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Purple sweet potato color attenuates oxidative stress and inflammatory response induced by d -galactose in mouse liver
Food and Chemical Toxicology 47 (2009) 496–501
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Purple sweet potato color attenuates oxidative stress and inflammatory response induced by D-galactose in mouse liver Zi-Feng Zhang a,1, Shao-Hua Fan a,1, Yuan-Lin Zheng a,*, Jun Lu a,b, Dong-Mei Wu a, Qun Shan a, Bin Hu a a
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Xuzhou Normal University, No. 101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Jiangsu Province, PR China b Institute of Molecular Medicine and Genetics Research Center, School of Basic Medical Science, Southeast University, Nanjing 210009, Jiangsu Province, PR China
a r t i c l e
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Article history: Received 21 May 2008 Accepted 3 December 2008
Keywords: PSPC D-gal Oxidative stress Inflammatory response Mouse liver
a b s t r a c t The hepatoprotective effects of purple sweet potato color (PSPC), which is natural anthocyanin food colors, have been well demonstrated in many studies. Nevertheless, little work has been done to clarify the detailed mechanism of hepatoprotective effects of PSPC. This study was designed to explore whether PSPC protected mouse liver from D-gal-induced injury by attenuating oxidative stress or suppressing inflammation. The histology changes of mouse liver was assessed by hematoxylin and eosin staining. The results showed that PSPC could effectively suppress the D-gal-induced histology changes including structure damage and leucocyte infiltration in mouse liver. Oxidative stress and antioxidant status in mouse liver were also analysed. The results showed that PSPC could largely attenuate the D-gal-induced MDA increasing and could markedly renew the activities of Cu, Zn-SOD, CAT and GPx in the livers of Dgal-treated mice. Furthermore, the results of western blot analysis showed that PSPC could inhibit the upregulation of the expression of NF-jB p65, COX-2 and iNOS caused by D-gal. In conclusion, our data suggested that PSPC could protect the mouse liver from D-gal-induced injury by attenuating lipid peroxidation, renewing the activities of antioxidant enzymes and suppressing inflammatory response. This study provided novel insights into the mechanisms of PSPC in the protection of the liver. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
1. Introduction Anthocyanins, a class of natural occurring polyphenol compounds, are widely distributed in fruits, beans, cereals, vegetables and they are responsible for much of the red, blue, and purple colors in fruits, vegetables, and ornamental crops. Many researches reported that anthocyanins have many potential biological and pharmacology functions, such as anti-oxidative (Shih et al., 2007), anti-inflammatory (Karlsen et al., 2007), and anti-tumor properties (Shih et al., 2005), and its ability to reduce the risk of cardiovascular diseases (Prior and Wu, 2006). Attention is now being focused on purple sweet potato because of its unique color, nutrition and health-promoting benefits (Mano et al., 2007; Goda et al., 1997). There is a high content of anthocyanin pigments in the tuber of some purple sweet potato cultivars. The anthocyanins from purple sweet potato are more stable than Abbreviations: PSPC, purple sweet potato color; D-gal, D-galactose; MDA, lipid peroxidation product malondialdehyde; SOD, superoxide dismutases; CAT, catalase; GPx, glutathione peroxidase; NF-jB, nuclear factor-jB; COX-2, cyclooxygenase-2; iNOS, inducible NO synthase. * Corresponding author. Tel./fax: +86 516 83500348. E-mail addresses: [email protected], [email protected] (Y.L. Zheng). 1 These authors contributed equally to this work.
the pigments of strawberry, red cabbage, perilla and other plants. So purple sweet potatos have been regarded as a good source of stable anthocyanins as a food colorant and purple sweet potato color (PSPC) could be recognized as a physiologically functional food factor. Many authors demonstrated that PSPC exhibited multiple physiological functions, such as antimutagenicity (Yoshimoto et al., 2001) and antihyperglycemic effect (Matsui et al., 2002). Not only in vitro but also in vivo, PSPC showed stronger free radicalscavenging activity than other pigments (red cabbage, grape skin, elderberry and purple corn) and ascorbic acid (Kano et al., 2005; Philpott et al., 2004). The strong antioxidative activity of PSPC also has been reported by many other papers (Konczak-Islam et al., 2003; Suda et al., 2002; Cho et al., 2003). There is increasing evidence that purple sweet potato anthocyanins can protect liver from injury induced by hepatoxins. It was reported that purple sweet potato beverage could decrease the serum levels of c-glutamyl transferase (GGT), aspertate aminotransferase (AST) and alanine aminotransferase (ALT) in healthy men with borderline hepatitis (Suda et al., 2008). In male rats fed a high-cholesterol diet, dark purple sweet potato flakes intake inhibited the increase of hepatic lipid peroxide level, furbished the hepatic glutathione level and renewed the activities of hepatic glutathione reductase and glutathione S-transferase (Han et al., 2007). Purple sweet potato
0278-6915/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.005
Z.-F. Zhang et al. / Food and Chemical Toxicology 47 (2009) 496–501
anthocyanins also reduced glutamic oxaloacetic transaminase (GOT) activity in hepatopathy rats induced by carbon tetrachloride (Kano et al., 2005). It was demonstrated that D-galactose (D-gal) treatment caused oxidative stress in mouse brain and ultimately resulted in neurodegeneration and cognitive dysfunction in mouse (Xu and Zhao, 2002; Cui et al., 2006; Lu et al., 2006, 2007). The D-gal-induced changes resembled accelerated aging in rodents (Li et al., 2005; Ho et al., 2003). The recent researches demonstrated that D-gal treatment caused oxidative stress and mitochondrial dysfunction in the livers of mice and rats (Ho et al., 2003; Ramana et al., 2006; Long et al., 2007). It has been well demonstrated that inflammation is one of a variety of biological phenomena caused by oxidative stress (Dambach et al., 2006). As previously described, many researches indicated that PSPC has hepatoprotective effects. Nevertheless, little work has been done to explore the underlying mechanism of hepatoprotective effects of PSPC. The object of this study was to explore whether PSPC protected mouse liver from D-gal-induced injury by attenuating oxidative stress or suppressing inflammation and to provide novel insights into the mechanisms of PSPC in the protection of the liver.
2. Materials and methods 2.1. Animals and treatments Eight-week-old male Kunming strain mice (30.1 ± 4.6 g; the Branch of National Breeder Center of Rodents, Shanghai, China) were used in the following experiments. The mice were maintained under constant conditions (23 ± 1 °C and 60% humidity) and had free access to rodent food and tap water. Eight mice were housed per cage on a 12-h light/dark schedule (lights on 08:30–20:30). After acclimatization to the laboratory conditions, as previously described (Lu et al., 2006), two groups of mice received daily subcutaneous injection of D-gal (Sigma–Aldrich, MO, USA) at dose of 500 mg/(kg/day) for 8 weeks, and the other two groups with injection of saline (0.9%) only. Then one group of D-gal-treated mice and one group with injection of saline (0.9%) received purple sweet potato color (PSPC; drug purity >90%, Qingdao Pengyuan Natural Pigment Research Institute, Qingdao, China) of 100 mg/(kg/day) in distilled water containing 0.1% Tween-80 (dH2O/0.1% Tween80) by oral gavage for another four weeks. Meanwhile, the other group of D-galtreated mice and the fourth group served as vehicle control were given dH2O/ 0.1% Tween-80 without PSPC. Then mice were sacrificed and the livers were immediately collected, respectively for experiments or stored at 70 °C for later use. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the respective university committees for animal experiments. 2.2. Histological evaluations The mice were perfused transcardially with 25 ml of normal saline (0.9%). The liver tissues were fixed in a fresh solution of 4% paraformaldehyde (pH 7.4) at 4 °C for 24 h, incubated overnight at 4 °C in 100 mM sodium phosphate buffer (pH 7.4) containing 30% sucrose; and embedded in optimal cutting temperature (OCT) compound (Leica, CA, Germany). Cryosections were collected on 3-aminopropyl-trimethoxysilane – coated slides (Sigma–Aldrich). The liver slices were stained with hematoxylin and eosin, and examined by an expert in liver pathology (S.M.) blinded to the type of treatment received by the animals. 2.3. Measurement of lipid peroxidation level Chemicals, including n-butanol, thiobarbutiric acid, 1,1,3,3-tetramethoxypropane and all other reagents, were purchased from Sigma Chemical Company (St. Louis, MO, USA). The level of malondialdehyde (MDA) in liver tissue homogenates was determined using the method of Uchiyama and Mihara (1978). Half a milliliter of each homogenate was mixed with 3 ml of H3PO4 solution (1%, v/v) followed by addition of 1 ml of thiobarbituric acid solution (0.67%, w/v). The mixture was incubated at 95 °C in a water bath for 45 min. The colored complex was extracted into nbutanol, and the absorption at 532 nm was measured using tetramethoxypropane as standard. MDA levels were expressed as nmol per milligram of protein. 2.4. Assay of Cu, Zn-SOD activity Chemicals used in the assay, including xanthine, xanthine oxidase, cytochrome c, bovine serum albumin (BSA) and SOD, were purchased from Sigma Chemical Company (St. Louis, MO, USA). Cu, Zn-SOD activity was measured using the method
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of McCord and Fridovich (1969). Solution A was prepared by mixing 100 ml of 50 mM PBS (pH 7.4) containing 0.1 mM EDTA and 2 lmol of cytochrome c with 10 ml of 0.001 N NaOH solution containing 5 lmol of xanthine. Solution B contained 0.2 U xanthine oxidase/ml and 0.1 mM EDTA. Fifty microliters of a tissue supernatant was mixed with 2.9 ml of solution A and the reaction was started by adding 50 ll of solution B. Change in absorbance at 550 nm was monitored in a spectrophotometer (Shimadzu UV-2501PC, Japan). A blank was run by replacing the supernatant with 50 ll of ultra pure water. Cu, Zn-SOD levels were expressed as units per mg protein with reference to the activity of a standard curve of bovine copper-, zinc-SOD under the same conditions. 2.5. Assay of CAT activity CAT activity was assayed by the method of Aebi (1984). In brief, to a quartz cuvette, 0.65 ml of the phosphate buffer (50 mmol/l; pH 7.0) and 50 ll sample were added, and the reaction was started by addition of 0.3 ml of 30 mM hydrogen peroxide (H2O2). The decomposition of H2O2 was monitored at 240 nm at 25 °C. CAT activity was calculated as nM H2O2 consumed/min/mg of tissue protein. 2.6. Assay of GPx activity The GPx activity assay was based on the method of Paglia and Valentine (1967). tert-Butylhydroperoxide was used as substrate. The assay measures the enzymatic reduction of H2O2 by GPx through consumption of reduced glutathione (GSH) that is restored from oxidized glutathione GSSG in a coupled enzymatic reaction by GR. GR reduces GSSG to GSH using NADPH as a reducing agent. The decrease in absorbance at 340 nm due to NADPH consumption was measured in a Molecular Devices M2 plate reader (Molecular Devices, Menlo. Park, CA). GPx activity was computed using the molar extinction coefficient of 6.22 mM 1 cm 1. One unit of GPx was defined as the amount of enzyme that catalyzed the oxidation of 1.0 lmol of NADPH to NADP+ per minute at 25 °C. 2.7. Protein and Western blot analysis Tissues were homogenized in 3 ml of ice cold RIPA lysis buffer (1 TBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide) combining 30 ll of 10 mg/ml PMSF solution, 30 ll of Na3VO4 and 30 ll of protease inhibitors cocktail per gram of tissue. Homogenates were sonicated four times for 30 s with 20 s intervals using a VWR Bronson Scientific sonicator, centrifuged at 5000g for 10 min at 4 °C, and then collected the supernatants and centrifuged again. The supernatants were collected. Protein levels in the supernatants were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Samples (60 lg each) were separated by denaturing SDS–PAGE and transferred to a PVDF membrane (Roche Diagnostics Corporation, Indianapolis, IN, USA) by electrophoretic transfer (BioRad Laboratories, Inc. USA). The membrane was pre-blocked with 5% non-fat milk and 0.1% Tween-20 in Tris-buffered saline (TBST), incubated overnight with the primary antibody (in TBST with 5% non-fat dried milk). Each membrane was washed three times for 15 min and incubated with the secondary horseradish peroxidaselinked antibodies (Santa Cruz Biotechnology, CA and Cell Signaling Technology, Beverly, MA, respectively). Quantitation of detected bands was performed with the Scion Image analysis software (Scion Co., Frederick, MD, USA). To prove equal loading, the blots were analysed for b-actin expression using an anti-b-actin antibody (Chemicon International Inc., Temecula, CA). Each density was normalized using each corresponding b-actin density as an internal control and averaged from three samples, and we standardized the density of vehicle control for relative comparison as 1.0 to compare other groups. 2.8. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. Lipid peroxidation level, antioxidant enzyme activity and Western blotting data were analyzed with Newman–Keuls or Tukey’s HSD post hoc test. Data were expressed as mean ± S.E.M. Statistical significance was set at P 6 0.05. ***P < 0.001, compared with the control group; ##P < 0.01, ### P < 0.001, vs. D-gal group.
3. Results 3.1. Effects of PSPC on histopathological changes of D-gal-treated mouse liver Liver histological studies were used to determine the protective effect of PSPC on D-gal-induced injury. As shown in Fig. 1, the results of histopathological evaluation showed that PSPC exhibited hepatoprotective effect against D-gal-induced liver injury. D-gal treatment (500 mg/kg/day) caused visible histology changes including structure damage and leucocyte infiltration in mice
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Fig. 1. Effect of PSPC on D-gal-induced histology changes in mouse liver. Representative light micrographs of liver sections from vehicle control mouse (A); mouse treated with PSPC at dose of 100 mg/(kg/day) only (B); mouse treated with D-gal at dose of 500 mg/(kg/day) (C); mouse treated with PSPC at dose of 100 mg/(kg/day) after D-gal injection at dose of 500 mg/(kg/day) (D). The arrow indicates congregated leucocytes and migratory leucocytes. Magnification 100.
livers (Fig. 1C). Whereas, PSPC (100 mg/kg/day) alleviated the liver damage in D-gal-treated mice (500 mg/kg/day), no appearance difference could be observed in the livers between the mice treated with PSPC (100 mg/kg/day) after d-gal injection (500 mg/kg/day) and the vehicle control mice (Fig. 1 D). Compared with normal mice, there were no visible histologic changes in the livers of the mice treated with PSPC only (Fig. 1B). 3.2. Effects of PSPC on lipid peroxidation level in D-gal-treated mouse liver
increase by 130% in hepatic level of MDA as compared with vehicle controls [F(3, 8) = 29.102, P < 0.001]. However, the hepatic MDA content of the mice treated with PSPC after D-gal injection was significant reduced by 52% as compared with D-gal-treated mice [F(3, 8) = 29.102, P < 0.001]. There was no significant difference with regard to the MDA content between the mice treated with PSPC after D-gal injection and the vehicle controls. No significant difference of MDA level could be seen in the livers from the mice treated with PSPC only as compared with vehicle controls. 3.3. Effects of PSPC on antioxidative status of D-gal-treated mouse liver
The results showed that PSPC could decrease D-gal induced lipid peroxidation level (Fig. 2). D-gal-treatment caused significant
Fig. 2. Effect of PSPC on the content of MDA in D-gal-treated mouse liver. Each value is expressed as mean ± S.E.M. ***P < 0.001, compared with the control group; ### P < 0.001, vs. D-gal group.
To determine whether PSPC can attenuate the increased oxidative damages in the livers of D-gal-treated mice, we measured the activities of major antioxidant enzymes, including Cu, Zn-SOD, CAT and GPx, in mouse liver. The results showed that PSPC could renew the activities of these antioxidant enzymes in the livers of D-gal-treated mice (Fig. 3). In D-gal-treated mice, hepatic Cu, Zn-SOD activities significantly decreased by 45% as compared with vehicle controls [F(3, 8) = 61.973, P < 0.001]. Interestingly, the hepatic Cu, Zn-SOD activities of the mice treated with PSPC after D-gal injection significantly increased by 64% as compared with D-gal-treated mice [F(3, 8) = 61.973, P < 0.001]. No significant difference in hepatic Cu, Zn-SOD activities was observed in the mice treated with PSPC after D-gal injection as compared with vehicle controls (Fig. 3A). Hepatic CAT activities significantly decreased by 51% in D-galtreated mice as compared with those in the vehicle controls [F(3, 8) = 21.876, P < 0.001]. In contrast, the treatment of PSPC caused a dramatic increase by 100.3% in hepatic CAT activities of D-gal-treated mice [F(3, 8) = 21.876, P < 0.001]. No significant difference in activities of CAT was observed in the livers between the mice treated with PSPC after D-gal injection and the control mice (Fig. 3B). Hepatic GPx activity levels were similar to Cu, Zn-SOD and CAT activity. Hepatic GPx activities were significantly decreased (62%) by the treatment of D-gal as compared with vehicle controls [F(3, 8) = 56.763, P < 0.001]. However, PSPC markedly increased (164%) the activities of GPx in D-gal-treated mice [F(3, 8) = 56.763, P < 0.001]. There was no significant difference in hepatic
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Fig. 3. Effect of PSPC on the activities of antioxidant enzymes in D-gal-treated mouse liver: (A) effect of PSPC on Cu, Zn-SOD activity in D-gal-treated mouse liver; (B) effect of PSPC on CAT activity in D-gal-treated mouse liver; and (C) effect of PSPC on GPx activity in D-gal-treated mouse liver. All values are expressed as mean ± S.E.M. ***P < 0.001, compared with the control group; ###P < 0.001, vs. D-gal group.
GPx activities between the mice treated with PSPC after D-gal injection and vehicle controls (Fig. 3C). Interestingly, there were no visible changes in hepatic activities of Cu, Zn-SOD, CAT and GPx between the mice treated with PSPC only and the vehicle controls. 3.4. Effects of PSPC on inflammatory response in D-gal-treated mouse liver NF-jB, COX-2 and iNOS were extensively studied in terms of their involvement in the inflammation. The expression levels of
NF-jB p65, COX-2 and iNOS were analysed by western blot in this study. As shown in Fig. 4, PSPC could suppress the D-gal-induced upregulation of the expression of NF-jB p65, COX-2 and iNOS. The expression levels of NF-jB p65 were markedly increased in the livers of D-gal-treated mice as compared with the vehicle controls [F(3, 8) = 56.784, P < 0.001]. However, the upregulation of NF-jB p65 expression was largely suppressed in the mice treated with PSPC after D-gal injection [F(3, 8) = 56.784, P < 0.001]. No significant changes of NF-jB p65 expression were seen in the mice treated with PSPC after D-gal injection as compared with the controls (Fig. 4B).
Fig. 4. Western blot analysis of the proteins in association with inflammation: (A) effect of PSPC on the expression of the proteins in association with inflammation in D-galtreated mouse liver; (B) relative density analysis of the NF-jB protein bands; (C) relative density analysis of the COX-2 protein bands; and (D) relative density analysis of the iNOS protein bands. b-Action was probed as an internal control. The relative density is expressed as the ratio (NF-jB/b-action, COX-2/b-action or iNOS/b-action) and the vehicle control is set as 1.0. Values are averages from three independent experiments. Each value is the mean ± S.E.M. ***P < 0.001, compared with the control group; ## P < 0.01, ###P < 0.001, vs. D-gal group.
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D-gal treatment caused marked upregulation of COX-2 expression in mice livers [F(3, 8) = 52.806, P < 0.001]. Whereas, PSPC largely suppressed the upregulation of COX-2 expression in D-gal-treated mice [F(3, 8) = 52.806, P < 0.001]. There were no significant changes of COX-2 expression between the mice treated with PSPC after D-gal injection and the controls (Fig. 4C). As compared with the vehicle controls, hepatic iNOS expression was markedly upregulated in D-gal-treated mice [F(3, 8) = 19.883, P < 0.001]. In comparison, the upregulation of iNOS expression was largely attenuated in the mice treated with PSPC after D-gal injection [F(3, 8) = 19.883, P < 0.01]. No marked difference of iNOS expression levels was seen in the livers between the mice treated with PSPC after D-gal injection and the controls (Fig. 4D). Interestingly, No visible expression changes of NF-jB p65, COX2 and iNOS were seen in the livers from the mice treated with PSPC only as compared with the controls.
4. Discussion Many hepatoxins (Majano et al., 2005; Kesteloot et al., 2007; Zhou et al., 2005) could induce liver injury, such as oxidative stress, necrosis, inflammation and fibrogenesis, in murines. At high levels, D-gal could cause the metabolism of sugar in disorder and led to the accumulation of reactive oxygen species (ROS), including superoxide, hydroxyl, and hydrogen peroxide (Xu and Zhao, 2002; Lu et al., 2006, 2007; Li et al., 2005). ROS could cause the damages of DNA, proteins and lipids within cells, which resulted in tissue injury. The histological changes of liver, such as structure damage, hepatocellular necrosis, leucocyte infiltration and massive hemorrhage, were observed in hepatoxin-treated mice (Chung et al., 2006; Wang et al., 2007; Zamara et al., 2007). In this research, the results showed that PSPC effectively suppressed the D-gal-induced histopathologic changes including structure damage and leucocyte infiltration in mouse liver (Fig. 1). These results suggested that PSPC could protect mouse liver from D-gal-induced injury. The recent studies showed that severe oxidative stress as indicated by elevated levels of oxidative biomarkers, could be observed in the livers of D-gal-treated rodents (Ho et al., 2003; Ramana et al., 2006). The level of MDA, which indicates the degree of lipid peroxidation, is an oxidative stress marker. It was reported that purple potato extract, which contains high levels of polyphenols, inhibited increase of MDA level induced by D-galactosamine in rat liver (Han et al.,2006a,b). It was also reported that the hepatic lipid peroxidation levels of rats fed with anthocyanin-rich purple potato (Hokkai No. 92) flakes were significantly lower than those in the controls. (Han et al., 2006a,b). These findings indicated that the inhibition of lipid peroxidation was involved in the hepatoprotective effects of polyphenol compounds including anthocyanins from potatos. In our study, PSPC markedly inhibited D-gal-induced increase of MDA content in mouse liver (Fig. 2). Our findings agreed with previous reports (Han et al.,2007a,b) and suggested that anthocyanins from purple sweet potatos could also attenuate oxidative stress by decreasing the lipid peroxide level in D-gal-treated mouse liver. The defense system of antioxidant enzymes contains SOD, CAT, GPx, and so on. It was reported that anthocyanin-rich purple potato flakes and red potato flakes could improve the antioxidant potentials in rats by enhancing hepatic Mn-SOD, Cu, Zn-SOD and GSH-Px mRNA expression (Han et al.,2006a,b; 2007a,b). These findings indicated that anthocyanins from potatos could enhance the expression of antioxidant enzymes in rats livers. Oxidative stress could enhance generation of free radical and could impair antioxidant enzymes. The present study showed that the activities of antioxidant enzymes, including Cu, Zn-SOD, CAT and GPx, in mouse liver were dramatically decreased by the treatment of D-
gal. Interestingly, PSPC could markedly renew the activities of those antioxidant enzymes in the livers of D-gal-treated mice (Fig. 3). Many investigators demonstrated that PSPC could improve the antioxidative activity within cell due to its strong free radicalscavenging activity and the increased level of glutathione (GSH) (Kano et al., 2005; Philpott et al., 2004; Han et al., 2007a,b). Those reports indicated that PSPC could act as antioxidant which could directly scavenge free radical. Our findings further suggested that PSPC could maintain the intracellular redox balance in the livers of D-gal-treated mice by renewing the activities of antioxidant enzymes. In addition to inducing direct cellular damage, oxidative stress could activate transcription factors including NF-jB which regulate the expression of various inflammatory genes implicated in hepatotoxicity (Dambach et al., 2006). NF-jB plays a key role in the process of inflammation. Activated NF-jB translocates to the nucleus and regulates the transcription of response genes encoding inflammation associated enzymes such as COX-2 and iNOS (Uwe, 2008). The present study showed that PSPC largely suppressed the upregulation of NF-jB p65 expression caused by D-gal in mouse liver (Fig. 4B). The results suggested that PSPC could inhibit the initiation of inflammatory response by suppressing NF-jB p65 expression. COX-2-induced production of prostanoids, which is often implicated in inflammatory diseases, can cause edema and tissue injury due to the release of many inflammatory cytokines and chemotactic factors, prostanoids, leukotrienes, and phospholipase (Müller-Decker et al., 2005; Yasuhito et al., 1997). iNOS is associated with both local and systemic inflammatory response (Michel and Feron, 1997). Induced iNOS can consistently release high levels of nitric oxide (NO) and results in deleterious effects in inflammatory processes. The present study showed that PSPC largely attenuated the upregulation of COX-2 and iNOS expression in D-gal-treated mice (Fig. 4C and D). The results implicated that PSPC could attenuate inflammatory processes by suppressing the expression of COX-2 and iNOS. These results suggested that PSPC could alleviate liver injury caused by D-gal through suppressing inflammatory response. As previously described, we observed that PSPC efficaciously suppressed leucocyte infiltration in the livers of D-galtreated mice (Fig. 1). Leucocytes and macrophages respond nonspecifically to foreign substances in immune system. Once tissue damage occurs, leucocytes rapidly migrate to sites of injury initiating an inflammatory response (Laskin and Laskin, 2001). So the results of histological analysis also substantiated that PSPC could suppress inflammatory response induced by D-gal in mouse liver. In conclusion, the results of this study indicated that PSPC has a protective effect against D-gal-induced hepatotoxicity in mice, through attenuating lipid peroxidation, renewing the activities of antioxidant enzymes and alleviating inflammatory response. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work is supported by Foundation for ‘‘863” Project of the Ministry of Science and Technology of PR China (No. 2004AA241180) and the Major Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 07KJA36029), Grants from Key Laboratory of Jiangsu Province, Grants from Qing Lan Project of Jiangsu Province, PR China and Grants from Natural Science Foundation by Xuzhou Normal University (No. 07XLA09).
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Purple sweet potato color attenuates oxidative stress and inflammatory response induced by D-galactose in mouse liver Zi-Feng Zhang a,1, Shao-Hua Fan a,1, Yuan-Lin Zheng a,*, Jun Lu a,b, Dong-Mei Wu a, Qun Shan a, Bin Hu a a
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Xuzhou Normal University, No. 101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Jiangsu Province, PR China b Institute of Molecular Medicine and Genetics Research Center, School of Basic Medical Science, Southeast University, Nanjing 210009, Jiangsu Province, PR China
a r t i c l e
i n f o
Article history: Received 21 May 2008 Accepted 3 December 2008
Keywords: PSPC D-gal Oxidative stress Inflammatory response Mouse liver
a b s t r a c t The hepatoprotective effects of purple sweet potato color (PSPC), which is natural anthocyanin food colors, have been well demonstrated in many studies. Nevertheless, little work has been done to clarify the detailed mechanism of hepatoprotective effects of PSPC. This study was designed to explore whether PSPC protected mouse liver from D-gal-induced injury by attenuating oxidative stress or suppressing inflammation. The histology changes of mouse liver was assessed by hematoxylin and eosin staining. The results showed that PSPC could effectively suppress the D-gal-induced histology changes including structure damage and leucocyte infiltration in mouse liver. Oxidative stress and antioxidant status in mouse liver were also analysed. The results showed that PSPC could largely attenuate the D-gal-induced MDA increasing and could markedly renew the activities of Cu, Zn-SOD, CAT and GPx in the livers of Dgal-treated mice. Furthermore, the results of western blot analysis showed that PSPC could inhibit the upregulation of the expression of NF-jB p65, COX-2 and iNOS caused by D-gal. In conclusion, our data suggested that PSPC could protect the mouse liver from D-gal-induced injury by attenuating lipid peroxidation, renewing the activities of antioxidant enzymes and suppressing inflammatory response. This study provided novel insights into the mechanisms of PSPC in the protection of the liver. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
1. Introduction Anthocyanins, a class of natural occurring polyphenol compounds, are widely distributed in fruits, beans, cereals, vegetables and they are responsible for much of the red, blue, and purple colors in fruits, vegetables, and ornamental crops. Many researches reported that anthocyanins have many potential biological and pharmacology functions, such as anti-oxidative (Shih et al., 2007), anti-inflammatory (Karlsen et al., 2007), and anti-tumor properties (Shih et al., 2005), and its ability to reduce the risk of cardiovascular diseases (Prior and Wu, 2006). Attention is now being focused on purple sweet potato because of its unique color, nutrition and health-promoting benefits (Mano et al., 2007; Goda et al., 1997). There is a high content of anthocyanin pigments in the tuber of some purple sweet potato cultivars. The anthocyanins from purple sweet potato are more stable than Abbreviations: PSPC, purple sweet potato color; D-gal, D-galactose; MDA, lipid peroxidation product malondialdehyde; SOD, superoxide dismutases; CAT, catalase; GPx, glutathione peroxidase; NF-jB, nuclear factor-jB; COX-2, cyclooxygenase-2; iNOS, inducible NO synthase. * Corresponding author. Tel./fax: +86 516 83500348. E-mail addresses: [email protected], [email protected] (Y.L. Zheng). 1 These authors contributed equally to this work.
the pigments of strawberry, red cabbage, perilla and other plants. So purple sweet potatos have been regarded as a good source of stable anthocyanins as a food colorant and purple sweet potato color (PSPC) could be recognized as a physiologically functional food factor. Many authors demonstrated that PSPC exhibited multiple physiological functions, such as antimutagenicity (Yoshimoto et al., 2001) and antihyperglycemic effect (Matsui et al., 2002). Not only in vitro but also in vivo, PSPC showed stronger free radicalscavenging activity than other pigments (red cabbage, grape skin, elderberry and purple corn) and ascorbic acid (Kano et al., 2005; Philpott et al., 2004). The strong antioxidative activity of PSPC also has been reported by many other papers (Konczak-Islam et al., 2003; Suda et al., 2002; Cho et al., 2003). There is increasing evidence that purple sweet potato anthocyanins can protect liver from injury induced by hepatoxins. It was reported that purple sweet potato beverage could decrease the serum levels of c-glutamyl transferase (GGT), aspertate aminotransferase (AST) and alanine aminotransferase (ALT) in healthy men with borderline hepatitis (Suda et al., 2008). In male rats fed a high-cholesterol diet, dark purple sweet potato flakes intake inhibited the increase of hepatic lipid peroxide level, furbished the hepatic glutathione level and renewed the activities of hepatic glutathione reductase and glutathione S-transferase (Han et al., 2007). Purple sweet potato
0278-6915/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.005
Z.-F. Zhang et al. / Food and Chemical Toxicology 47 (2009) 496–501
anthocyanins also reduced glutamic oxaloacetic transaminase (GOT) activity in hepatopathy rats induced by carbon tetrachloride (Kano et al., 2005). It was demonstrated that D-galactose (D-gal) treatment caused oxidative stress in mouse brain and ultimately resulted in neurodegeneration and cognitive dysfunction in mouse (Xu and Zhao, 2002; Cui et al., 2006; Lu et al., 2006, 2007). The D-gal-induced changes resembled accelerated aging in rodents (Li et al., 2005; Ho et al., 2003). The recent researches demonstrated that D-gal treatment caused oxidative stress and mitochondrial dysfunction in the livers of mice and rats (Ho et al., 2003; Ramana et al., 2006; Long et al., 2007). It has been well demonstrated that inflammation is one of a variety of biological phenomena caused by oxidative stress (Dambach et al., 2006). As previously described, many researches indicated that PSPC has hepatoprotective effects. Nevertheless, little work has been done to explore the underlying mechanism of hepatoprotective effects of PSPC. The object of this study was to explore whether PSPC protected mouse liver from D-gal-induced injury by attenuating oxidative stress or suppressing inflammation and to provide novel insights into the mechanisms of PSPC in the protection of the liver.
2. Materials and methods 2.1. Animals and treatments Eight-week-old male Kunming strain mice (30.1 ± 4.6 g; the Branch of National Breeder Center of Rodents, Shanghai, China) were used in the following experiments. The mice were maintained under constant conditions (23 ± 1 °C and 60% humidity) and had free access to rodent food and tap water. Eight mice were housed per cage on a 12-h light/dark schedule (lights on 08:30–20:30). After acclimatization to the laboratory conditions, as previously described (Lu et al., 2006), two groups of mice received daily subcutaneous injection of D-gal (Sigma–Aldrich, MO, USA) at dose of 500 mg/(kg/day) for 8 weeks, and the other two groups with injection of saline (0.9%) only. Then one group of D-gal-treated mice and one group with injection of saline (0.9%) received purple sweet potato color (PSPC; drug purity >90%, Qingdao Pengyuan Natural Pigment Research Institute, Qingdao, China) of 100 mg/(kg/day) in distilled water containing 0.1% Tween-80 (dH2O/0.1% Tween80) by oral gavage for another four weeks. Meanwhile, the other group of D-galtreated mice and the fourth group served as vehicle control were given dH2O/ 0.1% Tween-80 without PSPC. Then mice were sacrificed and the livers were immediately collected, respectively for experiments or stored at 70 °C for later use. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the respective university committees for animal experiments. 2.2. Histological evaluations The mice were perfused transcardially with 25 ml of normal saline (0.9%). The liver tissues were fixed in a fresh solution of 4% paraformaldehyde (pH 7.4) at 4 °C for 24 h, incubated overnight at 4 °C in 100 mM sodium phosphate buffer (pH 7.4) containing 30% sucrose; and embedded in optimal cutting temperature (OCT) compound (Leica, CA, Germany). Cryosections were collected on 3-aminopropyl-trimethoxysilane – coated slides (Sigma–Aldrich). The liver slices were stained with hematoxylin and eosin, and examined by an expert in liver pathology (S.M.) blinded to the type of treatment received by the animals. 2.3. Measurement of lipid peroxidation level Chemicals, including n-butanol, thiobarbutiric acid, 1,1,3,3-tetramethoxypropane and all other reagents, were purchased from Sigma Chemical Company (St. Louis, MO, USA). The level of malondialdehyde (MDA) in liver tissue homogenates was determined using the method of Uchiyama and Mihara (1978). Half a milliliter of each homogenate was mixed with 3 ml of H3PO4 solution (1%, v/v) followed by addition of 1 ml of thiobarbituric acid solution (0.67%, w/v). The mixture was incubated at 95 °C in a water bath for 45 min. The colored complex was extracted into nbutanol, and the absorption at 532 nm was measured using tetramethoxypropane as standard. MDA levels were expressed as nmol per milligram of protein. 2.4. Assay of Cu, Zn-SOD activity Chemicals used in the assay, including xanthine, xanthine oxidase, cytochrome c, bovine serum albumin (BSA) and SOD, were purchased from Sigma Chemical Company (St. Louis, MO, USA). Cu, Zn-SOD activity was measured using the method
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of McCord and Fridovich (1969). Solution A was prepared by mixing 100 ml of 50 mM PBS (pH 7.4) containing 0.1 mM EDTA and 2 lmol of cytochrome c with 10 ml of 0.001 N NaOH solution containing 5 lmol of xanthine. Solution B contained 0.2 U xanthine oxidase/ml and 0.1 mM EDTA. Fifty microliters of a tissue supernatant was mixed with 2.9 ml of solution A and the reaction was started by adding 50 ll of solution B. Change in absorbance at 550 nm was monitored in a spectrophotometer (Shimadzu UV-2501PC, Japan). A blank was run by replacing the supernatant with 50 ll of ultra pure water. Cu, Zn-SOD levels were expressed as units per mg protein with reference to the activity of a standard curve of bovine copper-, zinc-SOD under the same conditions. 2.5. Assay of CAT activity CAT activity was assayed by the method of Aebi (1984). In brief, to a quartz cuvette, 0.65 ml of the phosphate buffer (50 mmol/l; pH 7.0) and 50 ll sample were added, and the reaction was started by addition of 0.3 ml of 30 mM hydrogen peroxide (H2O2). The decomposition of H2O2 was monitored at 240 nm at 25 °C. CAT activity was calculated as nM H2O2 consumed/min/mg of tissue protein. 2.6. Assay of GPx activity The GPx activity assay was based on the method of Paglia and Valentine (1967). tert-Butylhydroperoxide was used as substrate. The assay measures the enzymatic reduction of H2O2 by GPx through consumption of reduced glutathione (GSH) that is restored from oxidized glutathione GSSG in a coupled enzymatic reaction by GR. GR reduces GSSG to GSH using NADPH as a reducing agent. The decrease in absorbance at 340 nm due to NADPH consumption was measured in a Molecular Devices M2 plate reader (Molecular Devices, Menlo. Park, CA). GPx activity was computed using the molar extinction coefficient of 6.22 mM 1 cm 1. One unit of GPx was defined as the amount of enzyme that catalyzed the oxidation of 1.0 lmol of NADPH to NADP+ per minute at 25 °C. 2.7. Protein and Western blot analysis Tissues were homogenized in 3 ml of ice cold RIPA lysis buffer (1 TBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide) combining 30 ll of 10 mg/ml PMSF solution, 30 ll of Na3VO4 and 30 ll of protease inhibitors cocktail per gram of tissue. Homogenates were sonicated four times for 30 s with 20 s intervals using a VWR Bronson Scientific sonicator, centrifuged at 5000g for 10 min at 4 °C, and then collected the supernatants and centrifuged again. The supernatants were collected. Protein levels in the supernatants were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Samples (60 lg each) were separated by denaturing SDS–PAGE and transferred to a PVDF membrane (Roche Diagnostics Corporation, Indianapolis, IN, USA) by electrophoretic transfer (BioRad Laboratories, Inc. USA). The membrane was pre-blocked with 5% non-fat milk and 0.1% Tween-20 in Tris-buffered saline (TBST), incubated overnight with the primary antibody (in TBST with 5% non-fat dried milk). Each membrane was washed three times for 15 min and incubated with the secondary horseradish peroxidaselinked antibodies (Santa Cruz Biotechnology, CA and Cell Signaling Technology, Beverly, MA, respectively). Quantitation of detected bands was performed with the Scion Image analysis software (Scion Co., Frederick, MD, USA). To prove equal loading, the blots were analysed for b-actin expression using an anti-b-actin antibody (Chemicon International Inc., Temecula, CA). Each density was normalized using each corresponding b-actin density as an internal control and averaged from three samples, and we standardized the density of vehicle control for relative comparison as 1.0 to compare other groups. 2.8. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. Lipid peroxidation level, antioxidant enzyme activity and Western blotting data were analyzed with Newman–Keuls or Tukey’s HSD post hoc test. Data were expressed as mean ± S.E.M. Statistical significance was set at P 6 0.05. ***P < 0.001, compared with the control group; ##P < 0.01, ### P < 0.001, vs. D-gal group.
3. Results 3.1. Effects of PSPC on histopathological changes of D-gal-treated mouse liver Liver histological studies were used to determine the protective effect of PSPC on D-gal-induced injury. As shown in Fig. 1, the results of histopathological evaluation showed that PSPC exhibited hepatoprotective effect against D-gal-induced liver injury. D-gal treatment (500 mg/kg/day) caused visible histology changes including structure damage and leucocyte infiltration in mice
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Fig. 1. Effect of PSPC on D-gal-induced histology changes in mouse liver. Representative light micrographs of liver sections from vehicle control mouse (A); mouse treated with PSPC at dose of 100 mg/(kg/day) only (B); mouse treated with D-gal at dose of 500 mg/(kg/day) (C); mouse treated with PSPC at dose of 100 mg/(kg/day) after D-gal injection at dose of 500 mg/(kg/day) (D). The arrow indicates congregated leucocytes and migratory leucocytes. Magnification 100.
livers (Fig. 1C). Whereas, PSPC (100 mg/kg/day) alleviated the liver damage in D-gal-treated mice (500 mg/kg/day), no appearance difference could be observed in the livers between the mice treated with PSPC (100 mg/kg/day) after d-gal injection (500 mg/kg/day) and the vehicle control mice (Fig. 1 D). Compared with normal mice, there were no visible histologic changes in the livers of the mice treated with PSPC only (Fig. 1B). 3.2. Effects of PSPC on lipid peroxidation level in D-gal-treated mouse liver
increase by 130% in hepatic level of MDA as compared with vehicle controls [F(3, 8) = 29.102, P < 0.001]. However, the hepatic MDA content of the mice treated with PSPC after D-gal injection was significant reduced by 52% as compared with D-gal-treated mice [F(3, 8) = 29.102, P < 0.001]. There was no significant difference with regard to the MDA content between the mice treated with PSPC after D-gal injection and the vehicle controls. No significant difference of MDA level could be seen in the livers from the mice treated with PSPC only as compared with vehicle controls. 3.3. Effects of PSPC on antioxidative status of D-gal-treated mouse liver
The results showed that PSPC could decrease D-gal induced lipid peroxidation level (Fig. 2). D-gal-treatment caused significant
Fig. 2. Effect of PSPC on the content of MDA in D-gal-treated mouse liver. Each value is expressed as mean ± S.E.M. ***P < 0.001, compared with the control group; ### P < 0.001, vs. D-gal group.
To determine whether PSPC can attenuate the increased oxidative damages in the livers of D-gal-treated mice, we measured the activities of major antioxidant enzymes, including Cu, Zn-SOD, CAT and GPx, in mouse liver. The results showed that PSPC could renew the activities of these antioxidant enzymes in the livers of D-gal-treated mice (Fig. 3). In D-gal-treated mice, hepatic Cu, Zn-SOD activities significantly decreased by 45% as compared with vehicle controls [F(3, 8) = 61.973, P < 0.001]. Interestingly, the hepatic Cu, Zn-SOD activities of the mice treated with PSPC after D-gal injection significantly increased by 64% as compared with D-gal-treated mice [F(3, 8) = 61.973, P < 0.001]. No significant difference in hepatic Cu, Zn-SOD activities was observed in the mice treated with PSPC after D-gal injection as compared with vehicle controls (Fig. 3A). Hepatic CAT activities significantly decreased by 51% in D-galtreated mice as compared with those in the vehicle controls [F(3, 8) = 21.876, P < 0.001]. In contrast, the treatment of PSPC caused a dramatic increase by 100.3% in hepatic CAT activities of D-gal-treated mice [F(3, 8) = 21.876, P < 0.001]. No significant difference in activities of CAT was observed in the livers between the mice treated with PSPC after D-gal injection and the control mice (Fig. 3B). Hepatic GPx activity levels were similar to Cu, Zn-SOD and CAT activity. Hepatic GPx activities were significantly decreased (62%) by the treatment of D-gal as compared with vehicle controls [F(3, 8) = 56.763, P < 0.001]. However, PSPC markedly increased (164%) the activities of GPx in D-gal-treated mice [F(3, 8) = 56.763, P < 0.001]. There was no significant difference in hepatic
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Fig. 3. Effect of PSPC on the activities of antioxidant enzymes in D-gal-treated mouse liver: (A) effect of PSPC on Cu, Zn-SOD activity in D-gal-treated mouse liver; (B) effect of PSPC on CAT activity in D-gal-treated mouse liver; and (C) effect of PSPC on GPx activity in D-gal-treated mouse liver. All values are expressed as mean ± S.E.M. ***P < 0.001, compared with the control group; ###P < 0.001, vs. D-gal group.
GPx activities between the mice treated with PSPC after D-gal injection and vehicle controls (Fig. 3C). Interestingly, there were no visible changes in hepatic activities of Cu, Zn-SOD, CAT and GPx between the mice treated with PSPC only and the vehicle controls. 3.4. Effects of PSPC on inflammatory response in D-gal-treated mouse liver NF-jB, COX-2 and iNOS were extensively studied in terms of their involvement in the inflammation. The expression levels of
NF-jB p65, COX-2 and iNOS were analysed by western blot in this study. As shown in Fig. 4, PSPC could suppress the D-gal-induced upregulation of the expression of NF-jB p65, COX-2 and iNOS. The expression levels of NF-jB p65 were markedly increased in the livers of D-gal-treated mice as compared with the vehicle controls [F(3, 8) = 56.784, P < 0.001]. However, the upregulation of NF-jB p65 expression was largely suppressed in the mice treated with PSPC after D-gal injection [F(3, 8) = 56.784, P < 0.001]. No significant changes of NF-jB p65 expression were seen in the mice treated with PSPC after D-gal injection as compared with the controls (Fig. 4B).
Fig. 4. Western blot analysis of the proteins in association with inflammation: (A) effect of PSPC on the expression of the proteins in association with inflammation in D-galtreated mouse liver; (B) relative density analysis of the NF-jB protein bands; (C) relative density analysis of the COX-2 protein bands; and (D) relative density analysis of the iNOS protein bands. b-Action was probed as an internal control. The relative density is expressed as the ratio (NF-jB/b-action, COX-2/b-action or iNOS/b-action) and the vehicle control is set as 1.0. Values are averages from three independent experiments. Each value is the mean ± S.E.M. ***P < 0.001, compared with the control group; ## P < 0.01, ###P < 0.001, vs. D-gal group.
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D-gal treatment caused marked upregulation of COX-2 expression in mice livers [F(3, 8) = 52.806, P < 0.001]. Whereas, PSPC largely suppressed the upregulation of COX-2 expression in D-gal-treated mice [F(3, 8) = 52.806, P < 0.001]. There were no significant changes of COX-2 expression between the mice treated with PSPC after D-gal injection and the controls (Fig. 4C). As compared with the vehicle controls, hepatic iNOS expression was markedly upregulated in D-gal-treated mice [F(3, 8) = 19.883, P < 0.001]. In comparison, the upregulation of iNOS expression was largely attenuated in the mice treated with PSPC after D-gal injection [F(3, 8) = 19.883, P < 0.01]. No marked difference of iNOS expression levels was seen in the livers between the mice treated with PSPC after D-gal injection and the controls (Fig. 4D). Interestingly, No visible expression changes of NF-jB p65, COX2 and iNOS were seen in the livers from the mice treated with PSPC only as compared with the controls.
4. Discussion Many hepatoxins (Majano et al., 2005; Kesteloot et al., 2007; Zhou et al., 2005) could induce liver injury, such as oxidative stress, necrosis, inflammation and fibrogenesis, in murines. At high levels, D-gal could cause the metabolism of sugar in disorder and led to the accumulation of reactive oxygen species (ROS), including superoxide, hydroxyl, and hydrogen peroxide (Xu and Zhao, 2002; Lu et al., 2006, 2007; Li et al., 2005). ROS could cause the damages of DNA, proteins and lipids within cells, which resulted in tissue injury. The histological changes of liver, such as structure damage, hepatocellular necrosis, leucocyte infiltration and massive hemorrhage, were observed in hepatoxin-treated mice (Chung et al., 2006; Wang et al., 2007; Zamara et al., 2007). In this research, the results showed that PSPC effectively suppressed the D-gal-induced histopathologic changes including structure damage and leucocyte infiltration in mouse liver (Fig. 1). These results suggested that PSPC could protect mouse liver from D-gal-induced injury. The recent studies showed that severe oxidative stress as indicated by elevated levels of oxidative biomarkers, could be observed in the livers of D-gal-treated rodents (Ho et al., 2003; Ramana et al., 2006). The level of MDA, which indicates the degree of lipid peroxidation, is an oxidative stress marker. It was reported that purple potato extract, which contains high levels of polyphenols, inhibited increase of MDA level induced by D-galactosamine in rat liver (Han et al.,2006a,b). It was also reported that the hepatic lipid peroxidation levels of rats fed with anthocyanin-rich purple potato (Hokkai No. 92) flakes were significantly lower than those in the controls. (Han et al., 2006a,b). These findings indicated that the inhibition of lipid peroxidation was involved in the hepatoprotective effects of polyphenol compounds including anthocyanins from potatos. In our study, PSPC markedly inhibited D-gal-induced increase of MDA content in mouse liver (Fig. 2). Our findings agreed with previous reports (Han et al.,2007a,b) and suggested that anthocyanins from purple sweet potatos could also attenuate oxidative stress by decreasing the lipid peroxide level in D-gal-treated mouse liver. The defense system of antioxidant enzymes contains SOD, CAT, GPx, and so on. It was reported that anthocyanin-rich purple potato flakes and red potato flakes could improve the antioxidant potentials in rats by enhancing hepatic Mn-SOD, Cu, Zn-SOD and GSH-Px mRNA expression (Han et al.,2006a,b; 2007a,b). These findings indicated that anthocyanins from potatos could enhance the expression of antioxidant enzymes in rats livers. Oxidative stress could enhance generation of free radical and could impair antioxidant enzymes. The present study showed that the activities of antioxidant enzymes, including Cu, Zn-SOD, CAT and GPx, in mouse liver were dramatically decreased by the treatment of D-
gal. Interestingly, PSPC could markedly renew the activities of those antioxidant enzymes in the livers of D-gal-treated mice (Fig. 3). Many investigators demonstrated that PSPC could improve the antioxidative activity within cell due to its strong free radicalscavenging activity and the increased level of glutathione (GSH) (Kano et al., 2005; Philpott et al., 2004; Han et al., 2007a,b). Those reports indicated that PSPC could act as antioxidant which could directly scavenge free radical. Our findings further suggested that PSPC could maintain the intracellular redox balance in the livers of D-gal-treated mice by renewing the activities of antioxidant enzymes. In addition to inducing direct cellular damage, oxidative stress could activate transcription factors including NF-jB which regulate the expression of various inflammatory genes implicated in hepatotoxicity (Dambach et al., 2006). NF-jB plays a key role in the process of inflammation. Activated NF-jB translocates to the nucleus and regulates the transcription of response genes encoding inflammation associated enzymes such as COX-2 and iNOS (Uwe, 2008). The present study showed that PSPC largely suppressed the upregulation of NF-jB p65 expression caused by D-gal in mouse liver (Fig. 4B). The results suggested that PSPC could inhibit the initiation of inflammatory response by suppressing NF-jB p65 expression. COX-2-induced production of prostanoids, which is often implicated in inflammatory diseases, can cause edema and tissue injury due to the release of many inflammatory cytokines and chemotactic factors, prostanoids, leukotrienes, and phospholipase (Müller-Decker et al., 2005; Yasuhito et al., 1997). iNOS is associated with both local and systemic inflammatory response (Michel and Feron, 1997). Induced iNOS can consistently release high levels of nitric oxide (NO) and results in deleterious effects in inflammatory processes. The present study showed that PSPC largely attenuated the upregulation of COX-2 and iNOS expression in D-gal-treated mice (Fig. 4C and D). The results implicated that PSPC could attenuate inflammatory processes by suppressing the expression of COX-2 and iNOS. These results suggested that PSPC could alleviate liver injury caused by D-gal through suppressing inflammatory response. As previously described, we observed that PSPC efficaciously suppressed leucocyte infiltration in the livers of D-galtreated mice (Fig. 1). Leucocytes and macrophages respond nonspecifically to foreign substances in immune system. Once tissue damage occurs, leucocytes rapidly migrate to sites of injury initiating an inflammatory response (Laskin and Laskin, 2001). So the results of histological analysis also substantiated that PSPC could suppress inflammatory response induced by D-gal in mouse liver. In conclusion, the results of this study indicated that PSPC has a protective effect against D-gal-induced hepatotoxicity in mice, through attenuating lipid peroxidation, renewing the activities of antioxidant enzymes and alleviating inflammatory response. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work is supported by Foundation for ‘‘863” Project of the Ministry of Science and Technology of PR China (No. 2004AA241180) and the Major Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 07KJA36029), Grants from Key Laboratory of Jiangsu Province, Grants from Qing Lan Project of Jiangsu Province, PR China and Grants from Natural Science Foundation by Xuzhou Normal University (No. 07XLA09).
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