槲皮素对四氯化碳引起的小鼠急性肝损伤治疗作用及其机理

Therapeutic detoxification of quercetin against carbon tetrachloride-induced acute liver injury in mice and its mechanism 槲皮素对四氯化碳引起的小鼠急性肝损伤治疗作用及其机理 1 2 first-author 1 2 2 2 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:lichenyue1307@126.com †E-mail:lichenyue1307@126.com lichenyue1307@126.com 1 MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China This study observes the therapeutic detoxification of quercetin, a well-known flavonoid, against carbon tetrachloride (CCl 4 ) induced acute liver injury in vivo and explores its mechanism. Quercetin decreased CCl 4 -increased serum activities of alanine and aspartate aminotransferases (ALT/AST) when orally taken 30 min after CCl 4 intoxication. The results of a histological evaluation further evidenced the ability of quercetin to protect against CCl 4 -induced liver injury. Quercetin decreased the CCl 4 -increased malondialdehyde (MDA) and reduced the glutathione (GSH) amounts in the liver. It also reduced the enhanced immunohistochemical staining of the 4-hydroxynonenal (4-HNE) in the liver induced by CCl 4 . Peroxiredoxin (Prx) 1, 2, 3, 5, 6, thioredoxin reductase 1 and 2 (TrxR1/2), thioredoxin 1 and 2 (Trx1/2), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) all play critical roles in maintaining cellular redox homeostasis. Real-time polymerase chain reaction (PCR) results demonstrated that quercetin reversed the decreased mRNA expression of all those genes induced by CCl 4 . In conclusion, our results demonstrate that quercetin ameliorates CCl 4 -induced acute liver injury in vivo via alleviating oxidative stress injuries when orally taken after CCl 4 intoxication. This protection may be caused by the elevation of the antioxidant capacity induced by quercetin. 研究目的 2 本研究旨在观察槲皮素对四氯化碳（CCl 4 ）诱导的肝损伤的治疗解毒作用及其机理。 创新要点 2 首次发现槲皮素对CCl4诱导的肝损伤有治疗作用，并且首次发现Prx和Trx家族参与其中。 研究方法 2 检测小鼠血清转氨酶含量，并检测肝组织中丙二醛（MDA）、谷胱甘肽（GSH）和4-羟基壬烯醛（4-HNE）含量，并用实时聚合酶链式反应（PCR）检测肝组织中Prx1-6、TrxR1/2、Trx1/2、Nrf2和HO-1的mRNA表达情况。 重要结论 2 CCl4造模成功后口服槲皮素对其造成的急性肝损伤有治疗作用，给药组小鼠血清中的转氨酶与模型组相比均有显著下降，通过MDA和免疫组化分析其机理可能和保护氧化应激损伤有关，通过实时PCR分析发现CCl 4 抑制了抗氧化酶Prx家族、TrxRd1、TrxRd2、Trx1、Trx2和Nrf2及其下游HO-1的基因表达，而槲皮素可以逆转CCl 4 降低的这些基因的表达。 Hepatotoxicity Oxidative stress Peroxiredoxin (Prx) Nuclear factor erythroid 2-related factor 2 (Nrf2) TrxR Trx HO-1 肝毒性 氧应激损伤 Prx Nrf2 TrxRd Trx HO-1 Introduction 1 Flavonoids are widely distributed through red wine, fruits, vegetables, herbal tea and medicines. As flavonoids have various pharmacological activities, such as antioxidant, anti-inflammation, and anti-tumor, the nutritional value of flavonoids is well-known to people (Hollman and Katan, 1999 1999 ; Park and Pezzuto, 2012 2012 ). Some epidemiological surveys report that a negative correlation can be observed between dietary intake of flavonoids and cardiovascular disease or cancer (Muldoon and Kritchevsky, 1996 1996 ; Yang et al., 2001 2001 ). However, nutraceuticals, including flavonoids, may also have toxic effects depending on the dose and the redox status of the cell, which is also called as hormesis (Brambilla et al., 2008 2008 ; Calabrese et al., 2010 2010 ). Quercetin is a well-known flavonoid, and it is widely distributed in various Chinese medicinal herbs, such as Flos sophorae Immaturus (Kelly, 2011 2011 ; Wu et al., 2012 2012 ). Various reports show that quercetin has neuro-protection, anti-tumor, anti-inflammation, anti-oxidant, and anti-diabetic effects (Chirumbolo, 2010 2010 ; Kelly, 2011 2011 ; Dajas, 2012 2012 ). Furthermore, some reports demonstrate that quercetin can ameliorate various toxin-induced liver injuries, such as ethanol and acetaminophen (Vicente-Sánchez et al., 2008 2008 ; Liu et al., 2010 2010 ; Yousef et al., 2010 2010 ; de David et al., 2011 2011 ; Domitrović et al., 2012 2012 ). However, the concrete-engaged signals in regulating the protection of quercetin against liver injuries are not very clear. Carbon tetrachloride (CCl 4 ), a classic hepatotoxin, has been widely used in the industry. CCl 4 can enter the body of humans and animals primarily through the respiratory and digestive tracts. Weber et al. ( 2003 2003 ) reported that CCl 4 could lead to a lipid peroxidation of unsaturated fatty membrane and organelle membrane after its metabolic activation by hepatic microsomal cytochrome P450, and thus lead to serious hepatocytes damage. CCl 4 -induced liver injury is one of the most widely used experimental models for exogenous toxins-induced hepatotoxicity (Ratziu and Zelber-Sagi, 2009 2009 ; Chen et al., 2012 2012 ). It have already demonstrated that pretreatment of mice or rats with quercetin for five consecutive days can prevent CCl 4 -induced acute liver injury (Janbaz et al., 2004 2004 ; Domitrović et al., 2012 2012 ; Cui et al., 2014 2014 ). However, whether quercetin can also alleviate CCl 4 -induced acute liver injury, when it is given after intraperitoneal (i.p.) injection of CCl 4 , is unknown. Peroxiredoxins (Prxs) and thioredoxins (Trxs) are important liver antioxidant proteins and play important roles in maintaining liver redox homeostasis (Watson et al., 2004 2004 ; Rhee et al., 2005 2005 ). The present study is designed to observe the therapeutic detoxification of quercetin against CCl 4 -induced liver injury when it is orally given after CCl 4 intoxication, and to further investigate the underlying mechanisms via focusing on Prxs, Trxs, and other antioxidant signals. Materials and methods 1 Drugs and reagents 2 Quercetin was purchased from Sigma (USA). Kits for analysis of alanine and aspartate aminotransferases (ALT/AST) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-4-hydroxynonenal (4-HNE) antibody was purchased from Abcam (Cambridge, UK). The DAKO EnVision™ detection system was purchased from DAKO Corporation (USA). The Trizol reagent was purchased from Life Technology (USA). PrimeScript RT master mix and SYBR Premix Ex Taq™ were all purchased from TaKaRa (Japan). Other reagents, unless noted, were purchased from Sigma (USA). Experimental animals 2 Specific pathogen free male ICR mice (18–22 g) were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Science (Shanghai, China). Animals were fed with a standard laboratory diet and given free access to tap water. The animal room was maintained at (22±1) °C and (65±5)% humidity, with a 12 h:12 h light-dark cycle. The animals received humane care in compliance with the institutional animal care guidelines approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine, China. Treatment of animals 2 Quercetin was dissolved in a sodium hydroxide solution and kept in a dark storage area. The solution was adjusted to pH 9.0. CCl 4 was dissolved in olive oil to a 25% concentration. Mice were randomly divided into four groups with eight mice each. The vehicle group (control) was first injected with olive oil i.p., and 30 min later was orally given a pH 9.0 vehicle solution. The CCl 4 -treated group was injected with CCl 4 (15 ml/kg; i.p.), and 30 min later was orally given a pH 9.0 vehicle solution. The quercetin-treated groups were first injected with CCl 4 solution i.p., and 30 min later were orally administered with quercetin 20 or 80 mg/kg, respectively. Mice were sacrificed 24 h after CCl 4 injection, and plasma and livers were collected. Analysis of ALT/AST activities 2 Fresh blood was obtained from mice in the four groups and put at room temperature for 60 min to clot. Serum was isolated following the centrifugation at 840 g for 15 min. Serum ALT and AST activities were determined by kits according to the manufacturer’s instructions. Histological examination 2 Live slices were fixed in 10% phosphate-buffered saline (PBS)-formalin and embedded in paraffin. Liver samples were subsequently sectioned (5 μm), stained with hematoxylin and eosin (H&E), and examined under a microscope (Olympus, Japan) to evaluate liver injuries. Determination of tissue lipid peroxidation (LPO) 2 Liver tissues were homogenized in cold PBS. LPO was determined according to the method described by Högberg et al. ( 1974 1974 ). MDA was formed as the product of LPO and served as an index for LPO. MDA reacts with 2-thiobarbituric acid to generate a pink-colored product, which has an absorbance at 532 nm. The MDA level was expressed as μmol/mg protein based on liver protein concentration. Determination of liver glutathione (GSH) 2 The amounts of liver GSH and oxidative GSH (GSSG) were measured as described in our previous report (Liang et al., 2011 2011 ). Immunohistochemical staining of 4-HNE 2 Paraffin-embedded liver sections (5 μm) were first deparaffinized in xylene, and then rehydrated in a gradient of ethanol to distilled water. After quenching the endogenous peroxidase activity using 3% hydrogen peroxide, liver sections were further incubated with 5% bovine serum albumin to alleviate nonspecific binding, and then incubated with 4-HNE antibody at 4 °C overnight. The antigen-antibody reactions were detected using DAKO EnVision™ detection kits. All sections were counterstained with hematoxylin. Pictures were taken with a microscope (Nikon, Japan). Liver expression of 4-HNE was determined by image information object definition (IOD) values analyzed by Image-Pro Plus 6.0 (Media Cybernetics, USA). Real-time polymerase chain reaction (PCR) 2 The total liver RNA was extracted by Trizol reagent as described by the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized according to the instruction of the PrimeScript RT master mix kits. Real-time PCR was performed with STEPONE Plus (Carlsbad, CA) by a SYBR green premix according to the manufacturer’s guide. Relative expressions of target genes were standardized to β-actin, evaluated by the 2 −ΔΔ C T method and given as a ratio to control the experiments. The primer sequences used in this study are shown in Table 1 1 . F: forward primer; R: reverse primer Target Primer sequence (5'→3') 2 Prx1 F: CACCCAAGAAACAAGGAGGA R: AAAAAGGCCCCTGAAAGAGA 2 Prx2 F: TGATGAGGGCATTGCTTACAGG R: GAGCGTCCCACAGGTAGGTCAT 2 Prx3 F: TTCCCACTTCAGTCATCTTGCC R: ATGCCAGCACTTTCCAACAGC 2 Prx4 F: TTCCTGTTGCGGACCGAAT R: TACACTTGTCCACCAGCGTAGAA 2 Prx5 F: GGAAGGCGACAGACTTATTATTGG R: CCTTCACTATGCCGTTGTCTATCAC 2 Prx6 F: GCACCACAGAACTTGGCAGAG R: CCAGGCAAGATGATCCTCAACA 2 Trx1 F: TCCCTCCCCGCAACAGCCAA R: ACCGGAGAACTCCCCCACCTTT 2 Trx2 F: TCGCCAAGCAGCACGGGAAG R: CGGTAGGCACAGCTGACACCTCA 2 TrxR1 F: TCGGCTCGCTGAACTGGGG R: GCGATGA GGAACCGCTCTGCTG 2 TrxR2 F: GATCGGTGGGGGATCCGGTG R: CCCACTTGGTGCCTCGGGGAG 2 HO-1 F: TCCCGAACATCGACAGCCCCA R: AGGGGCAGTATCTTGCACCAGG 2 Nrf2 F: TCTCCTCGCTGGAAAAAGAA R: AATGTGCTGGCTGTGCTTTA 2 Actin F: TTCGTTGCCGGTCCACACCC R: GCTTTGCACATGCCGGAGCC Statistical analysis 2 All experimental data were expressed as mean±standard error of the mean (SEM). Significant differences were determined by one-way analysis of variance (ANOVA). P < 0.05 was considered as a statistically significant difference. Results 1 Quercetin ameliorates CCl 4 -induced acute liver injury 2 Serum activities of ALT and AST were increased in the CCl 4 group compared with the control group ( P < 0.001; Fig. 1 1 ). After treatment with quercetin (80 mg/kg), CCl 4 -increased ALT and AST activities were decreased ( P < 0.05 and P < 0.01, respectively). Quercetin (20 mg/kg) had no effect on CCl 4 -increased serum ALT or AST activity. In contrast to the control group, the mice in the CCl 4 group showed serious liver damage, indicated by obvious hepatocyte necrosis and shrinkage nuclei (Fig. 2b 2b ). After treatment with quercetin (20 and 80 mg/kg), these abnormal changes were all alleviated (Figs. 2c and 2d 2c and 2d ). Data are shown as mean±SEM ( n =8). *** P < 0.001 vs. control; # P < 0.05 vs. CCl 4 -treated group (a) Vehicle group; (b) CCl 4 group; (c) CCl 4 +20 mg/kg quercetin group; (d) CCl 4 +80 mg/kg quercetin group Quercetin alleviates CCl 4 -induced liver oxidative injury 2 As the product of LPO, the MDA amount can reflect the liver LPO level (Högberg et al., 1974 1974 ). Fig. 3a 3a shows that the liver MDA amount increased significantly ( P < 0.05) in the CCl 4 -treated mice when compared with the control group. Quercetin (80 mg/kg) reduced the increased MDA amount induced by CCl 4 ( P < 0.05). Quercetin (20 mg/kg) also reduced the increased MDA amount, but it showed no significant difference when compared with the CCl 4 group ( P > 0.05). Data are shown as mean±SEM ( n =8). * P < 0.05 vs. control; # P < 0.05 vs. CCl 4 -treated group GSH is a cellular important antioxidant and it can protect cells against reactive oxygen species (ROS)-induced liver injury (Yuan and Kaplowitz, 2009 2009 ). As shown in Fig. 3b 3b , the liver GSH level in CCl 4 -treated mice was enhanced compared with the control group ( P < 0.05). Quercetin (20 or 80 mg/kg) decreased the CCl 4 -increased liver GSH level ( P < 0.05). In addition, our results showed that the number of 4-HNE-stained hepatocytes increased in the CCl 4 -treated mice (Fig. 4b 4b ). Quercetin (20 or 80 mg/kg) reduced the CCl 4 -increased 4-HNE-stained cells (Figs. 4c and 4d 4c and 4d ). The statistical result in Fig. 4e 4e showed that CCl 4 enhanced the density of the immunohistochemical staining of 4-HNE ( P < 0.001), while quercetin (20 and 80 mg/kg) decreased this increase induced by CCl 4 ( P < 0.001 and P < 0.05, respectively). Typical images selected from each group: (a) vehicle control; (b) CCl 4 ; (c) CCl 4 +20 mg/kg quercetin; (d) CCl 4 +80 mg/kg quercetin. (e) IOD values of the density of 4-HNE staining analyzed in every section of at least three random fields. Data are expressed as mean(SEM ( n =4). *** P < 0.001 vs. control; # P < 0.05, ### P < 0.001 vs. CCl 4 -treated group Effects of quercetin on Prx 1–6 mRNA expression 2 As shown in Fig. 5 5 , the liver mRNA expressions of Prx 1–3, 5, 6 in CCl 4 -treated mice all decreased remarkably compared with the control group ( P < 0.001). After treatment with quercetin (20 mg/kg), the mRNA expressions of Prx 1, Prx 2, Prx 3, Prx 5, and Prx 6 all increased when compared with the CCl 4 -treated mice ( P < 0.01, P < 0.001, P < 0.05, P < 0.01, and P < 0.01, respectively). The mRNA expressions of Prx 1, Prx 2, and Prx 6 in 80 mg/kg quercetin-treated mice also increased when compared with the CCl 4 -treated mice ( P < 0.05, P < 0.05, and P < 0.001, respectively). Data are shown as mean±SEM ( n =5–6). *** P < 0.001 vs. control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. CCl 4 -treated mice Effects of quercetin on Trx1/2 and TrxR1/2 mRNA expression 2 The liver mRNA expressions of other antioxidant enzymes, such as Trx1/2 and TrxR1/2, all decreased in the CCl 4 -treated mice when compared with the control group ( P < 0.01 and P < 0.001, respectively) (Fig. 6 6 ). After treatment with 20 mg/kg quercetin, the mRNA expressions of Trx1/2 and TrxR1/2 were all elevated when compared with the CCl 4 -treated mice ( P < 0.05 and P < 0.001, respectively). After treatment with 80 mg/kg quercetin, the mRNA expression of TrxR2 increased when compared with the CCl 4 -treated mice ( P < 0.05). Data are shown as mean±SEM ( n =5–6). ** P < 0.01, *** P < 0.001 vs. control; # P < 0.05, ### P < 0.001 vs. CCl 4 -treated mice Effects of quercetin on the mRNA expression of HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) 2 The liver mRNA expressions of HO-1 and Nrf2 in CCl 4 -treated mice decreased when compared with the control group ( P < 0.001 and P < 0.05, respectively) (Fig. 7 7 ). After treatment with 20 mg/kg quercetin, the liver mRNA expressions of HO-1 and Nrf2 increased when compared with the CCl 4 -treated mice ( P < 0.01 and P < 0.05, respectively). Data are shown as mean±SEM ( n =5–6). * P < 0.05, *** P < 0.001 vs. control; # P < 0.05, ## P < 0.01 vs. CCl 4 -treated mice Discussion 1 CCl 4 -induced acute liver injury in mice is widely used as an experimental animal model for screening hepato-protective drugs (Morio et al., 2001 2001 ; Yuan et al., 2008 2008 ). Some previous studies have already demonstrated that quercetin can prevent CCl 4 -induced acute liver injury when given several days before CCl 4 intoxication (Janbaz et al., 2004 2004 ; Domitrović et al., 2012 2012 ; Cui et al., 2014 2014 ). From the results of ALT/AST analysis and histological observations, our study demonstrates that quercetin can ameliorate CCl 4 -induced acute liver injury when it is given 30 min after CCl 4 intoxication. Our study shows the potential therapeutic effects of quercetin against CCl 4 -induced acute liver injury, and its scientific significance is far more important than indicated in previous reports. Generally, oxidative stress liver injuries are the main reason for CCl 4 -induced hepatotoxicity. 4-HNE and MDA are both the end-products of LPO (Högberg et al., 1974 1974 ; Vatsyayan et al., 2011 2011 ). Our results of liver MDA level and immunohistochemical staining of 4-HNE show that quercetin can decrease CCl 4 -induced LPO, which indicates that quercetin can ameliorate CCl 4 -induced liver oxidative injuries. GSH is the key antioxidant in liver, and its homeostasis is associated with various toxin-induced liver injuries (Kretzschmar and Klinger, 1990 1990 ; Han et al., 2006 2006 ). Further results show that CCl 4 increased the cellular GSH level, which may contribute to the elevation of the body’s defensive capacity to protect CCl 4 -induced oxidative stress injuries. After treatment with quercetin, the increased liver GSH level was reduced, which suggests that oxidative stress injuries were ameliorated. There are already existing reports that state that quercetin has a free radical scavenging activity (Pascoal et al., 2011 2011 ; di Meo et al., 2013 2013 ), so we can speculate that quercetin itself may have the capacity to scavenge free radicals, and thus substitute for the function of GSH, which leads to a decrease of GSH levels in quercetin-treated mice. The Prx family contains Prx 1–6, which are all antioxidant enzymes and have the capacity of scavenging free radicals in cells (Rhee et al., 2005 2005 ). Previous reports have demonstrated the changed expression of the Prx family in exogenous toxin-induced liver injuries such as alcoholic liver injury and pyrazole-induced liver injury (Cohen et al., 2011 2011 ; Bae et al., 2012 2012 ). In addition, Hong et al. ( 2011 2011 ) reported that an alkaline water named ENA actimineral resource A (ENA-A) could prevent CCl 4 -induced liver injury, and Prx1 is an increased component in the ENA-A group. Our results showed that the expressions of Prx 1–6 mRNAs in the CCl 4 -treated group all decreased when compared with the vehicle group. Except Prx 4, the mRNA expressions of all the other members of the Prx family increased in quercetin-treated mice. Our study suggests that quercetin can ameliorate liver oxidative injuries via elevating the expressions of Prx 1, 2, 3, 5, 6. Trxs, including Trx1 and Trx2, are cellular important antioxidant proteins (Watson et al., 2004 2004 ). Trx1/2 and Trx reductase 1/2 (TrxR1/2) constitute the Trx system, which plays a partially overlapping and highly complementary role to the GSH antioxidant system, and contributes to the maintenance of the cellular reducing environment (Williams et al., 2000 2000 ). Our results show that the mRNA expressions of Trx1/2 and TrxR1/2 decreased in the CCl 4 -treated mice, but this decrease was reversed in the quercetin-treated mice. These results suggest that quercetin can ameliorate liver oxidative injuries via elevating the expression of the Trx system. HO-1 is the inducible isoform of heme oxygenase, and it is reported to protect acute liver injuries (Sass et al., 2012 2012 ). Our results showed that the mRNA expression of HO-1 decreased in the CCl 4 -treated mice, but this decrease was reversed in the quercetin-treated mice. This result suggests that the increased HO-1 expression may contribute to the alleviation of quercetion against CCl 4 -induced liver injury. It is well-known that HO-1/biliverdin reductase axis catalyzes the degradation of heme, and it can also reduce biliverdin to bilirubin, which is a free radical scavenger and exerts cytoprotective effects (Barone et al., 2009 2009 ; Mancuso and Barone, 2009 2009 ; Mancuso et al., 2012 2012 ). Whether quercetin will affect the amounts of liver bilirubin needs further investigation. Nrf2 is a critical transcription factor for regulating the expression of some antioxidant enzymes, such as HO-1, Prxs, and Trx1 (Young et al., 2010 2010 ; Yang et al., 2011 2011 ; Ke et al., 2013 2013 ). Our results show that the mRNA expression of Nrf2 decreased in the CCl 4 -treated mice, but this decrease was reversed in the quercetin-treated mice. The above results suggest that CCl 4 can regulate the expression of Nrf2 and quercetin can ameliorate the liver oxidative injuries via elevating the expression of Nrf2, which may contribute to the increased expression of its downstream antioxidant enzymes. Moreover, the increased expressions of these antioxidant enzymes by quercetin may not provide the appropriate regulation through Nrf2, which needs further investigation. In conclusion, the present study demonstrates the therapeutic detoxification of quercetin against CCl 4 -induced acute liver injury in vivo . 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All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1400104 Project supported by the “Shu Guang” Project from Shanghai Municipal Education Commission and Shanghai Education Development Foundation (No. 13SG43), the National Natural Science Foundation of China (No. 81322053), and the Program for New Century Excellent Talents in University (No. NCET-11-1054), China 2014-04-14 2014-11-14 2014-07-20 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 15