Article pubs.acs.org/JAFC
Suppression of Hepatic Cyp1a2 by Total Ginsenosides in Lipopolysaccharide-Treated Mice and Primary Mouse Hepatocytes Haiyan Sun,† Yijing Yan,† Chenshu Xu,§ Hongxia Wan,† and Dong Liu*,† †
Department of Applied Biotechnology, Shenzhen Polytechnic, Shenzhen, Guangdong 518055, People’s Republic of China Department of Pharmacy, School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, People’s Republic of China
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ABSTRACT: The roots of Panax ginseng (ginseng) have been extensively used in traditional Chinese medicine. However, herb− drug interactions between ginseng and other co-administered drugs are not fully understood concerning the effect of ginseng on drug metabolism and clearance. The current study aimed to elucidate the effect of total ginsenosides, a typical ginseng extract, on the regulation of Cyp1a2, a key enzyme to regulate drug metabolism under the normal and inflammatory conditions in mice. Female C57BL/6J mice treated with vehicle and lipopolysaccharide (LPS) were intragastrically administered ginseng extract for 7 days before hepatic P450 expression was analyzed. Primary mouse hepatocytes were also employed to further explore the effects of total ginsenosides on Cyp1a2 expression. The results showed that total ginsenosides in P. ginseng extract exhibited a concentration-dependent suppression on Cyp1a2 mRNA and protein level in both mice and primary mouse hepatocytes. Notably, the inhibitory effects of total ginsenosides on Cyp1a2 mRNA and protein expression were further enhanced following LPS treatment. Therefore, future research is warranted to investigate the role of ginsenosides in the regulation of hepatic CYP450s. Moreover, consumption of ginseng as food or supplement should be monitored for patients on combinational therapy, especially those with inflammatory diseases. KEYWORDS: ginsenosides, Cyp1a2, lipopolysaccharide, C57BL/6J mice, murine primary hepatocyte
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INTRODUCTION Ginseng, the root of Panax ginseng in the Araliaceae family, has been widely used as both medicine and food in Asia since ancient times. Serving as a stimulant, tonic, diuretic, and digestive aid, ginseng is expected to boost energy, sharpen the mind, reduce stress, treat impotence, and extend life span. For other medical purposes, ginseng is also applied to boost the immune system, control blood pressure, regulate blood glucose levels, and protect the cardiovascular system.1 As the consumption of herbal products has grown dramatically over the past several decades worldwide, there is an increasing need for understanding possible herb−drug or food−drug interactions.2 The cytochrome P450 (CYP) superfamily is a group of enzymes that catalyze the oxidation and reduction of a wide variety of endogenous and exogenous compounds. P450 enzymes are highly expressed in the liver and responsible for the bioactivation and/or detoxification of numerous drugs and toxic chemicals.3 CYP1A2, one of the most important isozymes, accounts for the biotransformation of about 5% of all clinically prescribed drugs in human. Because ginseng is broadly used all over the world, it is important to elucidate its effect on cytochrome P450s such as CYP1A2. Previous study indicated that ginseng extract elevated the catalytic activity of CYP1A1, but had no effect on the mRNA expression of CYP1A2 in rat.4 Another study demonstrated that ginseng extract inhibited the catalytic activity of CYP1A1/2.5 These results exhibit different effects of ginseng extract on the expression or activity of P450 isozymes, and some conclusions are inconsistent. Additionally, the effect of ginseng extract on most cytochrome P450 isoforms remains uncertain. Particularly, whether the infection or inflammatory state influences the effect of ginseng extract on © 2016 American Chemical Society
the cytochrome P450 superfamily has not been investigated. Given the common occurrence of inflammation in cancer patients with a high prevalence of ginseng consumption, the demand for this knowledge is increasing.6 A number of studies indicate that stimulation of the immune system during chronic infectious or inflammatory disease such as tumor and diabetes mellitus results in impairment of cytochrome P450 expression and metabolic activity in the liver in various species including humans, rats, and mice.7,8 The infection- or inflammation-induced reduction in drug metabolism and clearance can result in elevated plasma drug concentrations. Alteration in the systemic exposure of drugs may lead to potentially severe adverse drug reactions, especially for drugs with a low therapeutic index, as seen in children with influenza A infections taking theophylline for their asthma.9,10 Therefore, it is highly necessary to verify the effect of total ginsenosides in ginseng extract on the cytochrome P450 superfamily not only in normal but also infectious or inflammatory states.11 In the current study, we evaluated the effects of total ginsenosides on the expression of Cyp1a2 in C57BL/6J mice and primary mouse hepatocytes with or without LPS treatment using RT-PCR and Western blotting. Understanding the possible regulation of CYP450s by P. ginseng will contribute to the rational application of P. ginseng as herbal medicine or supplement in clinical practice. Received: Revised: Accepted: Published: 2386
December 31, 2015 February 26, 2016 February 28, 2016 February 28, 2016 DOI: 10.1021/acs.jafc.5b06170 J. Agric. Food Chem. 2016, 64, 2386−2393
Article
Journal of Agricultural and Food Chemistry
Figure 1. Representative chromatogram of Panax ginseng extract solution.
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extract groups, mice were given different doses of P. ginseng extract (15, 30, and 60 mg/kg of BW) for 7 days. On day 6, mice were injected intraperitoneally with 1 mg/kg of LPS dissolved in sterile saline and then sacrificed 24 h later; livers were harvested, rinsed in cold 1.15% potassium chloride, and weighed. The liver tissues were then portioned, flash-frozen, and stored at −80 °C for subsequent RNA preparation. All procedures were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People’s Republic of China (http://www.most. gov.cn). The animal study protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Shenzhen Polytechnic. Hepatocyte Isolation and Treatment. Mouse hepatocytes were isolated by a two-step in situ collagenase perfusion procedure as described previously.9 After anesthesia with ketamine/xylazine solution, the livers of female C57BL/6J mice were perfused via the portal vein with 120−150 mL of Krebs−Ringer−bicarbonate buffer followed by 50−70 mL of 0.3 mg/mL of collagenase type IV at a flow rate of 14−15 mL/min. Hepatocytes with at least 80% viability were then plated on 12-well collagen-coated plates (BD Biosciences, San Jose, CA, USA) with WE containing 10 mM HEPES, pH 7.4, 150 nM insulin, 50 nM dexamethasone, 10 mg/mL penicillin/streptomycin, and 10% FBS. After 4 h, cells were overlaid with fresh WE containing 0.23 mg/mL Matrigel and allowed to incubate overnight. Cultures were maintained at 37 °C in 5% carbon dioxide with regular medium changes. For the LPS groups, cells were treated with LPS (2 μg/mL) for 24 h. For the LPS + P. ginseng extract groups, cells were incubated with different concentrations of P. ginseng extract (15, 60, and 240 μg/ mL) for 24 h prior to the addition of LPS (2 μg/mL) + PGE for another 24 h. For cell harvest, all cells were washed twice and scraped in ice-cold PBS. Cells were then pelleted, and the supernatant was discarded. The cell lysates were resuspended in 200 μL of cell lysis buffer (50 mM Tris, pH 7.4, 0.1% SDS, 0.5% Nonidet-P40, 1% EDTA) and stored at −80 °C. Cytotoxicity Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to determine the cytotoxicity of P. ginseng extract on hepatocytes as previously described.12 The drug was dissolved in water, diluted in culture medium, and added to the cultures 24 h after cell seeding. Cells were exposed to the drug at different concentrations (15−300 μg/mL) in culture medium for 48 h. The hepatocytes were further incubated for 4 h after 0.05 mg of MTT was added. Supernatants were then removed, and the purple precipitate was dissolved in 100 μL of DMSO. The absorbance of
MATERIALS AND METHODS
Materials and Reagents. Williams E medium (WE), fetal bovine serum (FBS), 0.02% trypsin ethylenediaminetetraacetic acid (EDTA), penicillin−streptomycin, phosphate-buffered saline (PBS), and nonessential amino acids (NEAA) were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies to Cyp1a2 and GADPH, lipopolysaccharides (LPS, Escherichia coli 0111:B4, ultrapure; Invitrogen), collagenase type IV, Matrigel, and protease inhibitor mixture were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS was reconstituted and stored according to the manufacturer specifications. Preparation and Determination of P. ginseng Extract. P. ginseng was purchased from Beijing Tongrentang (TRT, Beijing PRC). Briefly, dried ginseng (P. ginseng C.A. Meyer) roots were cut into small pieces and refluxed in methanol for 4 h. After removal of the methanol, water-saturated butanol was used to elute three times. A total ginsenosides stock solution was concentrated by a rotary evaporator. High-performance liquid chromatography (HPLC) was used to determine the content of major ginsenosides in the stock. Ginsenoside Re was chosen as the standard to quantify the major ginsenosides in the freeze-dried extracts using the vanillin colorimetric method in accordance with the China Pharmacopoeia. Quality control (QC) samples, containing different concentrations of Re, were used for preparing the calibration curves. Final concentrations of low, medium, and high QC samples were 50, 100, and 200 μg/mL, respectively. These samples were prepared on the day of analysis in the same way as calibration standards. The performance of the vanillin colorimetric method was assessed by determining the intra- and interday accuracy and precision, in which 15 QC samples (5 of each in low, medium, and high concentrations) were analyzed on a single assay day and on each of five consecutive assay days, respectively. Animals and Treatments. Female C57B/6J mice, 8 weeks old, were purchased from Guangdong Medical Laboratory Animal Center. The animals were fed rodent chow and accessed water freely in an environment of 25 ± 1 °C, 55 ± 5% relative humidity, and diurnal light supply. Mice were housed in groups of four in a cage and acclimatized to the animal facility for at least 1 week before the experiment. There were eight groups with four animals per treatment group: control group, mice were orally given saline alone; P. ginseng extract groups, different doses of P. ginseng extract (15, 30, and 60 mg/ kg of BW) were administered orally to mice for 7 days; LPS-treated groups, mice were given an intraperitoneal injection of 1 mg/kg LPS dissolved in sterile saline and sacrificed 24 h later; LPS + P. ginseng 2387
DOI: 10.1021/acs.jafc.5b06170 J. Agric. Food Chem. 2016, 64, 2386−2393
Article
Journal of Agricultural and Food Chemistry
Figure 2. Structures of ginsenoside Rb1, Rb3, Re, and Rg1 in P. ginseng extract. Student’s t test or one-way analysis of variance followed by Dunnett’s test. The criterion of significance was set at p < 0.05. All statistical analysis was performed with SPSS software (SPSS Inc., Chicago, IL, USA).
formazan, a metabolite of MTT, was measured at a wavelength of 595 nm using a microplate reader (Tecan Instrument Inc., Research Triangle Park, NC, USA). RNA Extraction and Reverse Transcriptase Real-Time PCR. Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentration was determined spectrophotometrically by measuring the absorbance at 260 nm. RNA purity and integrity were confirmed by formaldehyde−agarose gel electrophoresis followed by visualization with ethidium bromide. Two micrograms of total RNA was used for cDNA synthesis with the High Capacity cDNA Archive kit (Takara). Primers were commercially synthesized by Sangon Biotech. Real-time PCR was carried out with SYBR Green PCR Master Mix (Applied Biosystems) using ABI 7300 reverse transcription PCR equipment. The transcription level of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was quantified for normalization. Primers for CYP450s and GAPDH are listed in Table 2. Analysis of real-time PCR was carried out by the ΔΔCt method. The expression level in control samples was arbitrarily set at 1. Protein Extraction and Immunoblotting. Livers of female C57B/6J mice were harvested and rinsed with cold 1.15% potassium chloride. Hepatocytes were incubated on ice in PBS with 1 mM EDTA for at least 20 min before harvest and then collected by centrifugation at 1000g for 5 min. The livers or hepatocytes were then homogenized in homogenization buffer (0.1 M potassium phosphate buffer, pH 7.4, containing 0.125 M potassium chloride, 1.0 mM EDTA, and a protease inhibitor mixture) or sonicated in cell lysis buffer (50 mM Tris, pH 7.5, 0.1% SDS, 0.5% Nonidet P-40, 1 mM EDTA containing a protease inhibitor mixture) and then centrifuged at 11000g for 10 min. The supernatant was used for immunoblot assays unless otherwise noted. SDS−polyacrylamide gel electrophoresis (PAGE) and Western blotting were carried out as described by Ferrari et al.13 AntiCyp1a2 (1:2000) and GADPH (1:5000) antibodies were incubated overnight at 4 °C, and then horseradish peroxidase-conjugated secondary antibody was incubated for 1 h at room temperature. Chemiluminescence was detected with enhanced chemiluminescence substrate (Pierce Chemical, Rockford, IL, USA) on X-ray film. Quantitative densitometric analyses of Western blot images were achieved using Image Lab software. Statistical Analysis. Data are presented as the mean ± SD. Control and experimental groups were compared either by unpaired
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RESULTS Determination of Total Ginsenosides in P. ginseng Extract. Different types and contents of ginsenosides in P. ginseng extract were determined with HPLC. Figure 1 shows that P. ginseng extract stock solution mainly consisted four kinds of ginsenosides, including Re (23.18%), Rg1 (34.26%), Rb1 (19.09%), and Rb3 (7.38%) (Figure 2). The vanillin colorimetric method was also validated for the determination of P. ginseng extract in accordance with the China Pharmacopoeia. The major ginsenosides in freeze-dried extracts were quantified by comparison with the standard Re. Calibration curves were linear over the concentration range of 40−280 μg with the mean correlation coefficients >0.998 (n = 8). For each point of the analytes on the calibration curves, the concentrations back-calculated from the equation of the regression analysis were within acceptable limits for accuracy and precision. A linear regression of the back-calculated concentrations versus the nominal values provided a unit slope and an intercept not significantly different from zero. The distribution of the residuals showed random variation and was normally distributed and centered on zero. The bias was not statistically different from zero, and the 95% confidence intervals included zero (data not shown). The differences between the theoretical and actual concentrations and the relative standard deviation were
