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Geniposide protects against acute alcohol-induced liver injury in mice via up-regulating the expression of the main antioxidant enzymes Junming Wang, Yueyue Zhang, Ruixin Liu, Xiaobing Li, Ying Cui, and Lingbo Qu
Abstract: Geniposide (GP) is one of main compounds in Gardenia jasminoides Ellis, with both medicinal and nutritional value. This study was designed to determine, for the first time, how GP from G. jasminoides protects against acute alcohol-induced liver injury, and the underlying mechanisms. Mice were orally administered alcohol (6.0 g/kg body mass) 2 h after intragastric administration of GP and bifendate, every day for 7 continuous days. Six hours after the alcohol was administered, levels of serum alanine/aspartate transaminase (ALT/AST), hepatic lipid peroxidation (LPO), glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxidase (GPx), copper- and zinc-containing superoxide dismutase (CuZn-SOD), and catalase (CAT), and mRNA expression of CuZn-SOD and CAT were assayed. The results demonstrated that GP (20.0, 40.0, or 80 mg/kg) significantly reversed the excessive, alcohol-induced elevation in both serum ALT/AST and hepatic LPO levels. Moreover, hepatic GSH, GST, GPx, CuZn-SOD, and CAT levels were all decreased in the alcohol-treated mice, whereas treatment with GP reversed these decreases. Further analysis indicated that hepatic mRNA expression of CuZn-SOD and CAT in the alcohol-treated mice was significantly down-regulated, whereas GP up-regulated such decreases. Taken together, this study shows that GP protects against acute alcohol-induced liver injury via up-regulating the expression of the main antioxidant enzymes, and thus ameliorates alcohol-induced oxidative stress injury in the liver. Key words: hepatoprotection, oxidative stress, Gardenia jasminoides Ellis, copper- and zinc-containing superoxide dismutase, catalase. Résumé : Le géniposide (GP), un des composés principaux de Gardenia jasminoides Ellis, possède une valeur médicinale et nutritionnelle. Cette étude a été conçue afin d'observer, pour la première fois, la protection exercée par le GP de G. jasminoides envers le dommage hépatique induit par l'alcool, ainsi que les mécanismes sous-jacents. Des souris ont reçu de l'alcool par voie orale a` une dose de 6,0 g/kg de masse corporelle, 2 heures après l'administration par voie intragastrique de GP et de bifendate, quotidiennement, pendant 7 jours consécutifs. Six après l'administration d'alcool, les niveaux d'alanine transaminase/aspartate transaminase (ALT/AST) sériques et la peroxidation lipidique (LPO) hépatique, les niveaux de glutathion (GSH), de glutathion S-transférase (GST), de glutathion peroxydase (GPx), de superoxide dismutase a` cuiver et zinc (CuZn-SOD) et de catalase (CAT), de même que l'expression de l'ARNm de CuZn-SOD et de CAT ont été mesurés. Les résultats ont démontré que le GP (20,0, 40,0 ou 80,0 mg/kg) renversait significativement l'élévation excessive d'ALT/AST dans le sérum et de LPO hépatique provoquée par l'alcool. De plus, les niveaux hépatiques de GSH, GST, GPx, CuZn-SOD et CAT diminuaient tous chez les souris traitées a` l'alcool, alors que le GP renversait une telle diminution. Des analyses plus poussées ont indiqué que l'expression hépatique de l'ARNm de CuZn-SOD et de CAT chez les souris traitées a` l'alcool était régulée a` la baisse de manière significative, alors que le GP contrecarrait cette diminution. Dans l'ensemble, l'étude présente montre que le GP protège le foie du dommage induit par l'alcool en régulant a` la hausse l'expression des principales enzymes antioxydantes et ce faisant, contrecarre le dommage oxydant provoqué par l'alcool dans le foie. [Traduit par la Rédaction] Mots-clés : protection hépatique, stress oxydant, Gardenia jasminoides Ellis, superoxyde dismutase a` cuivre et zinc, catalase.
Introduction Alcohol abuse is one of the main causes of liver disease and is a serious social problem worldwide (Mandayam et al. 2004). The incidence of alcoholic liver disease has increased in China with the increased frequency of drinking, and is becoming another important risk factor for morbidity and mortality in addition to viral hepatitis (Li 2010). However, until now, there has been no
satisfactory treatment for alcoholic liver disease except for the combination of abstinence from alcohol and supportive care (Frazier et al. 2011). There is increasing evidence that oxidative stress plays a key role in the pathogenesis of alcoholic liver disease (Sid et al. 2013; Galligan et al. 2014; Grasselli et al. 2014; Wang et al. 2014). Reactive oxygen species (ROS), which are generated during alcohol-induced
Received 20 December 2014. Accepted 7 January 2015. J. Wang. Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, East Jinshui Road & Boxue Road, Zhengzhou 450046, China; College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, China. Y. Zhang, X. Li, and Y. Cui. Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, East Jinshui Road & Boxue Road, Zhengzhou 450046, China. R. Liu. The First Affiliated Hospital, Henan University of Traditional Chinese Medicine, Zhengzhou, China. L. Qu. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, China; School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou, China. Corresponding author: Junming Wang (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 93: 1–7 (2015) dx.doi.org/10.1139/cjpp-2014-0536
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oxidative stress, are highly reactive and may modify and inactivate lipids, proteins, DNA, and RNA, and thus induce cell dysfunction. The antioxidant system, which includes low-molecular-mass antioxidants such as glutathione, alpha-tocopherol, ascorbic acid, and the main antioxidant enzymes (including copper- and zinc-containing superoxide dismutase (CuZn-SOD), catalase (CAT), etc.), functions to protect the body from ROS-induced cell injury. Extensive studies have indicated that geniposide (GP), one of the main bioactive constituents in the dried ripe fruits of Gardenia jasminoides (Zhizi), not only protects against oxidative stress injuries, such as memory deficits, probably via the suppression of mitochondrial oxidative stress (Lv et al. 2014), but also prevents PC12 cells from oxidative damage via the mitogen-activated protein kinase (MAPK) pathway (Liu et al. 2007), elicits neuroprotection against hydrogen peroxide induced PC12 cells oxidative damage involved in the phosphatidylinositol-3 kinase (PI3K) signal pathway (Liu et al. 2009), and also provides hepatoprotection from many types of liver injury, including nonalcoholic steatohepatitis (NASH) (Ma et al. 2011), aflatoxin B1 (AFB1)-induced hepatotoxicity (Wang et al. 1991), and hepatic ischemia–reperfusion (I/R) injury, by reducing oxidative stress and apoptosis (Kim et al. 2013). However, as for its protection against alcohol-induced acute liver injury, there have been no experimental reports until now. Furthermore, whether the protection of GP against alcohol-induced acute liver injury is associated with the liver antioxidant system was not known. This study is designed to determine, for the first time, the protective effects of GP against alcohol-induced acute liver injury, and to investigate the underlying mechanisms with a focus on the antioxidant system.
Materials and methods Experimental animals Male Kunming (KM) mice (weighing 18–22 g) were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, China). Animals were provided with laboratory rodent chow and water ad libitum, and maintained under controlled conditions with a temperature of 22 ± 1 °C, relative humidity of 60% ± 10% and a 12 h (light) − 12 h dark cycle (lights on at 0800 h). All of the procedures were in strict accordance with the legislation from the People’s Republic of China on the use and care of laboratory animals, and with the guidelines established by the Institute for Experimental Animals of Henan University of Traditional Chinese Medicine, and were approved by the University Committee for Animal Experiments.
Materials and methods Geniposide (GP) was provided by Shanghai Jinsui Biotechnology (Shanghai, China) with a purity >98%, as assayed using highperformance liquid chromatography (HPLC). Bifendate was obtained from the Zhejiang Medicine Company (Xinchang Pharmaceutical Factory, Xinchang, China). Hot Start Fluorescent PCR Core Reagent Kits (SYBR Green I) were obtained from BBI (Kitchener, Ontario, Canada). The RevertAid First Strand cDNA Synthesis Kit was from Thermo Scientific (Shanghai, China). A Bradford protein assay kit, Trizol reagent, and all primers were purchased from Sangon Biotech (Shanghai, China). Unless indicated, other reagents and materials were purchased from Sangon Biotech (Shanghai, China). Treatment protocol Male mice were randomly distributed among the different groups of mice (n = 10 mice per group). Mice in the treatment groups (excluding the normal (no alcohol) group) were orally administered alcohol (6.0 g/kg body mass) 2 h after intragastric administration (Du et al. 2010) of either GP (20.0, 40.0, or 80.0 mg/kg) or the positive control (bifendate, 150.0 mg/kg), every day for
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7 consecutive days. The dose selection was based on the results from our preparatory experiments. Mice in the normal and control (alcohol only) groups received daily oral doses of 0.5% sodium carboxyl methyl cellulose (CMC-Na; 0.2 mL/10.0 g body mass). The peripheral blood samples from the different groups were collected (6 h after alcohol administration) for the determination of serum biomarkers indicating protective effects against acute, alcohol-induced liver injury, and liver tissues were sampled to investigate the mechanism of protection. Assaying serum biomarkers for liver injury The blood samples were obtained from the mice in all of the groups (10 mice per group) for the determination of serum biomarkers for liver injury. Serum ALT and AST were assayed according to a previously reported method (Kamei et al. 1986). Assay for levels of liver lipid peroxidation Liver tissues were homogenized in cold phosphate-buffered saline. Lipid peroxidation (LPO) was determined using a previously reported method (Hogberg et al. 1974). Malondialdehyde (MDA) forms as an end-product of LPO and so served as an index of the concentration of LPO. MDA reacts with thiobarbituric acid (TBA) to generate a pinkcolored product with absorbance at 532 nm. The standard curve for MDA was constructed over the concentration range of 0–40 mol/L. The level of lipid peroxides is expressed in micromoles of MDA per milligram of protein, based on the tissue protein concentration as measured using a Bradford protein assay kit. Measurement of GSH levels Hepatic GSH levels were assayed using the 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) assay according to a previously reported method (Liang et al. 2011). Enzymatic assays Tissues were homogenized in cold phosphate-buffered saline, and then centrifuged at 5000g for 5 min, and the supernatant was transferred to new tubes for assaying. The liver tissue activities of glutathione-S-transferase (GST), glutathione peroxidase (GPx), CuZnSOD, and CAT were determined using previously published methods (Rotruck et al. 1973; Marklund and Marklund 1974; Habig and Jakoby 1981; Aebi 1984), respectively, and the results were calculated based on tissue protein concentrations as measured using the Bradford protein assay kit. Fluorescent quantitative reverse-transcription PCR (FQ-RT–PCR) Total RNA was extracted from hepatic tissue using Trizol reagent, following the manufacturer's instructions. Reverse transcription (RT) was performed using a cDNA synthesis kit according to the manufacturer's instructions. The house-keeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an internal control. Sequences of the PCR primers were as follows: CuZn-SOD, forward 5=-AAGGCCGTGTGCGTGCTGAA-3=, reverse 5=-CAGGTCTCCAACATGCCTCT-3= (246 bp product) (El Mouatassim et al. 1999); CAT, forward 5=-GCAGATACCTGTGAACTGTC-3=, reverse 5=-GTAGAATGTCCGCACCTGAG-3= (229 bp product) (El Mouatassim et al. 1999); and GAPDH, forward 5=-GACCCCTTCATTGACCTCAACT-3=, reverse 5=-GTTTGTGATGGGTGTGAACCA-3= (200 bp product) (Hougardy et al. 2005). FQ-RT–PCR was performed using Hot Start Fluorescent PCR Core Reagent kits (SYBR Green I) on a real-time PCR instrument (ABI StepOnePlus; Applied Biosystems). PCR thermal cycling parameters were as follows: the denaturing step at 94 °C for 4 min, followed by 40 cycles of the annealing step at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. All amplifications and detections were carried out in a MicroAmp optical 96-well reaction plate with Published by NRC Research Press
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Fig. 1. Chemical structure of geniposide (GP) and its effects on serum alanine (ALT) and aspartate transaminase (AST) levels in mice. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with normal (non-alcohol treated) group; #, p < 0.05 and ##, p < 0.01 compared with the control (alcohol only) group.
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Results Effects of GP on serum biomarkers for alcohol-induced liver injury Serum ALT and AST activities are biomarkers for liver injury, and if they are significant elevated, this often reflects liver injury
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(Kamei et al. 1986). In the current study, ALT and AST were both found to be significantly elevated (from 47.93 ± 2.33 and 91.15 ± 2.47 U/L in the normal group, to 116.93 ± 6.62 and 171.62 ± 6.11 U/L in mice treated with alcohol only; Figs. 1B and 1C), demonstrating that acute, alcohol-induced liver injury was successfully reproduced. After intragastric administration of GP (20.0, 40.0, or 80.0 mg/kg) or bifendate (150.0 mg/kg) for 7 consecutive days, the increase in levels of ALT and AST was significantly reversed (Figs. 1B and 1C). These results demonstrate that both GP and bifendate protect against acute, alcohol-induced liver injury. Effects of GP on liver LPO levels MDA is one of the main end products of LPO (Hogberg et al. 1974). As shown in Fig. 2A, MDA levels increased from 0.34 ± 0.06 mol/mg protein in the normal group to 0.88 ± 0.10 mol/mg protein in the livers of mice treated with alcohol only, whereas the significant increase in MDA was inhibited in the mice treated Published by NRC Research Press
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Fig. 2. Effects of geniposide (GP) on hepatic malondialdehyde (MDA) and glutathione levels in mice. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with the normal (no alcohol) group; #, p < 0.05 and ##, p < 0.01 compared with control (alcohol only) group.
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Fig. 3. Effects of geniposide (GP) on hepatic glutathione-S-transferase (GST) and glutathione peroxidase (GPx) activities. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with normal (no alcohol) group; #, p < 0.05 and ##, p < 0.01 compared with the control (alcohol only) group. 250
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Fig. 4. Effects of geniposide (GP) on the enzymatic activities and mRNA expression of hepatic CuZn-SOD and CAT. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with normal (no alcohol) group; #, p < 0.05 and ##, p < 0.01 compared with the control (alcohol only) group. 300
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Effect of GP on liver glutathione levels Glutathione, as an antioxidant, helps protect cells against ROS such as free radicals and peroxides (Liang et al. 2011). If glutathione levels are significantly reduced, this can result in oxidative stress injury. In our study, the liver glutathione levels decreased significantly (from 75.4 ± 6.5 nmol/mg protein in normal group to 37.7 ± 4.1 nmol/mg protein) in the livers of mice treated with alcohol only, whereas the decrease in glutathione levels was significantly reversed in the mice treated with GP (80.0 mg/kg) or bifendate (150 mg/kg) (Fig. 2B). These results suggested that GP and bifendate protect against alcohol-induced disturbance of the balance between cellular oxidants and antioxidants by inhibiting the reduction in glutathione levels, and thus would be likely to protect against oxidative stress injury of the liver. Effect of GP on liver glutathione related enzyme activity GST and GPx, as intracellular glutathione-related enzymes, cooperate with glutathione in working against oxidative stress injury (Rotruck et al. 1973; Habig and Jakoby 1981). Our study shows that alcohol significantly reduced hepatic GST and GPx activities from 205.1 ± 18.6 and 75.1 ± 6.9 U/mg protein in the normal group, to 125.7 ± 13.1 and 42.7 ± 3.5 U/mg protein, respectively, in mice treated with alcohol only, whereas treatment with GP (80.0 mg/kg) or bifendate (150.0 mg/kg) inhibited these decreases (Figs. 3A and 3B), suggesting that GST and GPx participated in the protective actions
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of GP and bifendate against liver oxidative stress injury induced by alcohol. Our results further confirmed that the balance between cellular oxidants and antioxidants was destroyed by alcohol, whereas treatment with GP or bifendate reversed the imbalance. Effects of GP on main hepatic antioxidant enzyme activities and mRNA expression CuZn-SOD and CAT are both principle intracellular antioxidant enzymes that participate in the process of oxidative stress (Aebi 1984; Zelko et al. 2002). Our results show that alcohol decreased the activities of CuZn-SOD and CAT, from 35.8 ± 3.5 and 221.5 ± 19.1 U/mg protein in the normal group, to 13.2 ± 1.2 and 143.4 ± 12.8 U/mg protein, respectively, in mice treated with alcohol only, whereas treatment with GP (40.0 or 80.0 mg/kg) or bifendate (150.0 mg/kg) inhibited these decreases (Figs. 4A and 4C). Further results (Figs. 4B and 4D) showed that the mRNA expression of CuZn-SOD and CAT in alcohol-treated mice decreased as compared with normal mice (no alcohol), whereas treatment with either GP (40.0 or 80.0 mg/kg) or bifendate (150.0 mg/kg) significantly reversed these decreases. These results indicate that the main antioxidant enzymes CuZn-SOD and CAT play a key role in the protective mechanisms of GP and bifendate against alcohol-induced hepatic oxidative stress injury.
Discussion ALT and AST are both sensitive and reliable biomarkers for liver injury (Kamei et al. 1986). Among them, AST can be found in liver, cardiac muscle, skeletal muscle, kidney, brain, pancreas, lung, Published by NRC Research Press
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leukocytes, and erythrocytes, whereas ALT mainly occurs in liver. The significantly elevated levels of serum ALT and AST indicate increased permeability and damage and (or) necrosis of hepatocytes (Kamei et al. 1986). The results of our study showed that acute alcohol administration induced liver injury, as evidenced by the increase in serum levels of ALT and AST, reflecting the early biochemical changes of alcoholic liver disease. Pretreatment with GP (20.0, 40.0, or 80.0 mg/kg) provided significant protection against acute alcohol-intoxication in mice by attenuating increases in ALT and AST levels in a dose-dependent manner. The role of oxidative stress during the development of alcoholic liver disease has been investigated since the early 1960s by Diluzio (1964), who observed that alcohol administration promoted the oxidative breakdown of cell membranes. Studies with the intragastric feeding model have indicated that alcohol-induced liver injury is associated with elevated LPO, the formation of lipid radicals, and decreased liver antioxidant defense, providing convincing evidence for the pathogenic role of oxidative stress (Rouach et al. 1997; Polavarapu et al. 1998). LPO is a process associated with free radicals (Romero et al. 1998). As one of the major end products of LPO, MDA has the property of cross-linking cellular macromolecules such as protein or DNA, and can cause widespread cellular damage (Hassan et al. 2005). The results in Fig. 2A show that GP significantly prevented alcohol-induced excess in MDA levels, suggesting that GP prevented alcohol-induced hepatic LPO injury. Glutathione, which is a non-enzymatic antioxidant, is important for protecting hepatocytes against exogenous toxins, and the depletion of cellular glutathione is associated with oxidative injury (Carbonell et al. 2000; Han et al. 2006). Our results show that GP significantly inhibited alcohol-induced exhaustion of glutathione, indicating that glutathione is involved in the protection mechanisms of GP against alcohol-induced hepatic oxidative injury. GST and GPx are cellular, glutathione-related antioxidant enzymes. Of the 2, the cytosolic GSTs exist in almost all aerobic species. GST, together with glutathione, catalyzes the conjugation of electrophilic compounds formed during oxidative stress (Habig and Jakoby 1981). GPx catalyzes hydrogen peroxide decomposition to the stable form of hydroxides, specifically using reduced glutathione as the electron provider (Rotruck et al. 1973). In the current study, GP significantly reverted the alcohol-induced decrease in the activities of hepatic GST and GPx in mice, which further confirms that hepatic glutathione-related antioxidant enzymes were involved in the protection of GP against alcoholinduced hepatic oxidative injury. SOD and CAT are both believed to play significant roles in the enzymatic defenses of the cells against oxidative stress injury. SOD, as a metalloenzyme, can convert O2, generated during oxidative stress, to hydrogen peroxide (Bocchetti and Regoli 2006). Three SOD isoenzymes exist in mammalian cells, including CuZnSOD (copper and zinc-containing SOD, basically cytosolic), Mn-SOD (manganese-containing SOD, located in the mitochondria), and EC-SOD (extracellular SOD, actually also CuZn-SOD) (Zelko et al. 2002). Of the above 3 isoenzymes, CuZn-SOD is probably the most important antioxidant enzyme, and it is well established that CuZn-SOD is an irreplaceable enzyme for aerobic life (Peskin 1997). CAT mainly exists in the peroxisomes of all aerobic cells, and serves to protect the cells against damage from hydrogen peroxide by catalyzing it into molecular oxygen and water without producing toxic free radicals (Bocchetti and Regoli 2006). As peroxisomes are abundant in proteins, where oxidative stress always happens, CAT is a classic biomarker for oxidative stress. Our results show that GP reversed acute, alcohol-induced decreases in the enzymatic activity and mRNA expression of CuZn-SOD and CAT, suggesting that GP protects against acute alcohol-induced hepatic oxidative stress injury, and that CuZn-SOD and CAT participated in this protection. In conclusion, our study shows that GP protects against acute alcohol-induced liver injury, mainly via inhibiting LPO-induced
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damage and depletion of the antioxidant GSH, enhancing the activities of the main antioxidant enzymes, and up-regulating mRNA expression of CuZn-SOD and CAT, and thus GP ameliorates alcohol-induced liver oxidative stress injury. In addition, as clearly emphasized by other authors (Tarantino et al. 2007, 2011a), mitochondrial dysfunction is the basis of non-alcoholic fatty liver disease (NAFLD) and drug-induced liver injury (DILI). Furthermore, the key role of the apoptosis has been mentioned by other authors (Tarantino et al. 2011b). For this reason, the main role of a substance released by mitochondria in humans has been analyzed, so has the imbalance between the processes of apoptosis and antiapoptosis. Moreover, as previously mentioned (Lv et al. 2014), GP may attenuate memory deficits through the suppression of mitochondrial oxidative stress. As to whether GP protects against alcohol-induced liver injury via attenuating mitochondrial dysfunction and helps to keep the balance between apoptosis and anti-apoptosis, could be future research object for our lab.
Acknowledgements This work was financially supported by the National Science Foundation for Post-doctoral Scientists of China (2012M521412), the Provincial Fundamental Research Fund in Henan University of Chinese Medicine (2014KYYWF-QN01), the Innovation Program for Science & Technology Leading Talents of Zhengzhou City in China's Henan Province (121PLJRC534), the National Natural Science Foundation of China (81473368), and the Program for Innovative Research Team (in Science and Technology) in the Henan University of Chinese Medicine (2011XCXTD01). Conflict of Interest: The authors declare that there is no conflict of interest associated with this study.
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ARTICLE
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Geniposide protects against acute alcohol-induced liver injury in mice via up-regulating the expression of the main antioxidant enzymes Junming Wang, Yueyue Zhang, Ruixin Liu, Xiaobing Li, Ying Cui, and Lingbo Qu
Abstract: Geniposide (GP) is one of main compounds in Gardenia jasminoides Ellis, with both medicinal and nutritional value. This study was designed to determine, for the first time, how GP from G. jasminoides protects against acute alcohol-induced liver injury, and the underlying mechanisms. Mice were orally administered alcohol (6.0 g/kg body mass) 2 h after intragastric administration of GP and bifendate, every day for 7 continuous days. Six hours after the alcohol was administered, levels of serum alanine/aspartate transaminase (ALT/AST), hepatic lipid peroxidation (LPO), glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxidase (GPx), copper- and zinc-containing superoxide dismutase (CuZn-SOD), and catalase (CAT), and mRNA expression of CuZn-SOD and CAT were assayed. The results demonstrated that GP (20.0, 40.0, or 80 mg/kg) significantly reversed the excessive, alcohol-induced elevation in both serum ALT/AST and hepatic LPO levels. Moreover, hepatic GSH, GST, GPx, CuZn-SOD, and CAT levels were all decreased in the alcohol-treated mice, whereas treatment with GP reversed these decreases. Further analysis indicated that hepatic mRNA expression of CuZn-SOD and CAT in the alcohol-treated mice was significantly down-regulated, whereas GP up-regulated such decreases. Taken together, this study shows that GP protects against acute alcohol-induced liver injury via up-regulating the expression of the main antioxidant enzymes, and thus ameliorates alcohol-induced oxidative stress injury in the liver. Key words: hepatoprotection, oxidative stress, Gardenia jasminoides Ellis, copper- and zinc-containing superoxide dismutase, catalase. Résumé : Le géniposide (GP), un des composés principaux de Gardenia jasminoides Ellis, possède une valeur médicinale et nutritionnelle. Cette étude a été conçue afin d'observer, pour la première fois, la protection exercée par le GP de G. jasminoides envers le dommage hépatique induit par l'alcool, ainsi que les mécanismes sous-jacents. Des souris ont reçu de l'alcool par voie orale a` une dose de 6,0 g/kg de masse corporelle, 2 heures après l'administration par voie intragastrique de GP et de bifendate, quotidiennement, pendant 7 jours consécutifs. Six après l'administration d'alcool, les niveaux d'alanine transaminase/aspartate transaminase (ALT/AST) sériques et la peroxidation lipidique (LPO) hépatique, les niveaux de glutathion (GSH), de glutathion S-transférase (GST), de glutathion peroxydase (GPx), de superoxide dismutase a` cuiver et zinc (CuZn-SOD) et de catalase (CAT), de même que l'expression de l'ARNm de CuZn-SOD et de CAT ont été mesurés. Les résultats ont démontré que le GP (20,0, 40,0 ou 80,0 mg/kg) renversait significativement l'élévation excessive d'ALT/AST dans le sérum et de LPO hépatique provoquée par l'alcool. De plus, les niveaux hépatiques de GSH, GST, GPx, CuZn-SOD et CAT diminuaient tous chez les souris traitées a` l'alcool, alors que le GP renversait une telle diminution. Des analyses plus poussées ont indiqué que l'expression hépatique de l'ARNm de CuZn-SOD et de CAT chez les souris traitées a` l'alcool était régulée a` la baisse de manière significative, alors que le GP contrecarrait cette diminution. Dans l'ensemble, l'étude présente montre que le GP protège le foie du dommage induit par l'alcool en régulant a` la hausse l'expression des principales enzymes antioxydantes et ce faisant, contrecarre le dommage oxydant provoqué par l'alcool dans le foie. [Traduit par la Rédaction] Mots-clés : protection hépatique, stress oxydant, Gardenia jasminoides Ellis, superoxyde dismutase a` cuivre et zinc, catalase.
Introduction Alcohol abuse is one of the main causes of liver disease and is a serious social problem worldwide (Mandayam et al. 2004). The incidence of alcoholic liver disease has increased in China with the increased frequency of drinking, and is becoming another important risk factor for morbidity and mortality in addition to viral hepatitis (Li 2010). However, until now, there has been no
satisfactory treatment for alcoholic liver disease except for the combination of abstinence from alcohol and supportive care (Frazier et al. 2011). There is increasing evidence that oxidative stress plays a key role in the pathogenesis of alcoholic liver disease (Sid et al. 2013; Galligan et al. 2014; Grasselli et al. 2014; Wang et al. 2014). Reactive oxygen species (ROS), which are generated during alcohol-induced
Received 20 December 2014. Accepted 7 January 2015. J. Wang. Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, East Jinshui Road & Boxue Road, Zhengzhou 450046, China; College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, China. Y. Zhang, X. Li, and Y. Cui. Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, East Jinshui Road & Boxue Road, Zhengzhou 450046, China. R. Liu. The First Affiliated Hospital, Henan University of Traditional Chinese Medicine, Zhengzhou, China. L. Qu. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, China; School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou, China. Corresponding author: Junming Wang (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 93: 1–7 (2015) dx.doi.org/10.1139/cjpp-2014-0536
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oxidative stress, are highly reactive and may modify and inactivate lipids, proteins, DNA, and RNA, and thus induce cell dysfunction. The antioxidant system, which includes low-molecular-mass antioxidants such as glutathione, alpha-tocopherol, ascorbic acid, and the main antioxidant enzymes (including copper- and zinc-containing superoxide dismutase (CuZn-SOD), catalase (CAT), etc.), functions to protect the body from ROS-induced cell injury. Extensive studies have indicated that geniposide (GP), one of the main bioactive constituents in the dried ripe fruits of Gardenia jasminoides (Zhizi), not only protects against oxidative stress injuries, such as memory deficits, probably via the suppression of mitochondrial oxidative stress (Lv et al. 2014), but also prevents PC12 cells from oxidative damage via the mitogen-activated protein kinase (MAPK) pathway (Liu et al. 2007), elicits neuroprotection against hydrogen peroxide induced PC12 cells oxidative damage involved in the phosphatidylinositol-3 kinase (PI3K) signal pathway (Liu et al. 2009), and also provides hepatoprotection from many types of liver injury, including nonalcoholic steatohepatitis (NASH) (Ma et al. 2011), aflatoxin B1 (AFB1)-induced hepatotoxicity (Wang et al. 1991), and hepatic ischemia–reperfusion (I/R) injury, by reducing oxidative stress and apoptosis (Kim et al. 2013). However, as for its protection against alcohol-induced acute liver injury, there have been no experimental reports until now. Furthermore, whether the protection of GP against alcohol-induced acute liver injury is associated with the liver antioxidant system was not known. This study is designed to determine, for the first time, the protective effects of GP against alcohol-induced acute liver injury, and to investigate the underlying mechanisms with a focus on the antioxidant system.
Materials and methods Experimental animals Male Kunming (KM) mice (weighing 18–22 g) were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, China). Animals were provided with laboratory rodent chow and water ad libitum, and maintained under controlled conditions with a temperature of 22 ± 1 °C, relative humidity of 60% ± 10% and a 12 h (light) − 12 h dark cycle (lights on at 0800 h). All of the procedures were in strict accordance with the legislation from the People’s Republic of China on the use and care of laboratory animals, and with the guidelines established by the Institute for Experimental Animals of Henan University of Traditional Chinese Medicine, and were approved by the University Committee for Animal Experiments.
Materials and methods Geniposide (GP) was provided by Shanghai Jinsui Biotechnology (Shanghai, China) with a purity >98%, as assayed using highperformance liquid chromatography (HPLC). Bifendate was obtained from the Zhejiang Medicine Company (Xinchang Pharmaceutical Factory, Xinchang, China). Hot Start Fluorescent PCR Core Reagent Kits (SYBR Green I) were obtained from BBI (Kitchener, Ontario, Canada). The RevertAid First Strand cDNA Synthesis Kit was from Thermo Scientific (Shanghai, China). A Bradford protein assay kit, Trizol reagent, and all primers were purchased from Sangon Biotech (Shanghai, China). Unless indicated, other reagents and materials were purchased from Sangon Biotech (Shanghai, China). Treatment protocol Male mice were randomly distributed among the different groups of mice (n = 10 mice per group). Mice in the treatment groups (excluding the normal (no alcohol) group) were orally administered alcohol (6.0 g/kg body mass) 2 h after intragastric administration (Du et al. 2010) of either GP (20.0, 40.0, or 80.0 mg/kg) or the positive control (bifendate, 150.0 mg/kg), every day for
Can. J. Physiol. Pharmacol. Vol. 93, 2015
7 consecutive days. The dose selection was based on the results from our preparatory experiments. Mice in the normal and control (alcohol only) groups received daily oral doses of 0.5% sodium carboxyl methyl cellulose (CMC-Na; 0.2 mL/10.0 g body mass). The peripheral blood samples from the different groups were collected (6 h after alcohol administration) for the determination of serum biomarkers indicating protective effects against acute, alcohol-induced liver injury, and liver tissues were sampled to investigate the mechanism of protection. Assaying serum biomarkers for liver injury The blood samples were obtained from the mice in all of the groups (10 mice per group) for the determination of serum biomarkers for liver injury. Serum ALT and AST were assayed according to a previously reported method (Kamei et al. 1986). Assay for levels of liver lipid peroxidation Liver tissues were homogenized in cold phosphate-buffered saline. Lipid peroxidation (LPO) was determined using a previously reported method (Hogberg et al. 1974). Malondialdehyde (MDA) forms as an end-product of LPO and so served as an index of the concentration of LPO. MDA reacts with thiobarbituric acid (TBA) to generate a pinkcolored product with absorbance at 532 nm. The standard curve for MDA was constructed over the concentration range of 0–40 mol/L. The level of lipid peroxides is expressed in micromoles of MDA per milligram of protein, based on the tissue protein concentration as measured using a Bradford protein assay kit. Measurement of GSH levels Hepatic GSH levels were assayed using the 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) assay according to a previously reported method (Liang et al. 2011). Enzymatic assays Tissues were homogenized in cold phosphate-buffered saline, and then centrifuged at 5000g for 5 min, and the supernatant was transferred to new tubes for assaying. The liver tissue activities of glutathione-S-transferase (GST), glutathione peroxidase (GPx), CuZnSOD, and CAT were determined using previously published methods (Rotruck et al. 1973; Marklund and Marklund 1974; Habig and Jakoby 1981; Aebi 1984), respectively, and the results were calculated based on tissue protein concentrations as measured using the Bradford protein assay kit. Fluorescent quantitative reverse-transcription PCR (FQ-RT–PCR) Total RNA was extracted from hepatic tissue using Trizol reagent, following the manufacturer's instructions. Reverse transcription (RT) was performed using a cDNA synthesis kit according to the manufacturer's instructions. The house-keeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an internal control. Sequences of the PCR primers were as follows: CuZn-SOD, forward 5=-AAGGCCGTGTGCGTGCTGAA-3=, reverse 5=-CAGGTCTCCAACATGCCTCT-3= (246 bp product) (El Mouatassim et al. 1999); CAT, forward 5=-GCAGATACCTGTGAACTGTC-3=, reverse 5=-GTAGAATGTCCGCACCTGAG-3= (229 bp product) (El Mouatassim et al. 1999); and GAPDH, forward 5=-GACCCCTTCATTGACCTCAACT-3=, reverse 5=-GTTTGTGATGGGTGTGAACCA-3= (200 bp product) (Hougardy et al. 2005). FQ-RT–PCR was performed using Hot Start Fluorescent PCR Core Reagent kits (SYBR Green I) on a real-time PCR instrument (ABI StepOnePlus; Applied Biosystems). PCR thermal cycling parameters were as follows: the denaturing step at 94 °C for 4 min, followed by 40 cycles of the annealing step at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. All amplifications and detections were carried out in a MicroAmp optical 96-well reaction plate with Published by NRC Research Press
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Fig. 1. Chemical structure of geniposide (GP) and its effects on serum alanine (ALT) and aspartate transaminase (AST) levels in mice. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with normal (non-alcohol treated) group; #, p < 0.05 and ##, p < 0.01 compared with the control (alcohol only) group.
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Results Effects of GP on serum biomarkers for alcohol-induced liver injury Serum ALT and AST activities are biomarkers for liver injury, and if they are significant elevated, this often reflects liver injury
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(Kamei et al. 1986). In the current study, ALT and AST were both found to be significantly elevated (from 47.93 ± 2.33 and 91.15 ± 2.47 U/L in the normal group, to 116.93 ± 6.62 and 171.62 ± 6.11 U/L in mice treated with alcohol only; Figs. 1B and 1C), demonstrating that acute, alcohol-induced liver injury was successfully reproduced. After intragastric administration of GP (20.0, 40.0, or 80.0 mg/kg) or bifendate (150.0 mg/kg) for 7 consecutive days, the increase in levels of ALT and AST was significantly reversed (Figs. 1B and 1C). These results demonstrate that both GP and bifendate protect against acute, alcohol-induced liver injury. Effects of GP on liver LPO levels MDA is one of the main end products of LPO (Hogberg et al. 1974). As shown in Fig. 2A, MDA levels increased from 0.34 ± 0.06 mol/mg protein in the normal group to 0.88 ± 0.10 mol/mg protein in the livers of mice treated with alcohol only, whereas the significant increase in MDA was inhibited in the mice treated Published by NRC Research Press
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Fig. 2. Effects of geniposide (GP) on hepatic malondialdehyde (MDA) and glutathione levels in mice. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with the normal (no alcohol) group; #, p < 0.05 and ##, p < 0.01 compared with control (alcohol only) group.
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Fig. 3. Effects of geniposide (GP) on hepatic glutathione-S-transferase (GST) and glutathione peroxidase (GPx) activities. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with normal (no alcohol) group; #, p < 0.05 and ##, p < 0.01 compared with the control (alcohol only) group. 250
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Fig. 4. Effects of geniposide (GP) on the enzymatic activities and mRNA expression of hepatic CuZn-SOD and CAT. Data presented are the mean ± SEM (n = 10 mice per group); *, p < 0.05 and **, p < 0.01 compared with normal (no alcohol) group; #, p < 0.05 and ##, p < 0.01 compared with the control (alcohol only) group. 300
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Effect of GP on liver glutathione levels Glutathione, as an antioxidant, helps protect cells against ROS such as free radicals and peroxides (Liang et al. 2011). If glutathione levels are significantly reduced, this can result in oxidative stress injury. In our study, the liver glutathione levels decreased significantly (from 75.4 ± 6.5 nmol/mg protein in normal group to 37.7 ± 4.1 nmol/mg protein) in the livers of mice treated with alcohol only, whereas the decrease in glutathione levels was significantly reversed in the mice treated with GP (80.0 mg/kg) or bifendate (150 mg/kg) (Fig. 2B). These results suggested that GP and bifendate protect against alcohol-induced disturbance of the balance between cellular oxidants and antioxidants by inhibiting the reduction in glutathione levels, and thus would be likely to protect against oxidative stress injury of the liver. Effect of GP on liver glutathione related enzyme activity GST and GPx, as intracellular glutathione-related enzymes, cooperate with glutathione in working against oxidative stress injury (Rotruck et al. 1973; Habig and Jakoby 1981). Our study shows that alcohol significantly reduced hepatic GST and GPx activities from 205.1 ± 18.6 and 75.1 ± 6.9 U/mg protein in the normal group, to 125.7 ± 13.1 and 42.7 ± 3.5 U/mg protein, respectively, in mice treated with alcohol only, whereas treatment with GP (80.0 mg/kg) or bifendate (150.0 mg/kg) inhibited these decreases (Figs. 3A and 3B), suggesting that GST and GPx participated in the protective actions
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with GP (20.0, 40.0, or 80.0 mg/kg) or bifendate (150.0 mg/kg) (Fig. 2A), suggesting that both GP and bifendate protect against alcohol-induced LPO injury in mice.
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of GP and bifendate against liver oxidative stress injury induced by alcohol. Our results further confirmed that the balance between cellular oxidants and antioxidants was destroyed by alcohol, whereas treatment with GP or bifendate reversed the imbalance. Effects of GP on main hepatic antioxidant enzyme activities and mRNA expression CuZn-SOD and CAT are both principle intracellular antioxidant enzymes that participate in the process of oxidative stress (Aebi 1984; Zelko et al. 2002). Our results show that alcohol decreased the activities of CuZn-SOD and CAT, from 35.8 ± 3.5 and 221.5 ± 19.1 U/mg protein in the normal group, to 13.2 ± 1.2 and 143.4 ± 12.8 U/mg protein, respectively, in mice treated with alcohol only, whereas treatment with GP (40.0 or 80.0 mg/kg) or bifendate (150.0 mg/kg) inhibited these decreases (Figs. 4A and 4C). Further results (Figs. 4B and 4D) showed that the mRNA expression of CuZn-SOD and CAT in alcohol-treated mice decreased as compared with normal mice (no alcohol), whereas treatment with either GP (40.0 or 80.0 mg/kg) or bifendate (150.0 mg/kg) significantly reversed these decreases. These results indicate that the main antioxidant enzymes CuZn-SOD and CAT play a key role in the protective mechanisms of GP and bifendate against alcohol-induced hepatic oxidative stress injury.
Discussion ALT and AST are both sensitive and reliable biomarkers for liver injury (Kamei et al. 1986). Among them, AST can be found in liver, cardiac muscle, skeletal muscle, kidney, brain, pancreas, lung, Published by NRC Research Press
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leukocytes, and erythrocytes, whereas ALT mainly occurs in liver. The significantly elevated levels of serum ALT and AST indicate increased permeability and damage and (or) necrosis of hepatocytes (Kamei et al. 1986). The results of our study showed that acute alcohol administration induced liver injury, as evidenced by the increase in serum levels of ALT and AST, reflecting the early biochemical changes of alcoholic liver disease. Pretreatment with GP (20.0, 40.0, or 80.0 mg/kg) provided significant protection against acute alcohol-intoxication in mice by attenuating increases in ALT and AST levels in a dose-dependent manner. The role of oxidative stress during the development of alcoholic liver disease has been investigated since the early 1960s by Diluzio (1964), who observed that alcohol administration promoted the oxidative breakdown of cell membranes. Studies with the intragastric feeding model have indicated that alcohol-induced liver injury is associated with elevated LPO, the formation of lipid radicals, and decreased liver antioxidant defense, providing convincing evidence for the pathogenic role of oxidative stress (Rouach et al. 1997; Polavarapu et al. 1998). LPO is a process associated with free radicals (Romero et al. 1998). As one of the major end products of LPO, MDA has the property of cross-linking cellular macromolecules such as protein or DNA, and can cause widespread cellular damage (Hassan et al. 2005). The results in Fig. 2A show that GP significantly prevented alcohol-induced excess in MDA levels, suggesting that GP prevented alcohol-induced hepatic LPO injury. Glutathione, which is a non-enzymatic antioxidant, is important for protecting hepatocytes against exogenous toxins, and the depletion of cellular glutathione is associated with oxidative injury (Carbonell et al. 2000; Han et al. 2006). Our results show that GP significantly inhibited alcohol-induced exhaustion of glutathione, indicating that glutathione is involved in the protection mechanisms of GP against alcohol-induced hepatic oxidative injury. GST and GPx are cellular, glutathione-related antioxidant enzymes. Of the 2, the cytosolic GSTs exist in almost all aerobic species. GST, together with glutathione, catalyzes the conjugation of electrophilic compounds formed during oxidative stress (Habig and Jakoby 1981). GPx catalyzes hydrogen peroxide decomposition to the stable form of hydroxides, specifically using reduced glutathione as the electron provider (Rotruck et al. 1973). In the current study, GP significantly reverted the alcohol-induced decrease in the activities of hepatic GST and GPx in mice, which further confirms that hepatic glutathione-related antioxidant enzymes were involved in the protection of GP against alcoholinduced hepatic oxidative injury. SOD and CAT are both believed to play significant roles in the enzymatic defenses of the cells against oxidative stress injury. SOD, as a metalloenzyme, can convert O2, generated during oxidative stress, to hydrogen peroxide (Bocchetti and Regoli 2006). Three SOD isoenzymes exist in mammalian cells, including CuZnSOD (copper and zinc-containing SOD, basically cytosolic), Mn-SOD (manganese-containing SOD, located in the mitochondria), and EC-SOD (extracellular SOD, actually also CuZn-SOD) (Zelko et al. 2002). Of the above 3 isoenzymes, CuZn-SOD is probably the most important antioxidant enzyme, and it is well established that CuZn-SOD is an irreplaceable enzyme for aerobic life (Peskin 1997). CAT mainly exists in the peroxisomes of all aerobic cells, and serves to protect the cells against damage from hydrogen peroxide by catalyzing it into molecular oxygen and water without producing toxic free radicals (Bocchetti and Regoli 2006). As peroxisomes are abundant in proteins, where oxidative stress always happens, CAT is a classic biomarker for oxidative stress. Our results show that GP reversed acute, alcohol-induced decreases in the enzymatic activity and mRNA expression of CuZn-SOD and CAT, suggesting that GP protects against acute alcohol-induced hepatic oxidative stress injury, and that CuZn-SOD and CAT participated in this protection. In conclusion, our study shows that GP protects against acute alcohol-induced liver injury, mainly via inhibiting LPO-induced
Can. J. Physiol. Pharmacol. Vol. 93, 2015
damage and depletion of the antioxidant GSH, enhancing the activities of the main antioxidant enzymes, and up-regulating mRNA expression of CuZn-SOD and CAT, and thus GP ameliorates alcohol-induced liver oxidative stress injury. In addition, as clearly emphasized by other authors (Tarantino et al. 2007, 2011a), mitochondrial dysfunction is the basis of non-alcoholic fatty liver disease (NAFLD) and drug-induced liver injury (DILI). Furthermore, the key role of the apoptosis has been mentioned by other authors (Tarantino et al. 2011b). For this reason, the main role of a substance released by mitochondria in humans has been analyzed, so has the imbalance between the processes of apoptosis and antiapoptosis. Moreover, as previously mentioned (Lv et al. 2014), GP may attenuate memory deficits through the suppression of mitochondrial oxidative stress. As to whether GP protects against alcohol-induced liver injury via attenuating mitochondrial dysfunction and helps to keep the balance between apoptosis and anti-apoptosis, could be future research object for our lab.
Acknowledgements This work was financially supported by the National Science Foundation for Post-doctoral Scientists of China (2012M521412), the Provincial Fundamental Research Fund in Henan University of Chinese Medicine (2014KYYWF-QN01), the Innovation Program for Science & Technology Leading Talents of Zhengzhou City in China's Henan Province (121PLJRC534), the National Natural Science Foundation of China (81473368), and the Program for Innovative Research Team (in Science and Technology) in the Henan University of Chinese Medicine (2011XCXTD01). Conflict of Interest: The authors declare that there is no conflict of interest associated with this study.
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