Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 1

ARTICLE

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

Quercetin ameliorates liver injury induced with Tripterygium glycosides by reducing oxidative stress and inflammation Junming Wang, Mingsan Miao, Yueyue Zhang, Ruixin Liu, Xaobing Li, Ying Cui, and Lingbo Qu

Abstract: Quercetin (Que) is one of main compounds in Lysimachia christinae Hance (Christina loosestrife), and has both medicinal and nutritional value. Glycosides from Tripterygium wilfordii Hook.f. (léi goˉng téng [the thunder duke vine]; TG) have diverse and broad bioactivities but with a high incidence of liver injury. Our previous study reported on the hepatoprotective properties of an ethanol extract from L. christinae against TG-induced liver injury in mice. This research is designed to observe, for the first time, the possible protective properties of the compound Que against TG-induced liver injury, and the underlying mechanisms that are involved in oxidative stress and anti-inflammation. The results indicated that TG caused excessive elevation in serum levels of alanine/aspartate transaminase (ALT/AST), alkaline phosphatase (ALP), gamma glutamyl transferase (␥-GT), and proinflammatory cytokine tumor necrosis factor-alpha (TNF-␣), as well as hepatic lipid peroxidation (all P < 0.01). On the other hand, following TG exposure, we observed significantly reduced levels of biomarkers, including hepatic glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxidase (GPx), and the anti-inflammatory cytokine interleukin (IL)-10, as well as the enzyme activity and mRNA expression of copper- and zinc-containing superoxide dismutase (CuZn-SOD) and catalase (CAT) (all P < 0.01). Nevertheless, all of these alterations were reversed by the pre-administration of Que or the drug bifendate (positive control) for 7 consecutive days. Therefore, this study suggests that Que ameliorates TG-induced acute liver injury, probably through its ability to reduce oxidative stress and its anti-inflammatory properties. Key words: hepatoprotection, oxidative stress, antioxidant enzymes, pro-inflammatory cytokines, anti-inflammatory cytokines. Résumé : La quercétine (Que), un des composés principaux de Lysimachia christinae Hance, possède des vertus médicinales et nutritionnelles. Les glycosides de Tripterygium spp. (GT) exercent des activités biologiques variées et étendues, mais leur utilisation s’accompagne d’une incidence élevée de dommage hépatique. Une étude précédente réalisée par les auteurs a rapporté l’effet hépatoprotecteur d’un extrait a` l’éthanol de L. christinae envers le dommage hépatique induit par les GT chez la souris. Cette recherche a été conçue afin d’examiner de manière plus approfondie l’effet protecteur possible de la Que envers le dommage hépatique induit par les GT, et les mécanismes sous-jacents impliqués dans les effets antioxydants and antiinflammatoires, pour la première fois. Les résultats ont indiqué que les GT provoquaient une élévation excessive d’alanine/ aspartate transaminase (ALT/AST), de phosphatase alcaline (PA) et de gamma-glutamyl transférase (␥-GT) sériques, de la peroxydation lipidique hépatique, et des niveaux du facteur nécrosant des tumeurs-alpha (TNF-␣), une cytokine pro-inflammatoire. Par contre, une réduction significative de biomarqueurs incluant le glutathion hépatique (GSH) et l’activité enzymatique et l’expression de l’ARNm de la glutathion-S-transférase (GST), la glutathion peroxydase (GPx), la superoxyde dismutase a` cuivre et zinc (CuZn-SOD) et la catalase (CAT), de même que la cytokine anti-inflammatoire interleukine-10, a été observée a` la suite d’une exposition aux GT (pour tous P < 0,01). Toutefois, toutes ces anomalies étaient renversées de manière évidente par une administration préalable de Que et de bifendate pendant 7 jours consécutifs. En conséquence, l’étude présente suggère que la Que peut améliorer le dommage hépatique aigu induit par les GT, probablement par l’intermédiaire d’une activité antioxydante et anti-inflammatoire. [Traduit par la Rédaction] Mots-clés : hépatoprotection, stress oxydant, enzymes antioxydantes, cytokines pro-inflammatoires, cytokines anti-inflammatoires.

Introduction Drug-induced liver injury (DILI), as a rare but severe adverse drug reaction that represents a major problem in pharmacotherapy, both clinically and economically. Nowadays, DILI has been the major cause of acute hepatic failure all over the world, with global incidence as high as about 14 per 100 000 patients per year (Urban et al. 2012). Beyond its cost in terms of patient morbidity and mortality, DILI is among the most frequent reasons for late

termination of drug development programs and regulatory actions including withdrawal of drugs from the market, resulting in large financial losses to the drug industry that are inevitably passed on to consumers (Urban et al. 2012). Moreover, diverse and complex predisposing factors, including individual susceptibility, age, gender, nutritional status, pre-existing and environmental factors, etc., make DILI difficult to predict (Tarantino et al. 2009), and thus more difficult to be treated in a timely fashion. Although several hepatoprotective agents have been used clinically, some of

Received 26 January 2015. Accepted 12 February 2015. J. Wang. Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, Zhengzhou 450046, China; College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China. M. Miao, 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, Zhengzhou 450046, China. R. Liu. The First Affiliated Hospital, Henan University of Traditional Chinese Medicine, Zhengzhou 450000, China. L. Qu. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China. Corresponding author: Junming Wang (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 93: 1–7 (2015) dx.doi.org/10.1139/cjpp-2015-0038

Published at www.nrcresearchpress.com/cjpp on xx xxx xxxx.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

2

them have caused adverse reactions, especially when administered chronically or subchronically (Muriel and Rivera-Espinoza 2008). On this basis, phytochemicals with natural antioxidants can provide us with effective and safe dietary possibilities that avoid DILI (Ray et al. 2006; Girish et al. 2009). Although plant extracts constitute potential candidate compounds that avoid DILI, they often contain highly complex mixtures of many compounds, many of which are incompletely understood. Therefore, more attention has been focused on understanding the bioactive compounds from plant drugs in recent years. Lysimachia christinae Hance (Primulaceae), commonly known as “Jin Qian Cao,” is distributed worldwide in temperate climates and is widely found in China (Houghton et al. 2005; Verpoorte et al. 2005). Lysimachia christinae is used safely and effectively in the treatment of many diseases. Many investigations have demonstrated the diverse properties of this plant, such as hepatoprotection, anticholecystitis, and cholagogic activity (Yang et al. 2011; Wang et al. 2012). Extensive chemical studies have indicated that quercetin (Que) is one of the main bioactive compounds from L. christinae. Que has diverse and broad bioactive properties, e.g., it has demonstrated anti-inflammatory (Caddeo et al. 2014), antiapoptotic, and antioxidative activity in the case of traumatic brain injury (Yang et al. 2014), and has protected against ischemia–reperfusion (IR) induced injury of the myocardium in rats (Liu et al. 2014), ameliorated insulin resistance, and up-regulated cellular antioxidants during oleic acid induced hepatic steatosis in HepG2 cells (Vidyashankar et al. 2013). Tripterygium glycosides (TG), as the main chemical constituents derived from T. wilfordii Hook.f. (léi goˉng téng [the thunder duke vine]), have a diverse and broad range of bioactivities. TG alleviates Freund’s complete adjuvant (FCA)-induced arthritis, improves pulmonary function in an adjuvant arthritis rat model, treats Behcet’s disease, and erosive oral lichen planus, etc. (Lin and Qi 2005; Song et al. 2010; Wan et al. 2013), but it is associated with a high incidence of liver injury (Peng et al. 2003; Li et al. 2011, 2012; Wan et al. 2012; Zhang et al. 2012). A previous study by our group showed that an ethanol extract of L. christinae could protect against TG-induced liver injury in mice (Wang et al. 2013). However, as far as we are aware, there have been no reports concerning the use of Que to protect against TG-induced liver injury. This study is designed to investigate the protective effects of Que against TG-induced acute liver injury, and to explore, for the first time, the underlying mechanisms behind Que’s antioxidant and anti-inflammatory properties.

Materials and methods

Can. J. Physiol. Pharmacol. Vol. 93, 2015

Canada). The RevertAid First Strand cDNA Synthesis kit was from Thermo Scientific (Shanghai, China). The Bradford protein assay kit, Trizol reagent, and all of the primers were purchased from Sangon Biotech (Shanghai, China). Unless indicated, other reagents and consumables were purchased from Sangon Biotech. Treatment protocol Male KM mice were distributed among 6 groups (10 mice per group). Mice were orally administered TG at 270 mg/kg body mass, 12 h after treatment with Que (10, 20, or 40, mg/kg), or bifendate (150 mg/kg) once a day for 7 consecutive days (intragastric administration; i.g.), except for the mice in the normal (non-TG treated) group (Peng et al. 2003). The normal mice and the mice treated with TG only were administered 0.5% CMC–Na (0.2 mL per 10 g body mass) daily, i.g. The blood samples from all of the groups were collected at 18 h after TG administration, for the measurement of serum biomarkers for hepatoprotection against liver injury induced with TG; the hepatic tissues for the analysis of the potential mechanisms were also harvested. Measurement of serum biomarkers for liver injury The blood samples were obtained from mice in all of the groups for the measurement of serum biomarkers for liver injury. The serum levels of alanine/aspartate transaminase (ALT/AST) and alkaline phosphatase (ALP), and gamma glutamyl transferase (␥-GT) activity were measured with kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions. Measurement of hepatic levels of MDA Hepatic tissues were homogenized in phosphate-buffered saline (PBS). Malondialdehyde (MDA) was measured using a previously reported method (Hogberg et al. 1974). MDA, which is generated as an end-product of lipid peroxidation (LPO), served as a sensitive index of LPO. MDA reacts with TBA to produce a pinkcolored product with an absorbance at 532 nm. The standard curve for MDA was established within the concentration range of 0–40 ␮mol/L. The LPO level is given in micromoles of MDA per milligram of protein, on the basis of hepatic protein concentration as determined with a Bradford protein assay kit. Measurement of GSH level Hepatic GSH level was assayed by the 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) assay according to a previously reported method (Liang et al. 2011).

Experimental animals Male Kunming (KM) mice (body mass 18–22 g) were obtained from the Laboratory Animal Center of Henan Province (Zhengzhou, China) with the license number SCXK (YU) 2010-0002. Animals were housed 10 per cage under standard conditions (12 h (light) – 12 h (dark) cycle; lights on at 0800 h; 22 ± 1 °C; 60% ± 10% relative humidity) with free access to rodent laboratory chow and water ad libitum. All the procedures were performed in accordance with the published guidelines of the China Council on Animal Care, and approved by the Animal Care Committee of Henan University of Traditional Chinese Medicine.

Measurement of TNF-␣ and IL-10 Liver tissue was obtained as described above and used to determine the levels of inflammatory cytokines using an enzymelinked immunosorbent assay (ELISA). Tumor necrosis factor-alpha (TNF-␣) and interleukin (IL)-10 levels were assayed using commercially available mouse cytokine ELISA kits from R&D Systems (Minneapolis, Minnesota, USA) according to the manufacturer’s instructions. The results are presented in picograms of cytokine per milliliter of protein solution.

Drug and reagents Quercetin (Que), with >98% purity (as analyzed by high-performance liquid chromatography; HPLC) was provided by the Shanghai Jinsui Biotechnology Company (Shanghai, China). Bifendate was obtained from the Zhejiang Medicine Company, Xinchang Pharmaceutical Factory (Xinchang, China). Tripterygium glycoside tablets were purchased from the Shanghai Fudan Forward S&T Company. The Hot Start Fluorescent PCR Core Reagent Kits (SYBR Green I) were obtained from BBI (Kitchener, Waterloo, Ontario,

Enzymatic analysis Tissues were homogenized in cold PBS, and then centrifuged at 5000g for 5 min, and the supernatant was transferred to new tubes for assaying. The liver tissue activities of GST, GPx, CuZn-SOD, and CAT were determined following previously decribed methods (Rotruck et al. 1973; Marklund and Marklund 1974; Habig and Jakoby 1981; Dai et al. 2014), respectively, and the results were calculated based on tissue protein concentrations measured using a Bradford protein assay kit. Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) Wang et al.

3

Fig. 1. Effects of quercetin and bifendate on serum levels of (A) ALT, (B) AST, (C) ALP, and (D) ␥-GT in mice. Data presented are the mean ± SD (n = 10 mice per group). *, P < 0.05 and **, P < 0.01 compared with the normal non-TG treated group; #, P < 0.05 and ##, P < 0.01 compared with the control group treated with TG only; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; ␥-GT, gamma glutamyl transferase; TG, glycosides from Tripterygium wilfordii. 140

A

**

100

**, # #

80

*, # # ##

60 40

AST activity (U/L)

ALT activity (U/L)

**, #

20 0 Normal Control

250

C

150 10 20 Bifendate Quercetin TG (270 mg/kg)

200 180 160 140 120 100 80 60 40 20 0

40 (mg/kg)

120

**, #

200 **, # #

150 ##

100

##

50

B

** **, # *, # # ##

##

Normal Control 140

**

γ-GT activity (U/L)

300

ALP activity (U/L)

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

120

D

150 10 20 Bifendate Quercetin TG (270 mg/kg)

40

** **

100

**, # #

80 60

##

##

40 20

0

0 Normal Control

150 10 20 Bifendate Quercetin TG (270 mg/kg)

40 (mg/kg)

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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The sequences for the PCR primers were as follows: CuZn-SOD, forward 5=-AAGGCCGTGTGCGTGCTGAA-3= and reverse 5=-CAGGTCTCCAACATGCCTCT-3= (246 bp product) (El Mouatassim et al. 1999); CAT, forward 5=-GCAGATACCTGTGAACTGTC-3= and reverse 5=-GTAGAATGTCCGCACCTGAG-3= (229 bp product) (El Mouatassim et al. 1999); and GAPDH, forward 5=GACCCCTTCATTGACCTCAACT-3= and 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 a 40 cycle 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 optical adhesive covers. Relative expression of mRNA (%) = 2⫺⌬CT共1,2兲 × 100%, where CT represents the threshold cycle, ⌬CT1 = CT(CuZn-SOD) – CT(GAPDH), and ⌬CT2 = CT(CAT) – CT(GAPDH).

Normal Control

150 10 20 Bifendate Quercetin TG (270 mg/kg)

40

flects hepatic injury (Eidi et al. 2013). In this research, significantly increased levels of ALT, AST, ALP, and ␥-GT in the mice treated with TG only (all P < 0.01) (Figs. 1A–1D), indicated that we had successfully induced hepatic injury with TG. After i.g. administration of Que (20 or 40 mg/kg) or the positive control bifendate (150 mg/kg), once daily for 7 d, such excessive increases in ALT, AST, ALP, and ␥-GT were significantly inhibited (all P < 0.01) (Figs. 1A– 1D). The administration of 10 mg/kg Que significantly inhibited the increases in ALT, AST, and ALP (all P < 0.05) but not ␥-GT (P > 0.05) (Figs. 1A–1D). These results demonstrate that Que and bifendate protect against the liver injury induced by TG. Effect of Que on hepatic levels of MDA level MDA is considered to be one of the main end-products of LPO (Hogberg et al. 1974). As demonstrated in Fig. 2A, MDA levels increased (P < 0.01) in the hepatic tissues of mice treated with TG only, whereas Que (10, 20, or 40 mg/kg) and bifendate (150 mg/kg) both prevented such excessive increases (all P < 0.01) (Fig. 2A), indicating that Que and bifendate protect against the increased LPO levels associated with hepatic injury induced by TG.

Results

Effect of Que on levels of hepatic glutathione As an antioxidant, glutathione helps to protect cells against ROS such as free radicals and peroxides (Liang et al. 2011). Thus, if the levels of glutathione are significantly reduced, this can result in oxidative stress injury. In this study, the liver levels of glutathione decreased significantly (P < 0.01) in mice treated with TG alone, whereas the administration of Que (20 or 40 mg/kg, but not 10 mg/kg (P > 0.05)) or bifendate (150 mg/kg) significantly reversed such a decrease (all P < 0.01) (Fig. 2B). The results suggest that Que and bifendate prevent TG destroying the balance between cellular oxidants and antioxidants by inhibiting the exhaustion of glutathione levels, and thus probably protect against oxidative stress injury in the liver.

Effect of Que on serum biomarkers for TG-induced liver injury Serum levels of ALT, AST, ALP, and ␥-GT are biomarkers for hepatic injury: if they are significantly elevated, this usually re-

Effect of Que on glutathione-related antioxidant enzyme activity in the liver As intracellular glutathione-related enzymes, GST and GPx interact with glutathione to cause oxidative stress injury (Rotruck

Statistical analysis The presented results are the mean ± SD. The differences among experimental groups were compared by one-way analysis of variance (ANOVA) followed by a Least Significant Difference (LSD) test using the SPSS (Statistics Package for Social Science) program version 11.5. Values for P < 0.05 were considered statistically significant.

Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 4

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

Fig. 2. Effects of quercetin and bifendate on the hepatic levels of (A) MDA and (B) glutathione in mice. Data presented are the mean ± SD (n = 10 mice per group). *, P < 0.05 and **, P < 0.01 compared with the normal non-TG-treated mice; ##, P < 0.01 compared with the control group treated with TG only; MDA, malondialdehyde; TG, glycosides from Tripterygium wilfordii.

et al. 1973; Habig and Jakoby 1981). This study showed that TG significantly reduced hepatic GST and GPx activity in mice (both P < 0.01), whereas Que (20 or 40 mg/kg) and bifendate (150 mg/kg) inhibited this decrease (all P < 0.01) (Figs. 3A and 3B). The administration of 10 mg/kg Que attenuated the significant reduction in GST (P < 0.05) but not GPx (P > 0.05). These findings suggest that GST and GPx participate in the protective actions of Que and bifendate against hepatic oxidative stress injury induced by TG. Our results further confirmed that the balance between cellular oxidants and antioxidants was destroyed by TG, whereas Que and bifendate reversed the damage to this balance. The effects of Que on the activity and mRNA expression of CuZn-SOD and CAT in the liver CuZn-SOD and CAT are both principle antioxidant enzymes in the cell, and are therefore involved in the process of oxidative stress (Aebi 1984; Zelko et al. 2002). Our results showed that TG decreased the activities of CuZn-SOD and CAT in mouse liver (both P < 0.01), whereas Que (20 or 40 mg/kg) and bifendate (150 mg/kg) inhibited this decrease (all P < 0.01) (Fig. 4A and 4B). The administration of 10 mg/kg Que significantly attenuated the reduction in activity for CAT (P < 0.05) but not for CuZn-SOD (P > 0.05). Further, the results (Figs. 4C and 4D) showed that the mRNA expression of CuZn-SOD and CAT in TG-treated mice decreased (both P < 0.01) as compared with the normal (no TG) mice, whereas Que (20 or 40 mg/kg) and bifendate (150 mg/kg) both significantly reversed such decreases in mRNA expression (P < 0.05, P < 0.01, P < 0.01, respectively, for both); however, the administration of 10 mg/kg Que did not (P > 0.05 for both CuZn-SOD and CAT). These results indicate that CuZn-SOD and CAT may play an important role in

Can. J. Physiol. Pharmacol. Vol. 93, 2015

Fig. 3. Effects of quercetin and bifendate on the activity of (A) GST and (B) GPx in the liver. Data presented are the mean ± SD (n = 10 mice per group). *, P < 0.05 and **, P < 0.01 compared with normal non-TG treated group; #, P < 0.05 and ##, P < 0.01 compared with control group treated with TG only; GST, glutathione-S-transferase; GPx, glutathione peroxidase; TG, glycosides from Tripterygium wilfordii.

the protective actions of Que and bifendate against TG-induced hepatic oxidative stress injury. Effects of Que on hepatic levels of TNF-␣ and IL-10 As intracellular inflammatory mediators, TNF-␣ and IL-10 contribute to the process of liver injury (Tilg et al. 2006). Our results indicate that TG significantly increased hepatic levels of the proinflammatory cytokine TNF-␣ (P < 0.01) and decreased levels of the anti-inflammatory cytokine IL-10 (P < 0.01) in mice, whereas Que (20 or 40 mg/kg) and bifendate (150 mg/kg) reversed such changes in the levels of inflammatory mediators (P < 0.05, P < 0.01, P < 0.01, respectively, for both). However, the administration of Que at 10 mg/kg did not (P > 0.05, for both) (Figs. 5A and 5B). These findings suggested that anti-inflammatory properties could be involved in the protective activities of Que and bifendate against TG-induced liver injury.

Discussion Elevation in the levels of ALT, AST, ALP, and ␥-GT are diagnostic indicators for acute liver injury (Eidi et al. 2013). As shown in Fig. 1, TG caused hepatic injury, as evidenced by the increased serum levels of ALT, AST, ALP, and ␥-GT, reflecting early biochemical changes in hepatic disease that were induced with TG, whereas pretreatment with Que at 10, 20, or 40 mg/kg clearly offered protection against hepatic injury induced with TG in mice by attenuating the serum increases in ALT, AST, ALP, and ␥-GT. Oxidative stress in liver cells manifests in an imbalance between oxidants and antioxidants; moreover, the levels of many antioxidant-related enzymes and non-enzymatic antioxidants Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) Wang et al.

5

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

Fig. 4. (A–D) Effects of quercetin and bifendate on the hepatic activity and mRNA expression of the enzymes CuZn-SOD and CAT. Data presented are the mean ± SD (n = 10 mice per group). *, P < 0.05 and **, P < 0.01 compared with normal non-TG treated group; #, P < 0.05 and ##, P < 0.01 compared with the with the control group treated with TG only; CuZn-SOD, copper/zinc superoxide dismutase; CAT, catalase; TG, glycosides from Tripterygium wilfordii.

may be changed during the pathogenesis of DILI (Han et al. 2010; Lucena et al. 2010; Wang et al. 2011; Jaeschke et al. 2012). Among them, LPO is a free-radical-mediated process. MDA, as one of the main end products of LPO, has the characterization of crosslinking cellular macromolecules, including protein or DNA, and causes widespread cellular damage (Hassan et al. 2005). The results in Fig. 2A showed that Que significantly prevented excessive levels of MDA from being induced by TG, suggesting that Que inhibits TG-induced hepatic LPO injury in mice. Glutathione, a non-enzymatic antioxidant, is important in protecting hepatocytes against exogenous toxins, and depletion of cellular glutathione is associated with oxidative injury (Liang et al. 2011). Our results showed that Que significantly inhibited the excessive exhaustion of glutathione induced with TG, indicating that glutathione could be involved in the protection of Que against TG-induced hepatic oxidative injury. GST and GPx are cellular glutathione-associated antioxidant enzymes. Of them, the cytosolic GSTs exist in almost all aerobic species. GSTs catalyze the conjugation of electrophilic compounds formed during the oxidative stress with glutathione (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, Que significantly restored the decreased activities of hepatic GST and GPx induced with TG in mice, which further confirmed that hepatic glutathione-related antioxidant enzymes were involved in the protective actions of Que against TG-induced hepatic oxidative injury. SOD and CAT are both believed to have principal 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), MnSOD (manganese-containing SOD; located in the mitochondrion), and EC-SOD (extracellular SOD; actually, also CuZn-SOD) (Rej

1978). 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). Of all the aerobic cells, CAT mainly exists in the peroxisomes, and serves to protect the cells from damage by hydrogen peroxide through catalyzing it into molecular oxygen and water, without producing toxic free radicals (Bocchetti and Regoli 2006). As peroxisomes are abundant in proteins, which is where oxidative stress always happens, CAT is a classical oxidative biomarker. Our results showed that Que reversed the acutely decreased enzymatic activity and mRNA expression of CuZn-SOD and CAT caused by TG, suggesting that Que exerts protection from acute TGinduced hepatic oxidative stress injury, and that CuZn-SOD and CAT participated in such protection. Cytokines, which are produced by virtually every nucleated cell in the body, are pleiotropic, regulatory peptides, and are present in all types of liver cells (Tilg et al. 2006). The cytokine family includes several subfamilies, such as the interleukins (ILs), the tumor necrosis factor (TNF) family, chemokines, colony-stimulating factors, and others (Tilg et al. 2006). Among the various cytokines, at least 2 different cytokines, from different cytokine families, namely the pro-inflammatory molecule TNF-␣ and the antiinflammatory cytokine IL-10, have emerged as key factors in various aspects of liver disease (Tilg et al. 2006). In the present study in mice, Que significantly reversed the TG-induced increase in the levels of TNF-␣, and reduced IL-10, suggesting that pro- and antiinflammatory cytokines may be involved in the protective effects of Que against TG-induced liver injury. However, whether other key cytokines such as IL-6, which has been reported to modulate the anti-inflammatory properties of Que (Guo et al. 2013), also participate in the protective effects of Que against the liver injury induced by TG, is unknown and is considered to be a limitation of this study. In conclusion, this study shows that Que protects against TGinduced acute liver injury, mainly by up-regulating mRNA of the principle antioxidant enzymes, including CuZn-SOD and CAT, Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 6

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

Fig. 5. Effect of quercetin and bifendate on the hepatic levels of (A) TNF-␣ and (B) IL-10 (inflammation-related cytokines). Data presented are the mean ± SD (n = 10 mice per group). *, P < 0.05 and **, P < 0.01 compared with normal non-TG treated group; #, P < 0.05 and ##, P < 0.01 compared with the control group treated with TG only. TNF-␣, tumor necrosis factor alpha; IL-10, interleukin 10; TG, glycosides from Tripterygium wilfordii.

and regulating the levels of pro- and anti-inflammatory cytokines (e.g., by reducing the level of TNF-␣ and increasing the level IL-10), and thus ameliorates TG-induced liver injury. Further studies are in progress at our laboratory to explore the protection of Que against liver injury induced with carbon tetrachloride and other drugs.

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 Funding Scheme for Young Key Teachers of Colleges and Universities in Henan Province (2014GGJS-072), the National Natural Science Foundation of China (81473368), and the Innovation Program for Science & Technology Leading Talents of Zhengzhou City in China’s Henan Province (121PLJRC534). Conflict of Interest: There authors declare that there is no conflict of interest associated with this work.

References Aebi, H. 1984. Catalse in vitro. Methods Enzymol. 105: 121–126. doi:10.1016/S00766879(84)05016-3. PMID:6727660. Bocchetti, R., and Regoli, F. 2006. Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea. Chemosphere, 65(6): 913–921. doi:10.1016/j.chemosphere.2006.03.049. PMID:16678235. Caddeo, C., Díez-Sales, O., Pons, R., Ferna`ndez-Busquets, X., Fadda, A.M., and Manconi, M. 2014. Topical anti-inflammatory potential of quercetin in lipid-

Can. J. Physiol. Pharmacol. Vol. 93, 2015

based nanosystems: in vivo and in vitro evaluation. Pharm. Res. 31(4): 959–968. doi:10.1007/s11095-013-1215-0. PMID:24297068. Dai, J., Liu, M., Ai, Q., Lin, L., Wu, K., Deng, X., et al. 2014. Involvement of catalase in the protective benefits of metformin in mice with oxidative liver injury. Chem. Biol. Interact. 216: 34–42. doi:10.1016/j.cbi.2014.03.013. PMID:24717679. Eidi, A., Mortazavi, P., Behzadi, K., Rohani, A.H., and Safi, S. 2013. Hepatoprotective effect of manganese chloride against CCl4-induced liver injury in rats. Biol. Trace Elem. Res. 155(2): 267–275. doi:10.1007/s12011-013-9784-7. PMID: 24037643. El Mouatassim, S., Guérin, P., and Ménézo, Y. 1999. Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation. Mol. Hum. Reprod. 5(8): 720–725. doi:10.1093/molehr/5.8.720. PMID:10421798. Girish, C., Koner, B.C., Jayanthi, S., Ramachandra Rao, K., Rajesh, B., and Pradhan, S.C. 2009. Fundam. Clin. Pharmacol. 23(6): 735–745. doi:10.1111/j. 1472-8206.2009.00722.x. PMID:19656205. Guo, X.D., Zhang, D.Y., Gao, X.J., Parry, J., Liu, K., Liu, B.L., et al. 2013. Quercetin and quercetin-3-O-glucuronide are equally effective in ameliorating endothelial insulin resistance through inhibition of reactive oxygen speciesassociated inflammation. Mol. Nutr. Food Res. 57(6): 1037–1045. doi:10.1002/ mnfr.201200569. PMID:23504962. Habig, W.H., and Jakoby, W.B. 1981. Assay for differentiation of glutathione S-transferases. Methods Enzymol. 77: 398–405. doi:10.1016/S0076-6879(81) 77053-8. PMID:7329316. Han, D., Shinohara, M., Ybanez, M.D., Saberi, B., and Kaplowitz, N. 2010. Signal transduction pathways involved in drug-induced liver injury. Handb. Exp. Pharmacol. 196: 267–310. doi:10.1007/978-3-642-00663-0_10. PMID:20020266. Hassan, L., Bueno, P., Ferrón-Celma, I., Ramia, J.M., Garrote, D., Muffak, K., et al. 2005. Time course of antioxidant enzyme activities in liver transplant recipients. Transplant. Proc. 37(9): 3932–3935. doi:10.1016/j.transproceed.2005.10. 088. PMID:16386589. Hogberg, J., Larson, R.E., Kristoferson, A., and Orrenius, S. 1974. NADPHdependent reductase solubilized from microsomes by peroxidation and its activity. Biochem. Biophys. Res. Commun. 56(3): 836–842. doi:10.1016/0006291X(74)90681-0. PMID:4151195. Hougardy, B.M., van der Zee, A.G., van den Heuvel, F.A., Timmer, T., de Vries, E.G., and de Jong, S. 2005. Sensitivity to Fas-mediated apoptosis in high-risk HPV-positive human cervical cancer cells: relationship with Fas, caspase-8, and Bid. Gynecol. Oncol. 97(2): 353–364. doi:10.1016/j.ygyno.2005. 01.036. PMID:15863130. Houghton, P.J., Hylands, P.J., Mensah, A.Y., Hensel, A., and Deters, A.M. 2005. In vitro tests and ethnopharmacological investigations: wound healing as an example. J. Ethnopharmacol. 100(1–2): 100–107. doi:10.1016/j.jep.2005.07.001. PMID:16040217. Jaeschke, H., McGill, M.R., and Ramachandran, A. 2012. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity. Drug Metab. Rev. 44(1): 88–106. doi:10.3109/03602532.2011.602688. PMID:22229890. Li, H.G., Ji, W., and Su, J.M. 2012. Literature research of the hepatotoxicity of glucoside tripterygium total and its synergism and toxicity reducing effects. Zhongguo Zhong Xi Yi Jie He Za Zhi, 32(3): 415–418. [Available from http:// www.cjim.cn/zxyjhcn/zxyjhcn/ch/reader/view_abstract.aspx?file_no= 20120337&flag=1. 22686096]. Li, J., Lu, Y., Xiao, C., Lu, C., Niu, X., He, X., et al. 2011. Comparison of toxic reaction of Tripterygium wilfordii multiglycoside in normal and adjuvant arthritic rats. J. Ethnopharmacol. 135(2): 270–277. doi:10.1016/j.jep.2011.03.007. PMID:21397001. Liang, Q.N., Sheng, Y.C., Jiang, P., Ji, L.L., Xia, Y.Y., Min, Y., et al. 2011. The difference of glutathione antioxidant system in newly weaned and young mice liver and its involvement in isoline-induced hepatotoxicity. Arch. Toxicol. 85(10): 1267–1279. doi:10.1007/s00204-011-0664-7. PMID:21327617. Lin, L.M., and Qi, X.M. 2005. Comparative observation on the effects of Radix Tripterygium hypoglaucum tablet and Tripterygium glycosides tablet in treating erosive oral lichen planus. Chin. J. Integr. Med. 11(2): 149–150. [Available from http://d.wanfangdata.com.cn/Periodical_zgzxyjh-e200502017.aspx]. 16150205. Liu, H., Guo, X., Chu, Y., and Lu, S. 2014. Heart protective effects and mechanism of quercetin preconditioning on anti-myocardial ischemia reperfusion (IR) injuries in rats. Gene, 545(1): 149–155. doi:10.1016/j.gene.2014.04.043. PMID: 24769323. Lucena, M.I., García-Martín, E., Andrade, R.J., Martínez, C., Stephens, C., Ruiz, J.D., et al. 2010. Mitochondrial superoxide dismutase and glutathione peroxidase in idiosyncratic drug-induced liver injury. Hepatology, 52(1): 303– 312. doi:10.1002/hep.23668. PMID:20578157. Marklund, S.L., and Marklund, G. 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47(3): 469–474. doi:10.1111/j.1432-1033.1974. tb03714.x. PMID:4215654. Muriel, P., and Rivera-Espinoza, Y. 2008. Beneficial drugs for liver diseases. J. Appl. Toxicol. 28: 93–103. doi:10.1002/jat.1310. PMID:17966118. Peng, B., Miao, M.S., and Wang, Y.L. 2003. Initial discussion of mice acute hepatic injury caused by Tripterygium glycosides. Zhongguo Zhong Yao Za Zhi, 28(11): 1067–1070. [Available from http://www.cjcmm.com.cn/cjcmmte/ch/reader/ Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 05/13/15 For personal use only.

Wang et al.

create_pdf.aspx?file_no=10489&flag=1&journal_id=cjcmmte&year_id=2003]. 15615419. Peskin, A.V. 1997. Cu,Zn-superoxide dismutase gene dosage and cell resistance to oxidative stress: a review. Biosci. Rep. 17(1): 85–89. doi:10.1023/A: 1027343519591. PMID:9171924. Ray, S.D., Patel, N., Shah, N., Nagori, A., Naqvi, A., and Stohs, S.J. 2006. Preexposure to a novel nutritional mixture containing a series of phytochemicals prevents acetaminophen-induced programmed and unprogrammed cell deaths by enhancing BCL-XL expression and minimizing oxidative stress in the liver. Mol. Cell. Biochem. 293(1–2):119–136. doi:10.1007/s11010-006-9235-2. PMID:16902808. Rej, R. 1978. Aspartate aminotransferase activity and isoenzymes proportions in human liver tissue. Clin. Chem. 24: 1971-1979. [Available from http:// www.clinchem.org/content/24/11/1971.full.pdf]. PMID:213206. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G., and Hoekstra, W.G. 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science, 179(4073): 588–590. doi:10.1126/science.179. 4073.588. PMID:4686466. Song, Q., Lu, J.Z., and Li, J. 2010. Effect of Tripterygium glycosides on serum interleukin-1beta, interleukin-2, tumor necrosis factor alpha, and interferongamma levels in patients with Behcet’s disease. Zhongguo Zhong Xi Yi Jie He Za Zhi, 30(6): 598–600. [Available from http://www.cnki.net/KCMS/detail/ detail.aspx?QueryID=6&CurRec=1&recid=&filename=ZZXJ201006012&dbname= CJFD2010&dbcode=CJFQ&pr=&urlid=&yx=&v=MDY4Njg3RGgxVDNxVHJXTTFG ckNVUkwrZlp1UnFGeXZtVXIzSlB6ZlRaTEc0SDlITXFZOUVab1I4ZVgxTHV4WVM

Quercetin ameliorates liver injury induced with Tripterygium glycosides by reducing oxidative stress and inflammation.

Quercetin (Que) is one of main compounds in Lysimachia christinae Hance (Christina loosestrife), and has both medicinal and nutritional value. Glycosi...
1MB Sizes 1 Downloads 12 Views