Article pubs.acs.org/JAFC
Protective Effects of Glycyrrhizic Acid and 18β-Glycyrrhetinic Acid against Cisplatin-Induced Nephrotoxicity in BALB/c Mice Chi-Hao Wu,† An-Zhi Chen,‡ and Gow-Chin Yen*,‡ †
School of Nutrition and Health Sciences, Taipei Medical University, 250 Wuxing Street, Taipei 110, Taiwan Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan
‡
S Supporting Information *
ABSTRACT: The clinical use of antineoplastic drug cisplatin (CP) is commonly complicated by nephrotoxic side effects that limit its application and therapeutic efficiency. This study used a model of CP-induced renal injury in male BALB/c mice to investigate the protective effects of the active components of licorice, glycyrrhizic acid (GA), and 18β-glycyrrhetinic acid (18βGA) against CP-induced nephrotoxicity, and the chemoprotectant, amifostine, was used as a control. Oral administration of GA or 18βGA significantly reduced CP-induced increases in the levels of blood urea nitrogen, creatinine, and lactate dehydrogenase. Hematoxylin and eosin staining revealed that GA and 18βGA delayed the progression of renal injury, including tubular necrosis, hyaline casts, and tubular degeneration in response to CP exposure. Oxidative status and inflammatory responses in CP-treated mice were restored to near-normal levels by treatment with GA or 18βGA. These protective effects might be associated with upregulation of nuclear factor E2-related protein (Nrf2) and downregulation of nuclear factor-κ-lightchain-enhancer of activated B cells (NF-κB) in the kidney. Notably, we demonstrated that GA and 18βGA rendered renal cells resistant to CP-induced HMGB1 cytoplasmic translocation and release. These findings suggest that GA and 18βGA might be act as the chemoprotectants against CP-induced nephrotoxicity. KEYWORDS: cisplatin, anti-inflammation, glycyrrhizic acid, 18β-glycyrrhetinic acid, nephrotoxicity
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18β-glycyrrhetinic acid (18βGA) by β-D-glucuronidase, which is produced by intestinal bacteria. Intravenously injected GA is first metabolized by β-D-glucuronidase in hepatic lysosomes into 3monoglucuronide glycyrrhetinic acid; after secretion into the intestines along with bile, it is further metabolized into 18βGA by intestinal bacteria and reabsorbed into the circulatory system.9 GA and 18βGA are known to be the major active components of licorice, and recent studies suggested that they have multiple health benefits.10 GA can improve ischemia/reperfusion-induced acute renal injury in rats,11 inhibit CCl4-induced hepatic fibrosis in rats,12 inhibit lipopolysaccharide-induced pulmonary injury in mice,13 and relieve chemodrug-induced genotoxicity,14 whereas administration of GA and 18βGA can prevent stress ulcers in mice.15 In a streptozotocin-induced diabetes mellitus rat model, oral administration of 18βGA [100 mg/kg of body weight (BW)] had a hypoglycemic effect similar to that of glibenclamide.16 Our previous study indicated that GA and 18βGA have excellent antioxidant and anti-inflammatory characteristics that can promote antioxidant enzyme activities in neuronal cells and inhibit reactive oxygen species (ROS)-induced oxidative damage and apoptosis.17 GA and 18βGA can also inhibit nuclear factor κB (NF-κB) and reduce proinflammatory cytokine secretion through regulating the PI3K pathway, thus achieving an antiinflammatory function.18 Other phytochemicals, such as quercetin and hesperidin, can relieve CP toxicity and exhibit renal protective functions by clearing ROS, maintaining
INTRODUCTION
Cisplatin (CP) is a platinum-containing anticancer drug that directly intercalates into DNA double strands to cause crosslinking of intra- or interstrands, twisting of the double helix, interference with nucleic acid replication and DNA synthesis, and eventual occurrence of apoptosis.1 CP is usually used in clinical settings to control numerous types of solid tumors, including tumors involved in head and neck cancers, esophageal cancer, genital cancer, and non-small cell lung cancer. The treatment effect of CP on testicular cancer has the greatest success rate at 90%. Therefore, it is an indispensable drug for chemotherapy.2,3 However, nephrotoxicity is a major side effect of CP, and the clinical characteristics of renal function abnormalities, such as an increase of serum blood urea nitrogen (BUN) and creatinine and reduction of creatinine clearance (Ccr), usually occur after 2 weeks of drug administration.4 Amifostine (AMF) is a thiophosphate preparation currently used to relieve the side effects of chemotherapy and radiotherapy. AMF eliminates free radicals and promotes antioxidant enzyme gene expressions and DNA repair, which reduce the toxicity of platinum-based or alkylating chemotherapeutic drugs. However, studies have shown that AMF may have unintended effects that increase the survival rate of certain tumor cells.5−7 Therefore, it is necessary to develop adjuvants from natural bioactives that are safe and do not influence treatment effects. Licorice is a traditional medicinal plant, and its roots and rhizomes, which are used for medicine, contain 4−20% triterpenoid saponins, of which glycyrrhizic acid (GA) is the major active component. GA is the source of the sweetness of licorice, and it is 50 times sweeter than sucrose.8 Studies have shown that oral administration of GA can be metabolized into © 2015 American Chemical Society
Received: Revised: Accepted: Published: 1200
November 13, 2014 January 11, 2015 January 14, 2015 January 14, 2015 DOI: 10.1021/jf505471a J. Agric. Food Chem. 2015, 63, 1200−1209
Article
Journal of Agricultural and Food Chemistry
Figure 1. Elevated levels of serum BUN, creatinine, and LDH were reduced by GA and 18βGA in BALB/c mice with CP-induced renal injury. (A) Chemical structures of GA and 18βGA. (B) Mice were gavaged daily with GA (25−100 mg/kg of BW) or 18βGA (10−50 mg/kg of BW) for 8 consecutive days, and a single IP injection of CP (30 mg/kg of BW) was applied on day 6. The positive control group was given a single IP injection of AMF (200 mg/kg of BW) at 0.5 h before the CP challenge. Mice were sacrificed on day 9. Data are presented as the mean ± SD (n = 8), and letters (a−c) indicate statistically significant differences in each group (p < 0.05). One-way ANOVA tests were used for the statistical analyses. NADPH), sodium azide (NaN3), and ethanol were obtained from Sigma-Aldrich (St. Louis, MO). The superoxide dismutase (SOD) assay kit was purchased from Faith Technology (Taichung, Taiwan); the GSH assay kit was purchased from Cayman Chemicals (Ann Arbor, MI); the protein assay kit was purchased from Bio-Rad (Hercules, CA); and the anti-nuclear factor E2-related protein (Nrf2), anti-heme oxygenase-1 (HO-1), anti-high-mobility group protein B1 (HMGB1), anti-nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB), anti-tumor necrosis factor (TNF)-α, anti-interleukin (IL)-1β, and anti-IL-6 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Experimental Animals and Procedures. The 6-week-old male BALB/c mice were purchased from the National Laboratory Animal
antioxidant enzyme activities, and reducing inflammatory responses.1,19 Because oxidative stress and inflammatory responses play key roles in CP-induced nephrotoxicity, this study employed an animal model of CP-induced renal injury to verify the protective effects of GA and 18βGA against chemically induced renal injury and the possible underlying mechanisms of such protective effects.
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MATERIALS AND METHODS
Chemicals. CP, GA, 18βGA, Kollisolv PEG E 400, AMF, 1-chloro2,4-dinitro-benzene (CDNB), glutathione (GSH), glutathione reductase (GSH-Rd), β-nicotinamide adenine dinucleotide phosphate (β1201
DOI: 10.1021/jf505471a J. Agric. Food Chem. 2015, 63, 1200−1209
Article
Journal of Agricultural and Food Chemistry Center (Taipei, Taiwan). The animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the National Chung Hsing University (IACUC approval number 98-76) and followed related regulations of the Animal Care and Use Committee. Animals were housed in animal rooms at the Department of Food Science and Biotechnology of National Chung Hsing University. The temperature and relative humidity (RH) of the animal rooms were maintained at constant levels (22 ± 2 °C and RH of 65 ± 5%). A 12 h light/dark cycle was maintained, and the light cycle was controlled by an automatic timer; the light period was at 06:00−18:00, and the dark period was at 18:00−06:00. After arrival, the experimental mice were immediately weighed, and after a statistical analysis, they were distributed to feeding cages according to a normal distribution. Animals were allowed ad libitum access to water and food (laboratory rodent diet 5001, Purina, St. Louis, MO). The animal groupings and treatments were modified from the methods by Mitazaki et al.20 and Arjumand et al.14 After adaptive feeding for 1 week, the 6-week-old male BALB/c mice were continuously fed the sample diets for 8 days (GA at 25, 50, or 100 mg/kg of BW or 18βGA at 10, 25, or 50 mg/kg of BW). After 30 min of sample administration on day 6, an intraperitoneal (IP) injection of CP (30 mg/kg of BW) was given to induce damage, and 30 min prior to the injection of CP on day 6, the positive control group was IP-injected with the chemotherapy protectant, AMF (200 mg/kg of BW). The mice were sacrificed on day 9 to collect samples. Analysis of Serum Biochemical Values. Serum biochemical values, including BUN, creatinine, and lactate dehydrogenase (LDH), were analyzed using the ADVIA Chemistry Urea Nitrogen Reagent, ADVIA Chemistry Enzymatic Creatinine-2 Reagent, and ADVIA Lactate Dehydrogenase L-P Reagent, respectively (Siemens, NY). The analyses were performed according to protocols in the manuals of the manufacturer. Histopathological Studies. Renal tissues of BALB/c mice were fixed in 10% formaldehyde immediately following sacrifice, processed for histological examination according to a conventional method, and stained with hematoxylin and eosin (H&E). The morphology of any observed lesions was classified and recorded according to the classification criteria by Shackelford et al.21 Analysis of the Activities of Antioxidant Enzymes. Catalase, SOD, glutathione peroxidase (GPx), and glutathione reductase (GRd) activities were measured using an assay kit (Cayman, Ann Arbor, MI) according to the instruction of the manufacturer. The GSH/oxidized glutathione (GSSG) ratio was analyzed using a GSH assay kit purchased from Cayman (Ann Arbor, MI). Measurement of Malondialdehyde (MDA). The MDA levels in kidney samples of experimental rats were determined using the thiobarbituric acid (TBA) method, with modifications.22 Briefly, following a preincubation, 0.5 mL of tissue homogenate was mixed with 1 mL of 15% trichloroacetic acid, 0.375% thiobarbituric acid reactive substances (TBARS), and 0.25 mM HCl and then heated in boiling water for 45 min. After centrifugation (2000g for 15 min), the absorbance of the butanol phase was read at 535 and 520 nm. The difference between the two values was used to calculate the MDA concentration. An MDA standard was prepared from 1,1,3,3,tetraethoxypropane. Total protein concentrations were determined by Lowry’s method using bovine serum albumin as the standard. The MDA concentration was normalized against the total protein concentration and is expressed in micromoles per milligram of protein. Immunohistochemical (IHC) Staining. Paraffin blocks of kidney tissues were cut using a microtome into 2 μm thick sections. Sections were floated on water to allow for the tissues to unfold and extend. Tissue sections were placed on 2-aminopropyltriethoxysilane-coated slides, dried at 37 °C, and deparaffinized using xylene. Deparaffinized slides were treated with 3% H2O2 and proteinase K (0.5 mg/mL) for 10 min, washed with distilled water 3 times, and incubated in blocking buffer (StartingBlock, Pierce, Rockford, IL) for 5 min. When the slides had slightly dried, they were stained with primary antibodies for 30 min at 40 °C (anti-Nrf2, anti-HO-1, anti-HMGB1, anti-NF-κB, anti-TNF-α, anti-IL-1β, and anti-IL-6 antibodies were used in this study). After the slides were washed with phosphate-buffered saline (PBS) 3 times, they
were incubated in EnVision-labeled polymer peroxidase-conjugated anti-immunoglobulin G (IgG, Dako, Glostrup, Denmark) at room temperature for 30 min. Slides were again washed with PBS 3 times and then incubated in diluted 3,3-diaminobenzidine tetrahydrochloride (DAB-4HCl, Dako) for color development. After development, slides were washed with PBS 3 times and counterstained with hematoxylin. Slides were then washed with distilled water 3 times, dehydrated, dried, and mounted. The intensity of antigen expression after staining was quantified using Image-Pro Plus 6.3 image analysis software (Media Cybernetics, Rockville, MD). Results are presented as the positively stained area (%). Statistical Analysis. All data are expressed as the mean ± standard deviation (SD). An analysis of variance (ANOVA) was used to evaluate differences among multiple groups. Significant differences were subjected to Duncan’s test to compare the means of two specific groups. A p value of
