Quercetin Prevents Pyrrolizidine Alkaloid Clivorine-Induced Liver Injury in Mice by Elevating Body Defense Capacity

Quercetin is a plant-derived flavonoid that is widely distributed in nature. The present study is designed to analyze the underlying mechanism in the protection of quercetin against pyrrolizidine alkaloid clivorine-induced acute liver injury in vivo. Serum transaminases, total bilirubin analysis, and liver histological evaluation demonstrated the protection of quercetin against clivorine-induced liver injury. Terminal dUTP nick end-labeling assay demonstrated that quercetin reduced the increased amount of liver apoptotic cells induced by clivorine. Western-blot analysis of caspase-3 showed that quercetin inhibited the cleaved activation of caspase-3 induced by clivorine. Results also showed that quercetin reduced the increase in liver glutathione and lipid peroxidative product malondialdehyde induced by clivorine. Quercetin reduced the enhanced liver immunohistochemical staining for 4-hydroxynonenal induced by clivorine. Results of the Mouse Stress and Toxicity PathwayFinder RT2 Profiler PCR Array demonstrated that the expression of genes related with oxidative or metabolic stress and heat shock was obviously altered after quercetin treatment. Some of the alterations were confirmed by real-time PCR. Our results demonstrated that quercetin prevents clivorine-induced acute liver injury in vivo by inhibiting apoptotic cell death and ameliorating oxidative stress injury. This protection may be caused by the elevation of the body defense capacity induced by quercetin.


Introduction
Pyrrolizidine alkaloid (PA) is a type of natural hepatotoxin and widely distributed in thousands of plants worldwide [1,2]. In Europe, South/North America, Japan, China, etc., PA intoxication of humans and livestocks via the consumption of traditional herbs, tonic prescription, tea, or other PA-contaminated foods, including milk, honey, or meat, have been reported [3]. PAinduced hepatotoxicity has been extensively investigated. However, no antidote has been developed for the PA-induced hepatotoxicity. The US FDA and British Medicines Healthcare Products Regulatory Agency proposed a series of research programs, instructions, and standards to alert people on the hepatotoxicity of PA and its related herbs and remedies.
Clivorine, an otonecine-type PA abundant in Ligularia hodgsonii Hook and Ligularia Dentata, have been generally used to treat cough, hepatitis, and inflammation in traditional Chinese medicine [4,5]. L. hodgsonii Hook and L. Dentata, which belong to genus Ligularia and the family Asteraceae, are native to China and Japan [6]. We already demonstrated in our previous studies the involvement of oxidative stress injury and apoptosis in clivorineinduced hepatotoxicity [7,8]. N-Acetyl-Cysteine and S-adenosyl methionine, which are the precursors for cellular GSH biosyn-thesis, can reverse clivorine-induced hepatotoxicity by increasing cellular GSH level [9,10]. Previous studies have indicated that antioxidant compounds may have potential protective action against PA-induced liver injury.
Flavonoids, a large group of natural polyphenolic compounds, are powerful antioxidants found in various fruits, vegetables, tea, red wine, and medicinal herbs. Flavonoids can scavenge free radicals and other oxidizing intermediates because of their phenolic hydroxyl groups and thus contribute to the counteraction of body against a great variety of diseases [11]. Quercetin (3,39,49,5,7-pentahydroxyflavone) is one of the most abundant flavonoids and widely distributed in nature. Given its well-known antioxidant ability, quercetin exhibits therapeutic potential against various liver injuries caused by toxins, which include carbon tetrachloride, ethanol, thioacetamide, paracetamol, etc. [12][13][14][15]. The present study is designed to analyze the mechanism underlying the protection of quercetin against PA clivorineinduced acute liver injury.

Ethics Statements
All animal experiments were performed according to the protocol approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine (Approved Number: 11002). All mice received humane care, and all efforts were made to minimize suffering.

Chemical compounds and reagents
Clivorine was isolated from L. hodgsonii Hook and identified by IR, NMR, and MS with 99.5% purity [4]. Quercetin was purchased from Sigma Chemical Co. (St. Louis, MO). Terminal dUTP nick end-labeling (TUNEL) staining system (TdT-FragEL DNA Fragmentation Detection Kit) was purchased from Merck Calbiochem (Darmstadt, Germany). The kits for determining serum alanine/aspartate transaminase (ALT/AST) activity, and total bilirubin (TB) level were obtained from the Shanghai Rongsheng Biotech Corporation (Shanghai, China). Anti-caspase-3 antibody was purchased from Cell Signaling Technology (Danver, MA). Peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) was purchased from Jackson ImmunoResearch (West Grove, PA). BCA Protein Assay Kit was purchased from Thermo Scientific (Rockford, IL). Anti-4 Hydroxynonenal (4-NHE) antibody was purchased from Abcam (Cambridge, UK). The DAKO EnVision detection system was purchased from DAKO Corporation (Carpinteria, CA). Trizol reagent was purchased from Life Technology (Carlsbad, CA). PrimeScriptH RT Master Mix and SYBR Premix Ex Taq were purchased from Takara (Shiga, Japan). RT 2 Profiler PCR array was purchased from Qiagen (Hilden, German). All other reagents were purchased from Sigma (St. Louis, MO), unless otherwise indicated.

Experimental animals
Specific pathogen-free male ICR mice (body weight: 16 g to 20 g) were purchased from Shanghai Laboratory Animal Center of Chinese Academy of Science (Shanghai, China). The mice were fed with standard laboratory diet and were given free access to tap water. The animal room was maintained at a temperature of 2261uC with a 12 h light-dark cycle (6:00 to 18:00) and 65%65% humidity.

ALT, AST, and TB Assay
Fresh blood was obtained from mice and put at room temperature for 60 min to clot. After centrifugation at 3 0006g for 15 min, the serum was collected in new tubes. Serum ALT and AST activities, as well as TB level, were determined by kits according to the manufacturer's instructions.

Histological observation
The liver samples were fixed in 10% PBS-formalin and embedded in paraffin. Samples were subsequently sectioned (5 mM), stained with hematoxylin and eosin, and examined under a light microscope (Olympus, Japan) to evaluate the liver damage.

TUNEL assay
Apoptotic hepatocytes were labeled in situ using a TUNEL peroxidase apoptosis detection kit according to the manufacturer's instructions. An inverted microscope (Nikon, Japan) was used to observe apoptotic cells. A dark brown DAB signal indicated positive staining (apoptotic cells). Apoptotic hepatocytes were counted manually in at least three random fields using a light microscope at 1006 magnification.

Western-blot analysis
Liver tissues (approximately 40 mg) were homogenized in icecold lysis buffer containing 50 mM Tris (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 20 mM NaF, 0.5% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 10 mg/mL pepstatin A and centrifuged at 3000 g for 5 min before transferring the supernatant to new tubes. Protein concentrations were analysis by BCA method, and every sample was normalized to the equal protein concentration. Proteins were separated by SDS-PAGE and blots were probed with appropriate combination of primary and HRP-conjugated secondary antibodies.

Measurement of tissue lipid peroxidation (LPO)
LPO in tissues was determined by a previously reported method [16]. Malondialdehyde (MDA) is an end product of LPO and generally serves as an index of the intensity of LPO. MDA reacts with 2-thiobarbituric acid to generate a pink-colored product, which has an absorbance at 532 nm. The tissue MDA level was calculated based on the tissue protein concentration.

Measurement of liver GSH level
Liver GSH and GSSG levels were determined by the 5, 5dithio-bis (2-nitrobenzoic acid) assay according to the reported method described in our previous published paper [17].

Immunohistochemical Analysis of 4-HNE
Paraffin-embedded liver sections (5 mm thickness) were deparaffinized in xylene, and rehydrated in a gradient of ethanol to distilled water. After quenching endogenous peroxidase activity with 3% hydrogen peroxide, tissue sections were incubated with 5% bovine serum albumin to minimize nonspecific binding. Monoclonal 4-HNE antibody was applied to sections at 4uC overnight. The antigen-antibody reactions on sections were detected using DAKO EnVision detection kits. All sections were counterstained with hematoxylin. Images were taken using an inverted microscope (Nikon, Japan). The 4-HNE expression was determined by image information object definition (IOD) values analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD).

RNA isolation and cDNA synthesis
Liver total RNA was isolated using Trizol reagent according to the manufacturer's instruction. The RNA content was determined by measuring the optical density at 260 nm, and cDNA was synthesized according to the manufacturer's instruction described in PrimeScriptH RT Master Mix kits.

RT 2 Profiler PCR array
Mouse Stress and Toxicity PathwayFinder RT 2 Profiler PCR array was performed for quantitative PCR in the ABI 7900 system (Applied Biosystems, USA) with the following cycling conditions: 10 min at 95uC, 15 s at 95uC, and 1 min 60uC for 40 cycles with a final 4uC hold. Five endogenous control genes, namely, glucuronidase b, hypoxanthine guanine, heat-shock protein 90, glyceraldehyde phosphate dehidrogenase, and b-actin, found on the PCR array were used for data normalization. Each replicate cycle threshold (Ct) was normalized to the average Ct value of 5 endogenous controls per plate. Results were calculated using the 2 2DDCt method. Heat map was generated using the web-based program of RT 2 Profiler PCR Array Data Analysis. Variations in the liver gene expression between control and quercetin-treated animals are shown as a fold of increase or decrease.

Real-time PCR analysis
Quantitative real-time PCR was used to further verify the mRNA expression levels of SOD1, SOD2, Hmox1, Hmox2, Fmo5, Ephx2, Polrk2, Cyp2b10, Cyp1b1, Cyp2a5, Cyp2b9, Cyp3a11, Cyp7a1, Hspa1l, Hspe1, Hspa1b, and Dnaja 1. Realtime PCR was performed with STEPONE Plus (Carlsbad, CA, USA) using a SYBR green premix according to the manufacturer's instructions. Relative expression of target genes was normalized to Actin, analyzed by 2 2DDCt method and given as ratio compared with the control. The primer sequences used in this study are shown in Table S1.

Statistical analysis
Data were expressed as means 6 standard error of the mean. The significance of differences between groups was evaluated by one-way ANOVA with LSD post hoc test, and P,0.05 was considered as statistically significant differences.

Quercetin prevents clivorine-induced apoptosis in liver
From the TUNEL assay, no apoptotic cells were found in the liver of the control mice (Fig. 3A). After clivorine administration (210 mg/kg), the liver demonstrated an increased amount of scattered TUNEL-positive apoptotic hepatocytes (Fig. 3B). Ad-ministration of various doses of quercetin reduced the TUNELpositive apoptotic cells enhanced by clivorine (Figs. 3C to E). No apoptotic cells were found in the livers of quercetin (90 mg/kg)treated mice (Fig. 3F). Apoptotic cell count showed that clivorine (210 mg/kg) obviously increased the number of apoptotic cells (P,0.05), while quercetin (40 and 90 mg/kg) decreased APincreased apoptotic cell number (P,0.05) (Fig. 3G). Furthermore, Western-blot results (Fig. 4) showed that clivorine decreased the expression of pro-caspase-3 and increased the expression of cleaved caspase-3, which indicates that clivorine can induce the cleaved activation of caspase-3. Quercetin (60, 90 mg/kg) reversed the decreased expression of pro-caspase-3 and the increased expression of cleaved caspase-3 induced by clivorine. The inhibition of quercetin on the cleaved activation of caspase-3 induced by clivorine further evidences that quercetin can inhibit clivorine-induced liver apoptosis.

Results of mouse stress & toxicity pathway Finder PCR array
The layout of the genes of PCR array is shown in Fig. 7A, and Fig. 7B is a heat map that shows the fold regulation expression data between quercetin (90 mg/kg) and the control group. Numerous red and purple data are shown in Fig. 7B, which indicates that the expression of such genes was up-regulated in the livers of quercetin-treated mice. The differentially expressed genes over 5-folds between control and quercetin-treated group are listed in Table 1. Fmo5, Polr2k, Sod2, Ephx2, Sod1, Hmox2, and Hmox1 belong to the family of oxidative stress. Cyp2b10, Cyp1b1, Cyp2a5, Cyp3a11, and Cyp7a1 belong to the family of metabolic stress. Hspa5, Hspa1l, Hspa1b, Dnaja1, and Hspe1 belong to the family of heat shock.

Results of real-time PCR analysis
Real-time PCR was used to further verify the up-regulated genes in the previous PCR array. In addition, we also observed whether clivorine altered the expression of those above genes and the potential effects of quercetin.
Cyp2b10 expression was highly up-regulated (P,0.05), but Cyp1b1 expression was weakly up-regulated (P,0.05), in querce- tin-treated mice (Fig. 8B). However, no obvious up-regulation was found in Cyp3a11, Cyp2b9, Cyp2a5, and Cyp7a1 expression in quercetin-treated mice, which was inconsistent with the results of the previous PCR array. In addition, clivorine decreased the expression of Cyp2b10, Cyp2b9, Cyp3a11, and Cyp7a1 (P,0.05, P,0.01). After treatment with quercetin (90 mg/kg), the decreased expression of all those above genes was not reversed by quercetin.
Liver Hspa1l, Hspa5, and Dnaja1 expression was up-regulated in quercetin-treated mice (P,0.05) (Fig. 8C), and no evident upregulation was found in the Hspa1b expression in quercetintreated mice. However, Hspe1 expression was down-regulated in quercetin-treated mice (P,0.05), which is inconsistent with the previous PCR array results. In addition, clivorine increased the expression of Hspal1 (P,0.05), and although quercetin has the tendency to decrease the increased Hspal1 expression, but there is no statistical significance (P.0.05).

Discussion
Drug-induced liver injury (DILI) is an important public health problem affecting thousands of people worldwide. Chinese herbal medicine is traditionally considered as having no obvious toxicity and side effect. With the commonly used herbal and dietary supplements, liver toxicity and side effect induced by herbal medicines are concomitantly reported and has aroused widespread concern. PA is a type of natural hepatotoxin distributed in a variety of plants, and PA-containing plants are the most common poisonous plants that affect livestock and humans because of their global distribution. Over 8000 cases of PA-induced hepatic sinusoidal obstruction syndrome (HSOS), previously called hepatic veno-occlusive disease, have been reported globally [18]. However, no strategy has been formulated on the detoxification of PAinduced hepatotoxicity.
Our results demonstrate that quercetin can prevent PA clivorine-induced acute liver injury in mice as indicated by the results from the analysis of serum ALT, AST enzymatic activity, TB level, and histological assessment. Quercetin is a well-known natural antioxidant and a commonly used flavonoid. Several herbal medicines, including Senecio cannabifolius var. integrilifolius and Gynura segetum, contain quercetin [19,20]. These two herbal medicines also contain a considerable amount of PA [18,21]. G. segetum, also called ''Tusanqi'' in Chinese medicine, was reported to be the major causes of HSOS in clinic when PA-containing herbal medicines are consumed [18]. Thus, our results not only provide the potential strategy for the detoxification of PA-induced liver injury, but also evidence for the advantage of Chinese medicines considering that the medicinal plant is entirely consumed but not as individual compounds, which will lead to the possibility of PA detoxification caused by the other compounds contained in Chinese medicines.
The mechanism of the pathogenesis of DILI has been extensively investigated, in which apoptotic cell death, oxidative or metabolic liver injury, and inflammation are reported to be involved in the process of DILI [22][23][24][25]. We found in our previous reports that PA clivorine can induce apoptotic cell death and oxidative stress injury in human normal liver L-02 cells [7,8]. The present results of TUNEL assay and Western-blot analysis of caspase-3 demonstrate that quercetin prevented clivorine-induced apoptotic cell death. Liver MDA levels and immunohistochemical staining of 4-HNE reflect that quercetin can prevent clivorineinduced LPO. GSH is an important endogenous antioxidant, and its homeostasis has been associated with liver injury induced by drugs, alcohol, and other exogenous toxins [26,27]. The present results show that clivorine induced the increase in cellular GSH level, which may be caused by the elevation of the body defense capacity to defend clivorine-induced oxidative stress injury. After quercetin treatment, the increased liver GSH level was reduced, which suggests that oxidative stress injury was ameliorated.
Further, the expression profile of 84 genes included in the Stress and Toxicity PathwayFinder PCR Array demonstrated that oxidative or metabolic stress-related genes, including Fmo5, Polr2k, Sod2, Ephx2, Sod1, Hmox2, Hmox1, Cyp2b10, Cyp1b1, Cyp2a5, Cyp3a11, and Cyp7a1, and heat shock-related genes, including Hspa5, Hspa1l, Hspa1b, Dnaja1, and Hspe1, were upregulated in quercetin-treated mice compared with control. The results indicate the potential involvement of those signals in regulating the protection of quercetin against clivorine-induced liver injury.
The up-regulated expression of Fmo5, Polr2k, Sod2, Ephx2, Sod1, Hmox2, and Hmox1 induced by quercetin was further confirmed by the real-time PCR analysis. In addition, clivorinedecreased expression of Hmox2 and Fmo5 was reversed by quercetin. Sod2, Ephx2, Sod1, Hmox2, Hmox1, and Fmo5 encode critical proteins or antioxidant enzymes involved in regulating cellular redox homeostasis [28][29][30][31]. A previous report has already demonstrated that flavin-dependent monooxygenase (FMO) in arctiid moth Tyria jacobaeae is critical for PA detoxification, and there are five members of FMO (FMO1-5) found in mammals [32]. The present study demonstrates that quercetin increased Fmo5 gene expression, and it also increased clivorinedecreased Fmo5 expression, which may contribute to the detoxification of quercetin against PA clivorine. The results further prove that quercetin can prevent clivorine-induced liver oxidative stress injury by elevating antioxidant capacity.
CYP450, especially members of CYP3A family, is related with the metabolic activation of various PAs such as senecionine, monocrotaline etc [33][34][35][36]. The up-regulated expression of Cyp2b10 and Cyp1b1 induced by quercetin was confirmed by the real-time PCR analysis. In addition, clivorine decreased the mRNA expression of Cyp2b10, Cyp2b9, Cyp3a11, and Cyp7a1, but quercetin had no effect on clivorine-decreased expression of those genes. Our previous studies have demonstrated that clivorine can induce cytotoxicity in human normal liver L-02 cells and kidney HEK293 cells, and those two cells are lack of CYP450 enzymes [4,37], which indicates that metabolic activation is not request for the hepatotoxicity induced by clivorine. In addition, there is no report about the involvement of Cyp2b10 or Cyp1b1 in the metabolic activation of pyrrolizidine alkaloids. Our results indicate that the protection of quercetin against clivorine-induced liver injury may not attribute to the increased expression of Cyp2b10 or Cyp1b1. Under sublethal stress, cellular heat shock proteins will be transiently overproduced and thereby develop tolerance to the stress injury, which is also related with the outcome of stress liver injury [38]. The up-regulated expression of Hspa1l, Hsp5, and Dnaja 1 was further confirmed by real-time PCR analysis. In addition, clivorine also increased the mRNA expression of Hspa1l. The present results indicate that quercetin can increase the capacity of body to defend trauma or stress injury induced by clivorine, and of which Hspa1l may exert important role.

Conclusion
The present study demonstrates that quercetin can prevent PA clivorine-induced acute liver injury in vivo by inhibiting apoptotic cell death and ameliorating oxidative stress injury. Quercetin can enhance the body defense capacity to prevent clivorine-induced hepatotoxicity by regulating the expression of genes related with oxidative homeostasis or heat shock. Our study provides the strategy and reference for the detoxification of PA-induced liver injury.