Ferulic acid prevents liver injury and increases the anti-tumor effect of diosbulbin B in vivo

Ferulic acid prevents liver injury and increases the anti-tumor effect of diosbulbin B in vivo 阿魏酸降低黄独素B肝毒性并提高其抗肿瘤活性 1 3 first-author 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:lichenyue1307@126.com †E-mail:lichenyue1307@126.com lichenyue1307@126.com 1 MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China The present study is designed to investigate the protection by ferulic acid against the hepatotoxicity induced by diosbulbin B and its possible mechanism, and further observe whether ferulic acid augments diosbulbin B-induced anti-tumor activity. The results show that ferulic acid decreases diosbulbin B-increased serum alanine transaminase/aspartate transaminase (ALT/AST) levels. Ferulic acid also decreases lipid peroxide (LPO) levels which are elevated in diosbulbin B-treated mice. Histological evaluation of the liver demonstrates hydropic degeneration in diosbulbin B-treated mice, while ferulic acid reverses this injury. Moreover, the activities of copper- and zinc-containing superoxide dismutase (CuZn-SOD) and catalase (CAT) are decreased in the livers of diosbulbin B-treated mice, while ferulic acid reverses these decreases. Further results demonstrate that the mRNA expressions of CuZn-SOD and CAT in diosbulbin B-treated mouse liver are significantly decreased, while ferulic acid prevents this decrease. In addition, ferulic acid also augments diosbulbin B-induced tumor growth inhibition compared with diosbulbin B alone. Taken together, the present study shows that ferulic acid prevents diosbulbin B-induced liver injury via ameliorating diosbulbin B-induced liver oxidative stress injury and augments diosbulbin B-induced anti-tumor activity. 研究目的 2 观察阿魏酸对黄独素B诱导肝毒性的抑制活性及其机理，同时探索阿魏酸对黄独素B抗肿瘤活性的增效作用。 创新要点 2 黄独素B为中药黄药子抗肿瘤的主要药效活性成分，但同时又是其致肝毒性的主要毒性成分。本研究立足于中医药配伍减毒增效理论，试图通过试验考察配伍阿魏酸对黄独素B肝毒性/抗肿瘤活性的减毒增效作用，为黄独素B与阿魏酸联合应用于抗肿瘤提供了一定的临床前试验依据。 研究方法 2 荷瘤小鼠（S180肉瘤）连续12天灌胃给药阿魏酸和黄独素B。通过对血清丙氨酸/天冬氨酸转氨酶活性（见图2）、肝脂质过氧化（见图3）和肝组织病理分析（见图4）考察阿魏酸对黄独素B肝毒性的抑制作用；通过对铜锌-超氧化物歧化酶和过氧化氢酶的活性（见图5a、5b）和基因表达分析（见图5c）探讨阿魏酸抑制黄独素B肝毒性的机理；通过对瘤重、抑瘤率的统计分析阿魏酸增加的黄独素B抗肿瘤活性。 重要结论 2 阿魏酸可以通过改善黄独素B诱导的氧应激损伤从而抑制其肝毒性，同时还可以协同增加黄独素B的抗肿瘤活性。 Ferulic acid Diosbulbin B Hepatotoxicity Oxidative stress injury Anti-tumor activity 阿魏酸 黄独素B 肝毒性 氧应激损伤 抗肿瘤 Introduction 1 Dioscorea bulbifera L. (Dioscoreaceae) rhizome is widely distributed throughout the tropics and temperate regions, especially in Asia, and in China, it has been traditionally used to treat thyroid disease and tumors (Gao et al., 2002 2002 ). In addition, in northern Bangladesh, it is used to treat tumors and leprosy (Murray et al., 1984 1984 ). Our laboratory studies have shown that D. bulbifera rhizome can induce hepatotoxicity (Wang et al., 2010 2010 ; 2011 2011 ), which emphasizes a potential toxicity in the clinical setting. Among the biologically active substances in D. bulbifera rhizome, a major class is the clerodane diterpenoids (Wang et al., 2009 2009 ; Liu et al., 2010 2010 ). Diosbulbin B is the major chemical compound of the clerodane diterpenoids in D. bulbifera rhizome (Kawasaki et al., 1968 1968 ), and has been found to have anti-tumor activity (Gao et al., 2002 2002 ; Wang et al., 2012 2012 ). Angelica sinensis (Oliv.) Diels root has been reported to prevent the hepatotoxicity induced by D. bulbifera rhizome (Liu et al., 2004 2004 ), while the active compound has not yet been elucidated. Ferulic acid is one of the main bioactive molecules in A. sinensis root, and always acts as a strong anti-oxidant; it is known for treating skin cancer, ageing, fatigue, and muscle wasting (Graf, 1992 1992 ; Alias et al., 2009 2009 ; Barone et al., 2009 2009 ; You et al., 2009 2009 ). There are reports that ferulic acid prevents noise-induced hearing loss and free radical-induced skin damage (Calabrese et al., 2008 2008 ; Fetoni et al., 2010 2010 ), but whether it affects the hepatotoxicity and anti-tumor activity of diosbulbin B is still unknown. Various reports have demonstrated that oxidative stress plays a vital role in liver toxicity induced by such substances as alcohol, carbon tetrachloride, chemotherapeutic agents, and acetaminophen (dos Santos et al., 2007 2007 ; Kostopanagiotou et al., 2009 2009 ; Moselhy and Ali, 2009 2009 ; Samuhasaneeto et al., 2009 2009 ). Reactive oxygen species (ROS), generated during oxidative stress, are extremely reactive and may modify and inactivate cellular lipids, proteins, DNA, and RNA, and thus cause cell damage. The cellular anti-oxidant system in the body prevents ROS-induced cell injury; the system includes low-molecular-mass anti-oxidants (van der Vliet et al., 1999 1999 ) such as glutathione, α-tocopherol, ascorbic acid, and the main anti-oxidant enzymes including copper- and zinc-containing superoxide dismutase (CuZn-SOD) and catalase (CAT). Our previous report demonstrated that oxidative stress plays an important role in liver injury induced by acetate extracts of D. bulbifera (Wang et al., 2011 2011 ); however, whether the protection by ferulic acid against liver injury induced by diosbulbin B is related to the liver anti-oxidant system has not been demonstrated. The present study is designed to demonstrate the protection by ferulic acid against the hepatotoxicity induced by diosbulbin B and the underlying mechanism, and investigate whether ferulic acid enhances diosbulbin B-inhibited tumor growth. Materials and methods 1 Experimental animals 2 ICR male mice (18–22 g) were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences, China. Animals were given rodent laboratory chow and water ad libitum , and maintained under controlled conditions at (22±1) °C, relative humidity (65±10)%, and a 12 h/12 h light/dark cycle (lights on at 7:00 am). All animals were dealt with in compliance with the institutional animal care guidelines of the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine, China. Cell lines 2 Mouse sarcoma S180 tumor cells were maintained in the peritoneal cavities of ICR mice in the Laboratory of Experimental Oncology of the Shanghai Laboratory Animal Center of Chinese Academy of Sciences, China. Reagents 2 5-Fluorouracil was purchased from Shanghai Bangcheng Chemical Co., Ltd., China. Unless indicated, other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Preparations of diosbulbin B and ferulic acid 2 D. bulbifera rhizome was collected in Qingyang County, Anhui Province, China and authenticated by Prof. Shou-jin LIU (Anhui University of Chinese Medicine, China). A. sinensis root was purchased from Minxian County, Gansu Province, China and authenticated by Prof. Li-hong WU (Shanghai University of Traditional Chinese Medicine, China). The voucher specimens of D. bulbifera rhizome and A. sinensis root were both deposited in the herbarium of the Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, China. Diosbulbin B and ferulic acid were isolated from D. bulbifera rhizome and A. sinensis root, respectively, in our laboratory as previously described (Kawasaki et al., 1968 1968 ; Sun et al., 2006 2006 ). After purification using silica gel column and gel chromatography, purities of diosbulbin B and ferulic acid were both more than 98% as determined by high-performance liquid chromatography (HPLC) with diode array detector. The chemical structures of diosbulbin B and ferulic acid are shown in Fig. 1 1 . Treatment protocol 2 Ascites of the mouse sarcoma S180 were drawn out from mice under aseptic conditions. Cells were counted promptly and then diluted to about 1×10 7 cells/ml with aseptic saline. The diluted S180 cell suspension was inoculated subcutaneously (0.1 ml per mouse) into the right armpit (Bezerra et al., 2008 2008 ). One day after inoculation, mice, except for the normal (non-tumor-inoculated) animals, were randomly divided into five groups of eight mice each. 5-Fluorouracil-treated mice were injected intraperitoneally with 5-fluorouracil at a dose of 25 mg/kg once every other day. Normal (non-tumor-inoculated) and control (tumor-inoculated) groups of mice received daily oral administration of 0.5% (5 g/L) sodium carboxyl methyl cellulose (CMC-Na; 0.2 ml/10 g). The other three groups received ferulic acid (8 mg/kg), diosbulbin B (32 mg/kg), or diosbulbin B (32 mg/kg) plus ferulic acid (8 mg/kg) by intragastric administration for 12 d starting from 24 h after tumor inoculation. After treatment, mice were sacrificed by cervical dislocation after peripheral blood samples, liver tissues and tumors were collected at 24 h after the last administration. Serum samples were collected for the determination of alanine transaminase/aspartate transaminase (ALT/AST), and liver tissues were used for the analysis of the lipid peroxide level, histological observation, determination of glutathione-related and anti-oxidant enzymes, and mRNA expression of the main anti-oxidant enzymes. The tumors were weighed, arrayed in line on paper, and taken pictures. The tumor inhibition ratio (IR) was calculated by the formula of IR=[( C − T )/ C ]×100%, where C and T are the mean tumor weights of the control group (CMC-Na) and the treated group, respectively. Assay for serum ALT and AST 2 Blood samples were obtained from mice of all groups (eight mice per group) for the determination of ALT and AST. Serum ALT and AST were assayed according to Kamei et al. ( 1986 1986 ). Assay for liver lipid peroxide level 2 Liver tissues were homogenized in cold phosphate buffered saline (PBS). Lipid peroxide was determined as previously described (Nkosi et al., 2006 2006 ). Malondialdehyde is an end product of lipid peroxide and serves as a means of quantifying lipid peroxide. Malondialdehyde reacts with 2-thiobarbituric acid to generate a pink-colored product, which has an absorbance at 532 nm. The standard curve of malondialdehyde was constructed over the concentration range of 0–40 μmol/L. Lipid peroxide level was expressed as micromoles of malondialdehyde per milligram of protein based on tissue protein concentration. Histological observation 2 After fixation in formaldehyde, the livers were examined for size, color changes, and hemorrhage. Slices of liver were cut into small pieces and histological sections were stained with hematoxylin and eosin (H&E) for the observation under light microscopy. Enzymatic assays 2 Tissues were homogenized in cold PBS, and then centrifuged at 5000× g for 5 min and the supernatant transferred to new tubes for assay. The liver tissue activities of CuZn-SOD and CAT were determined as previously described (Marklund and Marklund, 1974 1974 ; Aebi, 1984 1984 ), and the results were all calculated based on tissue protein concentrations. Reverse transcription-polymerase chain reaction (RT-PCR) analysis 2 Total RNA was extracted from the liver tissue using Trizol (Life Technologies, USA) according to the manufacturer’s protocol. To synthesize single-strand cDNA, reverse transcription of 2 μg total RNA was carried out using 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT) and reaction mixture (2.5 mmol/L dNTP, 100 pmol/L oligo(dT) primer, RT buffer, 50 U RNA inhibitor) according to the manufacturer’s protocol. Transcripts of the gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as an internal control. Sequences of the PCR primers were as follows: CuZn-SOD forward 5'-AAG GCC GTG TGC GTG CTG AA-3', reverse 5'-CAG GTC TCC AAC ATG CCT CT-3' (246 bp product) (El Mouatassim et al., 1999 1999 ); CAT forward 5'-GCA GAT ACC TGT GAA CTG TC-3', reverse 5'-GTA GAA TGT CCG CAC CTG AG-3' (229 bp product) (El Mouatassim et al., 1999 1999 ); and, GAPDH forward 5'-GAC CCC TTC ATT GAC CTC AAC T-3', Reverse 5'-GTT TGT GAT GGG TGT GAA CCA-3' (200 bp product) (Hougardy et al., 2005 2005 ). The PCR protocol consisted of denaturation at 94 °C for 1 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C (CuZn-SOD) or 55 °C (CAT) for 1 min, extension at 72 °C for 1 min, and final extension at 72 °C for 15 min. The protocol for GAPDH was the same except for annealing at 65 °C for 1 min and amplification for 25 cycles. The PCR products were electrophoresed in a 2% agarose gel and stained with ethidium bromide. The bands were automatically analyzed by Smart View Bio-electrophoresis Image Analysis system (Version FR-980, FURI Science and Technology Co. Ltd., Shanghai, China). Statistical analysis 2 All experimental data were expressed as mean±standard error of mean (SEM). Significant differences among experimental groups were compared by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) ( P < 0.05) using the Statistics Package for Social Science (SPSS) program Version 11.5. Results 1 Ferulic acid prevents diosbulbin B-induced liver injury 2 Serum ALT and AST activities are liver injury biomarkers, a significant elevation of which often reflects liver injury (Kamei et al., 1986 1986 ). In the present study, ALT and AST were both found to be raised in mice treated with diosbulbin B alone for 12 consecutive days compared with CMC-Na controls ( P < 0.001), while ferulic acid inhibited this diosbulbin B-induced increase ( P < 0.001) (Fig. 2 2 ). FA inhibited diosbulbin B (DB)-induced increase of ALT/AST in tumor-bearing mice. Data are presented as mean±SEM ( n =8). ∆∆∆ P < 0.001 vs. normal; ### P < 0.001 vs. CMC-Na; *** P < 0.001 vs. DB As shown in Fig. 3 3 , malondialdehyde, one of the main end-products of lipid peroxide activity (Nkosi et al., 2006 2006 ), was increased in the livers of mice treated with diosbulbin B compared with CMC-Na controls ( P < 0.05), while ferulic acid inhibited this increase ( P < 0.001). FA inhibited diosbulbin B (DB)-increased MDA amount. Data are presented as mean±SEM ( n =8). ∆∆ P < 0.01 vs. normal; # P < 0.05 vs. CMC-Na; *** P < 0.001 vs. DB Further, histological evaluation of the livers removed from mice treated with diosbulbin B (32 mg/kg) showed hydropic degeneration of hepatocytes (Fig. 4c 4c ). After treatment with ferulic acid (8 mg/kg), these abnormal changes disappeared (Fig. 4d 4d ). After treatment, livers were removed, fixed, sectioned (5 μm), and processed for H&E staining. Typical images were chosen from each experimental group (original magnification 200×) Ferulic acid reverses diosbulbin B-decreased CuZn-SOD and CAT enzymatic activities and mRNA expression 2 CAT and CuZn-SOD are both important intracellular anti-oxidant enzymes, participating in the process of oxidative stress (Aebi, 1984 1984 ; Zelko et al., 2002 2002 ). Our results showed that diosbulbin B decreased the enzymatic activities of CuZn-SOD and CAT compared with CMC-Na controls ( P < 0.05 and P < 0.01, respectively), while ferulic acid reversed this decrease (both P < 0.01) (Figs. 5a and 5b 5a and 5b ). Further results (Fig. 5c 5c ) showed that in the CMC-Na control group, mRNA expressions of CAT and CuZn-SOD were decreased compared with the normal ( P < 0.05 and P < 0.001, respectively). The mRNA expressions of CAT and CuZn-SOD in diosbulbin B-treated mice were further decreased compared with the CMC-Na group (both P < 0.01), while ferulic acid clearly reversed this decrease ( P < 0.05 and P < 0.01, respectively). FA reversed diosbulbin B (DB)-decreased liver CuZn-SOD (a) and CAT (b) enzymatic activity and mRNA expression (c). Data are presented as mean±SEM, with n =8 (a, b) or n =6 (c). ∆ P < 0.05, ∆∆ P < 0.01, ∆∆∆ P < 0.001 vs. normal; # P < 0.05, ## P < 0.01 vs. CMC-Na; * P < 0.05, ** P < 0.01 vs. DB Ferulic acid enhances diosbulbin B-induced anti-tumor activity 2 The effect of ferulic acid on the anti-tumor activity of diosbulbin B in transplanted S180 sarcoma is shown in Fig. 6 6 . Our results showed that diosbulbin B decreased tumor weight of tumor-bearing mice ( P < 0.01) by 51.8%. After treatment with ferulic acid, diosbulbin B-decreased tumor weight was further decreased by 75.5% compared with diosbulbin B alone ( P < 0.01). Meanwhile, ferulic acid itself had no demonstrable inhibition of tumor growth. FA augmented diosbulbin B (DB)-induced anti-tumor activity in vivo . After treatment, the tumors were excised and weighed. (a) Pictures of tumor; (b) Tumor weight and the inhibitory ratio of tumor growth. Data are presented as mean±SEM ( n =8). ## P < 0.01 vs. CMC-Na; ** P < 0.01 vs. DB Discussion 1 Much attention has been paid to the prevention of cancer (Glade, 1999 1999 ). In addition to western therapies for cancer prevention, there are also studies on anti-tumor effects of herbal medicines such as D. bulbifera , Tripterygium wilfordii , and Andrographis paniculata (Shamon et al., 1997 1997 ; Gao et al., 2002 2002 ; Kumar et al., 2004 2004 ). Compatibility is a main principle of Chinese medicinal herbs, and some reports have demonstrated that compatibility may lead to eliminating toxicity or reinforcing bioactivities (Kim et al., 2007 2007 ; Gao et al., 2009 2009 ). Ferulic acid is the main bioactive component of A. sinensis root, and our results from ALT/AST assays and histological observations are the first to demonstrate that ferulic acid prevents diosbulbin B-induced liver injury. Furthermore, the results show that ferulic acid enhances the diosbulbin B-induced anti-tumor effect. All these results indicate that the compatibility of A. sinensis root with D. bulbifera may relieve the hepatotoxicity caused by D. bulbifera , and provide the possibility of clinical applications of these two drugs for cancer therapy. The imbalance of oxidants and anti-oxidants will lead to oxidative stress in the liver. Further, many anti-oxidant enzymes and non-enzymatic anti-oxidants may be changed during this process (Łuczaj and Skrzydlewska, 2004 2004 ; Dadkhah et al., 2006 2006 ; Limón-Pacheco and Gonsebatt, 2009 2009 ). Among them, lipid peroxide is free radical-related (Romero et al., 1998 1998 ), and one of the main end-products of lipid peroxide is malondialdehyde. The results show that ferulic acid inhibited the diosbulbin B-induced increase in malondialdehyde, indicating that ferulic acid can prevent diosbulbin B-induced lipid peroxide injury in the liver. CAT and SOD are believed to play key roles in the enzymatic defense of cells against oxidative stress injury. CAT mainly exists in the peroxisomes of aerobic cells and serves to protect against damage from hydrogen peroxide by catalyzing it into molecular oxygen and water without producing toxic free radicals (Bocchetti and Regoli, 2006 2006 ; Yilmaz et al., 2006 2006 ). The metalloenzyme, SOD, can convert two molecules of superoxide, produced during the oxidative stress, to hydrogen peroxide (Bocchetti and Regoli, 2006 2006 ). There are three SOD isoenzymes in mammalian cells: CuZn-SOD (copper- and zinc-containing SOD, mainly cytosolic), Mn SOD (manganese-containing SOD, located in the mitochondria), and EC SOD (extracellular SOD, actually also CuZn-SOD) (Zelko et al., 2002 2002 ); of these, CuZn-SOD is probably the most important anti-oxidant enzyme (Peskin, 1997 1997 ). Our results showed that ferulic acid reversed diosbulbin B-decreased enzymatic activities and mRNA expressions of CAT and CuZn-SOD. The results suggest that ferulic acid can prevent diosbulbin B-induced oxidative stress injury, while CAT and CuZn-SOD may also participate in this protection. In conclusion, the present study shows that ferulic acid can prevent hepatotoxicity induced by diosbulbin B isolated from D. bulbifera rhizome, mainly via preventing liver oxidative stress injury. This study also demonstrates that ferulic acid can enhance the anti-tumor activity of diosbulbin B in mice. 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Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression Compliance with ethics guidelines Jun-ming WANG, Yu-chen SHENG, Li-li JI, and Zheng-tao WANG declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1300250 Project supported by the Program for New Century Excellent Talents in University (No. NCET-11-1054), the National Natural Science Foundation of China (No. 81322053), and the Program for Changjiang Scholars and Innovative Research Team in University (No. PCSIRT-IRT1071), China 2013-09-16 2014-05-22 2014-03-17 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 15