis [
10,
11]. Therefore, liver diseases are among the most serious health problems worldwide. Our previous investigation found that
Sasa veitchii leaf extract (SE) prevented carbon tetrachloride (CCl
4)-induced hepatotoxicity in mice [
12], suggesting that SE might maintain liver homeostasis. Although CCl
4 is commonly used in animal models to study chemical toxin-induced liver injury [
13,
14], exposure to CCl
4 does not reflect a real-life scenario. The most common etiologies of liver damage in real life are acute viral hepatitis A and B, drug-related liver injury, and the alcohol-acetaminophen syndrome (AAS). In terms of drug-related liver injury, acetaminophen (APAP) is well known. In addition, the main cause of acute liver failure in industrialized countries is APAP overdose. APAP is recognized as a popular analgesic and antipyretic drug at therapeutic doses. However, APAP can cause severe liver injury in animals and humans through its side effects [
15]. The main mechanism of APAP-induced hepatotoxicity has been studied in detail [
16‐
19] and occurs in multiple steps. First, APAP is majority metabolized by Cyp2e1 and minority by Cyp1a2. Cyp2e1 is the main Cyp enzyme that bio-activates APAP at low doses [
16,
20]. In contrast, at a high dose, Cyp1a2 was shown to contribute to the bio-activation and toxicity of APAP [
21]. Cyp-produced
N-acetyl-p-benzoquinone imine (NAPQI) depletes glutathione (GSH) and covalently binds to proteins. Loss of GSH then permits increased formation of reactive oxygen and nitrogen species. As a result, oxidative stress is increased, leading to alterations in calcium homeostasis and initiation of signal transduction responses causing mitochondrial permeability transition. Mitochondrial permeability transition causes additional oxidative stress and inhibits ATP synthesis. Insufficient ATP triggers cell necrosis. Peripheral to these essential events, a number of inflammatory mediators such as cytokines and chemokines are induced that can modify toxicity.
Currently, the best antidote against APAP-induced hepatic injury is
N-acetylcysteine (NAC). NAC effectively minimizes APAP-induced toxicity when injected within a short time after APAP overdose [
22]. However, clinical studies have revealed that injection of NAC often causes undesirable side effects [
23]. Therefore, a need exists for compounds or chemicals that offer maximum protection against APAP-induced hepatotoxicity without NAC’s side effects. The search for new drugs and novel therapeutic intervention strategies increasingly includes testing plant extracts and other natural products. It is generally accepted that natural products have multiple activities including anticancer and antioxidant [
24,
25]. Owing to the critical and urgent need to explore the therapeutic potential of natural products and other compounds for prevention and treatment of hepatotoxicity, we evaluated the protective effects of SE against APAP-induced hepatic injury.
Methods
Animal experimentation
Six-week-old male ddY mice were purchased from Japan SLC (Shizuoka, Japan). They were housed under standard conditions of controlled temperature (24 ± 1 °C), humidity (55 ± 5%), and light (12-h/12-h light/dark cycle) and provided food and water ad libitum. All experiments were approved by the Institutional Animal Care and Experimentation Committee of Kinjo Gakuin University (No. 129).
Preparation of SE
SE was obtained from Sunchlon Co., Ltd. (Nagano, Japan) and was prepared using the method described previously [
9,
12]. One milliliter of SE was obtained from 2.82 g of
S. veitchii leaves according to the company’s data. The diluted SE solution contained minerals, carbohydrates, fat, protein, and silicic acid, as shown in Table
1.
Component | mg/mL |
Cu-chlorophyll | 250.00 |
Carbohydrate | 8.17 |
Fat | 3.58 |
Protein | 3.08 |
Ash content | 0.83 |
Silicic acid | 0.42 |
Water | 940.80 |
Minerals | μg/mL |
Na | 2250.00 |
Fe | 5.67 |
Zn | 1.83 |
Cu | 733.33 |
Mn | 0.18 |
Ca | 11.67 |
Mg | 15.00 |
K | 86.67 |
P | 18.33 |
Treatment protocol
Mice were pretreated with 0.2 mL of SE or saline as a control once a day for a week by oral gavage. Mice from each group were fasted for 16 h, and 550 mg/kg APAP dissolved in saline/polyethylene glycol emulsion or vehicle only (as a control) was i.p. injected at 10 mL/kg body weight. The mice were euthanized and bled to obtain plasma 2, 6, 24, or 72 h following the injection. The resulting plasma samples were stored at −80 °C. The livers were harvested and stored at −80 °C or fixed in 15% phosphate-buffered neutral formalin (pH 7.4).
Plasma biochemical analysis
Plasma activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the Transaminase CII kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s instructions, as previously described [
26]. Plasma levels of tumor necrosis factor (TNF)-α (eBioscience, San Diego, CA, USA) were determined using commercially available ELISA kits, according to the manufacturer’s instructions. For relative quantification, calibration curves were prepared using standard solutions.
Histopathological findings
For histological analysis, a portion of the left liver lobe from each animal was perfused with 15% phosphate-buffered neutral formalin (pH 7.4), dehydrated, and embedded in paraffin. Embedded tissues were sectioned at 4 μm.
For immunohistochemistry, paraffin-embedded sections were deparaffinized and rehydrated in a grated ethanol series. After antigen retrieval by proteinase K (Wako Pure chemical) and quenching of endogenous peroxidase by hydrogen peroxide (Wako Pure chemical), sections were incubated with rat anti-TNFα monoclonal antibody (Biolegend, San Diego, CA, USA) as primary antibodies (1:250 dilution) at 4 °C for overnight. The color reaction was developed with peroxidase-conjugated anti-rat IgG (Sigma-Aldrich, St. Louis, MO, USA) as secondary antibody (1:750 dilution). In addition, sections were counterstained with hematoxylin.
For the detection of nuclear DNA strand breaks, paraffin-embedded sections were stained with the terminal dUTP nick-end labeling (TUNEL) technique using an in situ apoptosis detection kit (Takara Bio, Shiga, Japan) according to the manufacture’s protocols. Sections were counterstained with hematoxylin.
For hematoxylin and eosin (H&E) stain, we previously described [
26].
Measurement of malondialdehyde and GSH levels in the liver
Total malondialdehyde (MDA) levels and total antioxidant capacity in the liver were examined using a colorimetric thiobarbituric acid reactive substances microplate assay kit and a colorimetric total antioxidant power assay kit, respectively (Oxford Biochemical Research, Oxford, MI, USA), according to the manufacturer’s protocols, as previously described [
27]. Hepatic GSH levels were measured using GSSG/GSH quantification kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions and as previously described [
28].
Western blot analysis
0.1-g liver sections were homogenized with 900 μL ice-cold phosphate-buffered saline containing protease inhibitor (Nacalai Tesque, Kyoto, Japan) and centrifuged at 18,000g for 20 min at 4 °C. The resulting supernatant for each sample was collected and protein level determined using the BCA protein kit (Nacalai Tesque). Protein samples (40 μg) were subjected to sodium dodecyl sulfate–polyacrylamide electrophoresis on a 10% gel and transferred to a polyvinylidene difluoride membrane. Rabbit anti-c-jun N-terminal kinase (JNK) polyclonal antibody, rabbit anti-phospho-JNK monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-Cyp2e1 polyclonal antibody (Enzo Life Sciences, NY, USA) were used as primary antibodies (1:1000 dilution) for immunoblotting. A peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) was used as secondary antibody (1:3000 dilution). Mouse anti-receptor-interacting protein (RIP)-1 monoclonal antibody (Santacruz, California, CA, USA) and mouse anti-β-actin monoclonal antibody (MBL, Aichi, Japan) were used as primary antibody (1:1000 dilution) for immunoblotting. A peroxidase-conjugated anti-mouse IgG (MBL) was used as secondary antibody (1:5000 dilution). The immunoreactive bands were visualized with the ECL system (BioRad, Hercules, CA, USA).
Isolation of total RNA and qRT-PCR assay
Total RNA was extracted from 0.1-g liver sections using the ISOGEN II kit (Nippon Gene, Tokyo, Japan). qRT-PCR was performed with One-Step SYBR PrimeScript PLUS RT-PCR kit (Perfect Real Time) (Takara Bio) using an Applied Biosystems 7300 system (Applied Biosystems, Foster City, CA, USA). PCR conditions were as previously described [
28]. Primer pairs are shown in Table
2. Relative expression of each messenger RNA (mRNA) was determined using the standard curve method. The amount of each target mRNA quantified was normalized against that of GAPDH-encoding mRNA.
Table 2
Oligonucleotide primer sequences and PCR conditions for real-time RT-PCR
Cyp1a2 | Forward | GAC ACC TCA CTG AAT GGC TTC | 103 |
(NM_009993) | Reverse | ACA CAA AGG GGT CTT TCC ACT G |
Cyp2e1 | Forward | CAT TCC TGT GTT CCA GGA GTA CAA G | 91 |
(NM_021282) | Reverse | GAT ACT TAG GGA AAA CCT CCG CAC |
GAPDH | Forward | TGG TGA AGG TCG GTG TGA AC | 98 |
(NM_008084) | Reverse | GTC GTT GAT GGC AAC AAT CTC C |
Statistical analysis
Statistical significance of the differences between two groups was estimated using a two-tailed Student t test. Multiple comparisons were made by one-way analysis of variance with post hoc Tukey–Kramer’s test. All statistical analyses were performed using the SPSS 19.0 software (Chicago, IL, USA). Values of p < 0.05 were considered statistically significant.
Discussion
This study demonstrated that pretreatment with SE can prevent APAP overdose-induced acute toxicity, as assessed by evaluating blood function markers, oxidative stress (MDA and GSH level), inflammatory responses, JNK activation, and RIP1 activation in the liver. In addition, SE also prevented APAP-induced necrotic and apoptotic cell death, respectively. Although apoptosis occurred by APAP treatment, ratio of TUNEL-positive cells was lower compared with necrosis area. Moreover, TUNEL-positive nucleus is observed within the area of necrosis. That suggests TUNEL-positive cells in APAP-induced cell death reflect necrosis-mediated DNA fragmentation not apoptotic cell death because caspase-3, that is well known to apoptosis marker, was not elevated by APAP treatment [
34,
35].
In the present study, although we cannot show whether TUNEL-positive cells were necrosis-mediated DNA fragmentation or apoptotic cell death, these are consistent with past literature that APAP-induced liver injury results from mainly necrotic rather than apoptotic cell death [
34,
36].
Since APAP-induced hepatotoxicity occurs in multiple steps, SE may have protective effects against several steps. Initially, we hypothesized that SE acts as an antioxidant. Oxidative stress is a mechanism that has been postulated to be important in the development of APAP-induced hepatotoxicity [
16,
37,
38]. Numerous studies have reported that antioxidants can prevent hepatic damage by counteracting free radicals and preventing lipid peroxidation [
39,
40]. We found that pretreatment of APAP-treated mice with SE reduced levels of hepatic MDA, a naturally occurring product of lipid peroxidation. Several literature reports have hypothesized that SE exhibits antioxidant activity because many new flavonoids and polyphenols have been discovered in
S. veitchii [
41,
42]. Our hypothesis is supported by the well-known antioxidant properties of such compounds [
43,
44], as well as our observation of SE-induced increase in hepatic antioxidant capacity. Chlorophyll might also be a candidate active molecule since it has demonstrated antioxidant activity and is present in abundance (250 mg/mL). Further, Serpeloni et al. [
45] have demonstrated that 13-day oral treatment with chlorophyll (0.5 mg/kg) suppressed cisplatin-induced oxidative stress in mice. Although the period of SE administration was shorter in our study, the chlorophyll content in our SE was much greater than 0.5 mg per day. Therefore, the chlorophyll in SE could have contributed to the suppression of APAP-induced oxidative stress in our study.
In clinical practice, NAC has been used to treat patients with APAP overdose since it has the potential to promote hepatic GSH synthesis [
46] and APAP is well known to deplete GSH [
47]. Moreover, GSH injection is effective in mice. However, GSH upregulation is not observed upon pretreatment with SE. Taken together, our data indicate that SE may protect against APAP toxicity by ameliorating the impairment of antioxidant systems except for GSH.
Another possible explanation for our results is that the protective effect of SE against APAP-induced toxicity may possibly reflect inhibition of some CYPs, which would in turn prevent the formation of toxic NAPQI. Cyp2e1 gene knockout mouse or Cyp2e1 inhibition by an inhibitor such as disulfiram or natural product such as resveratrol and schisandra attenuated APAP-induced hepatotoxicity in mice [
48‐
50]. In addition, Cyp1a2 knockout mice showed reduced APAP-induced toxicity [
51,
52]. We used qRT-PCR to investigate the effect of SE on
Cyp2e1 and
Cyp1a2 mRNA expression and showed that SE significantly decreased
Cyp2e1 gene expression. Furthermore, the same tendency was also observed in Cyp2e1 protein levels. These might provide an additional explanation of the protective mechanism of SE against APAP toxicity. In addition, we observed hepatic Cyp2e1 level 24 h after APAP treatment. Although APAP group and SE + APAP groups indicated a tendency of Cyp2e1 decrease, these two groups in Cyp2e1 level were comparable. Several literatures reported Cyp2e1 was degraded after administration of toxic doses of APAP, possibly through interaction between Cyp2e1 and NAPQI or other metabolites produced from Cyps-mediated metabolism of APAP. Our data was consistent with these reports [
53,
54]. On the other hand, in SE + APAP group, we cannot prove Cyp2e1 decrease was whether depending on APAP-induce degradation or inhibition potential by SE. Further investigation is needed to elucidate this question in the future research.
An additional explanation is that SE protects against APAP-induced hepatotoxicity via its anti-inflammatory potential. It is well known that drug toxicity such as that due to APAP causes hepatocyte necrosis, which results in neutrophil and monocyte infiltration. It is reported that Kupffer cells activate the expression of pro-inflammatory cytokines such as TNFα and IL-6 [
55,
56]. Our study also showed that APAP administration significantly induced TNFα, which was inhibited by SE pretreatment. Nuclear factor (NF)-κB is an important transcription factor for inducible inflammatory cytokine expression [
57,
58]. In vitro assays from other groups have shown that SE inhibited NF-κB activity [
8], suggesting that our explanation is plausible. However, in the current study, we cannot elucidate whether repression of inflammatory response is due to direct effect or upper event such as Cyp inhibition and ROS decrease since inflammatory response is latter event in APAP-induced hepatotoxicity. Hence, further investigation might be needed to prove whether SE’s anti-inflammatory potential contributes to protection against APAP-induced hepatotoxicity or not.
Acknowledgements
The authors thank Dr. Nobuhiko Miura (Japan National Institute of Occupational Safety and Health, Japan) for his kind suggestions. We are grateful to Sunchlon Co., Ltd., for providing the SE.